WO2005019471A2 - Periostin-like factor: compositions and methods for making and using the same - Google Patents

Periostin-like factor: compositions and methods for making and using the same Download PDF

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Publication number
WO2005019471A2
WO2005019471A2 PCT/US2004/025969 US2004025969W WO2005019471A2 WO 2005019471 A2 WO2005019471 A2 WO 2005019471A2 US 2004025969 W US2004025969 W US 2004025969W WO 2005019471 A2 WO2005019471 A2 WO 2005019471A2
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plf
seq
periostin
subject
amino acid
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PCT/US2004/025969
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French (fr)
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WO2005019471A3 (en
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Judith Daniels
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Temple University Of The Commonwealth System Of Higher Education
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators

Definitions

  • Periostin is expressed in the periosteum and periodontal ligament in adult mice, suggesting that this molecule is also potentially involved in the maintenance of bone and tooth structure.
  • TGF ⁇ l a growth factor responsible for bone formation
  • EST means expressed sequence tag.
  • FCS means fetal calf serum.
  • FMCM means fetal mouse cardiac myocytes.
  • G3PDH means glycerol-3 -phosphate dehydrogenase.
  • h means human.
  • IFN ⁇ means interferon gamma.
  • IL-l ⁇ means interleukin 1 beta.
  • IVT means in vitro transcription.
  • L means lower percentile.
  • LVAD means left-ventricular assist device.
  • LVEDD means left ventricular end-diastolic dimension.
  • m means mouse or murine.
  • MV means median cell volume.
  • OSF-2 means osteoblast-specific factor 2 (also called Periostin).
  • pc means post conception.
  • PCR means polymerase chain reaction.
  • PDGF platelet-derived growth factor.
  • PEF Periostin-Like Factor.
  • RTD is a differential display technique.
  • RT-PCR means reverse transcriptase polymerase chain reaction.
  • TGF ⁇ means transforming growth factor beta.
  • U means upper percentile.
  • VMHC 1 means ventricle myosin heavy chain- 1.
  • VSMC means vascular smooth muscle cell. Definitions The definitions used in this application are for illustrative purposes and do not limit the scope of the invention.
  • the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.
  • amino acid as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids.
  • Standard amino acid means any of the twenty L-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid residues means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source.
  • synthetic amino acid also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change a peptide's circulating half life without adversely affecting activity of the peptide.
  • amino acid is used interchangeably with “amino acid residue,” and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.
  • Amino acids have the following general structure:
  • Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxy lie (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group.
  • the nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue.
  • an "effective amount” or “therapeutically effective amount” of a compound of the invention or an antibody directed against PLF or a fragment, derivative, or homolog of PLF is an amount sufficient to inhibit progression of a PLF-associated disorder in a subject.
  • An “effective amount” of an inhibitor of PLF is an amount sufficient to inhibit the activity or effect of PLF.
  • a “fragment" of a nucleic acid can be at least about 20 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; preferably at least about 100 to about 500 nucleotides, more preferably at least about 500 to about 1000 nucleotides, even more preferably at least about 1000 nucleotides to about 1500 nucleotides; particularly, preferably at least about 1500 nucleotides to about 2500 nucleotides; most preferably at least about 2500 nucleotides.
  • fragment as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide.
  • a “fragment" of a protein or peptide can be at least about 20 amino acids in length; for example at least about 50 amino acids in length; more preferably at least about 100 amino acids in length, even more preferably at least about 200 amino acids in length, particularly preferably at least about 300 amino acids in length, and most preferably at least about 400 amino acids in length.
  • a "homolog" of PLF includes any nonpurposely generated peptide which, in its entirety or in part, comprises a substantially similar amino acid sequence to SEQ ID NO: 11 (mouse PLF), or SEQ ID NO:27 (human PLF) and has PLF biological activity. Homologs can include paralogs, orthologs, and naturally occurring alleles or variants of PLF.
  • homologous refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position.
  • the activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%.
  • isoform refers to proteins having similar sequences or regions of similar sequences, and includes members of a family which differ due to various processes such as alternative splicing of messenger RNA. "Isolated” means altered or removed from the natural state through the actions of a human being.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a "nucleic acid” refers to a polynucleotide and includes poly- ribonucleotides and poly-deoxyribonucleotides.
  • oligonucleotide typically refers to short polynucleotides of about 50 nucleotides or less in length.
  • oligonucleotide includes both oligomers of ribonucleotide i.e., oligoribonucleotides, and oligomers of deoxyribonucleotide i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides”).
  • oligonucleotide and “oligodeoxynucleotide” include oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or intemucleotide linkages is chemically modified.
  • oligonucleotide is thus understood to also include oligomers which may properly be designated as “oligonucleosides" because of modification of the intemucleotide phosphodiester bond.
  • modified oligonucleotides include, for example, the alkylphosphonate oligonucleosides, discussed below.
  • phosphorothioate oligonucleotide means an oligonucleotide wherein one or more of the intemucleotide linkages is a phosphorothioate group, as opposed to a phosphodiester group which is characteristic of unmodified oligonucleotides.
  • peptide polypeptide
  • protein protein
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence.
  • Periodin refers to a protein originally named
  • Periostin-Like Factor refers to a Periostin isoform containing an amino acid segment having the sequence SEQ ID NO: 14 or SEQ ID NO: 30, but not containing an amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16.
  • a "PLF-associated disease or disorder,” as used herein refers to a disease or disorder in which there is an association between aberrant expression or activity of PLF in a subject and abnormal embryonic development, cell proliferation, cell adhesion, or cell migration.
  • a PLF- associated disorder may include a Periostin-associated disorder.
  • PLF-associated diseases and disorders include cancers, myocardial diseases and disorders, bone diseases and disorders, cell adhesion disorders, cell migration disorders, cell proliferation disorders, embryonic development disorders and other such disorders wherein PLF expression or levels is aberrant.
  • PLF, or fragments, derivatives, or homologs thereof is used interchangeably herein with “PLF polypeptides” and with “PLF proteins.”
  • PLF nucleic acids refers to a DNA or RNA sequence encoding PLF, or a fragment, derivative, or homolog of PLF.
  • “Pharmaceutically acceptable” means physiologically tolerable, for either human or veterinary applications.
  • pharmaceutical compositions include formulations for human and veterinary use.
  • a “preventive” or “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a PLF- associated disorder.
  • a prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with a PLF-associated disorder or Periostin-associated disorder.
  • a “sample,” as used herein, refers to a biological sample from a subject, including normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, or urine.
  • a sample can also be any other source of material obtained from a subject which contains cells or tissue of interest.
  • a "subunit" of a nucleic acid molecule is a nucleotide
  • a “subunit” of a polypeptide is an amino acid.
  • substantially purified refers to a peptide or nucleic acid sequence which is substantially homogenous in character due to the removal of other compounds (e.g., other peptides, nucleic acids, carbohydrates, lipids) or other cells originally present. “Substantially purified” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or formulation into a pharmaceutically acceptable preparation.
  • a "substantially homologous amino acid sequence” includes those amino acid sequences which have at least about 90% homology, preferably at least about 95% homology, more preferably at least about 96% homology, even more preferably at least about 97% homology, even more preferably at least about 98% homology, and most preferably at least about 99% or more homology to an amino acid sequence of a reference peptide chain.
  • Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the present invention.
  • substantially homologous nucleic acid sequence means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur.
  • the substantially similar nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence.
  • the percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more.
  • nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm.
  • Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; preferably in 7% (SDS), 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C; preferably 7% SDS, 0.5 M NaPO 4 , 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; and more preferably in 7% SDS, 0.5 M NaPO 4 , 1 mM
  • Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package (Devereux et al., 1984 Nucl. Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al., J. Mol. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res. 25:3389- 3402). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the present invention.
  • treat refers to administering a PLF, an inhibitor or stimulator of PLF activity, antibodies, agents, or compounds to reduce the frequency with which the effects or symptoms of a PLF associated disorder are experienced, to reduce the severity of symptoms, or to prevent effects or symptoms from occurring.
  • Treatment can restore the effect of PLF function or activity which has been lost or diminished in a PLF associated disorder.
  • Treatment can also include inhibiting or reducing PLF function or activity where PLF function or activity has increased in a PLF associated disorder.
  • FIGURES IA to 1C comprise an amino acid sequence alignment and comparison of mouse PLF (SEQ ID NO: 10) and human PLF (partial sequence; (SEQ ID NO:27) to the amino acid sequences of a mouse Periostin protein (mPeriostin/OSF-2; GenBank Accession Nos. BC031449 and AAH31449; SEQ ID NO), mouse Periostin cloned by "Takeshita” (derived from Takeshita et al., Biochem J. 294:271-8, 1993; GenBank Accession Nos.
  • Takeshita derived from Takeshita et al., Biochem J. 294:271-8, 1993; GenBank Accession Nos.
  • FIGURES ID and IE depict a sequence alignment in which the amino acid residues (810; SEQ ID NO: 10) and nucleic acid residue coding region (SEQ ID NO:l 1) of mouse PLF are aligned.
  • FIGURE IF is a schematic comparing the predicted amino acid sequence of mouse PLF (PLF; 810 amino acids, 2430 bases) (upper) with mouse Periostin (811 amino acids, 2433 bases) (lower).
  • FIGURE 2A is a northern blot analysis of PLF expression during mouse embryonic development.
  • FIGURE 3 A is a northern blot analysis of the developmental regulation of PLF expression in mice. Upper panel- Total RNA from whole mouse embryos, days 9.5 pc. to day 18.5 pc, was probed with P-labeled PLF cDNA probe. Lower panel- the ethidium bromide stained formaldehyde-denatured gel of the RNA probed for PLF expression in the upper panel.
  • FIGURE 3B is a graphic representation of a densitometric analysis of the northern blot of FIGURE 3 A. Blots were stripped and re-probed with P- labeled 18S cDNA as a control for loading and transfer.
  • FIGURE 4 A demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in the uterine wall (day 8.5) (40X magnification). The area enclosed in a box is shown at higher magnification in FIGURE 4B.
  • FIGURE 4B demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in the uterine wall (day 8.5) (200X magnification).
  • FIGURE 4C demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in the uterine wall (day 9.5) (40X magnification).
  • FIGURE 4D demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis (day 10.5) in the uterine wall (20X magnification).
  • FIGURE 4E demonstrates by in situ hybridization the spatial expression of PLF mRNA in the atrial wall during early mouse embryogenesis (day 12.5) (400X magnification).
  • FIGURE 4F demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in a whole embryo at day 10.5 post-conception.
  • FIGURE 5 A demonstrates by in situ hybridization in a 16.5 day post- conception mouse embryo that PLF is expressed in bone and heart (20X magnification). The area demarcated by the box on the left is further magnified in FIG. 5C.
  • FIG. 5F The area demarcated by the box on the right is further magnified in FIG. 5F.
  • FIGURE 5B demonstrates PLF expression by in situ hybridization in the atrial wall of a 16.5 day post-conception mouse embryo (400X magnification).
  • AL atrial lumen.
  • FIGURE 5C demonstrates PLF expression by in situ hybridization in the region of developing vertebrae outlined in the left (dorsal) box of FIGURE 5A, (100X magnification).
  • C cartilage.
  • FIGURE 5D demonstrates PLF expression by in situ hybridization in developing pre-osteoblasts, but not chondrocytes, in a 16.5 day embryonic mouse vertebra (400X magnification).
  • Preosteoblasts migrating away from the body of the vertebra, to form the vertebral processes are indicated by arrowheads.
  • C cartilage.
  • FIGURE 5E demonstrates PLF expression by in situ hybridization in preosteoblasts, but not chondrocytes, of a presumptive rib (400X).
  • C cartilage.
  • FIGURE 5F demonstrates PLF expression by in situ hybridization of the region shown in the right box (ventral) of FIGURE 5A, at a higher magnification (400X). Cells indicated by the arrows in 5F are preosteoblasts migrating from the cartilaginous rib into the sternum.
  • C cartilage.
  • FIGURE 6 A demonstrates by in situ hybridization PLF expression in the mesenchymal primordia of the head region of a 16.5 day post-conception mouse embryo (20X magnification). The asterisk is located at the tip of the tongue (T). The region outlined by the box is shown at higher magnification in FIGURE 6D.
  • FIGURE 6B demonstrates by in situ hybridization PLF expression in the upper and lower jaws and the tongue of a 16.5 day mouse embryo (40X magnification). The arrow indicates a region of the hard palate shown at higher magnification in FIGURE 6C. The region outlined by the box is shown at a higher magnification in FIGURE 6E.
  • FIGURE 6C demonstrates by in situ hybridization PLF expression in pre-osteoblasts in the hard palate (arrow) and presumptive taste buds in the tongue (arrow head).
  • FIGURE 6C is a higher magnification of the image marked by an arrow in FIGURE 6B.
  • FIGURE 6D demonstrates by in situ hybridization PLF expression in pre-osteoblasts in the hard palate (arrow) and presumptive taste buds in the tongue (arrow head) in cells in the region comprising the transition from the hard palate to the soft palate (100X magnification).
  • FIGURE 6D is a higher magnification of the image outlined by the box in FIGURE 6 A.
  • FIGURE 6E is the region outlined by the box in FIGURE 6B, shown at a higher magnification, demonstrating PLF expression in the upper and lower jaws and the tongue of a 16.5 day mouse embryo (100X magnification).
  • FIGURE 7A is an in situ hybridization analysis of PLF expression in neonatal mouse heart tissue and is a sagittal section of the neonatal mouse heart shown at low magnification. The region enclosed in the box is shown at a higher magnification in FIG. 7B.
  • FIGURE 7B is an in situ hybridization analysis of PLF expression in neonatal mouse heart tissue. The image is the region enclosed in the box in FIG. 7B, shown at a higher magnification (400X).
  • FIGURE 8 A is a northern blot analysis of PLF expression in a series of normal human hearts.
  • the upper panel represents PLF expression and the lower panel depicts the same blot stripped and reprobed with an 18S probe.
  • FIGURE 8B is a northern blot analysis of PLF expression in a series of idiopathic human hearts.
  • the upper panel represents PLF expression and the lower panel depicts the same blot stripped and reprobed with an 18S probe.
  • FIGURE 8C is a northern blot analysis of PLF expression in a series of ischemic human hearts.
  • the upper panel represents PLF expression and the lower panel depicts the same blot stripped and reprobed with an 18S probe.
  • FIGURE 8D is a graph demonstrating the PLF/18S expression ratios in normal human heart (columns 1-6), idiopathic human heart (columns 7-13), and ischemic human hearts (columns 14-20).
  • FIGURE 8E is a spotfire visualization graph depicting the average mean expression intensity value (ordinate) versus tissue sample set (abscissa) of PLF mRNA examined via hybridization of total neonatal mouse heart mRNA to the human HU_95 (60K) gene chip set.
  • FIGURE 8F is a graph demonstrating a non-paired analysis of PLF mRNA expression in LVAD-treated human patients.
  • FIGURE 10A demonstrates, by in situ hybridization, PLF expression in the periosteum and endosteum of long bone in 2-week old neonatal mutant osteopetrotic rats (20X magnification). The region enclosed by the box is depicted at a higher magnification in FIG. 10B. H + zone of hypertrophic cells.
  • FIGURE 10B demonstrates, by in situ hybridization, PLF expression in the periosteum and endosteum of long bone in 2-week old neonatal mutant osteopetrotic rats (100X magnification).
  • FIGURE 12C is a photomicrograph of an immunohistochemical analysis of PLF protein expression in idiopathic human heart (1,000X). The tissue was stained with anti-PLF antiserum.
  • FIGURE 12D is a western blot analysis of PLF expression in mouse embryos. Proteins extracts from 13.5 day embryonic calvaria, 15.5 day embryonic long bone, 17.5 day embryonic long bone, 19.5 day embryonic long bone, and 2-day-old neonatal long bones were subjected to 8% SDS- PAGE. The proteins were transferred to nitrocellulose and probed with anti- PLF antiserum. The inset shows an image of the 15.5 day lane at a higher exposure where a fourth isoform is visible.
  • FIGURE 13 represents an RT-PCR analysis of PLF in bone, heart, spleen, lung, and brain embryonic tissue at day 13.5 post-conception, to detect different isoforms of PLFs.
  • the left panel utilized primers flanking the 672- 700 amino acid region.
  • the right panel utilized primers flanking the 785-813 amino acid region.
  • Upper and lower tissue specific bands are indicated by arrows.
  • FIGURE 14 is a northern blot analysis of PLF mRNA expression in fetal mouse cardiac myocytes treated with antisense PLF oligonucleotides to determine whether PLF expression was needed for differentiation.
  • FIGURE 15A, 15B, and 15C are micrographs of stage 4, stage 5, and stage 7 chicken embryos, respectively, treated with antisense oligonucleotide to PLF and photographed 24 hours post-treatment.
  • FIGURE 15D is a micrograph of the embryo of FIG. 15C subjected to in situ hybridization for expression of the ventricle specific marker VMHC1.
  • FIGURE 15E, 15F, 15G, and 15H are micrographs of a chicken embryos treated with antisense oligonucleotide against PLF at stage 8 and subjected to in situ hybridization for VMHCl expression 24 hours post- treatment.
  • FIGURE 151 is a micrograph of a chicken embryo treated with antisense oligonucleotide against PLF at stage 9 and subjected to in situ hybridization for VMHC 1 expression 24 hours post-treatment.
  • FIGURE 15 J is a micrograph of a control chicken embryo treated with DMSO, but not an antisense oligonucleotide against PLF, at stage 7 and subjected to in situ hybridization for VMHCl expression 24 hours post- treatment.
  • FIGURE 15K is a micrograph of a stage 4 chicken embryo at the time of treatment with antisense oligonucleotide against PLF (zero hours post- treatment).
  • FIGURE 15L is a graph summarizing the anomalies found in the studies described in FIGS.
  • FIGURE 16 is an RT-PCR analysis of PLF expression in MC3T3-E1 osteoblast cells treated with antisense oligonucleotide against PLF. PLF expression was compared to G3PDH expression, a gene not regulated by differentiation.
  • FIGURE 17 is an RT-PCR analysis of differentiation marker expression in MC3T3-E1 osteoblast cells treated with antisense oligonucleotide against PLF.
  • the differentiation markers include osteopontin, osteocalcin, collagen I, AL-PH, and Cbfa 1.
  • AS 1 antisense oligonucleotide 1;
  • AS2 antisense oligonucleotide 2.
  • FIGURE 18 is an RT-PCR analysis of differentiation marker gene expression in MC3T3-E1 osteoblast cells treated with anti-PLF antibody transfected into cells using CHARIOT. Cells were transfected with an antibody/Chariot mix and RT-PCR analysis was performed 7 and 21 days post-transfection.
  • the differentiation markers include osteopontin, osteocalcin, collagen I, and AL-PH.
  • D7 7 days post-treatment;
  • D21 21 days post-treatment.
  • the present invention is based, in part, on the discovery and characterization of a nucleic acid sequence which encodes a protein which appears to be a member of the Periostin family.
  • Periostin proteins are involved in various cellular functions, including cell differentiation, cell migration, cell adhesion, and metastases.
  • This new protein called "Periostin- Like Factor” or "PLF” herein, uniquely encodes a 27 amino acid segment not present in mouse or human Periostin.
  • PLF also lacks a 28 amino acid segment, which is present in human and mouse Periostin.
  • amino acid sequence of human PLF (SEQ ID NO:27), comprising amino acid residue positions 669-831 (FIG. IA to 1C), as with mouse PLF, contains a 27 amino acid segment not present in Periostin, and does not contain a 28 amino acid segment which is present in Periostin.
  • primer oligonucleotides are provided for cloning
  • Linking groups suitable for use in the present invention include, for example, cyclic compounds capable of connecting an amino- terminal portion and a carboxyl terminal portion of SEQ ID NOS: 11 or 27. Techniques for generating derivatives are also described in U.S. patent 6,030,942 the entire disclosure of which is herein incorporated by reference (derivatives are designated "peptoids" in the 6,030,942 patent). Examples of derivatives according to the present invention include, for example, synthetic variants of PLF. PLF derivatives also include fusion peptides in which a portion of the fusion peptide has a substantially similar amino acid sequence to SEQ ID NOS: 11 or 27.
  • Such fusion peptides can be generated by techniques well-known in the art, for example by subcloning nucleic acid sequences encoding SEQ ID NOS: 11 or 27 and a heterologous peptide sequence into the same expression vector, such that the PLF and the heterologous sequence are expressed together in the same protein.
  • the heterologous sequence can also comprise a peptide leader sequence that directs entry of the expressed protein into a cell.
  • leader sequences include "protein transduction domains" or "PTDs,” which are discussed in more detail below.
  • PLF key structural elements of PLF can be identified, for example, by evaluating the various portions of PLF for the ability to stimulate or inhibit genes expressed during cardiac muscle cell differentiation, osteoblast differentiation, or to inhibit normal embryogenesis as measured by cell migration and heart development (see Examples 8-11 below).
  • PLF key structural elements can be determined using nuclear magnetic resonance (NMR), crystallographic, and/or computational methods which permit the electron density, electrostatic charges or molecular structure of certain portions of PLF or fragments thereof to be mapped.
  • PLF key structural elements comprise the primary, secondary and tertiary structure of the amino acid sequence of SEQ ID NOS:l 1 and 27.
  • the compounds of the invention which comprise polypeptides can be synthesized de novo using conventional solid phase synthesis methods.
  • the peptide chain is prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
  • N-protecting groups e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group
  • various coupling reagents e.g., dicyclohexylcarbodiimide or carbonyldiimidazole
  • various active esters e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide
  • the various cleavage reagents e.g., trifluoroactetic acid (TFA), HCI in dioxane, boron tris-(trifluoracetate) and cyanogen bromide
  • reaction in solution with isolation and purification of intermediates are methods well- known to those of ordinary skill in the art.
  • a preferred peptide synthesis method follows conventional Merrifield solid phase procedures well known to those skilled in the art. Additional information about solid phase synthesis procedures can be had by reference to Steward and Young, Solid Phase Peptide Synthesis, W.H. Freeman & Co., San Francisco, 1969; the review chapter by Merrifield in Advances in Enzymology 32:221-296, F.F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins 2:61-64 (1990), the entire disclosures of which are incorporated herein by reference. Crude peptide preparations resulting from solid phase syntheses may be purified by methods well known in the art, such as preparative HPLC.
  • the amino-terminus may be protected according to the methods described for example by Yang et al., (1990 FEBS Lett 272:61-64), the entire disclosure of which is herein incorporated by reference.
  • the compounds of the invention which comprise PLF peptides can also be produced by biological synthesis. Biological synthesis of peptides is well known in the art, and includes the transcription and translation of a synthetic nucleic acid encoding a PLF protein, or a fragment, derivative, or homolog of PLF.
  • Bio syntheses of PLF, or fragments, derivatives, or homologs thereof can be based on the mouse PLF nucleic acid sequence (SEQ ID NO:4) or amino acid sequence (SEQ ID NO: 11), or on the human PLF nucleic acid sequence (SEQ ID NO:24) or amino acid sequence ( SEQ ID NO:27).
  • the techniques of recombinant DNA technology are within the skill in the art.
  • PLF and fragments, derivatives, and homologs thereof can be prepared utilizing recombinant DNA techniques, which can comprise combining a nucleic acid encoding the peptide in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the peptide produced by the resulting host cell, and purifying the polypeptide recovered.
  • the nucleic acids encoding PLF peptides may be operatively linked to one or more promoter and/or regulatory regions. Regulatory regions include promoters, polyadenylation signals, translation initiation signals (Kozak regions), termination codons, peptide cleavage sites, and enhancers.
  • the regulatory sequences used must be functional within the cells into which they are transfected. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Suitable promoters include both constitutive promoters and regulated (inducible) promoters, and can be prokaryotic or eukaryotic, depending on the host.
  • prokaryotic (including bacteriophage) promoters useful for practice of this invention are: lac, T3, T7, lambda Pr' PI' and tip promoters.
  • eukaryotic (including viral) promoters useful for practice of this invention are: ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue- specific promoters (e.g. actin promoter in smooth muscle cells), promoters which respond to a stimulus (e.g.
  • Suitable polyadenylation signals that can be used in the present invention include SV40 polyadenylation signals and LTR polyadenylation signals.
  • the compounds of the invention can be modified with other substances prior to use in the present methods, using techniques known in the art.
  • the compounds of the invention can be modified with a label (e.g., substances which are magnetic resonance active; radiodense; fluorescent; radioactive; detectable by ultrasound; detectable by visible, infrared or ultraviolet light).
  • a label e.g., substances which are magnetic resonance active; radiodense; fluorescent; radioactive; detectable by ultrasound; detectable by visible, infrared or ultraviolet light.
  • Suitable labels include, for example, fluorescein isothiocyanate, peptide chromophores such as phycoerythrin or phycocyanin and the like; bioluminescent peptides such as the luciferases originating from Photinus pyrali; or fluorescent proteins originating from Renilla reniformi.
  • the compounds of the invention which comprise peptides can also be cyclized via cysteine-cysteine linkages, which is known to enhance the biological activities of a variety of peptides.
  • the compounds of the invention can be derivatized with functional groups or linked to other molecules to facilitate their delivery to specific sites of action or to potentiate their activity.
  • the compounds of the invention can also be covalently or non-covalently linked to other pharmaceuticals, bioactive agents, or other molecules. Such derivatizations should not significantly interfere with the ubiquitin ligase or other biological properties of the compounds.
  • Carriers and derivatizations of the compounds of the invention should also be designed or chosen so as not to exert toxic or undesirable activities on animals or humans treated with these formulations.
  • PLF and fragments, derivatives, and homologs thereof, as well as antibodies against PLF
  • the compounds of the invention can be encapsulated in a liposome prior to being administered.
  • the encapsulated compounds are delivered directly into the abnormally proliferating cells by fusion of the liposome to the cell membrane.
  • Reagents and techniques for encapsulating the present compounds in liposomes are well known in the art, and include, for example, the ProVectinTM Protein Delivery Reagent from Imgenex.
  • the peptide compounds of the invention are modified by associating the compounds with a peptide leader sequence known as a "protein transduction domain” or "PTD.”
  • PTD protein transduction domain
  • These sequences direct entry of the compound into abnormally proliferating cells by a process known as “protein transduction” (Schwarze et al., 1999, Science 285:1569-1572).
  • PTDs are well-known in the art, and can comprise any of the known PTD sequences including, for example, arginine-rich sequences such as a peptide of nine to eleven arginine residues optionally in combination with one to two lysines or glutamines as described in Guis et al. (1999, Cancer Res.
  • sequences of eleven arginine residues or the NH 2 -terminal 11 - amino acid protein transduction domain from the human immunodeficiency vims TAT protein are preferred.
  • Other suitable leader sequences include, but are not limited to, other arginine-rich sequences; e.g., 9 to 10 arginines, or six or more arginines in combination with one or more lysines or glutamines.
  • Such leader sequences are known in the art; see, e.g., Guis et al. (1999), supra.
  • the PTD is designed so that it is cleaved from the compound upon entry into the cell.
  • a PLF associated disorder is characterized by decreased levels of PLF nucleic acid, PLF protein, or PLF protein activity.
  • This disorder is treated by administering to a subject an isolated PLF protein, or an isolated nucleic acid comprising a nucleic acid sequence encoding a PLF protein, either alone or in combination with other compounds.
  • the PLF protein completely or partially corrects the PLF associated disorder.
  • PLF protein is used to increase PLF proteins levels in PLF associated disorders where there are reduced levels of PLF nucleic acid, PLF protein, or PLF protein activity.
  • Periostins play a role in cell adhesion and migration, and loss of PLF disrupts cell adhesion and migration.
  • the invention provides a method of treating a PLF-associated disorder in a subject in need of such treatment.
  • the method comprises administering an effective amount of a PLF protein, or fragment, derivative, or homolog analog thereof, to the subject, such that biological processes which have been inhibited because of the PLF associated disorder are restored.
  • a PLF-associated disorder is characterized by increased levels of PLF nucleic acid, PLF protein or PLF protein activity.
  • Such disorders include idiopathic heart disease, ischemic heart disease, and osteopetrosis (see Examples 3 and 4).
  • This disorder is treated by administering to a subject an antisense oligonucleotide directed against a PLF nucleic acid, a nucleic acid comprising a nucleic acid sequence encoding an antisense oligonucleotide complementary to a PLF nucleic acid sequence, or an antibody directed against PLF.
  • PLF can be applied as a coating to devices such as stents to encourage migration of endothelial, smooth muscle, or other cells. The effect of treatment can be monitored using many cellular, molecular, and clinical techniques, which are known to those of ordinary skill in the art.
  • the assay is designed to measure the ability of a compound of the invention to stimulate cell adhesion or migration
  • assays are known in the art which can be used to measure cell adhesion or migration in vitro and in vivo.
  • Other methods useful for measuring cell adhesion or migration are known to those of skill in the art (also see Example 9).
  • the number of cells associated with a PLF associated disorder in a subject's body can be determined by direct measurement, or by estimation from the size of primary or metastatic tumor masses.
  • the number of cells associated with a PLF associated disorder in a subject can be readily determined by immunohistological methods, flow cytometry, or other techniques designed to detect the characteristic surface markers of a given cell type.
  • a PLF associated disorder is cancer, or another disorder capable of being subjected imaging techniques.
  • the size of a tumor mass can be ascertained by direct visual observation, or by diagnostic imaging methods such as X-ray, magnetic resonance imaging, ultrasound, and scintigraphy. Diagnostic imaging methods used to ascertain size of the tumor mass can be employed with or without contrast agents, as is known in the art.
  • the size of a tumor mass can also be ascertained by physical means, such as palpation of the tissue mass or measurement of the tissue mass with a measuring instrument such as a caliper. For prostate tumors, a preferred physical means for determining the size of a tumor mass is the digital rectal exam.
  • the PLF associated disorder is a bone disorder.
  • an effective amount of a compound of the invention or an antibody directed against PLF can be based on the approximate weight of a tumor mass to be treated.
  • the approximate weight of a tumor mass can be determined by calculating the approximate volume of the mass, wherein one cubic centimeter of volume is roughly equivalent to one gram.
  • An effective amount of the compounds of the invention based on the weight of a tumor mass can be at least about 10 micrograms/gram of tumor mass. More preferably, the effective amount is at least about 100 micrograms/gram of tumor mass. Particularly preferably, the effective amount is at least about 500 micrograms/gram of tumor mass. It is preferred that an effective amount based on the weight of the tumor mass be injected directly into the tumor.
  • an effective amount of the compounds of the invention or an antibody directed against PLF can also be based on the approximate or estimated body weight of a subject to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described below.
  • an effective amount of the nucleic acids of the invention administered to a subject can range from about 5-500 ⁇ g/kg of body weight, or between about 500-1000 ⁇ g/kg of body weight, or is greater than about 1000 ⁇ g/kg of body weight.
  • dosages of PLF protein, or fragments, derivatives, or homologs of PLF, or antibodies directed against PLF protein, or fragments, derivatives, or homologs of PLF are between about 0.001 mg/kg and about 100 mg/kg body weight.
  • dosages are between about 0.01 mg/kg and about 60 mg/kg body weight. In other embodiments, dosages are between about 0.05 mg/kg and about 5 mg/kg body weight.
  • an effective amount of a compound of the invention or antibody directed against PLF can be administered to the subject once (e.g., as a single injection or deposition).
  • the compounds of the invention can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days.
  • the compounds of the invention are administered once a day for seven days.
  • the effective amount can comprise the total amount of compound or antibody directed against PLF administered over the entire dosage regimen.
  • the compounds of the invention and antibodies directed against PLF can be administered to a subject by any means suitable for delivering the compounds to cells of the subject, for example by any suitable enteral or parenteral administration route.
  • Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g.
  • Transfection methods for eukaryotic cells include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.
  • cells can be transfected with a liposomal transfer compound, e.g., DOTAP (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methylsulfate, Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN.
  • DOTAP N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methylsulfate, Boehringer-Mannheim
  • nucleic acid used is not critical to the practice of the invention; acceptable results can be achieved with 0.1-100 micrograms of nucleic acid/10 5 cells. For example, a ratio of about 0.5 micrograms of plasmid vector in 3 micrograms of DOTAP per 10 5 cells can be used.
  • a nucleic acid comprising sequences encoding PLF, or a fragment, derivative, or homolog of PLF, can be obtained using a number of standard techniques. Such nucleic acids can, for example, be chemically synthesized or recombinantly produced using methods known in the art as described above.
  • the nucleic acid sequence of mouse PLF cDNAs is provided herein (SEQ ID NOS:4 and 13).
  • sequences are merely different lengths (3012 bases and 3290 base, respectively) and each includes the entire region (SEQ ID NO:12) encoding the amino acid sequence (SEQ ID NO:l 1) of mouse PLF. More than one nucleic acid sequence is capable of encoding a particular amino acid sequence. Degenerate sequences are degenerate within the meaning of the genetic code in that nucleotides can be replaced by other nucleotides in some instances without resulting in a change of the amino acid sequence originally encoded. Nucleic acid sequences comprising sequences encoding PLF protein, or fragments, derivatives, or homologs of PLF can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • Suitable promoters for expressing nucleic acid sequences from a plasmid include the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids suitable for use in the present invention can also comprise inducible or regulatable promoters for expression of nucleic acids in cells associated with a PLF associated disorder. Selection of plasmids suitable for expressing the PLF nucleic acid, methods for inserting nucleic acid sequences for expressing the PLF nucleic acid into the plasmid, and methods of delivering the recombinant plasmid to cells associated with a PLF associated disorder are within the skill in the art.
  • the recombinant viral vectors of the invention can comprise any suitable promoter for expressing the nucleic acid sequences in cells associated with a PLF associated disorder.
  • suitable promoters include, for example, the
  • Suitable AAV vectors, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61 :3096-3101; Fisher K. J. et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
  • Liposomes may also be used to deliver antisense oligonucleotides complementary to a PLF nucleic acid sequence. Liposomes can also increase the blood half-life of the nucleic acids.
  • the compounds of the invention, or nucleic acids comprising sequences encoding a PLF protein or fragment, derivative, or homolog of PLF are encapsulated in liposomes prior to administration to the subject.
  • Liposomes suitable for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol such as cholesterol.
  • Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable.
  • the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide.
  • the opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups.
  • the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof.
  • Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes.”
  • the opsonization-inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques.
  • an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl- ethanolamine lipid-soluble anchor, and then bound to a membrane.
  • a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive animation using Na(CN)BH 3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60°C.
  • Liposomes modified with opsonization-inhibiting moieties remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth” liposomes.
  • Stealth liposomes are known to accumulate in tissues fed by porous or "leaky” micro vasculature. Thus, tissue characterized by such microvasculature defects, for example solid tumors, will efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18:6949-53.
  • the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation of the liposomes in the liver and spleen.
  • the cells of the subject are transfected by administering an isolated nucleic acid comprising sequences which encode PLF or fragments, derivatives, or homologs of PLF, and a plasmid expression vector to the subject.
  • the cells being transfected have been isolated from the subject.
  • the cells are reimplanted to purge or displace remaining PLF associated disorder cells or to purge cells predisposed to developing a PLF associated disorder.
  • the compounds and antibodies of the invention can be administered to a subject by any means suitable for delivering the compounds to cells of the subject, for example by any suitable enteral or parenteral administration route.
  • Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery.
  • Suitable parenteral administration routes include intravascular administration (e.g.
  • intravenous bolus injection intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature
  • peri- and intra-tissue injection e.g., peri- tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection
  • subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation.
  • the compounds of the invention are administered by injection or infusion.
  • compounds of the invention are delivered locally to the site of the disorder.
  • the isolated nucleic acid comprising sequences encoding the PLF protein, or fragment, derivative or homolog of the PLF sequence is preferably administered by direct injection into the tumor.
  • an effective amount of an isolated nucleic acid comprising a sequence encoding a PLF protein, or fragments, derivatives, or homologs thereof, to be administered to a given subject by taking into account factors such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic.
  • a preferred method of delivering the isolated nucleic acid to the cells associated with a PLF associated disorder is by transfection.
  • the present invention provides antisense oligonucleotides directed against nucleic acids encoding PLF.
  • the antisense oligonucleotides of the present invention can be in the form of RNA or DNA, e.g., cDNA, genomic DNA, or synthetic RNA or DNA.
  • the antisense oligonucleotides can be used as primers in sequencing experiments, or they can be generated for use in dismpting functions because they bind to a specific nucleic acid sequence and block its function.
  • the antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonucleotide synthesis methods, including methods to generate more stable and efficient oligonucleotides. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology, VCH Verlagsgesellschaft mbH (H. Ibelgaufts trans. 1987). Any of the known methods of oligonucleotide synthesis may be utilized in preparing the instant antisense oligonucleotides.
  • antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers, for example, the Applied Biosystems 380B DNA Synthesizer. Because the nucleotide sequences of DNA complementary to mouse and human PLF mRNA transcripts are described herein, antisense oligonucleotides hybridizable with any portion of these mRNA transcripts may be prepared by the oligonucleotide synthesis methods known to those skilled in the art.
  • one of the characteristics that distinguishes PLF from other Periostins is a deletion in the mRNA of the nucleic acid sequence of Periostin encoding the peptide region comprising amino acids 785-812 (see FIGS. IA to 1C and Examples).
  • a novel sequence arises when the deletion occurs and amino acid residue 784 of Periostin is followed by the amino acid residue previously at position 813 (based on the alignment as shown in FIGS. IA to 1C).
  • the 27 amino acid residue segment (SEQ ID NO: 14) contained at position 673 of PLF, is novel relative to those Periostins which do not have the fragment.
  • antisense oligonucleotides can be directed against a nucleic acid sequence which spans some nucleic acid residues from the insert to the common sequences both 5' and 3' to the nucleic acid encoding the inserted fragment.
  • Such an antisense can be prepared which shares little homology with nucleic acid sequences which are not PLF nucleic acid sequences.
  • the following oligodeoxynucleotides are complementary to the PLF mRNA transcript: SEQ ID NOS :21-23.
  • the invention also includes antisense oligonucleotides complementary to the region of spliced PLF mRNA where the nucleic acid sequence encoding the peptide fragment having the sequence of amino acid residues 785-812 of Periostin has been deleted.
  • an oligonucleotide is prepared to span the site where a splice occurs. Oligomers of 8-40 nucleotides are preferred.
  • the invention includes antisense oligonucleotides to the nucleic acid sequence encoding the peptide inserted between amino acids 672 and 700 of Periostin to form PLF.
  • alkyl phosphonates includes but is not limited to the ethyl or methyl phosphonate analogs disclosed by U.S. Pat. No. 4,469,863.
  • Non-ionic oligonucleotides are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences.
  • the alkylphosphonates in particular are stable to nuclease cleavage and soluble in lipid.
  • the preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Pat. No. 4,469,863.
  • oligodeoxyribonucleotides While PLF mRNA translation can be inhibited by administering either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred over oligoribonucleotides in the practice of the present invention.
  • the antisense oligonucleotides of the present invention will have a sequence which is completely complementary to the target portion of a PLF mRNA. Absolute complementarity is not however required, particularly in larger oligomers.
  • the antisense oligonucleotides against PLF are believed particularly useful in blocking expression and overexpression of PLF.
  • An antisense oligonucleotide against PLF may also be useful in blocking Periostin expression, if the PLF sequence which the antisense oligonucleotide is directed to is substantially homologous to a Periostin sequence.
  • the antisense oligonucleotides of the invention may be prepared for use as generally described herein for nucleic acids. That is, they may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • a pharmaceutical carrier such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • the liquid vehicles and excipients are conventional and commercially available.
  • the actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors. Those skilled in the art should be readily able to derive suitable dosages and schedules of administration to suit the specific circumstance.
  • the daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient.
  • the present invention further provides antibodies directed against PLF.
  • Antibodies to PLF can be obtained, for example, using as an antigen the product of a PLF expression vector or PLF isolated from a natural source.
  • Anti-PLF antibodies can be produced using antigenic PLF epitope- bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention comprise a sequence of at least nine amino acids, preferably at least 10 to about 15 amino acids, or more preferably at least about 15 to about 30 amino acids contained within SEQ ID NO:l l.
  • a polyclonal antibody has been prepared against the final, e.g., carboxy terminal end, 22 amino acids (LysLysIleProAlaAsnLysArgValGlnGlyProArgArgArgSerArgGluGlyArgSer Gin; SEQ ID NO:29) of the mouse PLF amino acid sequence (SEQ ID NO:l 1).
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with PLF.
  • a sequence can be chosen that spans a junction.
  • a peptide sequence of about 20-24 amino acids in length which spans 10-12 amino acid residues on each side of a junction, can be used as an immunogen to generate an antibody which recognizes the region of the particular target splice junction.
  • junction sequences for PLF compared to other Periostins, because there is a junction at each end of the two fragments, and when the fragments are deleted, a new junction and new sequence is formed for each. Antibodies generated against these junctions are useful in distinguishing isoforms of the Periostin family.
  • the peptide segment contained in PLF (SEQ ID NO: 14 for mouse; SEQ ID NO:30 for human), but not in Periostin, comprises an antigenic determinant not present in Periostin.
  • An antibody can be produced which is directed against such an antigenic determinant.
  • Potential antigenic sites in PLF can be identified using the Jameson- Wolf method (Jameson and Wolf, CABIOS 4:181, 1988), as implemented by the Protean program, version 3.1 (DNASTAR; Madison, Wis.). The Jameson- Wolf method predicts potential antigenic determinants by combining six major subroutines for protein stmctural prediction.
  • Polyclonal antibodies to a recombinant PLF protein or to PLF isolated from natural sources can be prepared using methods well known to those of skill in the art. Antibodies can also be generated using a PLF-glutathione transferase fusion protein, which is similar to a method described by Burrus and McMahon (Exp. Cell. Res. 220:363 1995). General methods for producing polyclonal antibodies are known to those of ordinary skill in the art (Green et al., in Immunochemical Protocols, Manson, ed., pages 1-5, Humana Press, New York, 1992.
  • the immunogenicity of a PLF polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of PLF or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like," such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • tetanus toxoid tetanus toxoid
  • polyclonal antibodies are typically raised in animals such as horse, cow, dog, chicken, rat, mouse, rabbit, goat, guinea pig, or sheep
  • an anti-PLF antibody of the present invention may also be derived from a subhuman primate.
  • monoclonal anti-PLF antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495 1975). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a PLF gene product and then verifying the presence of antibody production by removing a semm sample. Then, the spleen is removed to obtain B-lymphocytes. The B-lymphocytes are fused with myeloma cells to produce hybridomas, the hybridomas are cloned, and then positive clones are selected which produce antibodies to the antigen.
  • an anti-PLF antibody of the present invention may be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • the antibodies of the invention are useful in assessing the levels of PLF protein, its fragments, derivatives, or homologs thereof.
  • the antibodies can be used in methods known in the art relating to localization and activity of the protein sequences of the normal or mutated PLF protein, for imaging these proteins, and for measuring levels thereof in samples derived from a test subject or from a control sample or subject.
  • a primary antibody is detected by detecting a label on a primary antibody which has bound to the desired immunogen.
  • a secondary antibody which has bound to a primary antibody, is detected by detecting a label on the secondary antibody.
  • Many assays are known in the art for labeling and detecting primary and secondary antibodies.
  • Suitable pharmaceutical excipients include stabilizers, . antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents.
  • Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA- bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
  • Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
  • solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, preferably 25%-75%, of a compound of the invention.
  • a pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably 1%-10% by weight, of the compound of the invention encapsulated in a liposome as described above, and a propellant.
  • a carrier can also be included as desired; e.g., lecithin for intranasal delivery.
  • the compounds of the present invention and antibodies directed against PLF can comprise a pharmaceutically acceptable salt.
  • Suitable acids which are capable of forming such salts with the compounds of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like.
  • compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Pharmaceutical compositions according to the present invention can be prepared in a manner fully within the skill of the art. When used in vivo, the PLF proteins, fragments, homologs, or derivatives are preferably administered as a pharmaceutical composition, and a pharmaceutically acceptable carrier.
  • Assays for determining protein levels include immunocytochemical and immunohistochemical techniques, electrophoretic separation and identification, western blot analysis, peptide digestion, and sequence analysis (see Examples). Other assays are known to those of skill in the art. Various immunoassays known in the art can be used to measure
  • Periostin isoform PLF protein, fragments, derivatives, or homologs include competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, gel diffusion precipitin reactions, western blots, precipitation assays, in situ immunoassays, immunocytochemical and immunohistochemical techniques, complement fixation assays, immunofluorescence assays, and immunoelectrophoretic assays.
  • a tissue sample is derived from a subject.
  • a tissue sample includes a biopsy of a tissue of interest.
  • the random-primer method may be used to incorporate the dTTP analogue 5-(N-(N-biotinyl- ⁇ - aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate into the probe molecule.
  • the thus biotinylated probe oligonucleotide can be detected by reaction with biotin binding proteins such as avidin, streptavidin, or anti-biotin antibodies coupled with fluorescent dyes or enzymes producing color reactions.
  • biotin binding proteins such as avidin, streptavidin, or anti-biotin antibodies coupled with fluorescent dyes or enzymes producing color reactions.
  • the relative number of PLF transcripts may also be determined by reverse transcription of mRNA followed by amplification in a polymerase chain reaction (RT-PCR), and comparison with a standard.
  • RT-PCR polymerase chain reaction
  • tissue samples are obtained from patients and the samples are embedded then cut to e.g., 3-5 ⁇ m, fixed, mounted and dried according to conventional tissue mounting techniques.
  • the fixing agent may advantageously comprise formalin.
  • the embedding agent for mounting the specimen may comprise, e.g., paraffin.
  • the samples may be stored in this condition. Following deparaffinization and rehydration, the samples are contacted with an immunoreagent comprising an antibody specific for PLF.
  • progression of a PLF associated disorder in a subject can be monitored by measuring the level of PLF nucleic acid, PLF protein, or activity of a PLF protein in the subject.
  • the method comprises measuring the level of PLF nucleic acid, PLF protein, or activity of PLF protein in a sample derived from the subject.
  • RNA was extracted from one day old neonatal mouse heart tissue solubilized in TRIZOL (Invitrogen). Oligo-dT primed first strand cDNA was generated from this RNA and used to amplify the full-length PLF cDNA.
  • the 3' oligonucleotide primer (5'GAGAAAAACATTTGTATTGCAAGAAGC; SEQ ID NO:2) was designed based on the READS EST sequence and the published mouse Periostin cDNA sequence (GenBank Accession No. NM_015784; SEQ ID NO:l). The 5' end oligonucleotide primer
  • NM_015784.1, G 7657428 Mus musculus osteoblast specific factor 2 (OSF2), also called Periostin.
  • Primers were designed to be homologous to the extreme ends of the GenBank sequence and used for RT- PCR to generate a cDNA from neonatal mouse heart total RNA derived from the same tissue that gave rise to the READS fragment. This cDNA was sequenced in both directions.
  • This newly discovered 3012 nucleotide mouse PLF cDNA (SEQ ID NO:4) is a unique and previously unreported form of Periostin, designated herein as Periostin-Like Factor (PLF).
  • PLF Periostin-Like Factor
  • the full length murine PLF protein is 810 amino acids in length (SEQ ID NO: 11) (see FIGURE 1).
  • the 2430 nucleotide sequence (SEQ ID NO: 12) encoding the 810 amino acid mouse PLF comprises the sequence starting with "atggttcctctc” and ending with "cgttctcag.” Further cloning with different primers (forward 5' primer- gattcgattcggctgaagatggttcctctctgc, SEQ ID NO:31 ; reverse 5' primer- ggatccggatccgagagaaaacatttgtattgcaagaagc, SEQ ID NO:32) yielded a mouse PLF cDNA comprising 3290 bases (SEQ ID NO: 13).
  • the human PLF partial cDNA (SEQ ID NO:24; 479 bases), just like the mouse PLF cDNA, was found to encode a protein (SEQ ID NO:27) which has a 27 amino acid segment inserted between amino acids 672 and 700 of Periostin, and is lacking the 785-812 amino acid segment, relative to Periostin (see FIG. 1C; discussed more fully below).
  • RT-PCR based sequence analysis for the regions 5' and 3' to the deletion found that the mouse and human PLF sequences share very high sequence identity.
  • mPeriostin is derived from a murine Periostin OSF2- like cDNA sequence isolated from a 10 month-old virgin mouse mammary tumor (GenBank Accession No. BC031449; SEQ ID NO:25).
  • the predicted amino acid sequence (GenBank Accession No. AAH31449; SEQ ID NO:26) for the mouse Periostin OSF2 protein encoded by SEQ ID NO:25 is listed in FIGS. IA to lC.
  • Another cDNA has also been sequenced from the 10 month- old virgin mouse mammary tumor (GenBank Accession No.
  • hPeriostin is that of human Periostin (GenBank Accession No. NP_006466; SEQ ID NO:9), encoded by SEQ ID NO:28 (GenBank Accession No. NM_006475).
  • the amino acid sequence labeled "hPLF” is a partial sequence of human PLF (SEQ ID NO:27), as predicted from the human PLF cDNA (SEQ ID NO:24) described herein. All protein sequences were determined based on translations of the corresponding cDNA sequence giving the longest open reading frame. These proteins, while still highly homologous, show differences.
  • a mouse Periostin cDNA (GenBank Accession No. BC007141) not analyzed in FIG.
  • the sequence for mouse Periostin region comprising amino acids 785-812 is Glu-Val-Ser-Lvs-Val-Thr-Lys-Phe-Ile-Glu- Gly-Gly-Asp-Gly-His-Leu-Phe-Glu-Asp-Glu-Glu-Ile-Lys-Arg-Leu-Leu-Gln- Gly (SEQ ID NO: 15).
  • the sequence for the human Periostin region comprising amino acids 785-812 is Glu-Val-Thr-Lys-Val-Thr-Lys-Phe-Ile- Glu-Gly-Gly-Asp-Gly-His-Leu-Phe-Glu-Asp-Glu-Glu-Ile-Lys-Arg-Leu-Leu- Gln-Gly (SEQ ID NO:16).
  • the sequences of FIGS. IA to 1C are aligned based on the amino acid sequence of mouse Periostin, which is 811 amino acids in length. Because several forms of Periostin and PLF are compared to one another in FIGS.
  • FIGS. IA and IB are aligned to account for all insertions or deletions of fragments or amino acid residues, the total number of positions indicated in each of FIGS. IA and IB is 839. Therefore, the region designated as positions 758 to 786 in FIG. 1C, where the deletion occurs in mouse PLF protein, corresponds to positions 785-813 in FIG. IB. These two designations are used interchangeably herein.
  • the schematic in FIGURE 1 F compares Periostin to PLF.
  • the mouse Periostin protein (GenBank Accession No. BAA02835.1 ; SEQ ID NO:6) is 811 amino acids in length (FIG. 1C).
  • the predicted murine PLF sequence of the invention was compared to that of mouse Periostin, and it can be seen that the region between amino acid residue 673 and residue 700 is present in mouse PLF, but not in mouse Periostin (see FIGS. 1A-1C).
  • the 28 amino acid sequence between amino acid residues 673 and 700 disclosed herein in mouse PLF is Thr-Thr-Lys-Ile-Ile-Thr-Lys-Val-Val-Glu-Pro-Lys- Ile-Lys-Val-Ile-Gln-Gly-Ser-Leu-Gln-Pro-Ile-Ile-Lys-Thr-Glu-Gly (SEQ ID NO: 14).
  • PLF is an isoform resulting from an alternately spliced gene.
  • the alterations in amino acid sequence may be probably functionally significant because the other proteins are highly conserved across species, and these regions when present are also highly conserved across species.
  • Example 2- PLF expression during mouse embryogenesis Northern blot analysis Tissues were collected from embryonic mice and solubilized in
  • RNA samples Ten ⁇ g of total RNA was separated on 1% formaldehyde-denatured agarose gels, transferred to Nytran membranes, and probed with radiolabeled full-length mouse PLF cDNA. The Nytran was exposed to x-ray film and the image analyzed by densitometric techniques to determine the level of PLF mRNA present in a given tissue. In order to adjust for equal loading of RNA in each lane, the blots were re-probed with an 18S rRNA radiolabeled cDNA probe, and the amount of PLF mRNA levels are represented as a ratio of PLF RNA/18S rRNA.
  • in situ hybridization To determine the spatial location of PLF in mouse and chicken embryos, in situ hybridization was used to detect PLF mRNA in mouse embryos on days 8.5, 9.5, 10.5, 12.5 and 16.5 post-conception (pc.) (staging described in Kaufman, The atlas of mouse development. Academic Press. Harcourt, Brace Jovanovich Publishers, 1992). In situ hybridization results On day 8.5 pc, signal was localized to the uterine wall of the mother mouse (FIGS. 4A and B), but was not detected in the embryo. However, from days 9.5 to 16.5 p.c, PLF was detected in the mouse embryo. In the 9.5 and 10.5 day p.c. mouse embryo (FIGS.
  • PLF mRNA was localized to the somites, body wall mesenchyme, ventricular wall, atrioventricular canal, and the endocardial cushions.
  • PLF mRNA was detected in the wall of the atrium (FIG. 4E), and to a lesser extent in the ventricular wall.
  • PLF expression was again detected in the atrial wall and at the atrial-ventricular junction (FIG. 5B).
  • PLF expression was highest in the neonatal heart, compared to other stages of heart development (FIGS. 2 A, 7 A, and 7B). PLF was localized to the myocardium and was seen in the heart valves but was not detected in the epicardium.
  • PLF mRNA was localized to the mesenchymal tissue containing the preosteoblasts that surround the cartilage primordia of the ribs (FIGS. 5C and 5D), vertebrae (FIGS. 5E and 5F) and the limb (FIGS. 5G and 5H). It can be seen in FIGS. 5E and 5F that pre-osteoblasts express PLF, but chondrocytes do not. PLF mRNA was also detected in cells comprising the cartilage primordia of the upper and lower jaws at day 16.5 pc. (FIGS. 6A-6E).
  • the expression values for these samples are first sorted in ascending order, generating a rank order R for each expression value. Criteria for Selection of Differentially Regulated Genes in Nonfailing, Ischemic or Idiopathic Human Myocardium
  • the fold-change analysis was performed using all known genes regardless of whether the gene fragment was considered "present” or "absent", with the confidence limit set to 95%.
  • the analysis results were tabulated in spreadsheet format and sorted according to fold-change value, fold-change p- value, and presence frequency. Those genes that showed expression level changes in either the up or down direction in the range of 2.5 to 100-fold were filtered and saved as gene sets, based upon a p-value where p ⁇ .0001.
  • Sectioned tissues were treated with 10 ⁇ g/ml proteinase K for 10 minutes at 37°C.
  • the embryos and sectioned tissues were re-fixed in glutaraldehyde, prehybridized and then hybridized with the digoxygenin labeled PLF anti-sense riboprobe (generated as recommended by manufacturer: Boehringer Mannheim Biochemica, Indianapolis, IN) at 55°C.
  • the substrate reaction stained embryos and sectioned tissues were photographed using a Nikon microscope. Results PLF expression in diseased versus nonfailing human myocardium Because some embryonic genes are re-expressed during adult heart disease, it was determined whether PLF may also be expressed in adult cardiac disease.
  • G3PDH glycerol-3 -phosphate dehydrogenase
  • a plasmid containing PLF was included as a control to identify PLF.
  • Densitometric analyses were performed for the PCR-amplified PCR product gels, and the data expressed as the ratio of PLF to G3PDH expression.
  • Northern blot analyses were performed to determine PLF expression in age-matched osteopetrotic mutant and normal rats at 2, 4, and 6 weeks of age. Northern blots were probed with labeled PLF cDNA, stripped, and the reprobed with labeled 18S. The resulting films from the northern blot analyses were subjected to densitometric analyses and PLF to 18S expression ratios were determined.
  • mutant (osteopetrotic) rat long bone was isolated from two week old neonatal rats and prepared for in situ hybridization as described above.
  • Results Northern blot and RT-PCR analyses demonstrate that PLF expression is up regulated in mutant rat osteopetrotic bone at 2 and 4 weeks, but not at 6 weeks of age, compared to age matched normal bone (FIGS. 9A-9C).
  • Electrophoretic analysis of RT-PCR products shows that at 2 weeks of age, PLF is expressed at much higher levels in the bones of osteopetrotic rats (FIG. 9A). Densitometric analyses of the gels of FIG.
  • the antisemm was also found to be specific for the expression of PLF and other Periostin isoforms in developing chicken hearts and mouse hearts (not shown). It was found by fluorescence microscopy that the antiserum against PLF recognized PLF in the cytoplasm of the MC3T3-E1 osteoblast cell line in vitro (not shown). Thus, this reagent is highly specific for PLF in more than one species of mammal. This antibody should also be useful for recognizing other isoforms, including
  • primers flanking the 785 to 813 amino acid region resulted in tissue specific bands at 300 (upper arrow), 250 (middle arrow) and 150 (lower arrow) bp.
  • Forward and reverse primers were located at 2306 bp and 2503 bp, respectively.
  • the various bands were excised from the gels. Sequence analysis will confirm the presence of isoforms of PLF and the differences between the isoforms.
  • FMCM Cell Culture Fetal mouse cardiac myocytes
  • an MTT cell viability assay was performed. Addition of MTT to living cells results in the formation of a purple formazan product, the intensity of which is detected spectrophotometrically by reading samples at 570 nm. Forty-eight hours post-transfection, cells were scraped into TRIZOL (GIBCO BRL) and total RNA isolated. RNA was DNase-treated and equal amounts of RNA were reverse transcribed to generate first strand cDNA. G3PDH and PLF-specific primers were used to amplify the respective mRNAs using the PCR reaction. Expression of PLF was normalized to that of G3PDH.
  • Chariots form a non-covalent complex with the antibody, are semm independent, are independent of the endosomal pathway, and upon intemalization protect the antibody from degradation.
  • a ⁇ -galactosidase control protein transfected with Chariot into MC3T3-E1 cells showed efficient transfection (not shown).
  • Control cells were treated with non- immune IgG.
  • differentiation factors were added to the media.
  • Cells were harvested on days 7 and 21 post-transfection and total RNA isolated using TRIZOL (Invitrogen). Data obtained from RT-PCR using gene- specific primers are shown in FIGURE 18.

Abstract

Methods and compositions are provided for Periostin-Like Factor (PLF) nucleic acids and proteins and their use in diagnosing PLF associated diseases and disorders. PLF comprises a novel Periostin protein, comprising and additional peptide fragment not present in other Periostins, and lacking a peptide fragment which is present in other Periostins. PLF expression is aberrant in various diseases, and its expression appears to be important for normal embryonic development and cell differentiation.

Description

PERIOSTIN-LIKE FACTOR: COMPOSITIONS AND METHODS FOR MAKING AND USING THE SAME
Cross-Reference to Related Application This application claims the benefit of copending U.S. Provisional Application Serial No. 60/494,730, filed August 13, 2003, the entire disclosure of which is herein incorporated by reference.
Field of the Invention The present invention relates to a novel isoform of Periostin protein, designated "Periostin-Like Factor" (PLF), and its use in the treatment and diagnosis of diseases associated with aberrant expression of the novel Periostin protein. The invention also relates to the use of the novel Periostin protein in the treatment and diagnosis of diseases associated with the Periostin protein family.
Background of the invention Osteoblast-specific factor 2 (OSF-2) was identified using subtractive hybridization techniques in the MC3T3-E1 calvarial osteoblast-like cell line (Takeshita et al., Biochem. J., 294:271-8, 1993). The name was later changed to Periostin. Periostin has been identified in mouse and humans, and is overexpressed by stromal cells in several human ovary, breast, colon, and brain tumors (Sasaki et al., Cancer Lett., 172:37-42, 2001; Lai et al., Cancer Res., 59:5403-7, 1999; Bao et al., Cancer Res., 59:2023-8, 1999). Periostin is an 811 amino acid protein that acts as a homophilic adhesion molecule in bone formation (Takeshita et al., Biochem. J., 294:271- 8, 1993; Horiuchi et al., J. Bone Miner. Res., 14:1239-49, 1999). It is highly homologous to Fasciclin I (Zinn et al., Cell, 53:577-87, 1988), βig-h3 (LeBaron et al., J. Invest. Dermatol., 104:844-9, 1995; Skonier et al., DNA Cell. Biol., 1 1 :511-22, 1992), MBP70 (Terasaka et al., Microbiol. Lett., 49:273-6, 1989; Ulstrup et al., Infect. Immun., 63:672-5, 1995) and midline Fasciclin (Hu et al., J. Neurobiol., 35:77-93, 1998). Periostin, MBP70, βig-h3, and Fasciclin have not been extensively characterized, but it is clear that they are integral to the adhesion and migration of cells. Fasciclin I is involved in axonal guidance providing a pathway from a neuron to its target along which the axon can grow and extend. Two forms of Fasciclin I are described, one which is associated with the bilayer of the cell membrane by a phosphatidyl lipid motif and a second which is a soluble form. Both forms of Fasciclin I vary in expression during embryonic development and are proposed regulators of neural cell adhesion (Hortsch et al., J. Biol. Chem., 265:15104-9, 1990, McAllister et al., Devel., 1 15:267-76, 1992). Fasciclin I and Periostin contain four potential N-glycosylation sites which do not appear to be glycosylated (Horiuchi, et al., J. Bone Miner. Res., 14:1239-49, 1999) and do not contain an obvious transmembrane domain (Takeshita et al., Biochem. J., 294:271-8, 1993). These glycoproteins contain four homologous domains of 150 amino acids each, referred to as 'Repeat Domains,' and an N-terminal signal sequence which was shown to be functional in MC3T3-E1 cells, i.e., the protein is secreted (Horiuchi et al., J. Bone Miner. Res., 14:1239-49, 1999). Within each Repeat Domain, two regions are highly conserved. Alternative protein forms of Periostin and Fasciclin are reported resulting in differentially regulated adhesion and attachment during development. Most recently, Periostin was identified in embryonic mouse hearts during valve formation (Kruzynska-Frejtag et al., Mech. Dev., 103:183-8, 2001) and in the heart of rats in a model of myocardial infarction (Stanton et al., Circ. Res., 86:939-45, 2000). Kruzynska-Frejtag et al. showed expression of Periostin in the endocardial cushions (which are formed by cells recruited from the endocardium of the developing mouse heart), post-epithelial- mesenchymal interactions, and in the fetal and adult heart valves, which are formed from the endocardial cushions (Mech. Dev., 103:183-8, 2001). Stanton et al. (Circ. Res., 86:939-45, 2000) showed that Periostin expression increased significantly 4 weeks after ligation of the coronary artery to induce myocardial infarction. This increase in Periostin may be a consequence of the ligation, wherein the cardiac myocytes respond by producing increased amounts of secreted proteins. The secreted proteins may be either structural extracellular matrix proteins or proteins integral to regulation of the disease process. Adult and congenital heart disease are leading contributors to morbidity and mortality. It has been clearly shown that, in adult heart disease cardiac myocytes begin to express proteins that were previously expressed during embryogenesis or neonatal development (Lim et al., J. Am. Coll. Cardiol., 38:1175-80, 2001; Liu et al., Dev. Biol., 234:497-509, 2001). This may be a purely compensatory mechanism of the myocytes to the disease state, or perhaps a causative factor of the adult heart disease. The cytokine-initiated activation of Vascular Smooth Muscle Cells (VSMCs) is the principal mediator of vascular proliferative diseases such as balloon angioplasty or transplant restenosis (Liu et al., Circulation, 79:1374- 87, 1989). VSMCs also provide a good model to study cellular differentiation because of their ability to dedifferentiate and reenter the cell cycle upon mitogen stimulation. Horiuchi et al. (J. Bone Miner. Res., 14:1239-49, 1999) showed that Periostin is expressed in the periosteum and periodontal ligament in adult mice, suggesting that this molecule is also potentially involved in the maintenance of bone and tooth structure. Using the MC3T3-E1 in vitro osteoblast model, it has been shown that Periostin is secreted, mediates cell adhesion and cell spreading, and that Periostin expression is stimulated by TGFβl, a growth factor responsible for bone formation (Marcelli et al., J. Bone Miner. Res., 5:1087-96, 1990). Periostin may also act as a ligand of integrins to help support adhesion and migration of cells. Thus, Periostin or members of the Periostin family may be involved in the regulation of embryonic development of cells in multiple tissues, and may be involved in diseases in at least several different tissues and organs in adults, including bone and heart. Alternative forms of Periostin, including, but not limited to splice variants, may also be involved in the processes of embryonic development and progression of diseases such as cancer. There is a need to discover other members of the Periostin family and there is a need for methods of treating and diagnosing diseases and disorders associated with aberrant expression or regulation of Periostin or members of the Periostin family. The present invention satisfies these needs.
Summary of the Invention The invention provides a novel isoform of Periostin, called Periostin- Like Factor (PLF). The invention provides an isolated nucleic acid comprising a nucleic acid sequence encoding a Periostin isoform, or a fragment, derivative, or homolog of such isoform, which isoform: (a) contains an amino acid segment not contained in Periostin, wherein the amino acid segment has the sequence SEQ ID NO: 14 or SEQ ID NO:30; and (b) does not contain a Periostin amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16. In one aspect, the isolated nucleic acid comprising a nucleic acid sequence encoding a Periostin isoform, encodes a Periostin isoform consisting of 810 amino acid residues. In one aspect, the invention provides an isolated nucleic acid comprising a Periostin isoform nucleic acid sequence comprising: (a) SEQ ID NO: 12 or a sequence that is substantially homologous to SEQ ID NO: 12; or (b) SEQ ID NO:24 or a sequence that is substantially homologous to SEQ ID NO:24. The invention also provides a nucleic acid sequence encoding a
Periostin isoform having the amino acid sequence SEQ ID NO: 11 or a sequence that is substantially homologous to SEQ ID NO:l l. The invention further provides an isolated polypeptide having the amino acid sequence SEQ ID NO: 11 or a sequence that is substantially homologous to SEQ ID NO: 11. The invention further provides an isolated nucleic acid encoding a
Periostin isoform having the amino acid sequence SEQ ID NO: 27 or a sequence that is substantially homologous to SEQ ID NO:27. The invention also provides an isolated polypeptide having the amino acid sequence SEQ ID NO:27 or a sequence that is substantially homologous to SEQ ID NO:27. The invention provides vectors comprising isolated nucleic acids encoding Periostin isoforms. The invention also provides host cells comprising isolated nucleic acids encoding Periostin isoforms and host cells comprising vectors encoding Periostin isoforms. The invention provides an antibody directed against a Periostin isoform, or a fragment, derivative, or homolog of the Periostin isoform. The invention also provides an antibody directed against a Periostin isoform amino acid sequence having the sequence SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO:29 or SEQ ID NO:30. The invention further provides an antibody directed against a Periostin isoform, wherein the antibody is directed against an antigenic determinant not contained in Periostin. The invention further provides a pharmaceutical composition for treating a PLF associated disorder, comprising the Periostin isoform, PLF protein, or a fragment, derivative, or homolog of a PLF protein, or an isolated nucleic acid comprising a nucleic acid sequence encoding a PLF protein, or an antisense oligonucleotide directed against PLF mRNA sequences, or an antibody directed against PLF, and a pharmaceutically acceptable carrier. PLF associated disorders which are treated by the methods of the invention include ischemic heart disease, idiopathic heart disease, restenosis, cardiac hypertrophy, osteopetrosis, osteoporosis, cell differentiation disorders, cell adhesion disorders, cell migration disorders, wound healing disorders, bone fractures, chronic rejection, asthma, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, stomach cancer metastases, cardia bifida, abnormal blood vessel development, head developmental anomalies, somite developmental anomalies, and developmental disorders. In one aspect, the subject is a human. The invention provides a method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and an isolated nucleic acid comprising a nucleic acid sequence encoding a Periostin isoform, or a fragment, derivative, or homolog of such isoform, which isoform: (a) contains an amino acid segment not contained in Periostin, wherein the amino acid segment has the sequence SEQ ID NO: 14 or SEQ ID NO:30; and (b) does not contain a Periostin amino acid segment having the sequence SEQ ID NO:15 or SEQ ID NO:16. The invention also provides a method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and a Periostin isoform, or a fragment, derivative, or homolog of such isoform, wherein the Periostin isoform has the amino acid sequence SEQ ID NO:l 1 or SEQ NO:27. The invention further provides a method of treating a PLF associated disorder, comprising administering to the subject an effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an antibody directed against a Periostin isoform, fragment, derivative, or homolog of a Periostin isoform protein. In one aspect, the antibody is directed against a Periostin isoform amino acid sequence having the sequence SEQ ID NO:l 1, SEQ ID NO: 14, SEQ ID NO:29 or SEQ ID NO:30. The invention provides a method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antisense oligonucleotide of about 8 nucleotides to about 40 nucleotides comprising a sequence complementary to a PLF mRNA sequence, and a pharmaceutically acceptable carrier. PLF mRNA encodes an amino acid segment not contained in Periostin, wherein the amino acid segment has the sequence SEQ ID NO: 14 or SEQ ID NO:30, but does not encode a Periostin amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16. The invention further provides treating a PLF associated disorder with an antisense oligonucleotide which is complementary to a PLF mRNA sequence comprising the translation initiation region of PLF mRNA. The invention also provides treating a PLF associated disorder with an antisense oligonucleotide selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23. The invention further provides treating with an antisense oligonucleotide complementary to a PLF mRNA sequence encoding an amino acid sequence comprising SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO:30. The invention also provides methods of diagnosing a PLF associated disorder in a subject. The method comprises measuring the expression level of PLF protein or PLF mRNA in a sample derived from the subject, wherein a higher or lower expression level of PLF protein or PLF mRNA in the sample relative to the expression level of PLF protein or PLF mRNA in a sample from a control subject not having the PLF associated disorder, indicates the presence of the PLF associated disorder. The invention provides a method of monitoring the progression of a PLF associated disorder in a subject. In one aspect, the method comprises measuring the expression level of PLF protein or PLF mRNA in a sample derived from the subject, wherein an increased level of PLF protein or PLF mRNA in the sample, relative to the level present in a sample derived from the subject at an earlier time, indicates progression of the PLF associated disorder. In another aspect, the method comprises measuring the expression level of PLF protein or PLF mRNA in a sample derived from the subject, wherein a lower level of PLF protein or PLF mRNA in the sample, relative to the level present in a sample derived from the subject at an earlier time, indicates progression of the PLF associated disorder. The invention further provides assays for diagnosing and monitoring the progression of a PLF associated disorder. The assays include western blot, immunocytochemical, immunohistochemical, radioimmunoassay, enzyme linked immunosorbent assay, sandwich immunoassay, gel diffusion precipitin reaction, complement fixation, immunofluorescence, immunoelectrophoretic, northern blot, reverse transcriptase polymerase chain reaction, in situ hybridization, and dot blot assays. Abbreviations and Short Forms The following abbreviations and short forms are used in this specification. "AL-PH" means alkaline phosphatase. "bFGF" means basic fibroblast growth factor. "bp" means base pair. "Cbfal" means core binding factor alpha- 1. "EST" means expressed sequence tag. "FCS" means fetal calf serum. "FMCM" means fetal mouse cardiac myocytes. "G3PDH" means glycerol-3 -phosphate dehydrogenase. "h" means human. "IFNγ" means interferon gamma. "IL-l β" means interleukin 1 beta. "IVT" means in vitro transcription. "L" means lower percentile. "LVAD" means left-ventricular assist device. "LVEDD" means left ventricular end-diastolic dimension. "m" means mouse or murine. "MCV" means median cell volume. "OSF-2" means osteoblast-specific factor 2 (also called Periostin). "pc." means post conception. "PCR" means polymerase chain reaction. "PDGF" means platelet-derived growth factor. "PLF" means Periostin-Like Factor. "READ" is a differential display technique. "RT-PCR" means reverse transcriptase polymerase chain reaction. "TGFβ" means transforming growth factor beta. "U" means upper percentile. "VMHC 1 " means ventricle myosin heavy chain- 1. "VSMC" means vascular smooth muscle cell. Definitions The definitions used in this application are for illustrative purposes and do not limit the scope of the invention. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. As used herein, each "amino acid" is represented by the full name thereof, by the three letter code corresponding thereto, or by the one-letter code corresponding thereto, as indicated in the following table:
Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D Glutamic Acid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr Y Cysteine Cys C Asparagine Asn N Glutamine Gin Q Serine Ser s Threonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L Isoleucine He I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan Tip W The expression "amino acid" as used herein is meant to include both natural and synthetic amino acids, and both D and L amino acids. "Standard amino acid" means any of the twenty L-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid residues" means any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or derived from a natural source. As used herein, "synthetic amino acid" also encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and substitutions. Amino acids contained within the peptides of the present invention, and particularly at the carboxy- or amino-terminus, can be modified by methylation, amidation, acetylation or substitution with other chemical groups which can change a peptide's circulating half life without adversely affecting activity of the peptide. Additionally, a disulfide linkage may be present or absent in the peptides of the invention. The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide. Amino acids have the following general structure:
H R C COOH NH2 Amino acids are classified into seven groups on the basis of the side chain R: (1) aliphatic side chains, (2) side chains containing a hydroxy lie (OH) group, (3) side chains containing sulfur atoms, (4) side chains containing an acidic or amide group, (5) side chains containing a basic group, (6) side chains containing an aromatic ring, and (7) proline, an imino acid in which the side chain is fused to the amino group. The nomenclature used to describe the peptide compounds of the present invention follows the conventional practice wherein the amino group is presented to the left and the carboxy group to the right of each amino acid residue. In the formulae representing selected specific embodiments of the present invention, the amino-and carboxy-terminal groups, although not specifically shown, will be understood to be in the form they would assume at physiologic pH values, unless otherwise specified. "Antibody," as used herein, includes polyclonal and monoclonal antibodies; recombinant antibodies; and chimeric, single chain, and humanized antibodies. "Antibody" includes not only intact antigen binding immunoglobulin molecules, but also fragments thereof which bind antigen, such as Fv, Fab, Fab', and F(ab')2 fragments, or the product of an immunoglobulin expression library. The term "amino acid segment," as used herein, refers to two or more consecutive amino acids in a peptide. The term "amino acid segment," is used interchangeably herein with peptide segment. "An antibody directed against," as used herein, refers to the molecule which was the target immunogen for producing the antibody. Thus, "an antibody directed against" a protein, polypeptide, or fragment or amino acid segment of such protein, binds to an epitope on the respective protein, polypeptide, or fragment or amino acid segment of such protein, and does not substantially recognize or bind other molecules in a sample. The term an "antibody directed against PLF" means an antibody directed against PLF or a fragment, derivative, or homolog of PLF. The term an "antibody directed against PLF," is used interchangeably herein with "anti- PLF antibody." An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules. An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules. The term "antisense," as used herein with respect to nucleic acids, refers to an oligonucleotide that is complementary to a target nucleic acid sequence. "Antisense" also refers to the specific hybridization of an oligonucleotide with its target nucleic acid. "An antisense oligonucleotide directed against," as used herein, refers to a reference nucleic acid or specific nucleic acid sequence which is complementary to the sequence of the antisense oligonucleotide. "Biologically active," as used herein with respect to PLF or fragments, derivatives, or homologs thereof, means that the PLF or fragments, derivatives, or homologs thereof, have the ability to function as a regulator of activities described herein such as embryonic development, cell growth, and cell adhesion, and cell migration. "Compound of the invention," as used herein, refers to isolated nucleic acids encoding PLF or fragments, derivatives, or homologs of PLF, to the polypeptides encoded by such nucleic acids, and to antisense oligonucleotides directed against PLF nucleic acid sequences. A protein or peptide "derivative," as used herein, includes any purposefully generated protein or peptide, which in its entirety, or in part, comprises a substantially similar amino acid sequence to PLF and has PLF biological activity. Derivatives of PLF may be characterized by single or multiple amino acid substitutions, deletions, additions, or replacements. These derivatives may include (a) derivatives in which one or more amino acid residues of SEQ ID NO: 11 (mouse PLF) are substituted with conservative or non-conservative amino acids; (b) derivatives in which one or more amino acids are added to SEQ ID NO:l 1; (c) derivatives in which one or more of the amino acids of SEQ ID NO:l 1 includes a substituent group; (d) derivatives in which SEQ ID NO: 11 or a portion thereof is fused to another peptide (e.g., serum albumin or protein transduction domain); (e) derivatives in which one or more nonstandard amino acid residues (i.e., those other than the 20 standard L-amino acids found in naturally occurring proteins) are incorporated or substituted into SEQ ID NO:l l; and (f) derivatives in which one or more nonamino acid linking groups are incorporated into or replace a portion of SEQ ID NO: 11. Derivatives also apply to human PLF (SEQ ID NO:27). The term "downstream" when used in reference to a direction along a nucleotide sequence means the 5' to 3' direction. Similarly, the term "upstream" means the 3' to 5' direction. As used herein, an "effective amount" or "therapeutically effective amount" of a compound of the invention or an antibody directed against PLF or a fragment, derivative, or homolog of PLF, is an amount sufficient to inhibit progression of a PLF-associated disorder in a subject. An "effective amount" of an inhibitor of PLF is an amount sufficient to inhibit the activity or effect of PLF. The term "expression," as used with respect to PLF mRNA, refers to transcription of the Periostin gene, resulting in synthesis and processing of Periostin mRNA to form PLF mRNA. "Expression," as used with respect to PLF protein, refers to translation of PLF protein. "Expression level," with respect to PLF mRNA or protein, means not only an absolute expression level, but also a relative expression level as determined by comparison with a standard level of PLF mRNA or protein. As used herein, the term "fragment," as applied to a nucleic acid, refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 20 nucleotides in length; for example, at least about 50 nucleotides to about 100 nucleotides; preferably at least about 100 to about 500 nucleotides, more preferably at least about 500 to about 1000 nucleotides, even more preferably at least about 1000 nucleotides to about 1500 nucleotides; particularly, preferably at least about 1500 nucleotides to about 2500 nucleotides; most preferably at least about 2500 nucleotides. As used herein, the term "fragment," as applied to a protein or peptide, refers to a subsequence of a larger protein or peptide. A "fragment" of a protein or peptide can be at least about 20 amino acids in length; for example at least about 50 amino acids in length; more preferably at least about 100 amino acids in length, even more preferably at least about 200 amino acids in length, particularly preferably at least about 300 amino acids in length, and most preferably at least about 400 amino acids in length. A "homolog" of PLF includes any nonpurposely generated peptide which, in its entirety or in part, comprises a substantially similar amino acid sequence to SEQ ID NO: 11 (mouse PLF), or SEQ ID NO:27 (human PLF) and has PLF biological activity. Homologs can include paralogs, orthologs, and naturally occurring alleles or variants of PLF. "Homologous" as used herein, refers to the subunit sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. By way of example, the DNA sequences 3ΑTTGCC5' and 3 'TATGGC are 50% homologous. As used herein, "homology" is used synonymously with "identity." The term "inhibit," as used herein, means to suppress or block an activity or function by at least ten percent relative to a control value. Preferably, the activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. The term "isoform," as used herein, refers to proteins having similar sequences or regions of similar sequences, and includes members of a family which differ due to various processes such as alternative splicing of messenger RNA. "Isolated" means altered or removed from the natural state through the actions of a human being. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. A "nucleic acid" refers to a polynucleotide and includes poly- ribonucleotides and poly-deoxyribonucleotides. The term "oligonucleotide" typically refers to short polynucleotides of about 50 nucleotides or less in length. It will be understood that when a nucleotide sequence is represented herein by a DNA sequence (i.e., A, T, G, and C), this also includes the corresponding RNA sequence (i.e., a, u, g, c) in which "u" replaces "T". As used herein, the term "oligonucleotide" includes both oligomers of ribonucleotide i.e., oligoribonucleotides, and oligomers of deoxyribonucleotide i.e., oligodeoxyribonucleotides (also referred to herein as "oligodeoxynucleotides"). The terms "oligonucleotide" and "oligodeoxynucleotide" include not only oligomers and polymers of the biologically significant nucleotides, i.e. nucleotides of adenine ("A"), deoxyadenine ("dA"), guanine ("G"), deoxyguanine ("dG"), cytosine ("C"), deoxycytosine ("dC"), thymine ("T") and uracil ("U"), but also oligomers and polymers hybridizable to a PLF nucleic acid sequence which may contain other nucleotides. Likewise, the terms "oligonucleotide" and "oligodeoxynucleotide" include oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or intemucleotide linkages is chemically modified. The term "oligonucleotide" is thus understood to also include oligomers which may properly be designated as "oligonucleosides" because of modification of the intemucleotide phosphodiester bond. Such modified oligonucleotides include, for example, the alkylphosphonate oligonucleosides, discussed below. The term "phosphorothioate oligonucleotide" means an oligonucleotide wherein one or more of the intemucleotide linkages is a phosphorothioate group, as opposed to a phosphodiester group which is characteristic of unmodified oligonucleotides. As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids which can comprise a protein's or peptide's sequence. "Periostin," as used herein, refers to a protein originally named
Osteoblast Factor-2 (Takeshita et al., Biochem. J., 294:271-8, 1993), comprising 811 amino acids, which is secreted, mediates cell adhesion, migration, and spreading, is stimulated by growth factors, and is expressed in bone, heart, blood vessels, and stromal cells of tumors. A "Periostin isoform," as used herein, refers to a protein having similar amino acid sequences or regions of similar sequences to Periostin, and includes Periostin isoforms which differ from Periostin due to various processes such as alternative splicing of messenger RNA, resulting in insertion or deletion of amino acid sequences, and substitution of amino acids. "Periostin-Like Factor (PLF)," as used herein, refers to a Periostin isoform containing an amino acid segment having the sequence SEQ ID NO: 14 or SEQ ID NO: 30, but not containing an amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16. A "PLF-associated disease or disorder," as used herein refers to a disease or disorder in which there is an association between aberrant expression or activity of PLF in a subject and abnormal embryonic development, cell proliferation, cell adhesion, or cell migration. A PLF- associated disorder may include a Periostin-associated disorder. PLF-associated diseases and disorders include cancers, myocardial diseases and disorders, bone diseases and disorders, cell adhesion disorders, cell migration disorders, cell proliferation disorders, embryonic development disorders and other such disorders wherein PLF expression or levels is aberrant. The term "PLF, or fragments, derivatives, or homologs thereof," is used interchangeably herein with "PLF polypeptides" and with "PLF proteins." The term "PLF nucleic acids," as used herein, refers to a DNA or RNA sequence encoding PLF, or a fragment, derivative, or homolog of PLF. "Pharmaceutically acceptable" means physiologically tolerable, for either human or veterinary applications. As used herein, "pharmaceutical compositions" include formulations for human and veterinary use. A "preventive" or "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs, or exhibits only early signs, of a PLF- associated disorder. A prophylactic or preventative treatment is administered for the purpose of decreasing the risk of developing pathology associated with a PLF-associated disorder or Periostin-associated disorder. A "sample," as used herein, refers to a biological sample from a subject, including normal tissue samples, diseased tissue samples, biopsies, blood, saliva, feces, or urine. A sample can also be any other source of material obtained from a subject which contains cells or tissue of interest. A "splice junction or region," as used herein, refers to the amino acid residue position or region of a peptide where a fragment can be inserted or deleted. It also refers to the nucleic acid sequence encoding the corresponding region of the peptide where the insertion or deletion occurs. A "subject," as used herein, can be a human or non-human animal. Non-human animals include, for example, livestock and pets, such as equine, ovine, bovine, porcine, canine, feline and murine mammals, as well as reptiles, birds and fish. Preferably, the subject is a human. As used herein, a "subunit" of a nucleic acid molecule is a nucleotide, and a "subunit" of a polypeptide is an amino acid. "Substantially purified" refers to a peptide or nucleic acid sequence which is substantially homogenous in character due to the removal of other compounds (e.g., other peptides, nucleic acids, carbohydrates, lipids) or other cells originally present. "Substantially purified" is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be present, for example, due to incomplete purification, addition of stabilizers, or formulation into a pharmaceutically acceptable preparation. As used herein, a "substantially homologous amino acid sequence" includes those amino acid sequences which have at least about 90% homology, preferably at least about 95% homology, more preferably at least about 96% homology, even more preferably at least about 97% homology, even more preferably at least about 98% homology, and most preferably at least about 99% or more homology to an amino acid sequence of a reference peptide chain. Amino acid sequence similarity or identity can be computed by using the BLASTP and TBLASTN programs which employ the BLAST (basic local alignment search tool) 2.0.14 algorithm. The default settings used for these programs are suitable for identifying substantially similar amino acid sequences for purposes of the present invention. "Substantially homologous nucleic acid sequence" means a nucleic acid sequence corresponding to a reference nucleic acid sequence wherein the corresponding sequence encodes a peptide having substantially the same structure and function as the peptide encoded by the reference nucleic acid sequence; e.g., where only changes in amino acids not significantly affecting the peptide function occur. Preferably, the substantially similar nucleic acid sequence encodes the peptide encoded by the reference nucleic acid sequence. The percentage of identity between the substantially similar nucleic acid sequence and the reference nucleic acid sequence is at least about 50%, 65%, 75%, 85%, 95%, 99% or more. Substantial similarity of nucleic acid sequences can be determined by comparing the sequence identity of two sequences, for example by physical/chemical methods (i.e., hybridization) or by sequence alignment via computer algorithm. Suitable nucleic acid hybridization conditions to determine if a nucleotide sequence is substantially similar to a reference nucleotide sequence are: 7% sodium dodecyl sulfate SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 2X standard saline citrate (SSC), 0.1% SDS at 50°C; preferably in 7% (SDS), 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C; preferably 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C; and more preferably in 7% SDS, 0.5 M NaPO4, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C. Suitable computer algorithms to determine substantial similarity between two nucleic acid sequences include, GCS program package (Devereux et al., 1984 Nucl. Acids Res. 12:387), and the BLASTN or FASTA programs (Altschul et al., 1990 Proc. Natl. Acad. Sci. USA. 1990 87:14:5509-13; Altschul et al., J. Mol. Biol. 1990 215:3:403-10; Altschul et al., 1997 Nucleic Acids Res. 25:3389- 3402). The default settings provided with these programs are suitable for determining substantial similarity of nucleic acid sequences for purposes of the present invention. The terms to "treat" or "treatment," as used herein, refer to administering a PLF, an inhibitor or stimulator of PLF activity, antibodies, agents, or compounds to reduce the frequency with which the effects or symptoms of a PLF associated disorder are experienced, to reduce the severity of symptoms, or to prevent effects or symptoms from occurring. Treatment can restore the effect of PLF function or activity which has been lost or diminished in a PLF associated disorder. Treatment can also include inhibiting or reducing PLF function or activity where PLF function or activity has increased in a PLF associated disorder.
Brief Description of the Figures FIGURES IA to 1C comprise an amino acid sequence alignment and comparison of mouse PLF (SEQ ID NO: 10) and human PLF (partial sequence; (SEQ ID NO:27) to the amino acid sequences of a mouse Periostin protein (mPeriostin/OSF-2; GenBank Accession Nos. BC031449 and AAH31449; SEQ ID NO), mouse Periostin cloned by "Takeshita" (derived from Takeshita et al., Biochem J. 294:271-8, 1993; GenBank Accession Nos. D13664 and BAA02835.1), human Periostin (hPeriostin; SEQ ID NO:9), and a consensus Periostin family sequence derived from this comparison. FIGURES ID and IE depict a sequence alignment in which the amino acid residues (810; SEQ ID NO: 10) and nucleic acid residue coding region (SEQ ID NO:l 1) of mouse PLF are aligned. FIGURE IF is a schematic comparing the predicted amino acid sequence of mouse PLF (PLF; 810 amino acids, 2430 bases) (upper) with mouse Periostin (811 amino acids, 2433 bases) (lower). Part of the region identified in the upper sequence between PI and P2 (amino acid residues 672 and 700) is present in mouse PLF but not in mouse Periostin. The region identified in the lower schematic sequence comprising the region between P3 and P4 (amino acid residues 758 to 786) is not present in mouse PLF. PI, P2, P3, and P4 represent regions where sequences were used to prepare primers for subsequent studies described herein. The alignment of the primers with the respective nucleic acid residue positions is indicated on each schematic. AA = amino acid; N = nucleotide. FIGURE 2A is a northern blot analysis of PLF expression during mouse embryonic development. Lane 1 : 7.5 day (post-conception) embryos; Lane 2: 13.5 day embryonic hearts; Lane 3: neonatal hearts; Lane 4: adult hearts. FIGURE 2B is a northern blot analysis of PLF expression during chicken embryonic development. Lane 1 : stage 4 anterior lateral plate; Lane 2: stage 8 anterior lateral plate; Lane 3: stage 12 heart neonatal heart; Lane 4: neonatal heart; Lane 5: adult heart. FIGURE 2C is a graphic representation of a densitometric analysis of the northern blot of FIGURE 2A. The graph represents the PLF/18S ratio as calculated in the densitometric analysis. FIGURE 2D is a graphic representation of a densitometric analysis of the northern blot of FIGURE 2B. The graph represents the PLF/18S ratio as calculated in the densitometric analysis. FIGURE 3 A is a northern blot analysis of the developmental regulation of PLF expression in mice. Upper panel- Total RNA from whole mouse embryos, days 9.5 pc. to day 18.5 pc, was probed with P-labeled PLF cDNA probe. Lower panel- the ethidium bromide stained formaldehyde-denatured gel of the RNA probed for PLF expression in the upper panel. FIGURE 3B is a graphic representation of a densitometric analysis of the northern blot of FIGURE 3 A. Blots were stripped and re-probed with P- labeled 18S cDNA as a control for loading and transfer. The graph represents the PLF/18S ratio as calculated in the densitometric analysis. FIGURE 4 A demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in the uterine wall (day 8.5) (40X magnification). The area enclosed in a box is shown at higher magnification in FIGURE 4B. FIGURE 4B demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in the uterine wall (day 8.5) (200X magnification). FIGURE 4C demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in the uterine wall (day 9.5) (40X magnification). FIGURE 4D demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis (day 10.5) in the uterine wall (20X magnification). FIGURE 4E demonstrates by in situ hybridization the spatial expression of PLF mRNA in the atrial wall during early mouse embryogenesis (day 12.5) (400X magnification). FIGURE 4F demonstrates by in situ hybridization the spatial expression of PLF mRNA during early mouse embryogenesis in a whole embryo at day 10.5 post-conception. HD = head; HT = heart. FIGURE 5 A demonstrates by in situ hybridization in a 16.5 day post- conception mouse embryo that PLF is expressed in bone and heart (20X magnification). The area demarcated by the box on the left is further magnified in FIG. 5C. The area demarcated by the box on the right is further magnified in FIG. 5F. FIGURE 5B demonstrates PLF expression by in situ hybridization in the atrial wall of a 16.5 day post-conception mouse embryo (400X magnification). AL = atrial lumen. FIGURE 5C demonstrates PLF expression by in situ hybridization in the region of developing vertebrae outlined in the left (dorsal) box of FIGURE 5A, (100X magnification). C = cartilage. FIGURE 5D demonstrates PLF expression by in situ hybridization in developing pre-osteoblasts, but not chondrocytes, in a 16.5 day embryonic mouse vertebra (400X magnification). Preosteoblasts migrating away from the body of the vertebra, to form the vertebral processes, are indicated by arrowheads. C = cartilage. FIGURE 5E demonstrates PLF expression by in situ hybridization in preosteoblasts, but not chondrocytes, of a presumptive rib (400X). C = cartilage. FIGURE 5F demonstrates PLF expression by in situ hybridization of the region shown in the right box (ventral) of FIGURE 5A, at a higher magnification (400X). Cells indicated by the arrows in 5F are preosteoblasts migrating from the cartilaginous rib into the sternum. C = cartilage. FIGURE 6 A demonstrates by in situ hybridization PLF expression in the mesenchymal primordia of the head region of a 16.5 day post-conception mouse embryo (20X magnification). The asterisk is located at the tip of the tongue (T). The region outlined by the box is shown at higher magnification in FIGURE 6D. FIGURE 6B demonstrates by in situ hybridization PLF expression in the upper and lower jaws and the tongue of a 16.5 day mouse embryo (40X magnification). The arrow indicates a region of the hard palate shown at higher magnification in FIGURE 6C. The region outlined by the box is shown at a higher magnification in FIGURE 6E. FIGURE 6C demonstrates by in situ hybridization PLF expression in pre-osteoblasts in the hard palate (arrow) and presumptive taste buds in the tongue (arrow head). FIGURE 6C is a higher magnification of the image marked by an arrow in FIGURE 6B. FIGURE 6D demonstrates by in situ hybridization PLF expression in pre-osteoblasts in the hard palate (arrow) and presumptive taste buds in the tongue (arrow head) in cells in the region comprising the transition from the hard palate to the soft palate (100X magnification). FIGURE 6D is a higher magnification of the image outlined by the box in FIGURE 6 A. FIGURE 6E is the region outlined by the box in FIGURE 6B, shown at a higher magnification, demonstrating PLF expression in the upper and lower jaws and the tongue of a 16.5 day mouse embryo (100X magnification). FIGURE 7A is an in situ hybridization analysis of PLF expression in neonatal mouse heart tissue and is a sagittal section of the neonatal mouse heart shown at low magnification. The region enclosed in the box is shown at a higher magnification in FIG. 7B. FIGURE 7B is an in situ hybridization analysis of PLF expression in neonatal mouse heart tissue. The image is the region enclosed in the box in FIG. 7B, shown at a higher magnification (400X). FIGURE 8 A is a northern blot analysis of PLF expression in a series of normal human hearts. The upper panel represents PLF expression and the lower panel depicts the same blot stripped and reprobed with an 18S probe. FIGURE 8B is a northern blot analysis of PLF expression in a series of idiopathic human hearts. The upper panel represents PLF expression and the lower panel depicts the same blot stripped and reprobed with an 18S probe. FIGURE 8C is a northern blot analysis of PLF expression in a series of ischemic human hearts. The upper panel represents PLF expression and the lower panel depicts the same blot stripped and reprobed with an 18S probe. FIGURE 8D is a graph demonstrating the PLF/18S expression ratios in normal human heart (columns 1-6), idiopathic human heart (columns 7-13), and ischemic human hearts (columns 14-20). FIGURE 8E is a spotfire visualization graph depicting the average mean expression intensity value (ordinate) versus tissue sample set (abscissa) of PLF mRNA examined via hybridization of total neonatal mouse heart mRNA to the human HU_95 (60K) gene chip set. FIGURE 8F is a graph demonstrating a non-paired analysis of PLF mRNA expression in LVAD-treated human patients. FIGURE 9A is an RT-PCR analysis of PLF expression in normal (N) and mutant (M) osteopetrotic calvaria of 2 week old rats using RT-PCR fragments. The lower panel depicts expression of the control G3PDH. P= a plasmid containing PLF used as a control; L= migration markers. FIGURE 9B is a bar graph representing the ratio of the densitometric analysis of the PCR-amplified PLF product from normal and mutant bone and the G3PDH lane loading control of the blot shown in FIG. 9A. FIGURE 9C is a graph depicting the PLF/18S ratios derived from a densitometric analysis of northern blots of normal and mutant osteopetrotic rat bone. Lanes 1, 3, and 5 depict PLF/18S ratios in mutant osteopetrotic bone in rats at 2, 4, and 6 weeks of age. Lanes 2, 4, and 6 depict PLF/18S ratios derived from normal rats at 2, 4, and 6 weeks of age. FIGURE 10A demonstrates, by in situ hybridization, PLF expression in the periosteum and endosteum of long bone in 2-week old neonatal mutant osteopetrotic rats (20X magnification). The region enclosed by the box is depicted at a higher magnification in FIG. 10B. H + zone of hypertrophic cells. FIGURE 10B demonstrates, by in situ hybridization, PLF expression in the periosteum and endosteum of long bone in 2-week old neonatal mutant osteopetrotic rats (100X magnification). The image is a higher magnification of the region enclosed by the box in FIG. 10A. H= zone of hypertrophic cells; P= zone of proliferating cells. The region between the arrows is further magnified in FIGURE IOC. FIGURE IOC is a 200X magnification of the region between the arrows shown in FIG. 10B. It demonstrates, by in situ hybridization, PLF expression in the periosteum and endosteum of long bone in 2 week old neonatal mutant osteopetrotic rats. PE= periosteum; EN= endosteum. FIGURES 11A and 11B are northern blot analyses of PLF and 18S expression, respectively, in human vascular smooth muscle cells grown under various conditions. Lanes: (1) starved; (2) 10 % fetal calf serum; (3) T-cell conditioned media; (4) dexamethasone; (5) differentiation media; (6) fibroblast growth factor; (7) interferon γ; (8) platelet derived growth factor; and (9) transforming growth factor β. FIGURE 11C is a graph depicting a densitometric analysis of PLF/18S expression of the blots depicted in FIGS. 1 1A and 1 IB. Lanes: (1) starved;
(2) 10% fetal calf serum; (3) T-cell conditioned media; (4) dexamethasone; (5) differentiation media; (6) fibroblast growth factor; (7) interferon γ; (8) platelet derived growth factor; and (9) transforming growth factor β. FIGURE 12A is a photomicrograph of an immunohistochemical analysis of PLF protein expression in non-failing human heart (600X). The tissue was stained with anti-PLF antiserum. FIGURE 12B is a photomicrograph of an immunohistochemical analysis of PLF protein expression in ischemic human heart (600X). The tissue was stained with anti-PLF antiserum. The asterisk (*) is located in the lumen of a blood vessel. FIGURE 12C is a photomicrograph of an immunohistochemical analysis of PLF protein expression in idiopathic human heart (1,000X). The tissue was stained with anti-PLF antiserum. FIGURE 12D is a western blot analysis of PLF expression in mouse embryos. Proteins extracts from 13.5 day embryonic calvaria, 15.5 day embryonic long bone, 17.5 day embryonic long bone, 19.5 day embryonic long bone, and 2-day-old neonatal long bones were subjected to 8% SDS- PAGE. The proteins were transferred to nitrocellulose and probed with anti- PLF antiserum. The inset shows an image of the 15.5 day lane at a higher exposure where a fourth isoform is visible. FIGURE 13 represents an RT-PCR analysis of PLF in bone, heart, spleen, lung, and brain embryonic tissue at day 13.5 post-conception, to detect different isoforms of PLFs. The left panel utilized primers flanking the 672- 700 amino acid region. The right panel utilized primers flanking the 785-813 amino acid region. Upper and lower tissue specific bands are indicated by arrows. FIGURE 14 is a northern blot analysis of PLF mRNA expression in fetal mouse cardiac myocytes treated with antisense PLF oligonucleotides to determine whether PLF expression was needed for differentiation. FIGURE 15A, 15B, and 15C are micrographs of stage 4, stage 5, and stage 7 chicken embryos, respectively, treated with antisense oligonucleotide to PLF and photographed 24 hours post-treatment. FIGURE 15D is a micrograph of the embryo of FIG. 15C subjected to in situ hybridization for expression of the ventricle specific marker VMHC1. FIGURE 15E, 15F, 15G, and 15H are micrographs of a chicken embryos treated with antisense oligonucleotide against PLF at stage 8 and subjected to in situ hybridization for VMHCl expression 24 hours post- treatment. FIGURE 151 is a micrograph of a chicken embryo treated with antisense oligonucleotide against PLF at stage 9 and subjected to in situ hybridization for VMHC 1 expression 24 hours post-treatment. FIGURE 15 J is a micrograph of a control chicken embryo treated with DMSO, but not an antisense oligonucleotide against PLF, at stage 7 and subjected to in situ hybridization for VMHCl expression 24 hours post- treatment. FIGURE 15K is a micrograph of a stage 4 chicken embryo at the time of treatment with antisense oligonucleotide against PLF (zero hours post- treatment). FIGURE 15L is a graph summarizing the anomalies found in the studies described in FIGS. 15A-K in DMSO-treated, control scrambled antisense oligonucleotide-treated, and antisense oligonucleotide against PLF- treated chicken embryos. The ordinate represents the number of embryos, and each group is labeled on the abscissa as either dead, beating heart, normal, cardia bifida, abnormal looping, or other abnormalities. FIGURE 16 is an RT-PCR analysis of PLF expression in MC3T3-E1 osteoblast cells treated with antisense oligonucleotide against PLF. PLF expression was compared to G3PDH expression, a gene not regulated by differentiation. FIGURE 17 is an RT-PCR analysis of differentiation marker expression in MC3T3-E1 osteoblast cells treated with antisense oligonucleotide against PLF. The differentiation markers include osteopontin, osteocalcin, collagen I, AL-PH, and Cbfa 1. AS 1 = antisense oligonucleotide 1; AS2 = antisense oligonucleotide 2. FIGURE 18 is an RT-PCR analysis of differentiation marker gene expression in MC3T3-E1 osteoblast cells treated with anti-PLF antibody transfected into cells using CHARIOT. Cells were transfected with an antibody/Chariot mix and RT-PCR analysis was performed 7 and 21 days post-transfection. The differentiation markers include osteopontin, osteocalcin, collagen I, and AL-PH. D7 = 7 days post-treatment; D21 = 21 days post-treatment.
Detailed Description of the Invention The present invention is based, in part, on the discovery and characterization of a nucleic acid sequence which encodes a protein which appears to be a member of the Periostin family. Periostin proteins are involved in various cellular functions, including cell differentiation, cell migration, cell adhesion, and metastases. This new protein, called "Periostin- Like Factor" or "PLF" herein, uniquely encodes a 27 amino acid segment not present in mouse or human Periostin. PLF also lacks a 28 amino acid segment, which is present in human and mouse Periostin. The putative DNA sequence complementary to the mRNA transcript of
PLF, both mouse and human, and predicted amino acid sequence are set forth herein. PLF comprises a unique nucleic acid and amino acid sequence compared to other members of the Periostin protein family. The numbering of amino acid positions and comparisons of members of the Periostin family, as used herein, are based on the alignment described in FIGURES IA to 1C, and as described in Example 1. It has been found that PLF mRNA and protein are upregulated in various disease states, cell growth and differentiation states, and during various stages of embryonic development. It has also been found that antisense oligonucleotides directed against various sequences of PLF mRNA and antibodies directed against PLF protein, results in inhibition of PLF expression and biological activity in cells. The present invention thus provides PLF nucleic acids and proteins useful for the diagnosis and treatment of Periostin-associated disorders. All nucleic acid sequences herein are given in the 5' to 3' direction unless otherwise indicated. A nucleic acid comprising a nucleic acid sequence encoding a naturally occurring isoform of Periostin has been isolated from mouse heart and from human heart. In one embodiment, the invention provides the mouse PLF cDNA, comprising the 3012 bases of SEQ ID NO:4. SEQ ID NO:4 encodes the entire mouse PLF protein amino acid sequence. The invention further provides a mouse PLF cDNA comprising the 3290 base SEQ ID NO: 13, which also encodes the entire mouse PLF protein. The nucleic acid sequence comprising only the coding region of the 810 amino acid mouse PLF protein (SEQ ID NO:l 1) is a 2430 base nucleic acid having the sequence SEQ ID NO: 12. In one embodiment, the invention provides a human PLF partial nucleic acid sequence. The partial sequence comprises a 479 base nucleic acid having the sequence SEQ ID NO:24. SEQ ID NO:24 encodes the human PLF amino acid sequence having the sequence SEQ ID NO:27, a peptide of 135 amino acid residues. The present invention provides the full length mouse PLF amino acid sequence (SEQ ID NO: 11). Mouse PLF contains a 27 amino acid segment (SEQ ID NO: 14) comprising amino acid residues 673-699, not present in mouse Periostin (SEQ ID NO:6). Mouse PLF does not contain a 28 amino acid (SEQ ID NO: 15) comprising amino acid residues 785-812, that is present in mouse Periostin. The present invention also provides human PLF. The partial cDNA sequence (SEQ ID NO:24) and partial amino acid sequence (SEQ ID NO:27) of human PLF are provided. The sequenced 135 amino acids of human PLF share 100%) sequence identity with the same 135 amino acid sequence region of mouse PLF (FIGS. IA to IF). Thus, the amino acid sequence of human PLF (SEQ ID NO:27), comprising amino acid residue positions 669-831 (FIG. IA to 1C), as with mouse PLF, contains a 27 amino acid segment not present in Periostin, and does not contain a 28 amino acid segment which is present in Periostin. In one embodiment, primer oligonucleotides are provided for cloning
PLF cDNA. Examples of such primers include SEQ ID NOS:2, 3, and 17-20 (Example 1; Figure IF). In one aspect, different primers complementary to other regions of a nucleic acid sequence encoding PLF can be prepared. Techniques for preparing, identifying, and sequencing cDNAs are known to those of ordinary skill in the art (Maniatis, Molecular Cloning, Cold Spring Harbor Laboratories, 1982; Sambrook (Molecular Cloning, Cold Spring Harbor Laboratories, Second Ed., 1989; Ausubel, Current Protocols in Molecular Biology, Wiley and Sons, 1987). The present invention encompasses all mammalian homologs of the PLF nucleic acids and polypeptides described herein. The present invention provides fragments of PLF. PLF fragments according to the invention can be obtained, for example, by chemical or enzymatic fragmentation of larger natural or synthetic PLF peptides, or by biological or chemical syntheses as described below. Derivatives of PLF can also be used in the present methods. The techniques for obtaining PLF derivatives are within the skill in the art and include, for example, standard recombinant nucleic acid techniques, solid phase peptide synthesis techniques and chemical synthetic techniques as described below. PLF derivatives can also be obtained by using linking groups to join PLF (especially SEQ ID NOS: 11 or 27) or PLF fragments to other peptides. Linking groups suitable for use in the present invention include, for example, cyclic compounds capable of connecting an amino- terminal portion and a carboxyl terminal portion of SEQ ID NOS: 11 or 27. Techniques for generating derivatives are also described in U.S. patent 6,030,942 the entire disclosure of which is herein incorporated by reference (derivatives are designated "peptoids" in the 6,030,942 patent). Examples of derivatives according to the present invention include, for example, synthetic variants of PLF. PLF derivatives also include fusion peptides in which a portion of the fusion peptide has a substantially similar amino acid sequence to SEQ ID NOS: 11 or 27. Such fusion peptides can be generated by techniques well-known in the art, for example by subcloning nucleic acid sequences encoding SEQ ID NOS: 11 or 27 and a heterologous peptide sequence into the same expression vector, such that the PLF and the heterologous sequence are expressed together in the same protein. The heterologous sequence can also comprise a peptide leader sequence that directs entry of the expressed protein into a cell. Such leader sequences include "protein transduction domains" or "PTDs," which are discussed in more detail below. Key structural elements of PLF can be identified, for example, by evaluating the various portions of PLF for the ability to stimulate or inhibit genes expressed during cardiac muscle cell differentiation, osteoblast differentiation, or to inhibit normal embryogenesis as measured by cell migration and heart development (see Examples 8-11 below). Alternatively, PLF key structural elements can be determined using nuclear magnetic resonance (NMR), crystallographic, and/or computational methods which permit the electron density, electrostatic charges or molecular structure of certain portions of PLF or fragments thereof to be mapped. Preferably, PLF key structural elements comprise the primary, secondary and tertiary structure of the amino acid sequence of SEQ ID NOS:l 1 and 27. The compounds of the invention which comprise polypeptides can be synthesized de novo using conventional solid phase synthesis methods. In such methods, the peptide chain is prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence. The use of various N-protecting groups, e.g., the carbobenzyloxy group or the t-butyloxycarbonyl group; various coupling reagents e.g., dicyclohexylcarbodiimide or carbonyldiimidazole; various active esters, e.g., esters of N-hydroxyphthalimide or N-hydroxy-succinimide; and the various cleavage reagents, e.g., trifluoroactetic acid (TFA), HCI in dioxane, boron tris-(trifluoracetate) and cyanogen bromide; and reaction in solution with isolation and purification of intermediates are methods well- known to those of ordinary skill in the art. A preferred peptide synthesis method follows conventional Merrifield solid phase procedures well known to those skilled in the art. Additional information about solid phase synthesis procedures can be had by reference to Steward and Young, Solid Phase Peptide Synthesis, W.H. Freeman & Co., San Francisco, 1969; the review chapter by Merrifield in Advances in Enzymology 32:221-296, F.F. Nold, Ed., Interscience Publishers, New York, 1969; and Erickson and Merrifield, The Proteins 2:61-64 (1990), the entire disclosures of which are incorporated herein by reference. Crude peptide preparations resulting from solid phase syntheses may be purified by methods well known in the art, such as preparative HPLC. The amino-terminus may be protected according to the methods described for example by Yang et al., (1990 FEBS Lett 272:61-64), the entire disclosure of which is herein incorporated by reference. The compounds of the invention which comprise PLF peptides can also be produced by biological synthesis. Biological synthesis of peptides is well known in the art, and includes the transcription and translation of a synthetic nucleic acid encoding a PLF protein, or a fragment, derivative, or homolog of PLF. Biological syntheses of PLF, or fragments, derivatives, or homologs thereof can be based on the mouse PLF nucleic acid sequence (SEQ ID NO:4) or amino acid sequence (SEQ ID NO: 11), or on the human PLF nucleic acid sequence (SEQ ID NO:24) or amino acid sequence ( SEQ ID NO:27). The techniques of recombinant DNA technology are within the skill in the art. General methods for the cloning and expression of recombinant molecules are described in Maniatis (Molecular Cloning, Cold Spring Harbor Laboratories, 1982), and in Sambrook (Molecular Cloning, Cold Spring Harbor Laboratories, Second Ed., 1989), and in Ausubel (Current Protocols in Molecular Biology, Wiley and Sons, 1987), the entire disclosures of which are herein incorporated by reference. For example, PLF and fragments, derivatives, and homologs thereof can be prepared utilizing recombinant DNA techniques, which can comprise combining a nucleic acid encoding the peptide in a suitable vector, inserting the resulting vector into a suitable host cell, recovering the peptide produced by the resulting host cell, and purifying the polypeptide recovered. The nucleic acids encoding PLF peptides may be operatively linked to one or more promoter and/or regulatory regions. Regulatory regions include promoters, polyadenylation signals, translation initiation signals (Kozak regions), termination codons, peptide cleavage sites, and enhancers. The regulatory sequences used must be functional within the cells into which they are transfected. Selection of the appropriate regulatory region or regions is a routine matter, within the level of ordinary skill in the art. Suitable promoters include both constitutive promoters and regulated (inducible) promoters, and can be prokaryotic or eukaryotic, depending on the host. Among the prokaryotic (including bacteriophage) promoters useful for practice of this invention are: lac, T3, T7, lambda Pr' PI' and tip promoters. Among the eukaryotic (including viral) promoters useful for practice of this invention are: ubiquitous promoters (e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g. MDR type, CFTR, factor VIII), tissue- specific promoters (e.g. actin promoter in smooth muscle cells), promoters which respond to a stimulus (e.g. steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovirus immediate-early, retroviral LTR, metallothionein, SV-40, El a, and MLP promoters. Tetracycline-regulated transcriptional modulators and CMV promoters are described in WO 96/01313, US 5,168,062 and US 5,385,839, the entire disclosures of which are incorporated herein by reference. Suitable polyadenylation signals that can be used in the present invention include SV40 polyadenylation signals and LTR polyadenylation signals. The compounds of the invention can be modified with other substances prior to use in the present methods, using techniques known in the art. Methods of modifying proteins and peptides with other substances, in particular labels, are well known to those skilled in the art. Modification of the proteins may alter their activity, for example by altering characteristics such as in vivo tissue partitioning, peptide degradation rate, or biological activity. The modifications may also confer additional characteristics to the compound, such as the ability to be detected, manipulated, or targeted. A modifying substance can be joined, for example, to the compound of the invention by chemical means (e.g., by covalent bond, electrostatic interaction, Van der Waals forces, hydrogen bond, ionic bond, chelation, and the like) or by physical entrapment. For example, the compounds of the invention can be modified with a label (e.g., substances which are magnetic resonance active; radiodense; fluorescent; radioactive; detectable by ultrasound; detectable by visible, infrared or ultraviolet light). Suitable labels include, for example, fluorescein isothiocyanate, peptide chromophores such as phycoerythrin or phycocyanin and the like; bioluminescent peptides such as the luciferases originating from Photinus pyrali; or fluorescent proteins originating from Renilla reniformi. Compounds of the invention may also be modified with polymeric and macromolecular structures (e.g., liposomes, zeolites, dendrimers, magnetic particles, and metallic beads) or targeting groups (e.g., signal peptide sequences, ligands, lectins, or antibodies). Peptides or peptide fragments can be further modified with end protecting groups at the carboxyl or amino- terminal ends, amino-acid side chain modifying groups, and the like. Modification of the peptides of the invention may alter their activity, for example by altering characteristics such as in vivo tissue partitioning, peptide degradation rate, or ligase activity. The modifications may also confer additional characteristics to the compound, such as the ability to be detected, manipulated, or targeted. Methods of modifying the compounds of the invention (in particular the compounds of the invention which comprise peptides) with other substances are well known to those skilled in the art. For example, methods of conjugating fluorescent compounds such as fluorescein isothiocyanate to a peptide are described in Danen et al., Exp. Cell Res., 238:188-86 (1998), the entire disclosure of which is incorporated herein by reference. Methods of radiolabeling peptides with I are disclosed by Sambrook et al. in Molecular Clonins: A Laboratory Manual. Cold Spring Harbor Laboratories, Second Ed., (1989), the disclosure of which is incorporated herein by reference. For example, functional groups which can be covalently linked to the compounds of the invention comprising peptides include amines, alcohols, or ethers. Functional groups covalently linked to the compounds of the invention which comprise peptides, and which can increase the in vivo half-life of the compounds include polyethylene glycols, small carbohydrates such as sucrose, or polypeptides. In one aspect of the invention, the proteins may contain aspartic acid (D) residues to promote their solubility. In another embodiment of the invention, the half-life in the blood stream of the compounds of the invention is enhanced by the addition of adducts such as sucrose or polyethylene glycol, production of peptide-IgG chimeras. The compounds of the invention which comprise peptides can also be cyclized via cysteine-cysteine linkages, which is known to enhance the biological activities of a variety of peptides. The compounds of the invention can be derivatized with functional groups or linked to other molecules to facilitate their delivery to specific sites of action or to potentiate their activity. The compounds of the invention can also be covalently or non-covalently linked to other pharmaceuticals, bioactive agents, or other molecules. Such derivatizations should not significantly interfere with the ubiquitin ligase or other biological properties of the compounds. Carriers and derivatizations of the compounds of the invention should also be designed or chosen so as not to exert toxic or undesirable activities on animals or humans treated with these formulations. PLF, and fragments, derivatives, and homologs thereof, as well as antibodies against PLF, can be modified to enhance their entry into cells associated with a PLF-associated disorder. For example, the compounds of the invention can be encapsulated in a liposome prior to being administered. The encapsulated compounds are delivered directly into the abnormally proliferating cells by fusion of the liposome to the cell membrane. Reagents and techniques for encapsulating the present compounds in liposomes are well known in the art, and include, for example, the ProVectin™ Protein Delivery Reagent from Imgenex. In a preferred embodiment, the peptide compounds of the invention are modified by associating the compounds with a peptide leader sequence known as a "protein transduction domain" or "PTD." These sequences direct entry of the compound into abnormally proliferating cells by a process known as "protein transduction" (Schwarze et al., 1999, Science 285:1569-1572). PTDs are well-known in the art, and can comprise any of the known PTD sequences including, for example, arginine-rich sequences such as a peptide of nine to eleven arginine residues optionally in combination with one to two lysines or glutamines as described in Guis et al. (1999, Cancer Res. 59:2577-2580, the disclosure of which is herein incorporated by reference). Preferred are sequences of eleven arginine residues or the NH2-terminal 11 - amino acid protein transduction domain from the human immunodeficiency vims TAT protein. Other suitable leader sequences include, but are not limited to, other arginine-rich sequences; e.g., 9 to 10 arginines, or six or more arginines in combination with one or more lysines or glutamines. Such leader sequences are known in the art; see, e.g., Guis et al. (1999), supra. Preferably, the PTD is designed so that it is cleaved from the compound upon entry into the cell. A PTD may be located anywhere on the compound that does not disrupt the compound's biological activity. For compounds of the invention comprising a peptide, the PTD is preferably located at the N-terminal end. Kits and methods for constructing fusion proteins comprising a protein of interest (e.g., PLF) and a PTD are known in the art; for example the TransVector™ system (Q-BIOgene), which employs a 16 amino acid peptide called "Penetratin™" corresponding to the Drosophila antennapedia DNA- binding domain; and the Voyager system (Invitrogen Life Technologies), which uses the 38 kDa VP22 protein from Herpes Simplex Virus- 1. PLF associated disorders include cell proliferation and differentiation disorders, cell adhesion disorders, cell migration disorders, cancer, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, metastasis, cardiac hypertrophy, ischemic heart disease, idiopathic heart disease, restenosis, diseases of overgrowth of bone (e.g., osteopetrosis) and/or destruction of bone (e.g., osteoporosis), and embryonic development disorders. PLF associated disorders of embryonic development include cardia bifida, abnormal vitelline vessel development, and head and somite anomalies (Example 9). PLF should be useful in promoting cell migration and adhesion during embryogenesis and during wound healing. In one embodiment, a PLF associated disorder is characterized by decreased levels of PLF nucleic acid, PLF protein, or PLF protein activity. This disorder is treated by administering to a subject an isolated PLF protein, or an isolated nucleic acid comprising a nucleic acid sequence encoding a PLF protein, either alone or in combination with other compounds. The PLF protein completely or partially corrects the PLF associated disorder. In the practice of the invention, PLF protein is used to increase PLF proteins levels in PLF associated disorders where there are reduced levels of PLF nucleic acid, PLF protein, or PLF protein activity. For example, as discussed above, Periostins play a role in cell adhesion and migration, and loss of PLF disrupts cell adhesion and migration. Without wishing to be bound by any theory, it is believed that in some cases cells associated with a PLF associated disorder do not express PLF mRNA or protein, or express mutant or aberrant levels of PLF, which results in decreased PLF function. Thus, the invention provides a method of treating a PLF-associated disorder in a subject in need of such treatment. The method comprises administering an effective amount of a PLF protein, or fragment, derivative, or homolog analog thereof, to the subject, such that biological processes which have been inhibited because of the PLF associated disorder are restored. In another embodiment, a PLF-associated disorder is characterized by increased levels of PLF nucleic acid, PLF protein or PLF protein activity. Such disorders include idiopathic heart disease, ischemic heart disease, and osteopetrosis (see Examples 3 and 4). This disorder is treated by administering to a subject an antisense oligonucleotide directed against a PLF nucleic acid, a nucleic acid comprising a nucleic acid sequence encoding an antisense oligonucleotide complementary to a PLF nucleic acid sequence, or an antibody directed against PLF. In one embodiment, PLF can be applied as a coating to devices such as stents to encourage migration of endothelial, smooth muscle, or other cells. The effect of treatment can be monitored using many cellular, molecular, and clinical techniques, which are known to those of ordinary skill in the art. Where the assay is designed to measure the ability of a compound of the invention to stimulate cell adhesion or migration, assays are known in the art which can be used to measure cell adhesion or migration in vitro and in vivo. Other methods useful for measuring cell adhesion or migration are known to those of skill in the art (also see Example 9). The number of cells associated with a PLF associated disorder in a subject's body can be determined by direct measurement, or by estimation from the size of primary or metastatic tumor masses. For example, the number of cells associated with a PLF associated disorder in a subject can be readily determined by immunohistological methods, flow cytometry, or other techniques designed to detect the characteristic surface markers of a given cell type. In one aspect of the invention, a PLF associated disorder is cancer, or another disorder capable of being subjected imaging techniques. The size of a tumor mass can be ascertained by direct visual observation, or by diagnostic imaging methods such as X-ray, magnetic resonance imaging, ultrasound, and scintigraphy. Diagnostic imaging methods used to ascertain size of the tumor mass can be employed with or without contrast agents, as is known in the art. The size of a tumor mass can also be ascertained by physical means, such as palpation of the tissue mass or measurement of the tissue mass with a measuring instrument such as a caliper. For prostate tumors, a preferred physical means for determining the size of a tumor mass is the digital rectal exam. In another aspect of the invention, the PLF associated disorder is a bone disorder. PLF associated bone disorders include osteopetrosis and osteoporosis. Diagnostic imaging methods can be used to ascertain bone densities and changes in bone associated with PLF associated bone disorders. Changes in bone density can also be determined using specialized techniques known to those of ordinary skill in the art. One skilled in the art can readily determine an effective amount of a compound of the invention or an antibody directed against PLF, or fragments, derivatives, or homologs of PLF, to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. For example, an effective amount of a compound of the invention or an antibody directed against PLF, can be based on the approximate weight of a tumor mass to be treated. The approximate weight of a tumor mass can be determined by calculating the approximate volume of the mass, wherein one cubic centimeter of volume is roughly equivalent to one gram. An effective amount of the compounds of the invention based on the weight of a tumor mass can be at least about 10 micrograms/gram of tumor mass. More preferably, the effective amount is at least about 100 micrograms/gram of tumor mass. Particularly preferably, the effective amount is at least about 500 micrograms/gram of tumor mass. It is preferred that an effective amount based on the weight of the tumor mass be injected directly into the tumor. An effective amount of the compounds of the invention or an antibody directed against PLF, can also be based on the approximate or estimated body weight of a subject to be treated. Preferably, such effective amounts are administered parenterally or enterally, as described below. For example, an effective amount of the nucleic acids of the invention administered to a subject can range from about 5-500 μg/kg of body weight, or between about 500-1000 μg/kg of body weight, or is greater than about 1000 μg/kg of body weight. Typically, dosages of PLF protein, or fragments, derivatives, or homologs of PLF, or antibodies directed against PLF protein, or fragments, derivatives, or homologs of PLF, are between about 0.001 mg/kg and about 100 mg/kg body weight. In some embodiments, dosages are between about 0.01 mg/kg and about 60 mg/kg body weight. In other embodiments, dosages are between about 0.05 mg/kg and about 5 mg/kg body weight. One skilled in the art can also readily determine an appropriate dosage regimen for the administration of the compounds of the invention or antibody directed against PLF for a given subject. For example, an effective amount of a compound of the invention or antibody directed against PLF, can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, the compounds of the invention can be administered once or twice daily to a subject for a period of from about three to about twenty-eight days, more preferably from about seven to about ten days. In a preferred dosage regimen, the compounds of the invention are administered once a day for seven days. Where a dosage regimen comprises multiple administrations, it is understood that the effective amount can comprise the total amount of compound or antibody directed against PLF administered over the entire dosage regimen. The compounds of the invention and antibodies directed against PLF, can be administered to a subject by any means suitable for delivering the compounds to cells of the subject, for example by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery. Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra- arterial infusion and catheter instillation into the vasculature); peri- and intra- tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by injection, a catheter, or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. Preferably, the compounds of the invention are administered by injection or infusion. Even more preferably, the compounds are administered locally to the site of the disorder. For the treatment of PLF associated disorders which involve solid tissue, the PLF nucleic acid, protein, or a fragment, derivative, or homolog thereof, or antisense oligonucleotide or antibody to Periostin-like is preferably administered by direct injection into the tissue. In one embodiment, an effective amount of the PLF protein, or fragment, derivative or homolog thereof, is administered to a subject by delivering an isolated nucleic acid comprising sequences encoding the PLF protein, or fragment, derivative or homolog thereof to a cell associated with a PLF-associated disorder. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors. For example, cells can be transfected with a liposomal transfer compound, e.g., DOTAP (N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methylsulfate, Boehringer-Mannheim) or an equivalent, such as LIPOFECTIN. The amount of nucleic acid used is not critical to the practice of the invention; acceptable results can be achieved with 0.1-100 micrograms of nucleic acid/105 cells. For example, a ratio of about 0.5 micrograms of plasmid vector in 3 micrograms of DOTAP per 105 cells can be used. A nucleic acid comprising sequences encoding PLF, or a fragment, derivative, or homolog of PLF, can be obtained using a number of standard techniques. Such nucleic acids can, for example, be chemically synthesized or recombinantly produced using methods known in the art as described above. The nucleic acid sequence of mouse PLF cDNAs is provided herein (SEQ ID NOS:4 and 13). These sequences are merely different lengths (3012 bases and 3290 base, respectively) and each includes the entire region (SEQ ID NO:12) encoding the amino acid sequence (SEQ ID NO:l 1) of mouse PLF. More than one nucleic acid sequence is capable of encoding a particular amino acid sequence. Degenerate sequences are degenerate within the meaning of the genetic code in that nucleotides can be replaced by other nucleotides in some instances without resulting in a change of the amino acid sequence originally encoded. Nucleic acid sequences comprising sequences encoding PLF protein, or fragments, derivatives, or homologs of PLF can be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing nucleic acid sequences from a plasmid include the U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art.
The recombinant plasmids suitable for use in the present invention can also comprise inducible or regulatable promoters for expression of nucleic acids in cells associated with a PLF associated disorder. Selection of plasmids suitable for expressing the PLF nucleic acid, methods for inserting nucleic acid sequences for expressing the PLF nucleic acid into the plasmid, and methods of delivering the recombinant plasmid to cells associated with a PLF associated disorder are within the skill in the art.
See, for example Zeng, et al. 2002, Molecular Cell 9: 1327-1333;
Brummelkamp et al. 2002, Science 296:550-553; Miyagishi, et al. 2002, Nat.
Biotechnol. 20:497-500; Paddison et al. 2002, Genes Dev. 16:948-958; Lee et al. 2002, Nat. Biotechnol. 20:500-505; and Paul et al. 2002, Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference. In a preferred embodiment, a plasmid according to the invention comprises a sequence encoding the PLF mRNA under the control of the CMV intermediate-early promoter. As used herein, "under the control" of a promoter means that the nucleic acid sequences encoding PLF are located 3' of the promoter, so that the promoter can initiate transcription of the PLF product coding sequences. A nucleic acid comprising sequences encoding PLF protein, or fragments, derivatives, or homologs thereof can also be expressed from recombinant viral vectors. The nucleic acids expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in cells associated with a
PLF associated disorder. The recombinant viral vectors of the invention can comprise any suitable promoter for expressing the nucleic acid sequences in cells associated with a PLF associated disorder. Suitable promoters include, for example, the
U6 or HI RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. The recombinant viral vectors suitable for use in the present invention can also comprise inducible or regulatable promoters for expression of PLF in a cell. Any viral vector capable of accepting and expressing nucleic acid sequences can be used; for example, vectors derived from adenovirus (AV); adeno-associated vims (AAV); retrovimses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia vims); herpes vims, and the like. The tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other vimses. For example, an AAV vector of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. Selection of recombinant viral vectors suitable for use in the invention, methods for inserting and expressing nucleic acid sequences, methods of delivering the viral vector to cells associated with a PLF associated disorder, and recovery of the expressed sequences are within the skill in the art. For example, see Dornburg, R., 1995, Gene Therap. 2:301-310; Eglitis, M.A. 1988, Biotechniques 6:608-614; Miller, A.D. 1990, Hum Gene Therap. 1:5- 14; and Anderson, W.F., 1998, Nature 392:25-30, the entire disclosures of which are herein incorporated by reference. Preferred viral vectors are those derived from AV and AAV. A suitable AV vector, a method for constmcting the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010, the entire disclosure of which is herein incorporated by reference. Suitable AAV vectors, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61 :3096-3101; Fisher K. J. et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference. In the present methods, an isolated nucleic acid comprising sequences encoding PLF protein, or a fragment, derivative, or homolog of PLF, can be administered to the subject in conjunction with a delivery reagent. Suitable delivery reagents for administration include the Mims Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes. In a preferred embodiment, liposomes are used to deliver isolated nucleic acids comprising sequences encoding PLF protein, or a fragment, derivative, or homolog of PLF, to a subject. Liposomes may also be used to deliver antisense oligonucleotides complementary to a PLF nucleic acid sequence. Liposomes can also increase the blood half-life of the nucleic acids. In the practice of this embodiment of the invention, the compounds of the invention, or nucleic acids comprising sequences encoding a PLF protein or fragment, derivative, or homolog of PLF, are encapsulated in liposomes prior to administration to the subject. Liposomes suitable for use in the invention can be formed from standard vesicle-forming lipids, which generally include neutral or negatively charged phospholipids and a sterol such as cholesterol. The selection of lipids is generally guided by consideration of factors such as the desired liposome size and half-life of the liposomes in the blood stream. A variety of methods are known for preparing liposomes. The liposomes encapsulating nucleic acids comprising sequences encoding PLF can comprise a ligand molecule that targets the liposome to a cell associated with a PLF associated disorder, such as heart, bone, muscle, or connective tissue. Ligands which bind to receptors prevalent in the cells of such tissues are preferred. The liposomes encapsulating isolated nucleic acids comprising sequences encoding PLF or a fragment, derivative, or homolog of PLF, can also be modified so as to avoid clearance by the mononuclear macrophage system ("MMS") and reticuloendothelial system ("RES"). Such modified liposomes have opsonization-inhibition moieties on the surface or incorporated into the liposome structure. In a particularly preferred embodiment, a liposome of the invention can comprise both opsonization- inhibition moieties and a ligand. Opsonization-inhibiting moieties for use in preparing the liposomes of the invention are typically large hydrophilic polymers that are bound to the liposome membrane. As used herein, an opsonization inhibiting moiety is "bound" to a liposome membrane when it is chemically or physically attached to the membrane, e.g., by the intercalation of a lipid-soluble anchor into the membrane itself, or by binding directly to active groups of membrane lipids. These opsonization-inhibiting hydrophilic polymers form a protective surface layer which significantly decreases the uptake of the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No. 4,920,016, the entire disclosure of which is herein incorporated by reference. Opsonization-inhibiting moieties suitable for modifying liposomes are preferably water-soluble polymers with a number-average molecular weight from about 500 to about 40,000 daltons, and more preferably from about 2,000 to about 20,000 daltons. Such polymers include polyethylene glycol (PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone; linear, branched, or dendrimeric polyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcojhol and polyxylitol to which carboxylic or amino groups are chemically linked, as well as gangliosides, such as ganglioside GMj. Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are also suitable. In addition, the opsonization inhibiting polymer can be a block copolymer of PEG and either a polyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, or polynucleotide. The opsonization inhibiting polymers can also be natural polysaccharides containing amino acids or carboxylic acids, e.g., galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic acid, carrageenan; aminated polysaccharides or oligosaccharides (linear or branched); or carboxylated polysaccharides or oligosaccharides, e.g., reacted with derivatives of carbonic acids with resultant linking of carboxylic groups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, or derivatives thereof. Liposomes modified with PEG or PEG-derivatives are sometimes called "PEGylated liposomes." The opsonization-inhibiting moiety can be bound to the liposome membrane by any one of numerous well-known techniques. For example, an N-hydroxysuccinimide ester of PEG can be bound to a phosphatidyl- ethanolamine lipid-soluble anchor, and then bound to a membrane. Similarly, a dextran polymer can be derivatized with a stearylamine lipid-soluble anchor via reductive animation using Na(CN)BH3 and a solvent mixture such as tetrahydrofuran and water in a 30:12 ratio at 60°C. Liposomes modified with opsonization-inhibiting moieties remain in the circulation much longer than unmodified liposomes. For this reason, such liposomes are sometimes called "stealth" liposomes. Stealth liposomes are known to accumulate in tissues fed by porous or "leaky" micro vasculature. Thus, tissue characterized by such microvasculature defects, for example solid tumors, will efficiently accumulate these liposomes; see Gabizon, et al. (1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, the reduced uptake by the RES lowers the toxicity of stealth liposomes by preventing significant accumulation of the liposomes in the liver and spleen. Thus, liposomes that are modified with opsonization-inhibition moieties are particularly suited to deliver nucleic acids comprising sequences encoding a PLF protein, or fragment, derivative, or homolog thereof to cells associated with a PLF associated disorder. In a preferred embodiment, the cells from a subject are transfected with a nucleic acid comprising sequences which encode PLF or fragments, derivatives, or homologs thereof, and a plasmid expression vector that stably integrates into the cell genome to provide long-term expression of a compound of the invention. This can be done for vectors expressing antisense sequences complementary to PLF nucleic acid sequences as well (see below). Stable integration and expression of transfected nucleic acids can be confirmed by techniques known in the art, such as a Southern blot of genomic DNA using PLF cDNA (or fragments thereof) as a probe. Stable expression of PLF mRNA can also be detected by standard northern blot techniques. In one embodiment, the cells of the subject are transfected by administering an isolated nucleic acid comprising sequences which encode PLF or fragments, derivatives, or homologs of PLF, and a plasmid expression vector to the subject. In another embodiment, the cells being transfected have been isolated from the subject. In one aspect, the cells are reimplanted to purge or displace remaining PLF associated disorder cells or to purge cells predisposed to developing a PLF associated disorder. The compounds and antibodies of the invention can be administered to a subject by any means suitable for delivering the compounds to cells of the subject, for example by any suitable enteral or parenteral administration route. Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery. Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri- tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. Preferably, the compounds of the invention are administered by injection or infusion. Even more preferably, compounds of the invention are delivered locally to the site of the disorder. For the treatment of PLF associated disorders which involve solid tumors, the isolated nucleic acid comprising sequences encoding the PLF protein, or fragment, derivative or homolog of the PLF sequence is preferably administered by direct injection into the tumor. One skilled in the art can readily determine an effective amount of an isolated nucleic acid comprising a sequence encoding a PLF protein, or fragments, derivatives, or homologs thereof, to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. A preferred method of delivering the isolated nucleic acid to the cells associated with a PLF associated disorder is by transfection. The present invention provides antisense oligonucleotides directed against nucleic acids encoding PLF. The antisense oligonucleotides of the present invention can be in the form of RNA or DNA, e.g., cDNA, genomic DNA, or synthetic RNA or DNA. The antisense oligonucleotides can be used as primers in sequencing experiments, or they can be generated for use in dismpting functions because they bind to a specific nucleic acid sequence and block its function. The specific hybridization of an oligomeric compound with its target nucleic acid, resulting in interference with the normal function of the nucleic acid, is generally referred to as "antisense." Antisense oligonucleotides disrupt DNA functions such as replication and transcription. RNA functions which are subject to disruption by antisense oligonucleotides include translocation of the RNA to it site of translation, splicing of the RNA to yield one or more mRNA species, catalytic activity which can be engaged in or facilitated by the RNA, and ultimately, expression of the encoded protein. The antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonucleotide synthesis methods, including methods to generate more stable and efficient oligonucleotides. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology, VCH Verlagsgesellschaft mbH (H. Ibelgaufts trans. 1987). Any of the known methods of oligonucleotide synthesis may be utilized in preparing the instant antisense oligonucleotides. The antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers, for example, the Applied Biosystems 380B DNA Synthesizer. Because the nucleotide sequences of DNA complementary to mouse and human PLF mRNA transcripts are described herein, antisense oligonucleotides hybridizable with any portion of these mRNA transcripts may be prepared by the oligonucleotide synthesis methods known to those skilled in the art. While any length oligonucleotide may be utilized in the practice of the invention, sequences shorter than about 8-12 nucleotides may be less specific in hybridizing to the target PLF mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by even a single base pair mismatch. Hence, oligonucleotides having about 8-12 or more nucleotides are preferred. Long sequences, particularly sequences longer than about 40 nucleotides, may be somewhat less effective in inhibiting PLF translation because of decreased uptake by the target cell. Thus, oligomers of about 8-40 nucleotides are preferred, preferably about 15-30 nucleotides. The slightly longer chains are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides. Oligonucleotides complementary to and hybridizable with any portion of a PLF mRNA transcript are, in principle, effective for inhibiting translation of the transcript, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-terminal region of a PLF mRNA transcript are preferred. The oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Particularly preferred are oligonucleotides hybridizable to a region of the PLF mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon. In one embodiment, the oligonucleotide is preferably directed to a novel site at a PLF splice junction. Where a splice occurs at the site of deletion of a fragment, or inclusion of a fragment not present in Periostin or members of the Periostin family, a novel sequence is formed at the junction(s). For example, one of the characteristics that distinguishes PLF from other Periostins is a deletion in the mRNA of the nucleic acid sequence of Periostin encoding the peptide region comprising amino acids 785-812 (see FIGS. IA to 1C and Examples). Thus, a novel sequence arises when the deletion occurs and amino acid residue 784 of Periostin is followed by the amino acid residue previously at position 813 (based on the alignment as shown in FIGS. IA to 1C). The 27 amino acid residue segment (SEQ ID NO: 14) contained at position 673 of PLF, is novel relative to those Periostins which do not have the fragment. Furthermore, antisense oligonucleotides can be directed against a nucleic acid sequence which spans some nucleic acid residues from the insert to the common sequences both 5' and 3' to the nucleic acid encoding the inserted fragment. Such an antisense can be prepared which shares little homology with nucleic acid sequences which are not PLF nucleic acid sequences. The following oligodeoxynucleotides are complementary to the PLF mRNA transcript: SEQ ID NOS :21-23. Thus, the invention also includes antisense oligonucleotides complementary to the region of spliced PLF mRNA where the nucleic acid sequence encoding the peptide fragment having the sequence of amino acid residues 785-812 of Periostin has been deleted. Preferably, an oligonucleotide is prepared to span the site where a splice occurs. Oligomers of 8-40 nucleotides are preferred. The invention includes antisense oligonucleotides to the nucleic acid sequence encoding the peptide inserted between amino acids 672 and 700 of Periostin to form PLF. The invention also includes antisense oligonucleotides to sequences spanning the splice junctions of the nucleic acid sequence encoding the peptide between amino acids 672 and 700 of PLF. Oligonucleotides hybridizable to a PLF mRNA transcript finding utility according to the present invention include not only native oligomers of the biologically significant nucleotides, i.e., A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility. The oligonucleotides may be any of a number of types, including those having a charged or uncharged backbone. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or sulfur atom for a phosphate oxygen in the intemucleotide phosphodiester linkage to form alkylphosphonate oligonucleotide or phosphorothioate oligonucleotides. The phosphorthioates, in particular, are stable to nuclease cleavage and soluble in lipid. They may be synthesized by known automatic synthesis methods. The oligonucleotide employed may represent an unmodified oligonucleotide or an oligonucleotide analog. One group of such analogs, the alkyl phosphonates, includes but is not limited to the ethyl or methyl phosphonate analogs disclosed by U.S. Pat. No. 4,469,863. Non-ionic oligonucleotides are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences. The alkylphosphonates in particular are stable to nuclease cleavage and soluble in lipid. The preparation of alkylphosphonate oligonucleosides is disclosed in U.S. Pat. No. 4,469,863. Methylphosphonate oligomers can be prepared by a variety of methods known in the art, both in solution and on insoluble polymer supports. Resistance to nuclease digestion may also be achieved by modifying the intemucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990). Phosphorothioate oligonucleotides contain a sulfur-for-oxygen substitution in the intemucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al., Proc. Natl. Acad. Sci. U.S.A. 86, 3474-3478 (1989). The general method for preparing phosphorothioate oligonucleotides are described by LaPlanche, et al. (Nucleic Acids Research 14, 9081 1986), Stec et al. (J. Am. Chem. Soc. 106, 6077 1984), and Stein et al. (Nucl. Acids Res., 16, 3209-3221 1988). Furthermore, recent advances in the production of oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-O-methylribonucleotides (Inoue et al., Nucleic Acids Res. 15, 6131 1987) and chimeric oligonucleotides that are composite RNA-DNA analogues (Inoue et al., FEBS Lett. 215, 327 1987). While PLF mRNA translation can be inhibited by administering either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides are preferred over oligoribonucleotides in the practice of the present invention. In general, the antisense oligonucleotides of the present invention will have a sequence which is completely complementary to the target portion of a PLF mRNA. Absolute complementarity is not however required, particularly in larger oligomers. Thus, reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA transcript" of PLF mRNA does not necessarily mean a sequence having 100% complementarity with the transcript. In general, any oligonucleotide having sufficient complementarity to form a stable duplex with PLF mRNA, that is, an oligonucleotide which is "hybridizable," is suitable. Thus, 100% complementarity is not required for an antisense compound to be specifically hybridizable when binding to its target nucleic acid. Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of a PLF mRNA. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides. One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target PLF nucleic acid sequence, based upon the melting point, and therefore the stability of the resulting duplex. Melting points of duplexes of a given base pair composition can be determined from standard texts, such as Molecular Cloning: A Laboratory Manual, (2nd edition, 1989), J. Sambrook et al., eds. In one embodiment of the invention, methods and assays are provided for testing the activity of the antisense oligonucleotides both in vitro and in vivo and to determine their efficacy as therapeutic agents. For example, the antisense oligonucleotides of the invention inhibit heart PLF mRNA and protein expression in vitro and in vivo (see Examples). These antisense oligonucleotides against PLF nucleic acid sequences inhibit osteoblast and heart cell differentiation in vitro and heart development in vivo. Thus, the antisense oligonucleotides of the invention are easily assayed and are believed useful in the treatment of any diseases and disorders characterized by an elevated PLF, including developmental disorders and heart and bone diseases and disorders. The antisense oligonucleotides against PLF are believed particularly useful in blocking expression and overexpression of PLF. An antisense oligonucleotide against PLF may also be useful in blocking Periostin expression, if the PLF sequence which the antisense oligonucleotide is directed to is substantially homologous to a Periostin sequence. For in vivo use, the antisense oligonucleotides of the invention may be prepared for use as generally described herein for nucleic acids. That is, they may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives. The liquid vehicles and excipients are conventional and commercially available. In addition to administration with conventional carriers, the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques. In one aspect of the invention, antisense oligonucleotides are transfected using agents such as oligofectamine, which form stable complexes with oligonucleotides and enhance the ability of the oligonucleotide to penetrate the cell membrane. For in vivo use, the PLF antisense oligonucleotides may be administered in an amount sufficient to result in extracellular concentrations approximating the above stated in vitro concentrations. Preferably, the intracellular concentration is in the range of from about 10 to about 100 μg/ml. The actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health and sex of the patient, the route of administration, and other factors. Those skilled in the art should be readily able to derive suitable dosages and schedules of administration to suit the specific circumstance. The daily dosage may range from about 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 1,000 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required. Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the patient. The present invention further provides antibodies directed against PLF. The antibodies can be used in diagnostic assays to detect and measure levels of PLF in various cells and tissues in a subject, or can be administered to a subject to inhibit PLF in a subject in need of such treatment. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. Polyclonal antibodies are obtained by injecting whole proteins, peptides, or fragments thereof into appropriate animals and then screening for antisera which recognize the desired antigen. Many variations and techniques are known to those of skill in the art in the preparation of polyclonal antibodies. In another embodiment, monoclonal antibodies are prepared which are directed against PLF or fragments, derivatives, and homologs thereof. Antibodies to PLF can be obtained, for example, using as an antigen the product of a PLF expression vector or PLF isolated from a natural source. Anti-PLF antibodies can be produced using antigenic PLF epitope- bearing peptides and polypeptides. Antigenic epitope-bearing peptides and polypeptides of the present invention comprise a sequence of at least nine amino acids, preferably at least 10 to about 15 amino acids, or more preferably at least about 15 to about 30 amino acids contained within SEQ ID NO:l l. For example, a polyclonal antibody has been prepared against the final, e.g., carboxy terminal end, 22 amino acids (LysLysIleProAlaAsnLysArgValGlnGlyProArgArgArgSerArgGluGlyArgSer Gin; SEQ ID NO:29) of the mouse PLF amino acid sequence (SEQ ID NO:l 1). However, peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with PLF. It is desirable that the amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production. The invention also provides antibodies directed against sequences of PLF in the regions where peptide segments are contained in Periostin, but not PLF, or where peptide segments are not contained in Periostin, but are contained in PLF, e.g., amino acid positions 672, 700, 784, and 813 (see FIGS. IA to 1C). Where each of the six junctions can occur, a sequence can be chosen that spans a junction. For example, a peptide sequence of about 20-24 amino acids in length, which spans 10-12 amino acid residues on each side of a junction, can be used as an immunogen to generate an antibody which recognizes the region of the particular target splice junction. There are six such junction sequences for PLF compared to other Periostins, because there is a junction at each end of the two fragments, and when the fragments are deleted, a new junction and new sequence is formed for each. Antibodies generated against these junctions are useful in distinguishing isoforms of the Periostin family. For example, the peptide segment contained in PLF (SEQ ID NO: 14 for mouse; SEQ ID NO:30 for human), but not in Periostin, comprises an antigenic determinant not present in Periostin. An antibody can be produced which is directed against such an antigenic determinant. Potential antigenic sites in PLF can be identified using the Jameson- Wolf method (Jameson and Wolf, CABIOS 4:181, 1988), as implemented by the Protean program, version 3.1 (DNASTAR; Madison, Wis.). The Jameson- Wolf method predicts potential antigenic determinants by combining six major subroutines for protein stmctural prediction. Polyclonal antibodies to a recombinant PLF protein or to PLF isolated from natural sources can be prepared using methods well known to those of skill in the art. Antibodies can also be generated using a PLF-glutathione transferase fusion protein, which is similar to a method described by Burrus and McMahon (Exp. Cell. Res. 220:363 1995). General methods for producing polyclonal antibodies are known to those of ordinary skill in the art (Green et al., in Immunochemical Protocols, Manson, ed., pages 1-5, Humana Press, New York, 1992. The immunogenicity of a PLF polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of PLF or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like," such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization. Although polyclonal antibodies are typically raised in animals such as horse, cow, dog, chicken, rat, mouse, rabbit, goat, guinea pig, or sheep, an anti-PLF antibody of the present invention may also be derived from a subhuman primate. Alternatively, monoclonal anti-PLF antibodies can be generated.
Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al., Nature 256:495 1975). Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising a PLF gene product and then verifying the presence of antibody production by removing a semm sample. Then, the spleen is removed to obtain B-lymphocytes. The B-lymphocytes are fused with myeloma cells to produce hybridomas, the hybridomas are cloned, and then positive clones are selected which produce antibodies to the antigen. The clones that produce antibodies to the antigen are cultured and the antibodies are purified from the culture medium using techniques known to those of ordinary skill in the art. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (Baines et al., in Methods in Molecular Biology, Vol. 10, pages 79-104, The Humana Press, Inc., New York, 1992). In addition, an anti-PLF antibody of the present invention may be derived from a human monoclonal antibody. Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge. Methods for obtaining human antibodies from transgenic mice are known to those of ordinary skill in the art (Green et al., Nature Genet. 7:13 1994, Lonberg et al., Nature 368:856 1994, and Taylor et al., Int. Immun. 6:579 1994). For particular uses, it may be desirable to prepare fragments of anti- PLF antibodies. Such antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. Alternatively, an anti-PLF antibody can be derived from a "humanized" monoclonal antibody. Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are known to those of skill in the art (Kelley, in Protein Engineering: Principles and Practice, Cleland et al., eds., pages 399-434, John Wiley & Sons, Inc., New York, 1996). Relatively pure antibody preparations are obtained from in vitro production techniques, which allow production to be scaled-up to yield large amounts of the desired antibodies. Techniques for bacterial cell, yeast cell, or mammalian cell cultivation are known in the art and include homogeneous suspension culture, e.g., in an airlift reactor or in a continuous stirrer reactor, or immobilized or entrapped cell culture, e.g. in hollow fibres, microcapsules, on agarose microbeads, or ceramic cartridges. Antibodies can be prepared by other techniques known to those of skill in the art, and include for example, standard recombinant nucleic acid techniques and chemical synthetic techniques. The purified antibodies or polyclonal antisera can then be assayed for biological activity according to the assay methods described in the Examples, as well as by methods known to those of skill in the art. Splice isoforms of PLF can serve as targets for diagnostic and/or therapeutic antibodies. The antibodies could be useful in the diagnosis and treatment of cancer, heart disease, vascular restenosis, atherosclerosis, calcified heart valves, calcified vasculature, bone disease, cancer, or other pathological conditions. The antibodies of the invention are useful in assessing the levels of PLF protein, its fragments, derivatives, or homologs thereof. The antibodies can be used in methods known in the art relating to localization and activity of the protein sequences of the normal or mutated PLF protein, for imaging these proteins, and for measuring levels thereof in samples derived from a test subject or from a control sample or subject. In one embodiment, a primary antibody is detected by detecting a label on a primary antibody which has bound to the desired immunogen. In another embodiment, a secondary antibody, which has bound to a primary antibody, is detected by detecting a label on the secondary antibody. Many assays are known in the art for labeling and detecting primary and secondary antibodies. One skilled in the art can readily determine an effective amount of protein to be administered to a given subject, by taking into account factors such as the size and weight of the subject; the extent of disease penetration; the age, health and sex of the subject; the route of administration; and whether the administration is regional or systemic. When a polypeptide is to be administered, one skilled in the art will determine the amount to be administered based on whether the protein is being administered as a diagnostic reagent or as a therapeutic reagent and whether it is a PLF protein or an anti-PLF antibody. For example, is the protein an antibody against PLF or another Periostin, or is the protein PLF or a fragment, derivative, or homolog of PLF. Those skilled in the art may derive appropriate dosages and schedules of administration to suit the specific circumstances and needs of the subject. For example, suitable doses to be administered can be estimated from age, sex or body weight. Typically, dosages of protein are between about 0.001 mg/kg and about 100 mg/kg body weight. In some embodiments, protein dosages are between about 0.01 mg/kg and about 60 mg/kg body weight. It is understood that the effective dosage will depend on the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. The most preferred dosage will be tailored to the individual subject, as is understood and determinable by one of skill in the art. In general, the schedule or timing of administration of a PLF protein, fragments, homologs, or derivatives, or antibodies against PLF, is according to the accepted practice for the procedure being performed. Compounds of the invention or antibodies, are preferably formulated as pharmaceutical compositions prior to administering to a subject, according to techniques known in the art. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. As used herein, "pharmaceutical formulations" include formulations for human and veterinary use. Methods for preparing pharmaceutical compositions of the invention are within the skill in the art, for example as described in Remington's Pharmaceutical Science, 17th ed., Mack Publishing Company, Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference. The present pharmaceutical formulations comprise at least one compound of the invention or at least one antibody directed against PLF (e.g., 0.1 to 90% by weight), or physiologically acceptable salts thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations of the invention can also comprise isolated nucleic acids comprising sequences encoding PLF protein or a fragment, derivative, or homolog of PLF, which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Preferred pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, . antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA- bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized. For solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For example, a solid pharmaceutical composition for oral administration can comprise any of the carriers and excipients listed above and 10-95%, preferably 25%-75%, of a compound of the invention. A pharmaceutical composition for aerosol (inhalational) administration can comprise 0.01-20% by weight, preferably 1%-10% by weight, of the compound of the invention encapsulated in a liposome as described above, and a propellant. A carrier can also be included as desired; e.g., lecithin for intranasal delivery. For in vivo applications, the compounds of the present invention and antibodies directed against PLF, can comprise a pharmaceutically acceptable salt. Suitable acids which are capable of forming such salts with the compounds of the present invention include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid and the like; and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid and the like. Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are preferably sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like. The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g. 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absoφtion enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Pharmaceutical compositions according to the present invention can be prepared in a manner fully within the skill of the art. When used in vivo, the PLF proteins, fragments, homologs, or derivatives are preferably administered as a pharmaceutical composition, and a pharmaceutically acceptable carrier. The proteins may be present in a pharmaceutical composition in an amount from 0.001 to 99.9 wt %, more preferably from about 0.01 to 99.0 wt %, and even more preferably from 0.1 to 50 wt %. To achieve good plasma concentrations, PLF proteins, fragments, homologs, or derivatives, may be administered, for example, by intravenous injection, as a solution comprising 0.1 to 1.0% of the active agent. The compounds of the invention or antibodies directed against PLF, or pharmaceutical compositions comprising these compounds or antibodies, may be administered by any method designed to allow compounds to have a physiological effect (see Examples). Parenteral administration is preferred. Particularly preferred parenteral administration methods include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature), peri- and intra-target tissue injection, subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps), intramuscular injection, and direct application to the target area, for example by a catheter or other placement device. Pharmaceutically acceptable carriers include physiologically tolerable or acceptable diluents, excipients, solvents, or adjuvants. The compositions are preferably sterile and nonpyrogenic. Examples of suitable carriers include, but are not limited to, water, normal saline, dextrose, mannitol, lactose or other sugars, lecithin, albumin, sodium glutamate, cysteine hydrochloride, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, ethoxylated isosteraryl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methahydroxide, bentonite, kaolin, agar-agar and tragacanth, or mixtures of these substances, and the like. The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary pharmaceutical substances or excipients and/or additives, such as wetting agents, emulsifying agents, pH buffering agents, antibacterial and antifungal agents (such as parabens, chlorobutanol, phenol, sorbic acid, and the like). Suitable additives include, but are not limited to, physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions (e.g., 0.01 to 10 mole percent) of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA or CaNaDTPA-bisamide), or, optionally, additions (e.g. 1 to 50 mole percent) of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). If desired, absorption enhancing or delaying agents (such as liposomes, aluminum monostearate, or gelatin) may be used. The compositions can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Compositions for intramuscular, subcutaneous or intravenous application are e.g. isotonic aqueous solutions or suspensions, optionally prepared shortly before use from lyophilized or concentrated preparations. Suspensions in oil contain as oily component the vegetable, synthetic or semi-synthetic oils customary for injection purposes. The pharmaceutical compositions may be sterilized and contain adjuncts, e.g. for conserving, stabilizing, wetting, emulsifying or solubilizing the ingredients, salts for the regulation of the osmotic pressure, buffer and/or compounds regulating the viscosity, e.g. sodium carboxycellulose, carboxymethylcellulose, sodium carboxymethylcellulose, dextran, polyvinylpyrrolidine or gelatin. Pharmaceutical compositions according to the present invention can be prepared in a manner fully within the skill of the art. Where the administration of the compounds of the invention or antibodies directed against the Periostin isoform PLF, is by injection or direct application, the injection or direct application may be in a single dose or in multiple doses. Where the administration of the compound is by infusion, the infusion may be a single sustained dose over a prolonged period of time or multiple infusions. The invention provides for diagnosing a PLF associated disorder. Detection of aberrant expression of a PLF nucleic acid or protein is an indication that a subject has a PLF associated disorder. Expression of the gene encoding PLF can be determined directly, for example by reverse transcriptase PCR (RT-PCR) of PLF mRNA, or by detection of the mRNA by northern blot analysis. RT-PCR and northern blotting techniques are within the skill in the art. Various assays also exist for measuring or detecting the Periostin isoform PLF protein levels and protein activity. Assays for determining protein levels are known in the art, include immunocytochemical and immunohistochemical techniques, electrophoretic separation and identification, western blot analysis, peptide digestion, and sequence analysis (see Examples). Other assays are known to those of skill in the art. Various immunoassays known in the art can be used to measure
Periostin isoform PLF protein, fragments, derivatives, or homologs. These assays include competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, gel diffusion precipitin reactions, western blots, precipitation assays, in situ immunoassays, immunocytochemical and immunohistochemical techniques, complement fixation assays, immunofluorescence assays, and immunoelectrophoretic assays. According to the practice of the invention, to diagnose a PLF associated disorder, a tissue sample is derived from a subject. A tissue sample includes a biopsy of a tissue of interest. The sample is then prepared for determination of Periostin isoform PLF expression level. A sample includes tissue or fluid samples. Determining the relative level of expression of the Periostin isoform PLF in a tissue sample comprises determining the relative number of the Periostin isoform PLF RNA transcripts, particularly mRNA transcripts in the sample tissue, or determining the relative level of the corresponding PLF protein in the sample tissue. Preferably, the relative level of PLF protein in the sample tissue is determined by an immunoassay whereby an antibody which binds PLF protein is contacted with the sample tissue. The relative PLF expression level in cells of the sampled tumor is conveniently determined with respect to one or more standards. The standards may comprise, for example, a zero expression level on the one hand and the expression level of the gene in normal tissue of the same patient, or the expression level in the tissue of a normal control group on the other hand. The standard may also comprise the PLF expression level in a standard cell line. The size of the decrement in PLF expression in comparison to normal expression levels is indicative of the future clinical outcome following treatment. Methods of determining the level of mRNA transcripts of a particular gene in cells of a tissue of interest are well-known to those skilled in the art. One such method is Northern blot analysis. Total or polyA selected RNA is derived from the sample, subjected to electrophoresis, and then transferred to nitrocellulose or other types of membranes. The RNA is immobilized on the membranes by heating. Detection and quantification of specific RNA is accomplished using appropriately labelled DNA or RNA probes complementary to the RNA in question (Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the disclosure of which is incorporated by reference). Other blotting techniques include dot blots or slot blots. In addition to blotting techniques, the mRNA assay test may be carried out according to the technique of in situ hybridization. The in situ technique involves depositing whole cells or pieces of tissue onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labelled cDNA or cRNA probes. The practice of the in situ hybridization technique is described in more detail in U.S. Pat. No. 5,427,916, the entire disclosure of which is incorporated herein by reference. The nucleic acid probes for the above RNA hybridization methods can be designed based upon the PLF cDNA sequences described herein. Either method of RNA hybridization, blot hybridization, or in situ hybridization, can provide a quantitative result for the presence of the target RNA transcript in the RNA donor cells. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning, supra, Chapters 10 and 11, incorporated herein by reference. The nucleic acid probe may be labeled with, e.g., a radionuclide such as 32P, 14C, or 35S; a heavy metal; or a ligand capable of functioning as a specific binding pair member for a labelled ligand, such as a labelled antibody, a fluorescent molecule, a chemiluminescent molecule, an enzyme or the like. Probes may be labelled to high specific activity by either the nick translation method (Rigby et al., J. Mol. Biol. 113: 237-251 1977) or by the random priming method (Feinberg et al., Anal. Biochem. 132: 6-13 1983). The latter is the method of choice for synthesizing 32P-labelled probes of high specific activity from single-stranded DNA or from RNA templates. Both methods are well known to those skilled in the art and will not be repeated herein. By replacing preexisting nucleotides with highly radioactive nucleotides, it is possible to prepare P-labelled DNA probes with a specific activity well in excess of 10 cpm/microgram according to the nick translation method. Autoradiographic detection of hybridization may then be performed by exposing filters to photographic film. Densitometric scanning of the filters provides an accurate measurement of mRNA transcripts. Where radionuclide labeling is not practical, the random-primer method may be used to incorporate the dTTP analogue 5-(N-(N-biotinyl-ε- aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate into the probe molecule. The thus biotinylated probe oligonucleotide can be detected by reaction with biotin binding proteins such as avidin, streptavidin, or anti-biotin antibodies coupled with fluorescent dyes or enzymes producing color reactions. The relative number of PLF transcripts may also be determined by reverse transcription of mRNA followed by amplification in a polymerase chain reaction (RT-PCR), and comparison with a standard. The methods for
RT-PCR and variations thereon are well known to those of ordinary skill in the art. According to another embodiment of the invention, the level of PLF expression in cells of a subject is determined by assaying the amount of the corresponding PLF protein. A variety of methods for measuring expression of PLF protein exist, including Western blotting and immunohistochemical staining. Western blots are n by spreading a protein sample on a gel, using an SDS gel, blotting the gel with a cellulose nitrate filter, and probing the filters with labeled antibodies. With immunohistochemical staining techniques, a cell or tissue sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, fluorescent labels, chemiluminescent labels, and the like. According to one embodiment of the invention, tissue samples are obtained from patients and the samples are embedded then cut to e.g., 3-5 μm, fixed, mounted and dried according to conventional tissue mounting techniques. The fixing agent may advantageously comprise formalin. The embedding agent for mounting the specimen may comprise, e.g., paraffin. The samples may be stored in this condition. Following deparaffinization and rehydration, the samples are contacted with an immunoreagent comprising an antibody specific for PLF. Antibodies can be prepared by many techniques known to those of skill in the art, which are described in greater detail above. The level of PLF expression in the tissue sample of a subject suspected of having a PLF associated disorder, can be prepared on a relative basis with the level of PLF expression of a sample derived from a control subject who does not have a PLF associated disorder. Other techniques for measuring PLF expression are also known to those of skill in the art and include various techniques useful for measuring the rates of transcription and rates of translation of PLF, as well as rates of degradation of PLF mRNA and PLF protein. A subject in need of treatment for a PLF associated disorder can be identified by obtaining a sample of cells or tissue associated with a PLF associated disorder, such as heart muscle, bone, or tumor samples (or cells suspected of being tumorigenic) from the subject, and determining whether the expression of PLF has changed in at least a portion of the cells or tissue, as compared to cells from normal tissue obtained from the subject. Alternatively, PLF expression in cells or tissue obtained from a subject can be compared to average expression levels of PLF in cells and tissues obtained from a population of normal subjects. A subject in heed of treatment for a PLF associated disorder can be readily identified by a physician using standard diagnostic techniques. The invention provides for monitoring the progression of a PLF associated disorder in a subject. Progression of a PLF associated disorder refers to an advance in the course of the disorder. Subjects who can be monitored include subjects being treated for a PLF associated disorder. In one embodiment where a decrease in PLF levels is associated with the disorder, progression of a PLF associated disorder in a subject can be monitored by measuring the level of PLF nucleic acid, PLF protein, or PLF protein activity in a sample derived from the subject. A decrease in the level of PLF nucleic acid, PLF protein, or PLF protein activity in the sample, relative to the level present in a sample derived from the subject at an earlier time, indicates that there has been progression the PLF associated disorder. In another embodiment, progression of a PLF associated disorder in a subject, where an increase in PLF expression or activity is associated with a PLF associated disorder, can be monitored by measuring the level of PLF nucleic acid, PLF protein, or activity of a PLF protein in the subject. The method comprises measuring the level of PLF nucleic acid, PLF protein, or activity of PLF protein in a sample derived from the subject. An increase in the level of mutant PLF nucleic acid, PLF protein, or PLF protein activity in the sample, relative to the level present in a sample derived from the subject at an earlier time, indicates progression of the PLF associated disorder. In accordance with the present invention, as described above or as discussed in the Examples below, there can be employed conventional clinical, chemical, cellular, histochemical, biochemical, molecular biology, microbiology, and recombinant DNA techniques which are known to those of ordinary skill in the art. Such techniques are fully explained in the literature. The invention should not be construed to be limited solely to the assays and methods described herein, but should be construed to include other methods and assays as well. One of skill in the art will know that other assays and methods are available to perform the procedures described herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize PLF nucleic acids, proteins, fragments, homologs, or derivatives, as well as antisense oligonucleotides to PLF nucleic acids and antibodies to PLF proteins, and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be constmed as limiting in any way the remainder of the disclosure.
Examples
Example 1- Identification of PLF Methods Mouse tissue Mouse embryos were isolated at 7.5 day post-conception (pc.) (Auda- Boucher, et al, Dev. Biol., 225:214-25, 2000; Downs, et al., Development, 118:1255-66, 1993) and carefully dissected away from the closely attached placental and uterine tissues. Hearts were collected from 13.5 day pc. embryos, neonatal and adult mice. Total RNA was prepared using TRIZOL (GibcoBRL, Grand Island, NY). These RNAs were used in the 'READS' differential display technique (Prashar and Weissman, Methods Enzymol. 303:258-272, 1999) to identify cDNAs that were differentially expressed in embryos at 7.5 day pc, 13.5 day pc, neonatal mouse heart, and adult mouse heart tissue. The cDNAs (READs ESTs) were sequenced and analyzed for homology in GenBank. Human tissue Human heart tissue was obtained for isolation of human PLF cDNAs. The tissue was derived from patients with non-failing hearts or diseased hearts. Cloning of the full-length mouse PLF cDNA The READS expressed sequence tag (EST) of 248 nucleotides was 99% homologous to the published sequence of mouse Periostin (GenBank Accession No. D 13664; SEQ ID NO:l). To obtain the full-length cDNA, total RNA was extracted from one day old neonatal mouse heart tissue solubilized in TRIZOL (Invitrogen). Oligo-dT primed first strand cDNA was generated from this RNA and used to amplify the full-length PLF cDNA. The 3' oligonucleotide primer (5'GAGAGAAAACATTTGTATTGCAAGAAGC; SEQ ID NO:2) was designed based on the READS EST sequence and the published mouse Periostin cDNA sequence (GenBank Accession No. NM_015784; SEQ ID NO:l). The 5' end oligonucleotide primer
(5'GGCTGAAGATGGTTCCTCTCCTGC; SEQ ID NO:3) was homologous to the sequence at the 5' end of the published Periostin sequence. For PCR amplification, the XL-Gene Amp PCR kit (Perkin-Elmer) was used with the following parameters: 94°C for 1 minute, followed by 94°C for 15 seconds, 66°C for 10 minutes for 25 cycles, and 72°C for 10 minutes. The full-length PLF cDNA (SEQ ID NO:4) was ligated into the pgem-3zf-vector (Promega). The full-length cDNA was sequenced in both directions to ensure that no errors occurred in deriving the nucleic acid sequence. Cloning human PLF cDNA The primers and techniques described above for cloning mouse PLF cDNA were used to isolate and clone human PLF cDNA from human heart tissue. Results Generation and analysis of full-length PLF cDNA from mouse neonatal heart A total of 756 differentially expressed cDNAs were identified (excluding duplicates), 36 of which were 'unknown' or whose sequence did not show homology to known sequences in GenBank. One of the 3'-cDNA fragments from the READS was identical (99% identity at the nucleotide level) to GenBank locus NM_015784 (GenBank Accession No. NM_015784.1, G 7657428), Mus musculus osteoblast specific factor 2 (OSF2), also called Periostin. Primers were designed to be homologous to the extreme ends of the GenBank sequence and used for RT- PCR to generate a cDNA from neonatal mouse heart total RNA derived from the same tissue that gave rise to the READS fragment. This cDNA was sequenced in both directions. This newly discovered 3012 nucleotide mouse PLF cDNA (SEQ ID NO:4) is a unique and previously unreported form of Periostin, designated herein as Periostin-Like Factor (PLF). The coding region for the mouse PLF cDNA extends from base 9 to 2441. Generation and analysis of a human PLF cDNA from human adult heart Using the methods described above for mouse tissue, a human PLF cDNA was derived from adult human heart tissue. A partial sequence, comprising 479 bases was obtained for the human PLF cDNA (SEQ ID
NO:24). A complete sequence will be obtained. Comparison of mouse and human PLF to Periostins The full length murine PLF protein is 810 amino acids in length (SEQ ID NO: 11) (see FIGURE 1). The 2430 nucleotide sequence (SEQ ID NO: 12) encoding the 810 amino acid mouse PLF comprises the sequence starting with "atggttcctctc" and ending with "cgttctcag." Further cloning with different primers (forward 5' primer- gattcgattcggctgaagatggttcctctcctgc, SEQ ID NO:31 ; reverse 5' primer- ggatccggatccgagagaaaacatttgtattgcaagaagc, SEQ ID NO:32) yielded a mouse PLF cDNA comprising 3290 bases (SEQ ID NO: 13). The mouse PLF 810 amino acid sequence (SEQ ID NO: 11) and its corresponding 2430 base nucleic acid coding sequence (SEQ ID NO: 12) and their alignment are provided in FIGS. ID and IE (also see FIGS. IA to 1C). A human PLF is also described herein, derived from cDNAs generated from heart tissue. Because analysis of different heart tissue samples identified identical electrophoretic patterns among various isolates, bands were excised from gels for different patients and the nucleic acid pooled for sequencing. The human PLF partial cDNA (SEQ ID NO:24; 479 bases), just like the mouse PLF cDNA, was found to encode a protein (SEQ ID NO:27) which has a 27 amino acid segment inserted between amino acids 672 and 700 of Periostin, and is lacking the 785-812 amino acid segment, relative to Periostin (see FIG. 1C; discussed more fully below). In addition, RT-PCR based sequence analysis for the regions 5' and 3' to the deletion, found that the mouse and human PLF sequences share very high sequence identity. For example, all 135 amino acid residues of the partial human PLF amino acid sequence (SEQ ID NO:24), including the region comprising amino acids 669 to 831, share 100%) homology with the same region in the mouse PLF protein (SEQ ID NO:l 1) (FIGS. IA to 1C). Furthermore, the human PLF nucleic acid sequence is more homologous to mouse PLF than it is to human Periostin or other isoforms of Periostin. Thus, PLF is disclosed herein to be present in at least rodents and humans. Figures IA to 1C show the alignment at the amino acid level of the newly described mouse and human PLF to three other proteins, and to a consensus sequence based on matches among the five proteins. The alignment demonstrated in these figures is used herein to described positional relationships of the amino acid residues of one Periostin isoform to another. The sequence labeled "mPeriostin" is derived from a murine Periostin OSF2- like cDNA sequence isolated from a 10 month-old virgin mouse mammary tumor (GenBank Accession No. BC031449; SEQ ID NO:25). The predicted amino acid sequence (GenBank Accession No. AAH31449; SEQ ID NO:26) for the mouse Periostin OSF2 protein encoded by SEQ ID NO:25 is listed in FIGS. IA to lC. Another cDNA has also been sequenced from the 10 month- old virgin mouse mammary tumor (GenBank Accession No. BC007141; SEQ ID NO: 5), but is not included in the analysis. It should be noted that referral to amino acid positions in FIGS. IA to 1C is based on positions of the amino acids following alignment of the sequences of the various proteins studied. The amino acid sequence labeled "Takeshita' s" is the mouse Periostin first cloned by Takeshita et al. (GenBank Accession No. BAA02835.1; SEQ ID NO:6), which is encoded by the nucleic acid of SEQ ID NO:7 (GenBank Accession No. D13664). The amino acid sequence labeled "mPLF" is the amino acid sequence of the mouse PLF described herein (SEQ ID NO: 11). The amino acid sequence labeled "hPeriostin" is that of human Periostin (GenBank Accession No. NP_006466; SEQ ID NO:9), encoded by SEQ ID NO:28 (GenBank Accession No. NM_006475). The amino acid sequence labeled "hPLF" is a partial sequence of human PLF (SEQ ID NO:27), as predicted from the human PLF cDNA (SEQ ID NO:24) described herein. All protein sequences were determined based on translations of the corresponding cDNA sequence giving the longest open reading frame. These proteins, while still highly homologous, show differences. A mouse Periostin cDNA (GenBank Accession No. BC007141) not analyzed in FIG. 1 , is frame shifted to produce an early stop codon. It is not known whether the resulting protein, tmncated at amino acid 199, is expressed. Another human Periostin/OSF2 like cDNA has been isolated from human placenta (HUMOSF2P1; cDNA GenBank Accession No. D13665; protein GenBank Accession No. BAA02836.1 ; SEQ ID NO: 10), but is not described in the analysis. The PLF 3012 base cDNA (SEQ ID NO:4) described herein encodes a peptide region (amino acid residues 673-699; SEQ ID NO. 14; FIGS. 1A-1C) that is absent from mPeriostin (FIG. 1C), but present in the mouse Periostin/OSF2 labeled "Takeshita' s and in human Periostin/OSF2 (labeled "Takeshita" in FIG. 1C). Further toward the COOH terminus of "Takeshita' s" Periostin and human Periostin is a peptide fragment comprising amino acid residues 785- 812 (see FIGS. IA to 1C) that is absent in the mouse and human PLF translated RT-PCR cDNA. As in the mouse and human PLF described herein, the same 28 amino acid sequence is also lacking in the mouse Periostin (SEQ ID NO:6; GenBank Accession No. AAH31449). Thus, human and mouse PLF are unique compared to other Periostins in that they comprise both an additional amino acid sequence inserted at Periostin position 673 and are missing an amino acid sequence beginning at Periostin position 785. The data also demonstrate a high degree of homology between mouse and human PLF, although only 135 amino acid residues of human PLF were used in the comparison. The sequence alignment and comparisons of FIGS. IA to 1C showed that both human and mouse PLF contain a 27 amino acid peptide between residues 672 and 770 that is lacking in other mouse Periostins. The comparison also reveals that both human and mouse PLF are lacking the 28 amino acid peptide between residues 784 and 813 of Periostin, that is present in human Periostin and the first mouse Periostin that was discovered (Takeshita et al., Biochem. J., 294:271-8, 1993). It can also be seen in FIGS IA to 1C that there is only one amino acid difference between human and mouse Periostin in the region where the 28 amino acid fragment is missing in human and mouse PLF, and that is at position 787. The sequence for mouse Periostin region comprising amino acids 785-812 (SEQ ID NO: 15) is Glu-Val-Ser-Lvs-Val-Thr-Lys-Phe-Ile-Glu- Gly-Gly-Asp-Gly-His-Leu-Phe-Glu-Asp-Glu-Glu-Ile-Lys-Arg-Leu-Leu-Gln- Gly (SEQ ID NO: 15). The sequence for the human Periostin region comprising amino acids 785-812 is Glu-Val-Thr-Lys-Val-Thr-Lys-Phe-Ile- Glu-Gly-Gly-Asp-Gly-His-Leu-Phe-Glu-Asp-Glu-Glu-Ile-Lys-Arg-Leu-Leu- Gln-Gly (SEQ ID NO:16). The sequences of FIGS. IA to 1C are aligned based on the amino acid sequence of mouse Periostin, which is 811 amino acids in length. Because several forms of Periostin and PLF are compared to one another in FIGS. IA and IB, and are aligned to account for all insertions or deletions of fragments or amino acid residues, the total number of positions indicated in each of FIGS. IA and IB is 839. Therefore, the region designated as positions 758 to 786 in FIG. 1C, where the deletion occurs in mouse PLF protein, corresponds to positions 785-813 in FIG. IB. These two designations are used interchangeably herein. The schematic in FIGURE 1 F compares Periostin to PLF. The mouse Periostin protein (GenBank Accession No. BAA02835.1 ; SEQ ID NO:6) is 811 amino acids in length (FIG. 1C). The predicted murine PLF sequence of the invention was compared to that of mouse Periostin, and it can be seen that the region between amino acid residue 673 and residue 700 is present in mouse PLF, but not in mouse Periostin (see FIGS. 1A-1C). The 28 amino acid sequence between amino acid residues 673 and 700 disclosed herein in mouse PLF is Thr-Thr-Lys-Ile-Ile-Thr-Lys-Val-Val-Glu-Pro-Lys- Ile-Lys-Val-Ile-Gln-Gly-Ser-Leu-Gln-Pro-Ile-Ile-Lys-Thr-Glu-Gly (SEQ ID NO: 14). Without wishing to be bound by any particular theory, it is possible that PLF is an isoform resulting from an alternately spliced gene. The alterations in amino acid sequence may be probably functionally significant because the other proteins are highly conserved across species, and these regions when present are also highly conserved across species.
Example 2- PLF expression during mouse embryogenesis Northern blot analysis Tissues were collected from embryonic mice and solubilized in
TRIZOL for isolation of RNA. Ten μg of total RNA was separated on 1% formaldehyde-denatured agarose gels, transferred to Nytran membranes, and probed with radiolabeled full-length mouse PLF cDNA. The Nytran was exposed to x-ray film and the image analyzed by densitometric techniques to determine the level of PLF mRNA present in a given tissue. In order to adjust for equal loading of RNA in each lane, the blots were re-probed with an 18S rRNA radiolabeled cDNA probe, and the amount of PLF mRNA levels are represented as a ratio of PLF RNA/18S rRNA. Northern blot results The differential display analysis of cDNAs showed that the cDNA homologous to Periostin, namely PLF, was most highly expressed in 13.5 day pc. mouse embryonic tissue and in neonatal mouse heart, relative to expression in embryos and heart at different stages (data not shown). These data were confirmed by northern blot analysis using the same total RNA that was analyzed by the READS analysis (FIGS. 2A and 2C). The data show that PLF increases during mouse embryogenesis and declines after birth. Because chicken embryos are often used to study heart development it was determined if PLF was expressed during chicken embryogenesis (FIGS. 2B and 2D). PLF was highly expressed in the neonatal chicken heart. However, PLF expression was lower in stage 4, stage 8, and stage 12 chicken embryo hearts and adult chicken hearts, than in the neonatal chicken heart (FIGS. 2B and 2D). More time points were then sampled during mouse embryogenesis (days 9.5 to 18.5 pc.) in order to determine the overall temporal pattern of expression of PLF during mouse embryogenesis. Mouse embryo total RNA was subjected to northern blot analyses and labeled with P-labeled PLF cDNA (FIGS. 3A and 3B, blot purchased from Seegene; Seoul, Korea). The data demonstrate a distinct pattern of expression during mouse embryogenesis, with significantly reduced levels of expression found at days 11.5 and 12.5 pc. in the whole mouse embryo (FIGS. 3A and 3B). In situ hybridization To determine the spatial location of PLF in mouse and chicken embryos, in situ hybridization was used to detect PLF mRNA in mouse embryos on days 8.5, 9.5, 10.5, 12.5 and 16.5 post-conception (pc.) (staging described in Kaufman, The atlas of mouse development. Academic Press. Harcourt, Brace Jovanovich Publishers, 1992). In situ hybridization results On day 8.5 pc, signal was localized to the uterine wall of the mother mouse (FIGS. 4A and B), but was not detected in the embryo. However, from days 9.5 to 16.5 p.c, PLF was detected in the mouse embryo. In the 9.5 and 10.5 day p.c. mouse embryo (FIGS. 4 C, D and F), PLF mRNA was localized to the somites, body wall mesenchyme, ventricular wall, atrioventricular canal, and the endocardial cushions. At day 12.5 pc, PLF mRNA was detected in the wall of the atrium (FIG. 4E), and to a lesser extent in the ventricular wall. At day 16.5 p.c. PLF expression was again detected in the atrial wall and at the atrial-ventricular junction (FIG. 5B). PLF expression was highest in the neonatal heart, compared to other stages of heart development (FIGS. 2 A, 7 A, and 7B). PLF was localized to the myocardium and was seen in the heart valves but was not detected in the epicardium. During cartilage and bone development at day 16.5 pc. of the mouse embryo, PLF mRNA was localized to the mesenchymal tissue containing the preosteoblasts that surround the cartilage primordia of the ribs (FIGS. 5C and 5D), vertebrae (FIGS. 5E and 5F) and the limb (FIGS. 5G and 5H). It can be seen in FIGS. 5E and 5F that pre-osteoblasts express PLF, but chondrocytes do not. PLF mRNA was also detected in cells comprising the cartilage primordia of the upper and lower jaws at day 16.5 pc. (FIGS. 6A-6E). PLF expression was detected in the mesenchymal/preosteoblasts in the hard palate as well as in the undifferentiated taste bud precursor cells (FIGS. 6B and 6C). PLF expression was also detected in ameloblasts and odontoblasts during tooth formation (FIGS. 6D and 6E). However, it can be seen in FIG. 6D that cells in the region comprising the transition from the hard to the soft palate do not express PLF. Example 3- PLF Expression in Failing Human Hearts Methods The convergence or divergence of gene expression patterns expressed in failing or nonfailing human myocardium was determined by microarray analysis on the Affymetrix GeneChip® platform using GeneExpress® analysis software Affymetrix, Inc., Santa Clara, CA). Sample sets consisting of nonfailing myocardium (n=25), ischemic cardiomyopathy (n=120), or non- ischemic cardiomyopathy (n=120) were created and saved by query of the GeneExpress® database. All average difference values were the result of hybridization of in vitro transcription (IVT) reaction products, and scanning of the human U_95 (60K) Affymetrix GeneChip® microarrays. Gene sets that significantly and reproducibly demonstrated dysregulated expression between nonfailing and ischemic or non-ischemic cardiomyopathy samples were created by performing a fold-change analysis and sorting of genes by p-value. PLF Affymetrix GeneChip® Analysis The average mean expression of PLF mRNA represented by multiple tissues was examined via hybridization of total neonatal mouse heart mRNA to the human HU_95 (60K) gene chip set as previously described (Prakash, et al., Proc. Natl. Acad. Sci. U S A., 99:7598-603, 2002). Sample sets consisting of human left ventricular myocardium from nonfailing, ischemic, or idiopathic diseased heart were compared to normal human tissues. Tissue sample set numbers were selected to represent a minimum of n=T0 samples in order to provide statistically significant gene signature data in terms of average mean expression intensity. Raw expression intensity values were formatted as one row per gene per sample, and imported into Spotfire Decision Site® for visualization. Determination of Data Expression Analysis using GeneExpress® Input is defined as a user-defined gene set and one or more sample sets, and reports the range of expression levels for each gene fragment in the gene set across each sample set, over all the samples with user specified present/absent call values. The interquartile range was reported using percentile values, with the upper and lower percentile levels U and L specified by the user. The default selection of U=100 and L=0, reports the maximum and minimum expression values over the entire range of selected samples. In this study (U = 75 and L = 25 were used respectively), the upper and lower quartile values were reported within this interquartile range. This analysis reported the average mean expression values from this interquartile range in order to simplify reporting output. Data Expression Analysis by Set For each gene fragment in the user-specified gene set, present and absent scores were computed by counting the numbers of present and absent calls for the samples in the given sample set, and dividing each count by the total number of samples that have expression data for the gene fragment. Samples with unknown or null calls are omitted and are not included in the total number of samples. The result is reported as a fraction in the tabular display (e.g., 17/22) and as a percentage value in the data expression output. For each gene fragment, the percentile and median values are computed over the samples with user-selected call values. The expression values for these samples are first sorted in ascending order, generating a rank order R for each expression value. Criteria for Selection of Differentially Regulated Genes in Nonfailing, Ischemic or Idiopathic Human Myocardium The fold-change analysis was performed using all known genes regardless of whether the gene fragment was considered "present" or "absent", with the confidence limit set to 95%. The analysis results were tabulated in spreadsheet format and sorted according to fold-change value, fold-change p- value, and presence frequency. Those genes that showed expression level changes in either the up or down direction in the range of 2.5 to 100-fold were filtered and saved as gene sets, based upon a p-value where pθ.0001. This gene set comprised > 270 genes (134 full-length gene fragments) regulated within this range for ischemic cardiomyopathy and > 290 genes (139 full- length gene fragments) regulated within this range for non-ischemic cardiomyopathy (these values include both full-length genes and ESTs). In Situ Hybridization For these experiments, the analysis was performed similar to the experiments described above. Embryos for whole mount in situ hybridization were fixed in 4% paraformaldehyde, dehydrated to 70% ethanol/PBT (phosphate buffered saline with 0.1% Triton X-100) and stored at -20°C for future use. Just prior to use these embryos were rehydrated. Older mouse embryos and rat neonatal bone were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin and sectioned. The 7 μm sections were deparaffinized and rehydrated. Hydrated whole embryos or sectioned tissues were processed as described by Wilkinson (Wilkinson, pp. 75-83, 1992. In Situ Hybridization: A Practical Approach. IRL Press at Oxford University Press). Briefly, day 10.5 and 12.5 mouse embryos were treated with 10 μg/ml proteinase K for 10 minutes at room temperature. Stage 16 chicken embryos were treated 10 μg/ml proteinase K for 10 minutes at room temperature (Redkar et al., 2001). Sectioned tissues were treated with 10 μg/ml proteinase K for 10 minutes at 37°C. The embryos and sectioned tissues were re-fixed in glutaraldehyde, prehybridized and then hybridized with the digoxygenin labeled PLF anti-sense riboprobe (generated as recommended by manufacturer: Boehringer Mannheim Biochemica, Indianapolis, IN) at 55°C. Following the substrate reaction stained embryos and sectioned tissues were photographed using a Nikon microscope. Results PLF expression in diseased versus nonfailing human myocardium Because some embryonic genes are re-expressed during adult heart disease, it was determined whether PLF may also be expressed in adult cardiac disease. Left ventricular wall tissue from six patients with non-failing adult hearts (accident victims), seven adults with advanced idiopathic, and seven hearts with ischemic cardiomyopathy were examined. The diseased hearts had a median cell volume (MCV) of > 50,000 cubic microns (normal MCV < 30,000) and a left ventricular end-diastolic dimension (LVEDD) of > 7.0 cm by echocardiography (normal LVEDD = 5.0 cm). These measures are indicators of cellular and organ hypertrophy, respectively. It was found that PLF mRNA was markedly up regulated in the myocardium of patients with ischemic cardiomyopathy (FIGS. 8A-D). These data were confirmed by hybridization of total neonatal mouse heart mRNA to the human HU_95 (60K) gene chip set (FIG. 8E). A 1.9 fold down regulation of PLF expression in patients with Left-ventricular Assist Device (LVAD) support was found, suggesting that PLF responds directly to enhanced stress by up-regulation and to decreased stress by down-regulation (unpaired analysis p=0.01478, 34 pre- LVAD and 28 post-LVAD patients compared). These data were also derived from a microarray analysis on the Affymetric Gene Chip® platform using Gene Express® analysis software. Example 4- PLF expression in normal and osteopetrotic rat bone Methods PLF expression in normal and diseased bone was examined in the "op" rat osteopetrotic bone model, where there is a generalized increase in bone mass (Dobbins et al., J. Bone Miner. Res., 17:1761-7, 2002; Popoff et al., Mol. Med. Today, 2:349-58, 1996). For northern blot and RT-PCR analyses, RNA was isolated from normal or mutant rat calvaria or long bone from neonatal rats at 2, 4, and 6 weeks of age. RT-PCR was performed using PLF primers from total RNA and the products were subjected to electrophoresis. G3PDH (glycerol-3 -phosphate dehydrogenase) was used as a lane loading control. A plasmid containing PLF was included as a control to identify PLF. Densitometric analyses were performed for the PCR-amplified PCR product gels, and the data expressed as the ratio of PLF to G3PDH expression. Northern blot analyses were performed to determine PLF expression in age-matched osteopetrotic mutant and normal rats at 2, 4, and 6 weeks of age. Northern blots were probed with labeled PLF cDNA, stripped, and the reprobed with labeled 18S. The resulting films from the northern blot analyses were subjected to densitometric analyses and PLF to 18S expression ratios were determined. For in situ hybridization studies, mutant (osteopetrotic) rat long bone was isolated from two week old neonatal rats and prepared for in situ hybridization as described above. Results Northern blot and RT-PCR analyses demonstrate that PLF expression is up regulated in mutant rat osteopetrotic bone at 2 and 4 weeks, but not at 6 weeks of age, compared to age matched normal bone (FIGS. 9A-9C). Electrophoretic analysis of RT-PCR products shows that at 2 weeks of age, PLF is expressed at much higher levels in the bones of osteopetrotic rats (FIG. 9A). Densitometric analyses of the gels of FIG. 2A, show that the normalized PLF/G3PDH expression ratio is less that than 20 in normal bone, but is almost 120 in mutant bone (FIG 2B). PLF/18S expression ratios derived from densitometric analyses of a series of northern blots for age-matched osteopetrotic mutant and normal rats at 2, 4, and 6 weeks of age show that PLF expression is up regulated in mutant osteopetrotic rat bone at 2 and 4 weeks, but not at 6 weeks of age, compared to age matched normal bone (FIG. 9C). In situ hybridization analyses of osteopetrotic mutant rats at 2 weeks of age show that, in long bone, PLF signal is localized to pre-osteoblasts in the endosteum and periosteum regions (FIGS. 10 A- IOC). In summary, the data show that PLF expression is regulated spatially and temporally in bone. Furthermore, regulation of PLF expression is disrupted in abnormal bone.
Example 5- PLF expression in human vascular smooth muscle cells Methods Cell Culture- Human coronary vascular smooth muscle cells (hVSMCs) were obtained as cryopreserved secondary cultures from Cascade Biologies (Portland, OR) and subcultured in growth medium as described previously (Autieri et al., Arterioscler. Thromb. Vase. Biol. 20:1737-44, 2000). Cells from passage 4 were used in the studies described here. The growth medium was changed every other day until cells approached confluence. Primary human VSMCs were challenged with a variety of proliferative, inflammatory and differentiation-inducing stimuli to determine their effects on PLF expression. Pre-confluent hVSMCs were cultured in reduced semm conditions (0.25% FCS) for 48 hours, then exposed to inflammatory or proliferative cytokines such as 10% fetal calf serum (FCS), 10 ng/ml basic Fibroblast Growth Factor (bFGF), 100 units/ml Interferon gamma (IFNγ), 20 ng/ml Interleukin 1 beta (IL-lβ), 20 ng/ml Platelet Derived Growth Factor AB (PDGF), 2 ng/ml Transforming Growth Factor beta (TGFβ), 1 x 10"8M dexamethasone or T cell conditioned medium for 48 hours, or differentiation medium (1% FCS plus heparin 30 μg/ml) for 5 days, at which time samples were processed for RNA isolation. Dexamethasone has been shown to induce calcium deposition in cultured VSMC (Mori et al., Arterioscler. Thromb. Vase Biol., 19:2112-8, 1999), and heparin and reduced semm has been shown to induce differentiation in cultured VSMC (Newcomb et al., J. Cell. Physiol., 155:385-93, 1993). T cell conditioned medium contains several immune soluble factors which are responsible for initiating the immediate VSMC response to injury and is probably the most physiologically relevant stimuli with which to challenge the smooth muscle cell response to injury. Some samples remained untreated and were used as controls. PDGF, bFGF, IFNγ, and TGFβ were purchased from GIBCO-BRL, (Bethesda, MD), IL-lβ from Boehringer Mannheim (Indianapolis, IN), and T cell conditioned media purchased from Fisher Biotech. Northern blot analyses Following stimulation of the human vascular smooth muscle cells in culture, RNA was isolated and subjected to Northern blot analyses as described above for PLF and 18S expression. Results hVSMCs are capable of phenotypic plasticity, and change from a differentiated, contractile phenotype to a synthetic, more fetal phenotype upon stimulation with certain cytokines (Ross et al., Nature, 362:801-9, 1993). hVSMCs were found to express PLF message at similar levels when starved or treated with growth medium, T-cell conditioned medium, dexamethasone, differentiation medium, FGF, or IFNγ (FIGs. 11A-11C, lanes 1-7). However, PDGF or TGFβl treatment of hVSMCs markedly stimulated PLF expression (FIG. 11C, lanes 8 and 9). Thus, northern blot analyses revealed that, not only is PLF expressed in hVSMCs, its expression is growth and differentiation state-dependent. Example 6- Production of a polyclonal antibody against PLF and Assay for Expression of PLF Protein in Human, Mouse, and Chicken Tissue Methods A polyclonal antibody was generated against PLF in rabbits using techniques known in the art. The antibody was generated against the hydrophilic region of PLF. This peptide comprises the last 22 amino acids of the 810 amino acid mouse PLF (SEQ ID NO:22). The hydrophilic region was chosen so that the antibody would recognize PLF and known isoforms of Periostin. Rabbits were immunized with the PLF hydrophilic region peptide. Serum was collected from rabbits immunized with the PLF hydrophilic region peptide. The antibody was tested for its ability to recognize PLF in human tissues and to determine if PLF expression was regulated in diseased heart tissue. To that end, adult human heart tissue (non-failing, ischemic, or idiopathic) was processed for immunostaining with the PLF antibody. Heart, long bone, and calvaria protein samples from day 13.5 pc. mouse embryos were subjected to 8% SDS-PAGE. Western blot analyses were performed. The specificity of the antisemm was analyzed by transferring proteins to nitrocellulose and probing with the new anti-PLF antisemm or with the new anti-PLF antisemm blocked with the peptide that was used as the immunogen. Results PLF protein expression is recognized in human cells by the anti-PLF antisemm It was shown with the antibody against PLF that PLF protein is expressed in human heart tissue (FIGS. 12A-12C). The data show increased immunostaining of ischemic and idiopathic human heart tissue, relative to non-failing human heart tissue (FIG. 12A-C). As shown above with the northern blot analysis of PLF mRNA expression in human heart, the PLF protein is also upregulated in diseased human heart, namely ischemic and idiopathic human heart (FIGS 8A-8F). PLF protein is recognized in mouse and chicken cells by the anti-PLF antisemm A highly specific antiserum against PLF was found which recognized a protein of about 90 kD, as well as other Periostin isoforms in embryonic and neonatal mouse bone (FIG. 12D). In addition, the antisemm failed to bind to the 90 kD protein or other isoforms when it was pre-incubated with the PLF peptide used to generate the antiserum. The antisemm was also found to be specific for the expression of PLF and other Periostin isoforms in developing chicken hearts and mouse hearts (not shown). It was found by fluorescence microscopy that the antiserum against PLF recognized PLF in the cytoplasm of the MC3T3-E1 osteoblast cell line in vitro (not shown). Thus, this reagent is highly specific for PLF in more than one species of mammal. This antibody should also be useful for recognizing other isoforms, including
OSF2/Periostin. The PLF antibody may be used to localize PLF expression in cells, and as a diagnostic tool in analyzing PLF expression in diseases and disorders associated with aberrant expression of PLF or Periostin.
Example 7- Identification of Various Isoforms of PLFs- Methods The sequence analysis described in Example 1 suggested that there may be alternatively spliced isoforms of PLFs. To this end, primers were generated flanking the 673 to 700 a.a. peptide insert, and the 785 to 812 a.a. deleted region. DNase-treated total RNA from 13.5 day pc. mouse embryonic tissues was subjected to RT-PCR using the primers and then subjected to electrophoretic analyses. The analyzed tissues included bone, heart, spleen, lung, brain, and liver. G3PDH expression was used as a control for lane loading. Expression levels of the isoforms were normalized to G3PDH. Results Electrophoretic analysis of the RT-PCR products yielded multiple bands. It was found that different isoforms of PLF were expressed in a tissue- specific manner in bone heart, spleen, lung, brain, and liver (FIG. 13). In the left panel of FIG. 13, primers flanking the 672 to 700 amino acid region resulted in tissue-specific bands at 250 bp (upper band arrow), particularly in bone, heart, and brain, and at 150 bp (lower band arrow). Forward and reverse primers were located at 2013 bp and 2222 bp, respectively. In the right panel, primers flanking the 785 to 813 amino acid region resulted in tissue specific bands at 300 (upper arrow), 250 (middle arrow) and 150 (lower arrow) bp. Forward and reverse primers were located at 2306 bp and 2503 bp, respectively. The various bands were excised from the gels. Sequence analysis will confirm the presence of isoforms of PLF and the differences between the isoforms.
Example 8- Antisense oligonucleotides against PLF inhibit PLF function required for heart cell differentiation Methods Antisense oligonucleotides It was next determined whether PLF was required for fetal cardiac myocyte differentiation. The antisense oligonucleotide
(gagaggaaccatcttcagccctgagctccg; SEQ ID NO:21) that was prepared was directed against 30 nucleotides, which included the translation start site of PLF. The oligonucleotide was a phosphorothioate-2'-O-methyl RNA chimera, which is highly nuclease resistant and forms RNase H sensitive hybrids with the RNA target, making it a better choice than the more commonly used phosphorothioated antisense oligonucleotides. The oligonucleotide did not display secondary stmcture at physiological temperature. Cell Culture Fetal mouse cardiac myocytes (FMCM) were isolated and cultured as described by (Deng et al., Circ Res. 2000 87:781-8). FMCM were treated with anti-sense oligonucleotides to PLF to determine the role of PLF in cardiac cell differentiation, (days 13.5 - 17.5 pc. were studied separately). First, FMCMs were transfected with varying amounts of antisense oligonucleotide (0.5, 1, 2, 3 and 5 μM) using oligofectamine to promote transfection (GIBCO-BRL). Controls included, untreated cells, cells treated with oligofectamine alone, and cells treated with control scrambled oligonucleotide. To ensure that the 5 μM concentration of antisense oligonucleotide did not have a toxic effect on the cells, an MTT cell viability assay was performed. Addition of MTT to living cells results in the formation of a purple formazan product, the intensity of which is detected spectrophotometrically by reading samples at 570 nm. Forty-eight hours post-transfection, cells were scraped into TRIZOL (GIBCO BRL) and total RNA isolated. RNA was DNase-treated and equal amounts of RNA were reverse transcribed to generate first strand cDNA. G3PDH and PLF-specific primers were used to amplify the respective mRNAs using the PCR reaction. Expression of PLF was normalized to that of G3PDH. Results PLF expression was not detectable in the group treated with 5 μM antisense PLF (FIG. 14). PLF expression was reduced by about 90% in the groups treated with 3 μM or 2 μM antisense PLF. PLF expression was reduced by about 50% in groups treated with 1 μM and 0.5 μM antisense PLF (FIG. 14), while a control-scrambled oligo had no effect on the expression of PLF (data not shown). The antisense oligonucleotide was determined to be effective in culture for approximately 3 days (data not shown). The MTT cell viability assay showed equally high levels of viability between cells treated with various concentrations of the antisense oligonucleotide, compared to the controls. To determine whether PLF is involved in the maintenance of the differentiated state of cardiac myocytes, differentiating FMCM were treated between days 13.5 and 17.5 pc. with 5 μM antisense PLF oligonucleotide and plated in 12-well dishes coated with laminin. Using immunofluorescence analysis, it was found that cells which express PLF and MHC protein, but not fibronectin (Fn), are cardiac myocytes (data not shown). Cells that did not express PLF, MHC, or Fn were cardiac myocytes in which PLF was reduced by the antisense oligonucleotide and the cells were not differentiated (data not shown). The cardiac myocytes which did not express PLF, also did not express MHC protein. Without wishing to be bound by any particular theory, the data indicate that PLF is involved in maintaining the differentiated state in cardiac myocytes.
Example 9- Inhibition of PLF with antisense oligonucleotides inhibits cell migration and heart development in vivo Methods To determine the role of PLF in vivo during embryogenesis, chicken embryos were treated at stage 4 (18 hours of development) through 9 with varying amounts of antisense oligonucleotide to PLF (SEQ ID NO:21 ; as prepared above) with 1% DMSO as described by Wei et al. (Development, 1996 1229:2779-89). Some embryos were fixed in 4% paraformaldehyde and examined for ventricle myosin heavy chain- 1 (VMHCl) mRNA, a ventricle marker, by in situ hybridization techniques. Other embryos were stained and subjected to microscopic examination to assess any developmental anomalies that might arise following treatment with the antisense oligonucleotide to PLF. Results Expression of PLF was reduced by -50%, at 20 μM antisense oligonucleotide, as determined by western blot analysis of treated embryonic hearts (data not shown). Twenty-four hours post-treatment phenotypic characteristics of the embryos were evaluated (FIGS. 15A to 15L). Antisense PLF oligonucleotide treatment of chicken embryos resulted in cardia bifida (FIGS. 15C and D), abnormal vitelline vessels (Fig. 15B) and head and somite anomalies (FIGS. 15B-D). Control embryos treated with scrambled oligo with DMSO developed normally (FIG. 15 J), as did other controls (DMSO alone or untreated embryos, data not shown). In chicken embryos with cardiac bifida, VMHCl mRNA (a ventricle tissue marker) was detected (FIG. 15D), although its pattern of expression appeared abnormal (patchy), which may be a result of a change in organization of the ventricular cells as a result of loss of PLF. Altematively, this may be due to only partial depletion of the PLF mRNA, or depletion of PLF mRNA in a subset of cells. In antisense oligonucleotide treated embryos with abnormal looping of the heart, expression of VMHCl (compare FIGS. 15E-G to FIG. 15J) appeared beyond the normal boundaries of VMHCl expression within the heart. This may be interpreted as (1) an expansion of the ventricular region of the heart, (2) abnormal migration of cells from the bilaterally located heart progenitors such that the normal proportion of ventricular/atrial progenitors has been altered or possibly (3) a change in potential of the cells to become either ventricular cells or atrial cells. Nonetheless, these findings indicate that PLF plays a role in regulating heart morphogenesis and that dismption of PLF expression results in abnormal embryonic development. The anomalies found in embryos treated with antisense oligonucleotides against PLF are summarized in FIG. 15L. The categories are: dead; beating heart; normal; cardia bifida; abnormal looping; and other abnormalities.
Example 10- The effect of inhibition of PLF by antisense oligonucleotides on osteoblast differentiation and function Methods Preparation of Antisense Oligonucleotides Different antisense oligonucleotides were used than the ones described above. A first antisense oligonucleotide (AS1; caggaacagcagcagcagagc; SEQ ID NO:22) was directed against a 21 nucleotide fragment, which included the translation start site of PLF and did not display secondary stmcture at physiological temperature. A second antisense oligonucleotide, AS2, was directed against a 25 nucleotide fragment between position 1394 and position 1419 of PLF (agtatctgtccattgtagaggtcgc; SEQ ID NO:23). Antisense oligonucleotides AS1 and AS2 are phosphorothioate-2'-O-methyl RNA chimeras, which are highly nuclease resistant. These oligonucleotides form RNase H sensitive hybrids with the RNA targe. The sequences of both AS1 and AS2 should allow both to block PLF and OSF2/Periostin translation. Treating cells First, MC3T3-E1 pre-osteoblast cells were transfected when 70% confluent with varying amounts of antisense oligonucleotides (1, 2, 3 and 5 μM) using oligofectamine to promote transfection into cells (GIBCO-BRL). As controls, cells were either untreated or treated with oligofectamine alone. Forty-eight hours post-transfection cells were scraped into TRIZOL (GIBCO BRL) and total RNA isolated. RNA was DNase-treated and equal amounts of RNA were reverse-transcribed to generate first strand cDNA. G3PDH and PLF specific primers were used to amplify the respective mRNAs using the PCR reaction. Expression of PLF was normalized to that of G3PDH. Then, MC3T3-E1 cells were treated with 5 μM of anti-sense PLF oligonucleotides AS1 (SEQ ID NO:22), or AS2 (SEQ ID NO:23), or AS 1 and 2 together. The differentiation markers assessed included alkaline phosphatase (AL-PH), a phosphotransferase that hydrolyzes inhibitors of mineral deposition, osteopontin and osteocalcin (known to play important roles in matrix mineralization), core binding factor alpha- 1 (Cbfa 1) (a transcription factor known to regulate osteoblast-specific genes), and collagen type I (a basic building block of the bone matrix fiber network, which is a major product synthesized by osteoblasts). Results Effect of antisense oligonucleotides to PLF on PLF expression in MC3T3-E1 osteoblast cells PLF expression was not detectable in MC3T3-E1 cells treated with 5 μM AS 1 , as measured by RT-PCR analysis. PLF expression was reduced by 90% at 3 and 2 μM, and by 50% at 1.0 and 0.5 μM (FIG. 16). The antisense oligonucleotides appear to be effective in culture for approximately 3 days (data not shown). Antisense oligonucleotide #2 (AS2) was not as effective in reducing the level of PLF mRNA (PLF mRNA reduced by -20%, data not shown). It was found that PLF expression increases with time in MC3T3-E1 cells induced to differentiate (data not shown). To ensure that the 5 μM concentration of antisense oligonucleotide did not have a toxic effect on the cells, an MTT viability assay was performed. This test showed equally high levels of viability between cells treated at the various concentrations of antisense oligonucleotides, compared to the controls (data not shown). Effect of antisense oligonucleotides against PLF on expression of differentiation markers in MC3T3-E1 osteoblast cells AS 1 treatment resulted in reproducibly reduced levels of PLF mRNA
(not shown) and appreciably lower levels of early and late stage markers of osteoblast differentiation (FIG. 17). AS2 was not effective in reducing the level of PLF mRNA (see above) and no reduction was detected in any of the differentiation markers (AL-PH, Cbfal, collagen type 1, osteocalcin, and osteopontin). Treatment with ASl and AS2 together resulted in a reduction in expression of differentiation markers similar to that seen with AS 1 treatment alone. The data suggest that ASl was most likely responsible for the reduction in level of the differentiation markers. Example 11- The effect of PLF inhibition in cells treated with anti-
PLF antibody Methods To confirm the findings described above using antisense oligonucleotides against PLF, another approach was taken to reduce the levels of available PLF protein and determine the effect on differentiation. MC3T3- El cells were cultured as described above and treated with anti-PLF antibody (described above) at a 1 :100 or 1:200 dilution (stock antibody concentration = 5 μg/ml). The cells were treated with the CHARIOT reagent as a carrier, as recommended by the manufacturer (Active Motif, Carlsbad, CA.). CHARIOT is a proprietary transfection reagent for the delivery of antibodies, peptides and proteins. Chariots form a non-covalent complex with the antibody, are semm independent, are independent of the endosomal pathway, and upon intemalization protect the antibody from degradation. A β-galactosidase control protein transfected with Chariot into MC3T3-E1 cells showed efficient transfection (not shown). Control cells were treated with non- immune IgG. On day 3 post-transfection, differentiation factors were added to the media. Cells were harvested on days 7 and 21 post-transfection and total RNA isolated using TRIZOL (Invitrogen). Data obtained from RT-PCR using gene- specific primers are shown in FIGURE 18. Results At 7 days post-transfection with anti-PLF antibody, AL-PH and collagen type I were markedly reduced, whereas osteocalcin and osteopontin were slightly reduced compared to controls. At day 21, the reduction in the level of the differentiation markers was not as pronounced. It appears that the Antibody-Chariot mix is effective up to at least the day 7 time point, after which it probably decreases over time. Cells were examined under bright field microscopy on days 7 and 21 (not shown). At both time points, the antibody- treated cells were observed to be hypertrophied. Collectively, these data suggest that PLF is necessary for the osteoblast phenotype and differentiation. The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims should be constmed to include all such embodiments and equivalent variations.

Claims

What is claimed is: 1. An isolated nucleic acid encoding a Periostin isoform which: (a) contains an amino acid segment not contained in Periostin, wherein the amino acid segment has the sequence SEQ ID NO: 14 or SEQ ID NO:30; and (b) does not contain a Periostin amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16.
2. The isolated nucleic of claim 1, which encodes a Periostin isoform consisting of 810 amino acid residues.
3. An isolated nucleic acid according to claim 1, wherein the isolated nucleic acid comprises a nucleic acid sequence comprising: (a) SEQ ID NO: 12 or a sequence that is substantially homologous to SEQ ID NO: 12; or (b) SEQ ID NO:24 or a sequence that is substantially homologous to SEQ ID NO:24.
4. An isolated nucleic acid according to claim 1, wherein the isolated nucleic acid comprises a nucleic acid sequence encoding a Periostin isoform having the amino acid sequence SEQ ID NO: 11.
5. An isolated nucleic acid according to claim 1, wherein the isolated nucleic acid comprises a nucleic acid sequence encoding a Periostin isoform having the amino acid sequence SEQ ID NO:27 or a sequence that is substantially homologous to SEQ ID NO:27.
6. An isolated Periostin isoform encoded by the nucleic acid of claim 1.
7. An isolated Periostin isoform having the amino acid sequence SEQ ID NO:l l .
8. An isolated Periostin isoform having the amino acid sequence SEQ ID NO:27.
9. A vector comprising the isolated nucleic acid of claim 1.
10. The vector of claim 9, further comprising a nucleic acid sequence comprising a promoter/regulatory sequence operably linked thereto.
11. A host cell comprising the isolated nucleic acid of claim 1.
12. A host cell comprising the vector of claim 10.
13. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the isolated nucleic acid of claim 1.
14. An antibody directed against an antigenic determinant of the Periostin isoform of claim 6.
15. The antibody of claim 14, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized monoclonal antibody, and an antibody fragment.
16. The antibody of claim 15, wherein the antibody is directed against an antigenic determinant of a Periostin isoform having the amino acid sequence SEQ ID NO:l 1 or SEQ ID NO:27.
17. The antibody of claim 15, wherein the antibody is directed against the antigenic determinant having the sequence SEQ ID NO:29.
18. The antibody of claim 15, wherein the antibody is directed against an antigenic determinant not contained in Periostin.
19. A method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the isolated nucleic of claim 1 and a pharmaceutically acceptable carrier.
20. The method of claim 19, wherein the subject is a human.
21. The method of claim 19, wherein the PLF associated disorder is selected from the group consisting of ischemic heart disease, idiopathic heart disease, restenosis, cardiac hypertrophy, osteopetrosis, osteoporosis, cell differentiation disorders, cell adhesion disorders, cell migration disorders, wound healing disorders, bone fractures, chronic rejection, asthma, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, stomach cancer, metastases, cardia bifida, abnormal blood vessel development, head developmental anomalies, somite developmental anomalies, and developmental disorders.
22. A method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the Periostin isoform according to claim 6, and a pharmaceutically acceptable carrier.
23. The method of claim 22, wherein the subject is a human.
24. The method of claim 22, wherein the PLF associated disorder is selected from the group consisting of ischemic heart disease, idiopathic heart disease, restenosis, cardiac hypertrophy, osteopetrosis, osteoporosis, cell differentiation disorders, cell adhesion disorders, cell migration disorders, wound healing disorders, bone fractures, chronic rejection, asthma, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, stomach cancer, metastases, cardia bifida, abnormal blood vessel development, head developmental anomalies, somite developmental anomalies, and developmental disorders.
25. A method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the antibody of claim 14 and a pharmaceutically acceptable carrier.
26. The method of claim 25, wherein the subject is a human.
27. The method of claim 25, wherein the PLF associated disorder is selected from the group consisting of ischemic heart disease, idiopathic heart disease, restenosis, cardiac hypertrophy, osteopetrosis, osteoporosis, cell differentiation disorders, cell adhesion disorders, cell migration disorders, wound healing disorders, bone fractures, chronic rejection, asthma, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, stomach cancer, metastases, cardia bifida, abnormal blood vessel development, head developmental anomalies, somite developmental anomalies, and developmental disorders.
28. A method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antibody of claim 16 and a pharmaceutically acceptable carrier.
29. A method of treating a PLF associated disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of a pharmaceutical composition comprising an antisense oligonucleotide of about 8 nucleotides to about 40 nucleotides comprising a sequence complementary to an mRNA sequence encoding the Periostin isoform of claim 6, and a pharmaceutically acceptable carrier.
30. The method of 29, wherein the antisense oligonucleotide is complementary to the region of said mRNA which includes the translation initiation region.
31. The method of claim 29, wherein the antisense oligonucleotide is an oligonucleotide selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.
32. The method of claim 29, wherein the antisense oligonucleotide is complementary to a Periostin isoform mRNA sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 16, or SEQ ID NO:30.
33. The method of claim 29, wherein the subject is a human.
34. The method of claim 29, wherein the PLF associated disorder is selected from the group consisting of ischemic heart disease, idiopathic heart disease, restenosis, cardiac hypertrophy, osteopetrosis, osteoporosis, cell differentiation disorders, cell adhesion disorders, cell migration disorders, wound healing disorders, bone fractures, chronic rejection, asthma, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, stomach cancer, metastases, cardia bifida, abnormal blood vessel development, head developmental anomalies, somite developmental anomalies, and developmental disorders.
35. The method of claims 19, 22, 25, and 29, wherein the administration is selected from the group consisting of intravascular, peri- and intra-tissue injection, subcutaneous injection, subcutaneous deposition, subcutaneous infusion, direct application, inhalation, oral, rectal, and intranasal administration.
36. A method of diagnosing a PLF associated disorder in a subject, comprising measuring the level of the Periostin isoform of claim 6 in a sample derived from the subject, wherein a higher or lower level of the Periostin isoform in the sample relative to the level of the Periostin isoform in a control sample, indicates the presence of the PLF associated disorder.
37. The method of claim 36, wherein the PLF associated disorder is selected from the group consisting of ischemic heart disease, idiopathic heart disease, restenosis, cardiac hypertrophy, osteopetrosis, osteoporosis, cell differentiation disorders, cell adhesion disorders, cell migration disorders, wound healing disorders, bone fractures, chronic rejection, asthma, ovarian cancer, breast cancer, bone cancer, colon cancer, brain cancer, stomach cancer, metastases, cardia bifida, abnormal blood vessel development, head developmental anomalies, somite developmental anomalies, and developmental disorders.
38. The method of claim 36, wherein the Periostin isoform comprises the amino acid sequence SEQ ID NO:27.
39. The method of claim 36, wherein the Periostin isoform comprises the amino acid sequence SEQ ID NO: 11.
40. The method of claim 36, wherein the level of the Periostin isoform is measured with an assay selected from the group consisting of western blot, immunocytochemical, immunohistochemical, radioimmunoassay, enzyme linked immunosorbent assay, sandwich immunoassay, gel diffusion precipitin reaction, complement fixation, immunofluorescence, and immunoelectrophoretic assays.
41. The method of claim 36, wherein the level of the Periostin isoform is measured by contacting a sample derived from the subject with an antibody directed against an antigenic determinant of the Periostin isoform having the amino acid sequence selected from the group consisting of SEQ ID NO:l l, SEQ ID NO:27, and SEQ ID NO:29.
42. The method of claim 41 , wherein the subject is a human.
43. A method of diagnosing a PLF associated disorder in a subject, comprising measuring the level of an mRNA encoding a Periostin isoform in a sample derived from the subject, which mRNA: (a) encodes an amino acid segment not contained in Periostin, wherein the amino acid segment has the sequence SEQ ID NO: 14 or SEQ ID NO:30; and (b) does not encode a Periostin amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16; wherein a higher or lower level of the mRNA in the sample relative to the level of the mRNA in a control sample, indicates the presence of the PLF associated disorder.
44. The method of claim 43, wherein the mRNA is measured with an assay selected from the group consisting of northern blot, reverse transcriptase polymerase chain reaction, in situ hybridization, and dot blot assays.
45. A method of monitoring the progression of a PLF associated disorder in a subject, comprising measuring the level of the Periostin isoform of claim 6 in a sample derived from the subject, wherein a higher or lower level of the Periostin isoform in the sample, relative to the level present in a sample derived from the subject at an earlier time, indicates progression of the PLF associated disorder.
46. The method of claim 45, wherein the subject is a human.
47. The method of claim 45, wherein the level of the Periostin isoform is measured with an assay selected from the group consisting of western blot, immunocytochemical, immunohistochemical, radioimmunoassay, enzyme linked immunosorbent assay, sandwich immunoassay, gel diffusion precipitin reaction, complement fixation, immunofluorescence, and immunoelectrophoretic assays.
48. A method of monitoring the progression of a PLF associated disorder in a subject, comprising measuring the level of an mRNA encoding a Periostin isoform in a sample derived from the subject, which mRNA: (a) encodes an amino acid segment not contained in Periostin, wherein the amino acid segment has the sequence SEQ ID NO: 14 or SEQ ID NO:30; and (b) does not encode a Periostin amino acid segment having the sequence SEQ ID NO: 15 or SEQ ID NO: 16; wherein a higher or lower level of the mRNA in the sample relative to the level present in a sample derived from the subject at an earlier time, indicates progression of the of the PLF associated disorder.
49. The method of claim 48, wherein the subject is a human.
50. The method of claim 49, wherein the level of an mRNA encoding a Periostin isoform is measured with an assay selected from the group consisting of northern blot, reverse transcriptase polymerase chain reaction, in situ hybridization, and dot blot assays.
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