WO2010144140A2 - Egfr and par2 regulation of intestinal permeability - Google Patents
Egfr and par2 regulation of intestinal permeability Download PDFInfo
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Definitions
- the present invention relates to the fields of cell biology and intestinal permeability.
- the present invention relates to EGFR and PAR2 regulation of intestinal permeability.
- IP intestinal permeability
- TJs intercellular tight junctions
- Vibrio cholerae zonula occludens toxin (Zot), a toxin that increases tight junction permeability, led to the identification of its eukaryotic counterpart, zonulin, as the only physiological mediator known to reversibly regulate intestinal permeability by modulating intercellular tight junctions (6, 7).
- Human zonulin is a ⁇ 47 kDa protein that increases intestinal permeability in non-human primate intestinal epithelia (7), participates in intestinal innate immunity (8), and is overexpressed in autoimmune disorders in which tight junction dysfunction is central, including celiac disease (CD) (9, 10) and type 1 diabetes (T1 D) (11).
- Haptoglobin is an acute-phase response protein, synthesised mainly in the liver as well as arterial walls, endometrium and peritoneum.
- the core function of haptoglobin is as a haemoglobin (Hb) binding protein, required for terminal processing and disposal of free haemoglobin, mostly in the reticular endothelial system of the liver. This system allows the iron present in the Hb moiety to be conserved.
- Haptoglobin has a tetrameric structure comprising two .alpha, and two .beta, chains, linked by disulphide linkages.
- the .beta, chain (245 amino acids) has a mass of about 40 kDa (of which approximately 30% w/w is carbohydrate) and is shared by all phenotypes.
- the .alpha, chain exists in two forms: . alpha.1 , (83 amino acids, 9 kDa) and .alpha.2 (142 amino acids, 17.3 kDa) and therefore haptoglobin occurs as three phenotypes, referred to as Hp1- 1 , Hp2-1 and Hp2-2.
- Hp1-1 contains two .alpha.1 chains
- Hp2-2 contains two .alpha.2 chains
- Hp2-1 contains one .alpha.1 and one .alpha.2 chain.
- Hp 1-1 has a molecular mass of 100 kDa, or 165 kDa when complexed with Hb.
- Hp1-1 exists as a single isoform, and is also referred to as Hp dimer.
- Hp2-1 has an average molecular mass of 220 kDa and forms linear polymers.
- Hp2-2 has an average molecular mass of 400 kDa and forms cyclic polymers. Each different polymeric form is a different isoform.
- Haptoglobin is a potential treatment for renal disorders caused by haemolysis.
- haptoglobin is considered difficult to isolate in large amounts whilst retaining its biological activity.
- the present invention fulfills this long standing need in the art.
- zonulin While zonulin's role as an intestinal permeating modulator in health and disease has been described functionally, its biochemical characterization has remained elusive.
- the present invention shows that through proteomic analysis of human sera, zonulin is identical to pre-haptoglobin (HP)2, a molecule that, to date, has only been regarded as the inactive precursor for HP2, one of the two genetic variants (together with HP1 ) of human pre- haptoglobins.
- the present invention demonstrates the functional characterization of zonulin as pre-haptoglobin 2, a multifunctional protein that, in its intact single chain precursor form, appears to regulate intestinal permeability by transactivating the EGFR via PAR 2 activation, while in its cleaved two-chain form acts as a Hb scavenger.
- a method of treating an autoimmune disease comprising the steps of increasing transepithelial electrical resistance leading to decreased cell permeability.
- a method of treating an autoimmune disease in an individual in need of such treatment comprising the steps of inhibiting epidermal growth factor receptor; and inhibiting PAR 2 .
- a method of treating celiac disease in an individual in need of such treatment comprising the steps of: administering an antibody directed against single chain zonulin thereby inhibiting epidermal growth factor receptor and inhibiting PAR 2 .
- FIG. 1 shows Western blotting using zonulin cross-reacting anti-Zot polyclonal Ab on CD patient sera that were depleted of albumin and immunoglobulins. Three main patterns were detected: sera showing a 18 kDa immunoreactive band and a fainter -45 kDa band
- FIGs 2A-2B shows Coomassie and Western immunoblotting (WB) of purified human homozygote HP1-1 and HP2-2 both untreated and after deglycosylation with PGNase.
- Figure 2A Coomassie staining of untreated HPs showed a shared glycosylated b chain migrating at a MW -52 kDa, while the a of HP1-1 (a1) and of HP2-2 (a2) migrated at the predicted MW of 8 and 18 kDa, respectively.
- Deglycosylation with PGNase caused a shift of the b chain to a MW of ⁇ 36 kDa (complete deglycosylation) or higher (incomplete deglycosylation).
- Figure 2B WB of purified human homozygote HP1-1 and HP2-2 both untreated and after deglycosylation with PGNase run in triplicate on a single gel, transferred, and then separately subjected to WB analysis using polyclonal anti-Zot (left panel), monoclonal anti- HP (center panel), or polyclonal anti-HP Ab (right panel).
- the three Ab tested recognized both the a1 and a2 chains (all panels, lanes 1 and 2) whose pattern of reactivity did not change after deglycosylation of both HP1-1 and HP2-2 protein preparations (lanes 3 and 4).
- Zonulin was applied to the luminal side of C57BL/6 WT intestinal segments at 5, 10, 25 and 50 ⁇ g/well. Trypsin-cleaved pre-HP2 was applied at 50 ⁇ g/well. Starting at 60 min post-exposure, zonulin induced significant drop in TEER when applied at concentrations > 10 ⁇ g/well (P value ranging from 0.03 to 0.036). Data are mean values ⁇ SEM from 4 independent experiments.
- Figures 4A-4D show the effect of zonulin on mouse gastrointestinal permeability in vivo.
- Zonulin closed bars
- (170 mg/mouse) increases both mouse small intestinal (Figure 4A) and gastroduodenal ( Figure 4B) permeability as compared to BSA-treated controls (open bars).
- lacman ratio small IP
- sucrose fractional excretion gastroduodenal permeability
- Mature two-chain HP2 (dotted bars) (170 mg/mouse) caused no changes in either small intestinal or gastroduodenal permeability.
- Figures 5A-5D show the effect of zonulin on EGFR phosphorylation.
- Figure 5A Zonulin at increasing concentrations was incubated on serum-starved Caco-2 cells. The cells were lysed, immunoprecipitated using anti-EGFR Ab, and processed for WB using anti- phospho EGFR (PY Plus) Ab. To ensure equal loading, the blots were stripped and re- probed for EGFR. Zonulin caused a dose-dependent increase in EGFR phosphorylation that reached a plateau at 3 ml/ml.
- Figure 5D The zonulin-induced EGFR phosphorylation was significantly reduced following treatment with two-chain mature HP2 (10 ml/ml) (lane 3) compared with single chain zonulin (lane 2). Lane 1 shows EGFR phosphorylation in cells treated with media alone.
- Figures 6A-6B illustrate the effects of zonulin on EGFR phosphorylation and IP.
- Figure 6A Zonulin-induced EGFR phosphorylation was decreased when PAR 2 was silenced. PAR 2 expression was silenced in Caco-2 using two different PAR 2 siRNAs. Cells were then treated with zonulin (10 mg/ml) or media control, lysed, immunoprecipitated using anti-EGFR Ab, and processed for WB with anti-phospho-EGFR PY-plus Ab. Zonulin- mediated EGFR phosphorylation was prevented by PAR 2 silencing. Equivalent protein loading and transfer was confirmed by stripping and reprobing the blots for EGFR.
- Ab Antibodies
- EGFR Epidermal Growth Factor Receptor
- HP Haptoglobin
- IP Intestinal Permeability
- PAR Proteinase Activating Receptor
- TJ Tight Junctions
- WB Western Blot
- CD celiac disease.
- a or "an”, when used in conjunction with the term
- the term “or” in the claims refers to “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or”.
- the term “contacting” refers to any suitable method of bringing one or more of the compounds described herein with or without one or more other therapeutic agents into contact with one or more cells. For in vivo applications, any known method of administration is suitable as described herein.
- the terms "effective amount”, “pharmacologically effective amount” or “therapeutically effective amount” are interchangeable and refer to an amount that results in an effect against cells in vitro or an improvement. Those of skill in the art understand that the effective amount may improve the patient's or subject's condition, but may not be a complete cure.
- the term "subject" refers to any target of the treatment.
- the present invention is directed to a method of treating an autoimmune disease, comprising the steps of increasing transepithelial electrical resistance leading to decreased cell permeability.
- This method would be applicable to any autoimmune disease in which decreased cell permeability is desired.
- Representative cells include but are not limited to small intestinal cells or gastroduodenal cells. In one aspect, such cell would have a decreased expression of zonulin mRNA.
- this method further comprises the step of inhibiting epidermal growth factor receptor. A person having ordinary skill in this art would readily recognize known techiques to inhibit epidermal growth factor receptor to use in this method.
- a preferred embodiment is in which epidermal growth factor receptor is inhibited by administering an antibody directed against single chain zonulin.
- this method further comprises the step of inhibiting PAR 2 .
- a person having ordinary skill in this art would readily recognize known techiques to inhibit PAR 2 .
- PAR 2 is inhibited using an antibody directed against single chain zonulin or using an siRNA.
- this method further comprises the step of avoiding zonulin release by gliadin through CXCR3 receptor binding.
- Representative autoimmune diseases which may be treating using this method of the present invention include but are not limited to T1D, systemic lupus erythematosus, celiac disease, ankylosing spondylitis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, and schizophrenia.
- the present invention is further directed to a method of treating an autoimmune disease in an individual in need of such treatment, comprising the steps of inhibiting epidermal growth factor receptor; and inhibiting PAR 2 .
- transepithelial electrical resistance is increased leading to decreased cell permeability.
- Cell permeability may be decreased in any cell including but not limited to small intestinal cells or gastroduodenal cells. Typically, such cell will exhibit decreased expression of zonulin mRNA.
- Epidermal growth factor receptor and PAR 2 may be inhibited as described above.
- this method further comprising the step of inhibiting gliadin using any technique known to those of ordinary skill in this art, including anti-gliadin antibodies.
- Representative diseases which may be treated using this method of the present invention include but are not limited to autoimmune disease such as T1 D, systemic lupus erythematosus, celiac disease, ankylosing spondylitis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, and schizophrenia.
- autoimmune disease such as T1 D, systemic lupus erythematosus, celiac disease, ankylosing spondylitis, multiple sclerosis, rheumatoid arthritis, Crohn's disease, and schizophrenia.
- the present invention is further directed to a method of treating celiac disease in an individual in need of such treatment, comprising the steps of: administering an antibody directed against single chain zonulin thereby inhibiting epidermal growth factor receptor and inhibiting PAR 2 .
- transepithelial electrical resistance is increased leading to decreased cell permeability.
- Representative cells include small intestinal cells or gastroduodenal cells but this method could be useful in many cell types.
- PAR 2 is further inhibited using an siRNA.
- this method may further comprise the step of inhibiting gliadin.
- IP intestinal permeability
- pre-HP2 haptoglobin-2
- the present invention demonstrates that the single chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via Proteinase Activated Receptor (PAR) 2 activation. Activation of these two receptors was coupled to increased intestinal permeability. siRNA-induced silencing of PAR 2 or the use of PAR 2 " ' " mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its a2 and b subunits neutralized its ability to both activate EGFR and increase intestinal permeability. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease.
- HP1-1 and HP2-2 extracted from human plasma were purchased from Sigma (St.
- HP SDS-PAGE both mono- and two-dimensional gel electrophoresis WB, and mass-spectrometry analyses were performed.
- HP deglycosylation was performed by addition of ⁇ /-glycosidase F (PNGase F) according to the manufacturer's instructions (Sigma,
- Proteins were either stained with SimplyBlue SafeStain solution (Invitrogen) or transferred onto a PVDF membrane (Millipore) and probed with either 5 mg/mL affinitypurified rabbit polyclonal anti-Zot IgG Ab, which were previously shown to cross-react with purified human zonulin (1) using the ImmunoPure IgG (Protein A) Purification Kit (PIERCE), or with 2 mg/mL mouse monoclonal anti-human HP (Sigma) or 1 mg/mL rabbit polyclonal anti-human HP (Sigma) as the primary Ab.
- 2-DE was performed using the ZOOM IPGRunner System (Invitrogen). Briefly, albumin and IgG depleted sera were added to the commercial sample rehydration buffer containing urea, detergent, reducing agent, ampholyte solution, and a dye (ReadyPrep Rehydration/Sample buffer; Bio-Rad) in a ratio of 1 :2 to rehydrate the ZOOM STRIP pH 5.3-6.3 (Invitrogen) for 1 h at room temperature (RT). The strips were then loaded in the ZOOM IPGRunner Cassette (Invitrogen) to perform the isoelectric focusing (IEF).
- ZOOM IPGRunner System Invitrogen
- an IEF step voltage protocol of 200 V for 20 min, 450 V for 15 min, 750 V for 15 min, and 2,000 V for 105 min was used.
- strips were equilibrated for 15 min in NuPAGE LDS Sample buffer (Invitrogen) containing NuPAGE Sample Reducing Agent and alkylated for 15 min in NuPAGE LDS Sample buffer containing freshly added iodoacetamide (125 mM; BioRad).
- 2-DE SDS/PAGE was run using NuNovex 4-20% Tris-GlycineZOOMGels (1.0 mm) in an immobilized pH gradient well (Invitrogen).
- Protein bands were visualized by SimplyBlue SafeStain solution (Invitrogen). Protein bands were transferred onto PVDF membrane (Millipore) and probed using affinity- purified [Immuno-Pure IgG (Protein A) Purification Kit; PIERCE] rabbit polyclonal zonulin cross-reacting anti-Zot IgG (5 mg/mL) as the primary Ab and anti-rabbit IgG (ECL Rabbit IgG, HRP-Linked; Amersham Biosciences) as the secondary Ab. Films were developed after exposure of the PVDF membrane with ECL detection reagent (Amersham Biosciences).
- In-gel tryptic digest for protein band identification was performed on gel bands prestained with SimplyBlue excised from the SDS/PAGE or 2-DE and analyzed by MS/MS to identify the protein using the protein sequencing/mass mapping facility at the Stanford Protein and Nucleic Acid Biotechnology Facility (Beckman Center, Stanford, CA).
- Human full-length cDNA clone encoding for the HP2 was purchased from OriGene (TC116954; accession no. NM_005143; OriGene Technologies, Inc.).
- Recombinant baculoviruses containing WT human zonulin cDNA, with a 6xHis tag at the C-terminus were constructed using pDEST ⁇ and the Bac-to-Bac baculovirus expression system (Invitrogen) according to the manufacturer's protocol. Zonulin was then transferred from the pENTR/D- TOPO vector into the pDEST ⁇ through recombination using Gateway technology (Invitrogen).
- DHIOBac cells carrying bacmid DNA were transformed with pDEST ⁇ -zonulin.
- Recombinant bacmid was isolated from DHIOBac cells and transfected into Spodoptera frugiperda (Sf9) cells using Cellfectin reagent (Invitrogen) to generate recombinant baculoviruses.
- Sf9 cells were used for expression of zonulin protein.
- Sf9 cells 3 x 10 7
- SFM-900 III medium Invitrogen
- Sf9 cells were collected by centrifugation for 10 min at 2,000 x g.
- phosphate buffer (pH 7.5) and NaCI were added to the conditioned medium to final concentrations of 20mMand 0.5 M, respectively (2).
- the solution was applied to a chelatingsepharose (His-bind resin; Novagen) column charged with Ni2 + and then eluted with 200 mM imidazole and dialyzed into PBS.
- the purified human zonulin was aliquoted and stored at -8O 0 C until use.
- zonulin/pre-HP2 The effect of zonulin/pre-HP2 on ex vivo intestinal permeability was monitored in the microsnapwell system as described (3). Briefly, segments of small intestine from C57BL/6 WT mice were mounted onto the microsnapwell system, and their luminal side was exposed for 30 min to medium alone or to the medium containing increasing concentrations of the purified recombinant zonulin. TEER was measured at time 0 and at 30-min time intervals for a period of 2 h using a planar electrode (Endohm SNAP electrode attached to an Evom-G WPI analyzer; World Precision Instruments) and expressed in ⁇ /cm 2 after normalization.
- a planar electrode Endohm SNAP electrode attached to an Evom-G WPI analyzer; World Precision Instruments
- the effect of zonulin on TEER was monitored both under basal conditions and after pretreatment with the EGFR tyrosine kinase inhibitor AG 1478.
- zonulin was tested both in C57BU6 WT and PAR2-/- mice.
- mice were randomized into 3 groups of 30 mice. They were acclimatized to the experimental techniques for 3 wk, by fasting the animals for 3 h, gavaging the animals with a sugar probe, and placing them in metabolic cages twice each wk. On the day of protein challenge, the animals received either 170 mg of the purified single-chain zonulin in a 60-mL solution or a similar amount of purified 2-chain cleaved HP2, together with the sugar gavage as described (4). Mice were placed in metabolic cages and offered drinking water ad libitum for the following 22 h; during this time, their urine was collected, and the mice were then returned to conventional cages. Two days after the drug challenge day, mice were again placed in metabolic cages to measure their recovery from the treatment.
- PAR2 expression in Caco-2 cells was silenced using 2 different PAR2 siRNAs [HSS103471 and HSS103473 (50 nM each); Invitrogen].
- the cells were transfected following the manufacturer's instructions with the PAR2 siRNAs using DharmaFECTI transfection reagent (Dharmacon) in a 10-cm plate in the presence of 5% FCS for 24 h.
- PAR2 knockdown efficiency was confirmed by bothWB and real-time PCR analysis.
- RNA was precipitated by adding 0.5 mL of isopropyl alcohol per 1 mL of TRizol Reagent used for the initial homogenization. Samples were incubated at RT for 10 min and centrifuged at 15,000 x g for 10 min at 4°C. After removing the supernatant, the RNA pellet was washed once with 75% ice-cold ethanol, adding at least 1 mL of 75% ethanol per 1mL of TRizol Reagent used for the initial homogenization. The pellet was air-dried for no more than 2 min, dissolved in 20 mL of RNase-free water, and stored at -80 0 C. The RNA concentration was read at 260 nm by spectrophotometer (DU530, UV/vis; Beckman Coulter). The 260:280 ratio was determined for each sample.
- PCR Amplification of HP in Human Intestinal Biopsies Aliquots of the cDNA were utilized for PCR of fragments specific to HP2 using the following primer pairs, which were specifically designed to cover different exons: forward primer (exon 5) ⁇ '-ATGGCTATGTGGAGCACTCG-S' (SEQ ID NO: 1) and reverse primer (exon 7) 5'-TACAGGGCTCTTCGGTGTCT-3' (SEQ ID NO: 2).
- PCR was performed with 0.1 mg of cDNA, 2.5 units of TaqDNA polymerase (Promega), 0.2 mM dNTP mix, 0.5 mM each primer, 5 mM MgCI2, and 1:10 volume of 10ml PCR standard buffer (Promega).
- the PCR was run in the thermal cycler (Thermo Electro Corporation). After an initial 1 min of denaturation at 94 0 C, 30 cycles comprising 30 sec at 94 0 C (denaturation), 30 sec at 58 0 C (annealing), and 30 sec at 72 0 C (extension) were completed, followed by a 10-min final extension at 72 0 C.
- the PCR products were then separated on a 2%agarose gel, stained with ethidium bromide, excised out of the gel, purified using a gel band purification kit (Amersham Biosciences), and sequenced by a 373OxI DNA Analyzer (Applied Biosystems).
- Samples of small-intestine mucosae were obtained from the second/third portion of the duodenum from subjects undergoing a diagnostic upper gastrointestinal (Gl) endoscopy. Subjects included were 10 healthy controls, 7 patients with active CD at diagnosis, 3 patients with CD on treatment with a gluten-free diet for at least 6 months. All patients had clinical indications for the procedure and gave their informed consent to undergo an additional biopsy for the purpose of this study. The study protocol was approved by the Ethics Committee of the University of Maryland. The small-intestine biopsies were immediately collected in RNA/ater RNA Stabilization Reagent (Qiagen, Valencia, CA, USA) and stored at -20 0 C until processed. Total RNA extraction, cDNA synthesis, and real time PCR are described above.
- zonulin is detected in human sera by a zonulin cross-reacting anti-Zot Ab (Ab)- based ELISA (7-10) and is increased in patients with CD compared to normal controls (10).
- Western analysis was initially used to detect zonulin immunoreactivity of proteins in albumin- and IgG-depleted sera from CD subjects. These sera displayed two major protein bands with apparent molecular weights of 18 and 9 kDa (Fig. 1). Three distinct patterns of reactivity were identified in CD sera: a 18 kDa protein band (Fig. 1 , lane 1), a 9 kDa protein band (Fig. 1 , lane 2), and both 9 and 18 kDa protein bands (Fig. 1 , lane 3).
- HP1-1 and HP2-2 proteins both before and after deglycosylation. Proteins were run simultaneously on a single gel and immunoblotted with polyclonal zonulin-cross reacting anti-Zot Ab (Fig. 2B, left panel), monoclonal anti-glycosylated b chain HP (Fig. 26, center panel), or polyclonal anti-HP Ab (Fig. 2S, right panel). Anti-Zot Ab reacted strongly with both the HP1-1 a1 chain and the HP2-2 a2 chain and revealed an additional band at ⁇ 45 kDa present in the HP2-2, but not in the HP1-1 preparations (Fig. 2S, left panel, lanes 2 and 1 , respectively).
- the monoclonal anti-HP antibody raised against the ⁇ 52 kDa HP b glycosylated subunit, recognized only the b chain of either HP1-1 or HP2-2 (Fig. 2B, center panel, lanes 1 and 2, respectively), while the polyclonal anti-HP Ab recognized epitopes of the a1 , a2 andb chains of both HP1-1 and HP2-2 (Fig. 2B, right panel, lanes 1 and 2, respectively).
- Figure 2B also shows immunoblotted HP1-1 and HP2-2 preparations after deglycosylation using the same three Ab.
- the 45 kDa band that was present only in the HP2-2 preparation and recognized by anti-Zot Ab did not show any change in gel mobility upon deglycosylation, but it appeared less intense (Fig. 2S, left panel, lane 4).
- MS/MS analysis and NH 2 -terminal sequencing of this 45 kDa protein band performed on two distinct samples analyzed at different times identified this protein as the human HP2 precursor (pre-HP2, accession no. P00738).
- the combined MS/MS analysis covered a total of 49.8% of non-overlapping protein and 13 unique peptides spanning the entire protein sequence. Therefore, in addition to a1 and a2 chains, the anZ-ot Ab recognize the uncleaved single chain pre-HP2, but not the b chain.
- the anti-HP polyclonal Ab that did not recognize the uncleaved pre-HP2, detected bands both in commercially purified HP1-1 and HP2-2 preparations.
- the anti-Zot Ab detect only the single chain pre-HP2, but not the two- chain mature HPs, further supporting the notion that the single chain pre-HP2, but not its cleaved two-chain mature form, corresponds to the zonulin molecule.
- the primary translation product of the mammalian HP2 mRNA transcript is a polypeptide that dimerizes co-translationally and is proteolytically cleaved while still in the endoplasmic reticulum by the serine protease, CrI LP (13).
- zonulin is detectable in human serum as uncleaved pre-HP2 (see above).
- recombinant pre- HP2 was expressed by inserting the pre-HP2 cDNA into an insect cell vector and expressed it using a baculovirus expression system.
- Recombinant pre-HP2 (from now on defined as zonulin) was applied to WT C57BL/6 murine small intestine segments mounted in microsnapwells.
- Recombinant single chain zonulin added to the mucosal (luminal) aspect of mouse intestinal segments decreased transepithelial electrical resistance (TEER), i.e., increased permeability, when applied at concentrations ⁇ 40 ⁇ g/ml (Fig. 3).
- TEER transepithelial electrical resistance
- Fig. 3 no consistent TEER changes were detected when the trypsin-cleaved two chain HP2 was tested (Fig. 3).
- mice were gavaged with the single chain recombinant pre-HP2 protein (170 mg/mouse), and gastroduodenal and small intestinal permeability tested using specific sugar probes (sucrose and lactulose/mannitol, respectively) as described (14).
- Zonulin/preHP2 increased both small intestinal (Fig. 4A) and gastroduodenal (Fig. 4S) permeability compared to bovine serum albumin (BSA)-treated controls.
- BSA bovine serum albumin
- Gastroduodenal and small intestinal permeability each returned to baseline within 48 h following exposure to zonulin/preHP2 (Fig. AC and 4D).
- zonulin mRNA expression was increased in the intestinal mucosae of CD subjects with active disease (3-fold increase, P ⁇ 0.05).
- Intestinal mucosae of three celiac subjects adhering to a gluten-free diet showed only 1.5 fold increase zonulin expression compared to controls.
- Recombinant zonulin increases tyrosine phosphorylation of EGFR Gliadin, a glycoprotein present in wheat and several other cereals and the environmental trigger responsible for the autoimmune damage of the small intestine typical of CD (15), fully reproduces the effects of EGF on the actin cytoskeleton (16), effects that are very similar of zonulin (7, 10, 16). Furthermore, structural analysis revealed that the pre- HP-2 b chain includes an EGF motif that contains 6 spatially conserved cysteine residues that form 3 intramolecular disulfide bonds necessary for EGF-like activity.
- zonulin can activate EGFR
- increasing concentrations of baculovirus-derived, recombinant zonulin were added to Caco-2 intestinal epithelial cells.
- the cells were lysed, immunoprecipitated with anti-EGFR Ab, and processed for phosphotyrosine immunoblotting (PY-Plus).
- PY-Plus phosphotyrosine immunoblotting
- concentrations ⁇ 3 mg/ml zonulin increased tyrosine phosphorylation of EGFR (Fig. 5A).
- both in vitro and ex vivo experiments described above were performed in the presence of the EGFR-selective PTK inhibitor, AG1478.
- Zot active peptide FCIGRL (AT1002) has structural similarities with the PAR 2 - Activating Peptide (AP) 1 SLIGRL, and causes PAR ⁇ -dependent changes in TEER (17), a finding that was demonstrated in WT, but not PAR2 " ' " mice. Further, several G protein coupled receptors (GPCR), including PAR 2 , transactivate EGFR (18). Since Zot and zonulin share a similar mechanism of action (6) and the zonulin protein sequence contains a Zot-like and PAR 2 AP-like motif in its b chain (FCAGMS), whether zonulin-induced EGFR activation might be PAR 2 -dependent was determined.
- GPCR G protein coupled receptors
- siRNA-induced silencing of PAR 2 in Caco-2 cells diminished EGFR Y1068 phosphorylation in response to recombinant zonulin (10 mg/ml)(Fig. 6>A), compatible with PARr-dependent transactivation of EGFR.
- small intestinal barrier function was studied in the microsnapwell system using segments isolated from either C57BL/6 WT or PAR 2 ' " mice. As anticipated, recombinant zonulin decreased TEER in intestinal segments from C57BL/6 WT mice, while it failed to reduce TEER in small intestinal segments from PAR 2 " ' " mice (Fig. 6S), so linking zonulin-induced PAR 2 -dependent transactivation of EGFR with barrier function modulation.
- the present invention identified zonulin as the precursor of HP2.
- Mature human HPs are heterodimeric plasma glycoproteins composed of a and b polypeptide chains that are covalently associated by disulfide bonds and in which only the b chain is glycosylated (19). Unlike the b chain (36 kDa), the a chain exists in two forms, i.e., GM ( ⁇ 9 kDa) and 0,2 ( ⁇ 18 kDa). The presence of one or both of the 2 chains results in the three phenotypes, HP1-1 , HP2-1 , and HP2-2.
- HP variants evolved from a mannose-binding lectin-associated serine protease (MASP) (12, 20), with the a chain containing a complement control protein and the b chain a catalytically dead chymotrypsin-like serine protease domain (21-24).
- MASP mannose-binding lectin-associated serine protease
- Other members of the MASP family include a series of plasminogen-related growth factors (EGF 1 HGF, etc.) involved in cell growth, proliferation, differentiation, migration, and disruption of intercellular junctions.
- EGF 1 HGF plasminogen-related growth factors
- HPs are unusual secretory proteins in that their precursor proteins, instead of being cleaved in the trans-Golgi complex, are proteolytically processed by complement C1 r-like protease (CrI LP) in the endoplasmic reticulum (13).
- CrI LP complement C1 r-like protease
- the endoplasmic reticulum fraction was the cellular fraction in which the highest zonulin concentrations were detected (9). Since the key biological effect of zonulin is to regulate intercellular TJ function (7, 9- 11), recombinant pre-HP2 was exanubed in intestinal permeability assays.
- Pre-HP2 dose- and time-dependently reduced TEER across murine small intestinal mucosa both ex vivo and in vivo.
- NHfc-terminal amino acid sequence of zonulin has striking similarities with the light chain of human g globulins (7), a similarity also noted for HP (26). Clearance of the HP- Hb complex can be mediated by the monocyte/macrophage scavenger receptor, CD163 (25). Clustal W dendogram analysis showed a region in the zonulin b chain just upstream of the CD163 binding site with the following gamma globulin-like consensus motif: QLVE — V — P. Discrepancies between the previously reported zonulin sequence and this pre-HP2 consensus motif may be due to intra-species differences.
- Zonulin contains growth factor-like repeats. Like zonulin, growth factors affect intercellular tight junction integrity (27, 28).
- the present invention shows that the single chain zonulin, but not its cleaved mature form, transactivates EGFR via PAR 2 and that its effect on TEER is prevented by pharmacological inhibition of EGFR or siRNA-induced PAR 2 silencing. This suggests that the growth factor motif in the single chain zonulin, but not in the mature two-chain HP2, has the molecular conformation required to induce tight junctions disassembly by indirect transactivation via PAR 2 .
- Gliadin the environmental trigger of CD, reportedly reproduces the effects of EGF on the actin cytoskeleton (16).
- zonulin is upregulated during the acute phase of CD (9, 10).
- the present invention reports for the first time the expression of zonulin mRNA in human intestine.
- real time PCR experiments showed that zonulin expression was increased in CD patients compared to normal controls.
- the enhanced expression of zonulin correlated with disease activity as CD patients who were on a gluten-free diet showed mean values for zonulin expression that were intermediate to active CD patients and normal controls.
- Papp and co-workers recently reported that a polymorphism in the HP gene represents a novel genetic risk factor for CD development and its clinical manifestations (35).
- the human plasma levels of pre-HPs are between 100 and 300 mg/100ml, with HP2-
- zonulin used herein are within physiological range and are most likely indicative of the signaling pathways activated when zonulin is upregulated during pathological processes.
- zonulin elevated levels of zonulin have been reported in other autoimmune diseases, including T1 D (1 1), systemic lupus erythematosus (38), and ankylosing spondylitis (39), further delineating the importance of the zonulin pathway in the pathogenesis of autoimmune diseases.
- Tract. 1 ed. in physiology of the Gastrointestinal Tract Edition IV, eds Johnson LR, Elsevier Academic Press, 2006:183-246.
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AU2010259213A AU2010259213A1 (en) | 2009-06-10 | 2010-06-10 | EGFR and PAR2 regulation of intestinal permeability |
CA2765075A CA2765075A1 (en) | 2009-06-10 | 2010-06-10 | Egfr and par2 regulation of intestinal permeability |
JP2012514946A JP2012529508A (en) | 2009-06-10 | 2010-06-10 | EGFR and PAR2 regulation of intestinal permeability |
EP10786502.4A EP2440246A4 (en) | 2009-06-10 | 2010-06-10 | Egfr and par2 regulation of intestinal permeability |
US13/323,100 US20120107329A1 (en) | 2009-06-10 | 2011-12-12 | EGFR and PAR2 Regulation of Intestinal Permeability |
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