WO2022137964A1 - Pharmaceutical composition for preventing or treating cartilage/bone/joint diseases, and method for screening drug for preventing or treating cartilage/bone/joint diseases - Google Patents

Pharmaceutical composition for preventing or treating cartilage/bone/joint diseases, and method for screening drug for preventing or treating cartilage/bone/joint diseases Download PDF

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WO2022137964A1
WO2022137964A1 PCT/JP2021/043187 JP2021043187W WO2022137964A1 WO 2022137964 A1 WO2022137964 A1 WO 2022137964A1 JP 2021043187 W JP2021043187 W JP 2021043187W WO 2022137964 A1 WO2022137964 A1 WO 2022137964A1
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plexin
cells
substance
test substance
semaphorin
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French (fr)
Japanese (ja)
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理行 西村
智彦 村上
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国立大学法人大阪大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers

Definitions

  • the present invention relates to a pharmaceutical composition for preventing or treating cartilage / bone / joint disease and a method for screening a drug for preventing or treating cartilage / bone / joint disease.
  • articular cartilage Destruction of articular cartilage is the most common feature of chronic cartilage, bone and joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA).
  • RA rheumatoid arthritis
  • OA osteoarthritis
  • Inflammatory-inducing cytokines include tumor necrosis factor- ⁇ (TNF ⁇ ), interleukin-1 ⁇ (IL-1 ⁇ ), interleukin 6 (IL-6), etc., which are matrix metalloproteinases (MMPs) and aggrecanases. Promotes expression directly and / or indirectly.
  • TNF ⁇ tumor necrosis factor- ⁇
  • IL-1 ⁇ interleukin-1 ⁇
  • IL-6 interleukin 6
  • MMPs matrix metalloproteinases
  • Patent Document 1 describes a prophylactic / therapeutic agent for bone diseases containing a binding inhibitor of semaphorin 4D and plexin B1 as an active ingredient, and bone diseases include bone fracture, bone defect, osteoporosis, and bone softening. , Osteoporosis, lumbar backache, bone paget disease, rigid spondylitis, rheumatoid arthritis and osteoarthritis. However, Patent Document 1 does not refer to signal transduction of semaphorin 4D and plexin B2.
  • the present invention has found an inflammatory cytokine that causes destruction of articular cartilage, a pharmaceutical composition for preventing or treating cartilage / bone / joint disease targeting the inflammatory cytokine, and a cartilage / bone targeting the inflammatory cytokine.
  • -It is an object to provide a screening method for a drug for preventing or treating joint diseases.
  • a pharmaceutical composition for preventing or treating cartilage, bone, and joint diseases which contains a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient.
  • the substance is a substance that inhibits the binding between plexin B2 and semaphorin 4D, a substance that inhibits the binding between c-Met and semaphorin 4D, a substance that inhibits the binding between plexin B2 and Traf2, or a substance that inhibits the binding between plexin B2 and c-.
  • the pharmaceutical composition according to the above [1] which is a substance that inhibits the binding of Met.
  • a method for screening a drug for preventing or treating cartilage, bone, or joint disease which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2.
  • the step of contacting semaphorin 4D with the test substance and the expression level of the reporter gene are measured by contacting the cells of a cultured cell or a genetically modified animal in which a reporter gene is inserted downstream of the gene whose expression is induced by semaphorin 4D.
  • the screening method according to [7] above comprising the step of selecting a test substance that reduces the expression level of the reporter gene as compared with a control that has not been contacted with the test substance.
  • [9] A step of contacting a cultured cell or a gene-modified animal cell in which a reporter gene is inserted downstream of an expression control region recognized by I ⁇ B ⁇ or CEBP ⁇ with a test substance, and measuring the expression level of the reporter gene.
  • the step of contacting plexin B2 and Traf2 with the test substance, the step of measuring the binding between plexin B2 and Traf2, and the step of measuring the binding between plexin B2 and Traf2 are reduced as compared with the control to which the test substance is not added.
  • the screening method according to the above [7] which comprises a step of selecting a test substance that reduces the expression level of CEBP ⁇ .
  • the present invention it is possible to provide a pharmaceutical composition for preventing or treating cartilage, bone and joint diseases containing a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient.
  • the present invention also provides a method for screening a drug for preventing or treating cartilage, bone, and joint diseases, which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2.
  • (B) is the result of MEK inhibitor U0126 and c-Met inhibitor SU11274,
  • (C) is the result of Rho kinase inhibitor RKI-1447 and ErbB2 inhibitor AG825, and
  • (D) is the result of Rho kinase inhibitor Y-27632.
  • the result of the Akt inhibitor MK-2206. It is a figure which shows the result of having confirmed the knockdown effect of c-Met in the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of the expression level of c-Met, and (B) is the result of the expression level of Mmp13. Is.
  • Anti-c-Met antibody in cytolytic fluid of articular cartilage surface cells treated with semaphorin 4D or hepatocellular proliferation factor (HGF) Anti-phosphorylated tyrosine antibody for immunoprecipitates, anti-phosphorylated Erkl / 2 antibody for cytolytic fluid
  • HGF hepatocellular proliferation factor
  • Cytolysis of articular cartilage surface cells treated with semaphorin 4D or interleukin-1 ⁇ (positive control) was fractionated into cytoplasmic and nuclear fractions, each fraction being anti-NF- ⁇ B (p65) antibody and anti. It is a figure which shows the result of the analysis by the immune blotting using the histone H3 antibody. Articular cartilage surface cells treated with semaphorin 4D or interleukin-1 ⁇ (positive control) were immunofluorescently stained with anti-NF- ⁇ B (p65) antibody and fluorescently labeled secondary antibody, and the results observed with a fluorescence microscope are shown. It is a figure.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of cartilage, bone and joint diseases (hereinafter referred to as "the composition of the present invention") containing a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient.
  • the present inventors have stated that macrophages that induce inflammation are involved in the destruction of articular cartilage, and that soluble semaphorin 4D secreted from macrophages that induce inflammation acts on chondrocytes to form a matrix metalloproteinase.
  • the invention is based on the finding that it induces the expression of (MMP) and that the expression of MMP in chondrocytes is induced by signal transduction by binding of soluble semaphorin 4D and chondrocyte plexin-B2. be. Furthermore, we found that semaphorin 4D-plexin B2 binding activates both the TRAF2-NF- ⁇ B signaling pathway and the c-Met-Ras-Erk l / 2 signaling pathway and is a transcription factor. It was found that it exerts its action by inducing I ⁇ B ⁇ and CEBP ⁇ . We also revealed that semaphorin 4D acts cooperatively with IL-6.
  • the cartilage / bone / joint disease which is the target disease of the therapeutic pharmaceutical composition of the present invention is not particularly limited as long as it is a disease accompanied by destruction of articular cartilage.
  • articular cartilage Specifically, for example, fracture, bone defect, osteoporosis, osteomalacia, bone loss, lumbar backache, Paget's disease of bone, rigid spondylitis, rheumatoid arthritis, osteoarthritis, osteodysplasia. , Osteomalacia and the like. Rheumatoid arthritis, osteoarthritis or osteoporosis are preferred.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between semaphorin B2 and semaphorin 4D.
  • the substance that inhibits the binding between plexin B2 and semaphorin 4D may be, for example, an antibody that specifically binds to plexin B2 and inhibits the binding between plexin B2 and semaphorin 4D.
  • it may be a small molecule compound or peptide that inhibits the binding of plexin B2 to semaphorin 4D.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between c-Met and semaphorin 4D.
  • the substance that inhibits the binding between c-Met and semaphorin 4D may be, for example, an antibody that specifically binds to c-Met and inhibits the binding between c-Met and semaphorin 4D. Alternatively, it may be a small molecule compound or peptide that inhibits the binding of c-Met to semaphorin 4D.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between plexin B2 and Traf2.
  • the substance that inhibits the binding between plexin B2 and Traf2 may be, for example, an antibody that specifically binds to plexin B2 and inhibits the binding between plexin B2 and Traf2.
  • it may be an antibody that specifically binds to Traf2 and inhibits the binding of plexin B2 to Traf2.
  • it may be a small molecule compound or peptide that inhibits the binding between plexin B2 and Traf2.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between plexin B2 and c-Met.
  • the substance that inhibits the binding between plexin B2 and c-Met may be, for example, an antibody that specifically binds to plexin B2 and inhibits the binding between plexin B2 and c-Met.
  • it may be an antibody that specifically binds to c-Met and inhibits the binding of plexin B2 to c-Met.
  • it may be a small molecule compound or peptide that inhibits the binding of plexin B2 to c-Met.
  • Each antibody exemplified above may be a polyclonal antibody or a monoclonal antibody.
  • the antibody may be a low molecular weight antibody to which an antibody fragment having an antigen-binding ability (for example, Fab, Fab', F (ab') 2, Fv, scFv, diabody, etc.) or a variable portion of the antibody is bound. good.
  • Polyclonal antibody and monoclonal antibody can be produced by a known method. Polyclonal antibody immunizes mammals (mouse, rat, rabbit, goat, horse, etc.) using, for example, an antigen dissolved in PBS and optionally mixed with an appropriate amount of a usual adjuvant (for example, Freund's complete adjuvant) as an immunogen.
  • a usual adjuvant for example, Freund's complete adjuvant
  • the immunization method is not particularly limited, but for example, a method of subcutaneous injection or intraperitoneal injection once or multiple times at appropriate intervals is preferable.
  • the monoclonal antibody can be produced, for example, by fusing immune cells (for example, splenocytes) obtained from the immunized mammal and myeloma cells to obtain a hybridoma, and collecting the antibody from the culture of the hybridoma. can.
  • an antibody gene from a hybridoma incorporate it into an appropriate vector, introduce it into a host cell, and use gene recombination technology to produce a recombinant monoclonal antibody.
  • monoclonal antibodies can also be prepared using the phage display method.
  • the antibody may be a human chimeric antibody or a humanized antibody.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a nucleic acid that inhibits the expression of plexin B2.
  • the nucleic acid that inhibits the expression of plexin B2 include siRNA (short interfering RNA), shRNA (short hairpin RNA), antisense oligonucleotide, and antisense mRNA of the plexin B2 gene.
  • the nucleotide sequence of the plexin B2 gene of the administration target animal can be obtained from a known database (NCBI, etc.).
  • a siRNA is a double-stranded RNA having a length of about 20 bases (for example, about 21 to 23 bases) or less, and by introducing such siRNA into a cell, the gene targeted by the siRNA (book). In the invention, the expression of plexin B2 gene) can be suppressed.
  • shRNA is a single-stranded RNA containing a partially palindromic base sequence, so that it has a double-stranded structure within the molecule and consists of a short hairpin structure with a protrusion at the 3'end of about 20 base pairs. Refers to the above molecules.
  • shRNA After being introduced into a cell, such shRNA is decomposed into a length of about 20 bases (typically, for example, 21 bases, 22 bases, and 23 bases) in the cell and becomes a target like siRNA.
  • the expression of a gene can be suppressed.
  • the siRNA and shRNA may be in any form as long as they can suppress the expression of plexin B2.
  • the siRNA or shRNA can be designed by a known method based on the base sequence of the target gene.
  • siRNA or shRNA can be artificially synthesized.
  • Antisense and sense RNA can also be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and T7 promoter.
  • the antisense oligonucleotide may be either DNA or RNA, as long as it is a nucleotide that is complementary or hybridizes to the consecutive 5 to 100 base sequences in the DNA sequence of the plexin B2 gene. Further, it may be modified as long as it does not interfere with the function.
  • the antisense oligonucleotide can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer.
  • the antisense mRNA may be an RNA that is complementary to or hybridizes to the base sequence of the mRNA of plexin B2. Antisense mRNA can be artificially chemically synthesized.
  • Antisense and sense RNA can also be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and T7 promoter. During synthesis, pseudouridine is used instead of uridine to impart a cap structure and poly-A signal.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the function of plexin B2.
  • Functions of plexin B2 include, but are not limited to, the ability to bind to semaphorin 4D, the ability to bind to Traf2, and the ability to bind to c-Met. Substances that inhibit the function of plexin B2, which will be discovered in the future, are also included in the substances that inhibit the signal transduction of semaphorin 4D-plexin B2.
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the expression of I ⁇ B ⁇ or CEBP ⁇ .
  • examples of the substance that inhibits the expression of I ⁇ B ⁇ or CEBP ⁇ include siRNA, shRNA, antisense oligonucleotide, and antisense mRNA of the I ⁇ B ⁇ gene or CEBP ⁇ gene.
  • the nucleotide sequence of the I ⁇ B ⁇ gene and the nucleotide sequence of the CEBP ⁇ gene of the administration target animal can be obtained from a known database (NCBI, etc.).
  • the substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the function of I ⁇ B ⁇ or CEBP ⁇ .
  • Functions of I ⁇ B ⁇ or CEBP ⁇ include, for example, DNA binding ability, transcriptional activation ability, and ability to bind to other transcription factors. Therefore, substances that inhibit the function of I ⁇ B ⁇ or CEBP ⁇ include, for example, substances that inhibit binding to DNA recognized by I ⁇ B ⁇ or CEBP ⁇ , substances that reduce the expression of genes involved in the expression control region recognized by I ⁇ B ⁇ or CEBP ⁇ , and the like. Can be mentioned.
  • the substance that inhibits semaphorin 4D-plexin B2 signal transduction may be a c-Met inhibitor.
  • the present inventors cultivated mouse femoral head cartilage, added semaphorin 4D or semaphorin 4D and the c-Met inhibitor crizotinib to the medium, and then added the c-Met inhibitor. It has been confirmed that the amount of aggrecan released into the medium is significantly suppressed, that is, cartilage destruction is suppressed (see Example 8). Therefore, c-Met inhibitors are useful as active ingredients in pharmaceutical compositions for the prevention or treatment of cartilage, bone and joint diseases.
  • the c-Met inhibitor is not particularly limited, and examples thereof include crizotinib, tepotinib, and capmatinib.
  • the pharmaceutical composition of the present invention contains a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient, and can be formulated according to conventional means.
  • formulations for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and the like. Examples include syrups, emulsions and suspending agents. These formulations are manufactured by known methods and contain carriers, diluents or excipients commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets.
  • injections, suppositories, etc. are used as preparations for parenteral administration, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, intra-articular injection. Including dosage forms such as.
  • Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily solution normally used for injections.
  • aqueous solution for injection for example, a physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, and the like are used, and appropriate solubilizing agents such as alcohol (for example, ethanol etc.) and polyalcohol (for example) are used.
  • suppositories used for rectal administration are prepared by mixing the active ingredient with a conventional suppository base.
  • the parenteral route of administration eg, intravenous, intramuscular, intradermal, as an injection or infusion formulated with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier Preferably administered intraperitoneally, subcutaneously or topically.
  • the active ingredient of the pharmaceutical composition of the invention is nucleic acid
  • it can be administered in the form of a non-viral vector or a viral vector.
  • a method of introducing a nucleic acid molecule using a liposome liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.
  • microinjection method microinjection method
  • gene gun GeneGun
  • siRNA or shRNA When siRNA or shRNA is administered in the form of a viral vector, detoxified retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, Sindobis virus, Sendai virus, SV40
  • a DNA expressing siRNA or shRNA into a DNA virus or RNA virus such as, and infecting cells or tissues with this recombinant virus, siRNA or shRNA can be introduced into cells or tissues.
  • the formulations thus obtained are safe and low toxicity, and therefore, for example, orally to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Alternatively, it can be administered parenterally.
  • the pharmaceutical composition of the present invention can contain 0.001 to 50% by mass, preferably 0.01 to 10% by mass, and more preferably 0.1 to 1% by mass of the active ingredient.
  • the dose of the pharmaceutical composition of the present invention is appropriately set in consideration of the severity of the disease, the age, weight, sex, medical history, type of active ingredient, and the like of the patient. For an average human having a body weight of about 65 to 70 kg, about 0.02 mg to 4000 mg per day is preferable, and about 0.1 mg to 200 mg is more preferable.
  • the total daily dose may be a single dose or a divided dose.
  • Screening method Provided is a method for screening a drug for preventing or treating cartilage, bone, or joint disease (hereinafter referred to as "the screening method of the present invention"), which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2. do.
  • the test substance used in the screening method of the present invention is not particularly limited, and for example, a single substance such as a natural compound, an organic compound, an inorganic compound, a nucleic acid, a nucleic acid oligo, a protein, or a peptide, a compound library, or a nucleic acid oligolive.
  • a single substance such as a natural compound, an organic compound, an inorganic compound, a nucleic acid, a nucleic acid oligo, a protein, or a peptide, a compound library, or a nucleic acid oligolive.
  • peptide library, gene library expression product, cell extract, cell culture supernatant, fermented microbial product, marine biological extract, plant extract, prokaryotic cell extract, eukaryotic single cell extract, animal cell extract Things can be mentioned.
  • the test substance may be a novel substance or a known substance. These test substances may form salts.
  • the first embodiment of the screening method of the present invention may include the following steps (1) to (3).
  • (2) Step of measuring the binding between plexin B2 and semaphorin 4D and (3) selecting a test substance that reduces the binding between plexin B2 and semaphorin 4D as compared with a control to which no test substance is added.
  • step (1) plexin B2, semaphorin 4D, and the test substance are brought into contact with each other.
  • the system in which plexin B2 and semaphorin 4D are brought into contact with the test substance include a solution system or a culture system.
  • a solution system for example, a system in which plexin B2, semaphorin 4D and a test substance are brought into contact with each other in a buffer solution suitable for a protein solution can be used.
  • a control is provided that is not in contact with the test substance.
  • buffer solution suitable for the protein solution examples include Tris buffer solution having a pH of 6 to 9, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, and a citrate buffer solution.
  • Plexin B2 and semaphorin 4D can be produced as recombinant proteins using known gene recombination techniques and recombinant protein expression techniques. Semaphorin 4D may be soluble semaphorin 4D.
  • the amino acid sequence information of plexin B2, the base sequence information of the gene encoding plexin B2, the amino acid sequence information of semaphorin 4D, and the base sequence information of the gene encoding semaphorin 4D can be obtained from a known database (NCBI, etc.). can.
  • accession numbers of the human and mouse base sequences and amino acid sequences are as follows.
  • NP_001268809.1 amino acid sequence
  • a culture system for example, a system in which semaphorin 4D or soluble semaphorin 4D and a test substance are added to a medium in which plexin B2-expressing cells are cultured, a system in which plexin B2-expressing cultured cells and soluble semaphorin 4D are added. It is possible to use a system in which the test substance is added to the medium in which the cells secreting the cells are co-cultured. Usually, a control without the addition of the test substance is provided.
  • plexin B2-expressing cells include articular cartilage surface cells, cultured cells into which a plexin B2 expression vector has been introduced, chondrocytes, osteoblasts, and limb blast cells.
  • cells that secrete semaphorin 4D include macrophages, lymphocytes, synovial cells, osteoclasts, etc. treated with LPS.
  • step (2) the binding between plexin B2 and semaphorin 4D is measured.
  • the method for measuring the binding between plexin B2 and semaphorin 4D is not particularly limited, and a known method can be appropriately selected and used.
  • a known method can be appropriately selected and used.
  • an ELISA method, a fluorescence polarization method, an immunostaining method, a mass spectrometry method, a structural analysis, a screening method using an HTS system using cells, or the like can be preferably used.
  • either one of the recombinant protein of soluble semaphorin 4D and the recombinant protein of the extracellular region of plexin B2 is immobilized, and the other and the test substance are added thereto to react with plexin B2.
  • the binding level of semaphorin 4D can be detected using the appropriate primary and secondary antibodies.
  • a test substance that reduces the binding between plexin B2 and semaphorin 4D as compared with the control to which the test substance is not added is selected. Criteria for selection are not particularly limited, but for example, the binding level is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less as compared with the binding level of the control to which the test substance is not added.
  • the test substance to be lowered may be selected.
  • the second embodiment of the screening method of the present invention may include the following steps (1) to (3).
  • Step of contacting plexin B2 and Traf2 with a test substance (2) A step of measuring the binding between plexin B2 and Traf2, and (3) a step of selecting a test substance that reduces the binding between plexin B2 and Traf2 as compared with a control to which no test substance is added.
  • step (1) plexin B2 and Traf2 are brought into contact with the test substance.
  • the system in which plexin B2 and Traf2 are brought into contact with the test substance include a solution system and a culture system.
  • a solution system for example, a system in which plexin B2 and Traf2 are brought into contact with a test substance in a buffer solution suitable for a protein solution can be used.
  • a culture system for example, a system in which Traf2 and a test substance are added to a medium in which plexin B2-expressing cells are cultured can be used.
  • a control is provided that is not in contact with the test substance.
  • Plexins B2 and Traf2 can be produced as recombinant proteins using known gene recombination techniques and recombinant protein expression techniques.
  • the sequence information of plexin B2 is as described above, and the amino acid sequence information of Traf2 and the base sequence information of the gene encoding Traf2 can be obtained from a known database (NCBI or the like).
  • NCBI National Center for Biotechnology Information
  • accession numbers of the human and mouse base sequences and amino acid sequences are as follows.
  • plexin B2-expressing cell Human: NM_021138.4 (base sequence), NP_066961.2 (amino acid sequence) Mouse: NM_001290413.1 (base sequence), NP_001277342.1 (amino acid sequence)
  • the cell exemplified in the first embodiment can be used.
  • the steps (2) and (3) of the second embodiment of the screening method of the present invention are the steps of the first embodiment except that Traf2 is used instead of semaphorin 4D in the first embodiment. It can be performed in the same manner as in 2) and (3).
  • the third embodiment of the screening method of the present invention may include the following steps (1) to (3).
  • (2) Step of measuring the binding between plexin B2 and c-Met may be used.
  • step (1) plexin B2, c-Met and the test substance are brought into contact with each other.
  • the system in which the plexin B2 and c-Met are brought into contact with the test substance include a solution system and a culture system.
  • a solution system for example, a system in which plexin B2, c-Met and a test substance are brought into contact with each other in a buffer solution suitable for a protein solution can be used.
  • a culture system for example, a system in which c-Met and a test substance are added to a medium in which plexin B2-expressing cells are cultured, and a system in which plexin B2 and a test substance are added to a medium in which c-Met-expressing cells are cultured.
  • a system in which a test substance is added to a medium in which plexin B2-expressing cells and c-Met-expressing cells are co-cultured can be used.
  • a control is provided that is not in contact with the test substance.
  • Semaphorins 4D and c-Met can be produced as recombinant proteins using known gene recombination techniques and recombinant protein expression techniques.
  • the sequence information of plexin B2 is as described above, and the amino acid sequence information of c-Met and the base sequence information of the gene encoding c-Met can be obtained from a known database (NCBI or the like).
  • NCBI National Center for Biotechnology Information
  • accession numbers of the human and mouse base sequences and amino acid sequences are as follows.
  • the cell exemplified in the first embodiment can be used.
  • the c-Met expressing cell include articular cartilage surface layer cells, cultured cells into which a c-Met expression vector has been introduced, chondrocytes, osteoblasts, and limb blast cells.
  • steps (2) and (3) of the third embodiment of the screening method of the present invention are the same as that of the first embodiment, except that c-Met is used instead of semaphorin 4D in the first embodiment. It can be carried out in the same manner as in steps (2) and (3).
  • the fourth embodiment of the screening method of the present invention may include the following steps (1) to (3). (1) A step of contacting a test substance with a cell expressing I ⁇ B ⁇ and / or CEBP ⁇ , (2) A step of measuring the expression level of I ⁇ B ⁇ and / or CEBP ⁇ , and (3) a step of selecting a test substance that reduces the expression level of I ⁇ B ⁇ and / or CEBP ⁇ as compared with a control not contacted with the test substance.
  • the test substance is brought into contact with cells expressing I ⁇ B ⁇ and / or CEBP ⁇ .
  • the test substance can be brought into contact with the test substance by adding the test substance to the medium of the cultured cells expressing I ⁇ B ⁇ and / or CEBP ⁇ .
  • a control is provided that is not in contact with the test substance.
  • cells expressing I ⁇ B ⁇ and / or CEBP ⁇ include articular cartilage superficial cells, chondrocytes, osteoblasts, limb blast cells and the like.
  • the expression levels of I ⁇ B ⁇ and / or CEBP ⁇ are measured.
  • the expression level may be measured by measuring the amount of protein or mRNA.
  • the protein can be extracted from the cell by a known method and quantified by using a known method for measuring the amount of protein.
  • Known methods for measuring the amount of protein include, for example, Western blotting, EIA method, ELISA method, RIA method, and a method using a protein measuring reagent.
  • RNA can be extracted from cells by a known method and quantified using a known method for measuring the amount of mRNA.
  • Known methods for measuring the amount of mRNA include Northern blotting, RT-PCR, quantitative RT-PCR, RNase protection assay and the like.
  • step (3) a test substance that reduces the expression level of I ⁇ B ⁇ and / or CEBP ⁇ as compared with a control that has not been contacted with the test substance is selected.
  • the criteria for selection are not particularly limited, but for example, the expression level is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less as compared with the expression level of the control to which the test substance is not added.
  • the test substance to be lowered may be selected.
  • the fifth embodiment of the screening method of the present invention may include the following steps (1) to (3).
  • I ⁇ B ⁇ or CEBP ⁇ and the test substance are brought into contact with cultured cells or genetically modified animal cells in which a reporter gene is inserted downstream of the expression control region recognized by I ⁇ B ⁇ or CEBP ⁇ .
  • cultured cells used in the step (1) include established cultured cells, articular superficial chondrocytes, chondrocytes, osteoblasts, and limb blasts.
  • genetically modified animal used in the step (1) include mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys and the like.
  • the reporter gene containing these gene expression control regions is introduced by a known transfection method, virus transfer method, or the like. Can be made. Genetically modified animals in which a reporter gene is inserted downstream of the expression control region recognized by I ⁇ B ⁇ or CEBP ⁇ should be produced by knocking in (inserting) the reporter gene into these gene expression regions by genome editing, homologous recombination, etc. Can be done.
  • the reporter gene is not particularly limited as long as it is generally used, but a reporter gene that is stable and whose activity can be easily quantified is preferable.
  • genes encoding luciferase, ⁇ -galactosidase (lacZ), ⁇ -glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), alkaline phosphatase (ALP), peroxidase (POD), green fluorescent protein (GFP), etc. can be mentioned.
  • the cultured cells When the cultured cells are used in the step (1), for example, by adding I ⁇ B ⁇ or CEBP ⁇ and the test substance to the medium of the cultured cells, the cells can be brought into contact with the I ⁇ B ⁇ or CEBP ⁇ and the test substance.
  • a genetically modified animal for example, by administering I ⁇ B ⁇ or CEBP ⁇ and the test substance to the genetically modified animal, the cells of the genetically modified animal can be brought into contact with I ⁇ B ⁇ or CEBP ⁇ and the test substance.
  • the route of administration is not particularly limited, and examples thereof include systemic administration such as oral administration, intravenous administration, and intraperitoneal administration, and local administration to a target organ or target tissue. Usually, a control is provided that is not in contact with the test substance.
  • the expression level of the reporter gene is measured.
  • the expression level of the reporter gene can be measured by appropriately selecting a known measuring method according to the reporter gene to be used.
  • the reporter gene is a CAT gene
  • the expression level of the reporter gene can be measured by detecting the acetylation of chloramphenicol by the gene product.
  • the reporter gene is the lacZ gene
  • the color development of the dye compound due to the catalytic action of the gene expression product is detected
  • the reporter gene is the luciferase gene
  • the luminescence of luciferin due to the catalytic action of the gene product is emitted.
  • the expression level of the reporter gene can be measured.
  • step (3) a test substance that reduces the expression level of the reporter gene as compared with a control that has not been contacted with the test substance is selected.
  • the criteria for selection are not particularly limited, but for example, the expression level is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less as compared with the expression level of the control to which the test substance is not added.
  • the test substance to be lowered may be selected.
  • the sixth embodiment of the screening method of the present invention may include the following steps (1) to (3).
  • step (1) of the sixth embodiment semaphorin 4D and the test substance are brought into contact with cultured cells or genetically modified animal cells in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D.
  • Examples of the cultured cells and genetically modified animals used in the step (1) include the cells and animals exemplified in the fifth embodiment.
  • Examples of the gene whose expression is induced by semaphorin 4D include Mmp13, Mmp3, IL-6, Nfikbz, C / ebpd and the like.
  • a known transfection method or virus transfer method is used to construct a construct in which a reporter gene is linked downstream of a gene whose expression is induced by semaphorin 4D. It can be produced by introducing a gene using such as.
  • a gene-modified animal in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D knocks in (inserts) a reporter gene downstream of a gene whose expression is induced by semaphorin 4D by genome editing, homologous recombination, etc. ) Can be produced.
  • the sixth embodiment of the screening method of the present invention is carried out in the same manner as the fifth embodiment, except that the cultured cells into which the reporter gene used in the step (1) is inserted or the cells of the genetically modified animal are different. be able to.
  • the present invention includes the following inventions.
  • a method for preventing or treating cartilage / bone / joint disease which comprises a step of administering a substance that inhibits signal transduction of semaphorin 4D-plexin B2 to a patient with cartilage / bone / joint disease.
  • a substance that inhibits signal transduction of semaphorin 4D-plexin B2 for use in the prevention or treatment of cartilage, bone and joint diseases.
  • Use of substances that inhibit semaphorin 4D-plexin B2 signaling to produce pharmaceutical compositions for the prevention or treatment of cartilage, bone and joint diseases.
  • Nfkbiz KO mice Nfkbiz flox / flox mice were mated with CAG-CRE transgenic mice to obtain Nfkbiz +/- mice.
  • Nfkbiz +/- mice were mated with Nfkbiz +/- mice to generate Nfkbiz -/- mice.
  • Sema4d knockout mice (hereinafter referred to as "Sema4d KO mice") were produced using the TAKE method (Technique for Animal Knockout system by Electroporation) based on the CRISPR / Cas9 system (SciRep 4, 6382, doi: 10.1038 / srep06382 (2014). )).
  • Guide RNA target sequence: 5'-AGGATGTGCGCCCCGTTAG-3', SEQ ID NO: 1 targeting around the start codon of the Sema4d gene is transcribed in vitro using Guide-it sgRNA In Vitro Transcription and Screening System (Takara Bio).
  • Sema4d KO mice Cas9 protein and mRNA that introduces a stop codon, were introduced into fertilized egg cells of C57BL / 6J mice by electroporation. Genomic DNA sequence analysis of the Sema4d gene was performed to confirm appropriate genome editing in Sema4d KO mice.
  • Plxnb2 flox / flox mice were generated using the two-step TAKE method.
  • a guide RNA targeting downstream of exon 22 encoding Plxnb2 is transcribed in vitro, and Cas9 protein, mRNA and single-stranded oligodeoxy are transcribed.
  • a guide RNA targeting the upstream of exon 21 encoding the Plxnb2 gene is transcribed in vitro, and Cas9 protein, mRNA and ssODN (5') are transcribed.
  • --ACAAGCTCATCCACGCCCGGGTAAGAAGACCCCTACATAACTTCGTATAGCATACATTATACGAAGTTATGGCTGGCATGGGGTCCCGGGTCGGGCGGGG -3', SEQ ID NO: 5 was introduced into fertilized egg cells of Plxnb2 LoxP transgenic mice by the electroporation method. The introduction of two LoxP sites was confirmed by DNA sequence analysis.
  • Plxnb2 flox / flox mice were mated with CAG-CRE transgenic mice to obtain Plxnb2 +/- mice.
  • Plxnb2 +/- mice were mated with Plxnb2 +/- mice to produce Plxnb2 knockout mice (hereinafter referred to as "Plxnb2 KO mice").
  • DNA sequence analysis confirmed that these exons were excised by mating with CAG-CRE transgenic mice and that the transmembrane domain of plexin-B2 was deleted.
  • the paws of each mouse were evaluated histologically.
  • the feet were fixed with 4% PFA / PBS and then decalcified with 10% EDTA for 4 weeks.
  • the cartilage was embedded in paraffin to prepare a section having a thickness of 5 ⁇ m. Sections were deparaffinized and stained with hematoxylin and eosin (HE). Joint lesions were evaluated and quantified according to the description of Maeda et al. (Nature Medicine 18, 405-U166, doi: 10.1038 / nm.2653 (2012)).
  • HEK293 cells were cultured in DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% FCS and penicillin / streptomycin / glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.). Bone marrow-derived by differentiating tibial and femoral bone marrow progenitor cells in RPMI (Fujifilm Wako Pure Drug) supplemented with 10% FCS, 25% L929 acclimatized medium, sodium pyruvate, HEPES, 2ME and penicillin-streptomycin. Macrophages (bone marrow derived macrophages, hereinafter referred to as "BMDM”) were obtained.
  • BMDM bone marrow derived macrophages
  • SFZ cells Articular cartilage surface layer (SFZ: superficial zone) cells
  • LPS 10 ng / ml
  • SFZ cells Articular cartilage surface layer cells
  • SFZ cells were isolated by the method described in Yasuhara et al. (Lab Invest 91, 1739-1752, doi: 10.1038 / labinvest.2011.144 (2011)). .. That is, the proximal end of the femur and the distal end of the tibia were cut from the knee joint of a 17.5 day embryo for the production of 4-day-old mice or Plxnb2 KO mice, and ligaments and tendons were carefully removed from their attachment sites.
  • Tissues were incubated with Hanks Balanced Salt Solution (HBSS) containing 0.25% trypsin for 1 hour, followed by digestion with DMEM containing 0.25% collagenase (Wako Pure Chemical Industries, Ltd.) for 1.5 hours.
  • Dissociated cells were seeded on BioCoat TM fibronectin plates (Sigma-Aldrich) and incubated for 20 minutes.
  • Non-adherent cells were rinsed twice with phosphate buffered saline (PBS) and adherent cells were used as SFZ cells.
  • SFZ cells were maintained in DMEM medium containing 10% FBS and penicillin / streptomycin / glutamine.
  • Deep zone (DZ: deep zone) cells were separated by additional digestion of residual cartilage tissue with collagenase.
  • DZ cells were maintained in DMEM medium containing 10% FBS and penicillin / streptomycin / glutamine.
  • Primary osteoblasts were obtained by continuous digestion of the calvaria of 4-day-old mice with collagenase. Osteoblasts were collected from the dispersion and cultured in ⁇ -MEM medium supplemented with 10% FBS and penicillin / streptomycin / glutamine.
  • Costal chondrocytes were isolated by digesting the ribs of 4-day-old mice with collagenase (Wako Pure Chemical Industries, Ltd.).
  • Costal chondrocytes were collected from the dispersion and cultured in DMEM medium containing 10% FBS and penicillin / streptomycin / glutamine. For the stimulus assay, medium was replaced with serum-free DMEM 2 hours prior to the assay and treated with each stimulant.
  • the VSV plasmid was purchased from Addgene.
  • Expression vectors for mouse I ⁇ B ⁇ and C / EBP ⁇ were purchased from GenScript. I ⁇ B ⁇ and C / EBP ⁇ were subcloned into pLVSIN-CMVPur Vector (Takara Bio). Each plasmid was introduced into cells using the transfection reagent X-tremeGENE 9 (Sigma-Aldrich).
  • Plxnb1 targeting shRNAs (Plxnb1 shRNA, TRCN0000078917, TRCN0000078916), Plxnb2 targeting shRNAs (Plxnb2 shRNA, TRCN0000078853, TRCN0000078857), and c-Met targeting shRNAs (c-Met shRNA, TRCN0000226122).
  • TRCN0000235 was cloned into the lentivirus pLKO.1 plasmid (Sigma-Aldrich). An empty vector was used as a negative control. Lentivirus particles were prepared and infected according to the manufacturer's (Sigma-Aldrich) protocol.
  • Lentiviruses expressing controls or shRNA were infected with SFZ cells and selected in media containing puromycin (2 ⁇ g / ml).
  • puromycin 2 ⁇ g / ml
  • pLVSIN-I ⁇ B ⁇ or pLVSIN-C / EBP ⁇ was introduced into Lenti-X 293T cells (Takara Bio) together with Lentiviral High Titer Packaging Mix (Takara Bio). Lentivirus particles were prepared and infected according to the manufacturer's (Takara Bio) protocol.
  • c-Met inhibitors As various inhibitors, c-Met inhibitors: SU11274 (Fujifilm Wako Pure Drug, 1 ⁇ M), K252a (abcam, 1 ⁇ M) and crizotinib (Sigma, 0.5 ⁇ M), MEK inhibitors: PD98059 (Cell Signaling Technology, 10 ⁇ M) and U0126 (Cell Signaling Technology, 10 ⁇ M), RAS Inhibitor: Salilacib (abcam, 10 ⁇ M), Rho Kinase Inhibitor: RKI-1447 (Sigma-Aldrich, 5 ⁇ M) and Y-27632 (Sigma-Aldrich, 10 ⁇ M), ErbB2 Inhibitor : AG825 (abcam, 1 ⁇ M), Akt inhibitor: MK-2206 (Selleck, 1 ⁇ M), Translation inhibitor: Cycloheximide (Fujifilm Wako Pure Drug, 100 ⁇ g / ml) and NF
  • the membrane was immunoblotted with a primary antibody, and anti-mouse IgG antibody (Jackson, 1: 5,000) to which Western wasabi peroxidase was bound, anti-rabbit antibody IgG antibody (Jackson, 1: 5,000), anti-goat antibody IgG antibody (Jackson, 1: 5,000). Visualization was performed using 5000) and an anti-sheep IgG antibody (Jackson, 1: 5000) and the peroxidase luminescent substrate Immuno Star LD (Fujifilm Wako Junyaku).
  • Anti-MMP13 antibody (ab39012, 1: 2000) and anti-Myc antibody (ab9132, 1: 2000) were purchased from Abcam as primary antibodies, and anti-c-Met antibody (82202, 1: 1000) and anti-NF- ⁇ B (p65).
  • cells washed with cold PBS are lysed buffer (10 mM HEPES, pH 7.5, 1 mM MgCl 2 , 10 mM KCl, 0.1% TritonX-100, 20) on ice for 10 minutes. % Glycerol, phosphatase inhibitors, protease inhibitors).
  • cytolytic solution was centrifuged at 3,000 g for 10 minutes at 4 ° C., and the supernatant (cytoplasmic fraction) was collected.
  • Organ culture Organ culture of articular cartilage was performed according to the description of Stanton et al. (Nat Protoc 6, 388-404, doi: 10.1038 / nprot.2010.179 (2011)). That is, the femoral head was isolated from 3-week-old ICR mice and cultured for 3 days in serum-free DMEM medium containing Sema4D (20 ⁇ g / ml) or IL-1 ⁇ (10 ng / ml), or serum-free DMEM medium containing neither. did. The femoral head cartilage was fixed with 4% paraformaldehyde and then decalcified with 10% EDTA for 2 weeks. The cartilage was embedded in paraffin to prepare a section having a thickness of 5 ⁇ m.
  • the sections were deparaffinized, washed with PBS and distilled water, and then stained with 0.2% fast green for 10 minutes and with 0.1% safranin O for 20 minutes. Aggrecan release into the medium was analyzed by the dimethylmethylene blue assay.
  • CM conditioned media
  • the supernatant is divided into anti-FLAG antibody (M185-3, 1: 400), anti-Plexin-B2 antibody (PA547880, 1:50) or anti-c-Met antibody (1:50) (3127, Cell Signaling) and 4 After incubating overnight at ° C, it was incubated with Dynabeads Protein G at 4 ° C for 1 hour. The beads were washed 5 times with NP-40 buffer. The bound protein was eluted with SDS sample buffer and then immunoblotting was performed. To identify the plexin-B2-binding protein, the bound protein was purified by immunoprecipitation using an anti-plexin-B2 antibody (1: 100) (abcam, ab193355). The bound protein was eluted with SDS sample buffer and then mass spectrometric analysis was performed.
  • RNA-Seq RNA-Seqing
  • Microarray Analysis Total RNA was isolated from SFZ cells using Nucleospin RNA Plus for RNA-Seq.
  • the library was prepared using the TruSeq stranded mRNA sample prep kit (Illumina) and according to the manufacturer's instructions. The sequence was run in single-ended mode with 101 bases on the Illumina NovaSeq 6000 platform. I used Illumina Casava 1.8.2 software for the base call. Sequence reads were mapped to the mouse reference genome sequence (mm10) using TopHat v2.0.13 in combination with Bowtie2 ver.2.2.3 and SAMtools ver.0.1.19.
  • FPKMs fragments per kilobase of exon per million mapped fragments
  • Cufflinks version 2.2.1 Bioinformatics analysis using iDEP (http://bioinformatics.sdstate.edu/idep/) was performed to evaluate the screened genes.
  • Luciferase Reporter Assay A luciferase reporter plasmid (Clontech) containing NF- ⁇ B responsible elements was introduced into SFZ cells using the transfection reagent X-tremeGENE 9 (Sigma-Aldrich). Twenty-four hours after transfection, cells were treated with Sema4D (2 ⁇ g / ml) for 12 hours. Cells treated with IL-1 ⁇ (10 ng / ml) were designated as positive controls, and untreated cells were designated as negative controls. Luciferase activity was measured using the Luciferase Assay System (Promega) according to the manufacturer's protocol.
  • SFZ cells cultured with LPS-treated BMDM CM did not express Mmp13
  • SFZ cells cultured with LPS-treated BMDM CM did not express Mmp13.
  • Remarkably induced Mmp13 expression Remarkably induced Mmp13 expression.
  • Example 1 Identification of Mmp13 expression-inducing factor present in CM of BMDM treated with LPS
  • Example 2 Shotgun Mass Spectrometry
  • shotgun mass spectrometry of proteins present in CM was performed, and 32 types of candidate proteins shown in Table 2 below were detected. ..
  • LPS-treated BMDM CM was fractionated by 50 kDa, and SFZ cells were cultured for 16 hours in CM ⁇ 50 kDa fraction, CM> 50 kDa fraction, and unfractionated CM. Then, the expression level of Mmp13 was analyzed by RT-qPCR. In addition, LPS-treated BMDM CM was fractionated by 100 kDa, SFZ cells were cultured for 16 hours in CM ⁇ 100 kDa fraction, CM> 100 kDa fraction, and unfractionated CM, and the expression level of Mmp13 was RT. -analyzed by PCR (two independent experiments).
  • Fig. 2 The results shown in Fig. 2 are the results when (A) is fractionated by 50 kDa and (B) is the result when fractionated by 100 kDa.
  • (A) Mmp13 was expressed in the> 50 kDa fraction, and Mmp13 was hardly expressed in the ⁇ 50 kDa fraction.
  • (B) the expression level of Mmp13 was significantly higher in the ⁇ 100 kDa fraction than in the> 100 kDa fraction. From this result, we focused on semaphorin 4D (Sema4D) among 32 types of candidate proteins by mass spectrometry.
  • Sema4D semaphorin 4D
  • Example 2 Effect of Sema4D on chondrocytes
  • a4D Induction of Mmp13 expression in SFZ cells by Sema4D
  • (A) is the result of RT-qPCR when culturing at three stages of Sema4D concentration
  • (B) is the result of RT-qPCR when culturing at three stages of culture time
  • (C) is the result of immune blotting. ..
  • Sema4D induced dose- and time-dependent expression of Mmp13 in SFZ cells (** P ⁇ 0.01, *** P ⁇ 0.001). Sema4D also significantly increased the MMP13 protein.
  • Sema4D induced the expression of Mmp13 not only in SFZ cells but also in costal cartilage cells and deep articular cartilage cells.
  • the induction of Mmp13 expression on osteoblasts was weak, and the expression of Mmp13 was not induced on macrophages.
  • the results of analysis of the expression level of cartilage matrix degrading enzyme are shown in Fig. 8.
  • (A) is the result of Mmp13
  • (B) is the result of Mmp3
  • (C) is the result of Adamts4
  • (D) is the result of Adamts5.
  • Sema4D has been shown to increase the expression level of cartilage matrix degrading enzymes in SFZ cells (*** P ⁇ 0.001, **** P ⁇ 0.0001).
  • Example 3 Effect of Sema4D on cartilage tissue
  • 3-1 Organ culture of mouse articular cartilage Tissue sections of mouse femoral head cartilage organ-cultured by the method described in Experimental Method (8) were stained with safranin O and fast green and observed under a microscope.
  • IL-1 ⁇ is a positive control.
  • the results are shown in Fig. 9.
  • (A) is the result of control (negative control)
  • (B) is the result of culturing in the medium containing Sema4D
  • (C) is the result of culturing in the medium containing IL-1 ⁇ .
  • the scale bar is 200 ⁇ m.
  • Sema4D reduced proteoglycans stained with safranin O in the superficial and intermediate layers of cartilage.
  • Sema4d KO mice scored significantly lower than wild-type mice (* P ⁇ 0.05, ** P ⁇ 0.01). The results of the erosion score in the tissue specimen are shown in Fig. 13. Sema4d KO mice scored significantly lower than wild-type mice (* P ⁇ 0.05).
  • Example 4 Search for molecules involved in Sema4D-dependent Mmp13 expression induction
  • 4-1 Expression level of Sema4D receptor in SFZ cells
  • the expression level of Sema4D receptor in SFZ cells was evaluated by RNA-Seq. The results are shown in FIG. It was shown that the expression level of Plexin-B2 (Plxnb2 in the figure) was higher than that of Plexin-B1 (Plxnb1 in the figure), Plexin-B3 (Plxnb3 in the figure) and CD72 (Cd72 in the figure) in SFZ cells. ..
  • Plexin-B2 knockdown SFZ cells are transfected with a Plexin-B1 targeting shRNA (Plxnb1 shRNA) expression vector, a Plexin-B2 targeting shRNA (Plxnb2 shRNA) expression vector, or an empty vector (Control). did.
  • a group treated with Sema4D and a group not treated with Sema4D were provided, respectively.
  • SFZ cells were treated with Sema4D (2 ⁇ g / ml) or Sema4D and various inhibitors for 12 hours. Untreated SFZ cells were used as controls.
  • the expression level of Mmp13 (n 2 each) was analyzed by RT-qPCR. The results are shown in FIGS. 17 (A) to 17 (D).
  • c-Met inhibitors, RAS inhibitors and MEK inhibitors suppressed Sema4D-dependent Mmp13 expression induction.
  • Rho-kinase inhibitors, ErbB2 inhibitors and Akt inhibitors did not suppress Sema4D-dependent Mmp13 expression induction.
  • c-Met knockdown SFZ cells were transfected with a c-Met-targeted shRNA (c-Met shRNA) expression vector or an empty vector (Control).
  • c-Met shRNA c-Met-targeted shRNA
  • Control a c-Met-targeted shRNA
  • Two clones (shcMet1 and shcMet2) from SFZ cells transfected with the c-Met shRNA expression vector were used in the experiment.
  • Analyzed by -qPCR The results are shown in FIG. (A) is the result of the expression level of c-Met, and (B) is the result of the expression level of Mmp13. Knockdown of c-Met markedly inhibited Sema4D-dependent induction
  • Immunoprecipitated SFZ cells with anti-c-Met antibody were treated with Sema4D or HGF (hepatocyte growth factor) for 1 or 5 minutes. Untreated SFZ cells were used as controls. Cytolysis was prepared, anti-c-Met antibody was added, and the immunoprecipitate was recovered. Phosphorylated c-Met in immunoprecipitated proteins and phosphorylated Erk l / 2 in cytolysis were analyzed by immunobrotting with anti-phosphorylated tyrosine and anti-phosphorylated Erk l / 2 antibodies. The results are shown in FIG. Sema4D has been shown to stimulate c-Met phosphorylation and Erk l / 2 phosphorylation downstream of c-Met in SFZ cells.
  • HGF hepatocyte growth factor
  • Semc4D transmits the effect on SFZ cells via both the plexin-B2 pathway and the c-Met-Ras-Erk l / 2 pathway.
  • Example 5 Search for molecules that mediate semaphorin 4D / plexin-B2 signaling in SFZ cells.
  • the semaphorin-plexin system is known to regulate cell morphology and migration by regulating the rearrangement of the actin cytoskeleton, primarily via RhoA, a member of the Rho family (Perrot et al). ., J Biol Chem 277, 43115-43120 (2002)).
  • Rho-kinase inhibitors did not suppress Sema4D-dependent Mmp13 expression induction (see FIGS. 17 (c) and 17 (d)). This result suggests that other signaling pathways mediate the signal of plexin-B2 in SFZ cells to regulate Mmp13 expression.
  • Shotgun mass spectrometry of plexin-B2-binding protein in 5-1 SFZ cells Shotgun mass spectrometry of plexin-B2-binding protein was performed by the methods described in Experimental Methods (10) and (11). The results are shown in Table 3. We found that TNF receptor-related factor 2 (TRAF2), a regulator of the NF- ⁇ B signaling pathway, binds to plexin-B2.
  • TNF receptor-related factor 2 a regulator of the NF- ⁇ B signaling pathway
  • HEK293T cells were introduced with either or both of the Plexin-B2 expression vector and the FLAG-TRAF2 expression vector.
  • HEK293T cells into which an expression vector had not been introduced were used as controls.
  • a cytolytic solution was prepared.
  • Anti-Plexin-B2 antibody or anti-FLAG antibody was added to the cytolytic solution to recover the immunoprecipitate.
  • Immunoprecipitated protein with anti-Plexin-B2 antibody, immunoprecipitated protein with anti-FLAG antibody, and post-cytolytic solution were subjected to immunoblotting and analyzed with anti-Plexin-B2 antibody and anti-FLAG antibody.
  • the results are shown in FIG. It was confirmed that Plexin-B2 and TRAF2 were associated in cells into which both the Plexin-B2 expression vector and the FLAG-TRAF2 expression vector were introduced.
  • each of the above cells was immobilized, immunofluorescently stained with an anti-NF- ⁇ B (p65) antibody as the primary antibody, and an anti-rabbit IgG antibody Alexa Fluor 568 as the secondary antibody, and observed with a fluorescence microscope.
  • the results of immune blotting are shown in FIG.
  • the results of immunofluorescent staining are shown in FIG. In Figure 22, DAPI shows the nucleus and the scale bar is 50 ⁇ m. From these results, it was clarified that Sema4D induces the translocation of NF- ⁇ B from the cytoplasm to the nucleus in SFZ cells.
  • Sema4D was shown to activate the TRAF2-NF- ⁇ B and c-Met-Ras-Erk l / 2 signaling pathways, it was activated by Sema4D using a MEK inhibitor.
  • a MEK inhibitor was investigated the role of the c-Met-Ras-Erk l / 2 signaling pathway in the NF- ⁇ B pathway. 2 ⁇ g / ml of recombinant Sema4D, Sema4D and MEK inhibitor PD98059 (10 ⁇ M) or Sema4D and MEK inhibitor U0126 (10 ⁇ M) were added to the medium of SFZ cells and cultured for 12 hours. Untreated SFZ cells were used as controls.
  • Each of the above cells was immobilized, immunofluorescently stained with anti-NF- ⁇ B (p65) antibody as the primary antibody and anti-rabbit IgG antibody Alexa Fluor 568 as the secondary antibody, and observed with a fluorescence microscope.
  • the results are shown in FIG. DAPI shows the nucleus and the scale bar is 50 ⁇ m.
  • Sema4D has been shown to induce the translocation of NF- ⁇ B to the nucleus by a MEK inhibitor. This result suggests that c-Met-Ras-Erk l / 2 signaling is required for NF- ⁇ B activation by Sema4D.
  • Example 6 Search for known inflammatory cytokines involved in Sema4D-dependent Mmp13 expression induction. Since Sema4D activated NF- ⁇ B signaling in SFZ cells and up-regulated inflammatory responsive genes, the following to determine whether Sema4D has a direct or indirect inflammatory effect on SFZ cells: Experiment was conducted.
  • Sema4D (1 ⁇ g / ml), IL-6 (1 ⁇ g / ml) or Sema4D and IL-6 were added to the medium of SFZ cells and cultured for 12 hours. Untreated SFZ cells were used as controls.
  • (A) is the result of the expression level of Mmp13
  • (B) is the result of the expression level of Mmp3.
  • Example 7 Search for transcription factors involved in Sema4D-dependent Mmp13 expression induction
  • RNA-Seq analysis of transcription factor genes highly expressed in Sema4D-treated SFZ cells
  • the results of the de novo protein synthesis inhibition experiment in Example 6 show that specific transcription factors are involved in the induction of Sema4D-dependent Mmp13 expression. It suggests that it is. Therefore, RNA-Seq was used to analyze transcription factor genes that were 10-fold or more highly expressed in Sema4D-treated SFZ cells as compared to untreated SFZ cells.
  • Table 4 Sema4D has been shown to increase the expression of Nfkbiz, which encodes I ⁇ B ⁇ , and Cebpd, which encodes C / EBP ⁇ , in SFZ cells.
  • Example 8 Suppression of agrecan release from articular cartilage by a c-Met inhibitor
  • Mouse femoral head cartilage was organ-cultured by the method described in Experimental Method (8). That is, the femoral head was isolated from 3-week-old ICR mice, Sema4D (20 ⁇ g / ml) or Sema4D and the c-Met inhibitor crizotinib (0.5 ⁇ M) were added to serum-free DMEM medium, cultured for 3 days, and then cultured into the medium. Aggrecan release was analyzed by dimethylmethylene blue assay. Controls were cultured in serum-free DMEM medium containing neither for 3 days. The results are shown in Figure 39.
  • the c-Met inhibitor significantly suppressed the aggrecan release increased by Sema4D treatment (* P ⁇ 0.05, **** P ⁇ 0.0001). This result indicates that c-Met inhibitors are effective in the prevention or treatment of cartilage destruction caused by Sema4D.
  • Mouse femoral head cartilage was organ-cultured by the method described in Experimental Method (8). That is, the femoral head was isolated from 3-week-old ICR mice, and Sema4D (20 ⁇ g / ml) or Sema4D and the NF- ⁇ B inhibitor BAY11-7082 (10 ⁇ M) were added to serum-free DMEM medium and cultured for 3 days. Controls were cultured in serum-free DMEM medium containing neither for 3 days. The femoral head cartilage was fixed with 4% paraformaldehyde and then decalcified with 10% EDTA for 2 weeks.
  • the cartilage was embedded in paraffin to prepare a section having a thickness of 5 ⁇ m.
  • the sections were deparaffinized, washed with PBS and distilled water, and then stained with 0.2% fast green for 10 minutes and with 0.1% safranin O for 20 minutes. Proteoglycan release into the medium was analyzed by the dimethylmethylene blue assay.
  • Fig. 40 The observation results of the articular cartilage section are shown in Fig. 40.
  • (A) is the result of control
  • (B) is the result of culturing in the medium containing Sema4D
  • (C) is the result of culturing in the medium containing Sema4D and BAY11-7082.
  • the scale bar is 200 ⁇ m. It was observed that Sema4D reduced the proteoglycan stained by safranin O in the superficial and intermediate layers of cartilage, but the addition of BAY11-7082 suppressed the reduction of proteoglycan.
  • the results of the dimethylmethylene blue assay are shown in Figure 41.
  • the NF- ⁇ B inhibitor significantly suppressed the aggrecan release increased by Sema4D treatment (** P ⁇ 0.01).

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Abstract

The present invention provides a pharmaceutical composition for preventing or treating cartilage/bone/joint diseases, the pharmaceutical composition containing, as active ingredients, a substance that inhibits the signal transduction of semaphorin 4D-plexin B2, for example: a substance that inhibits the binding of plexin-B2 and semaphoring 4D; a substance that inhibits the binding of c-Met and semaphorin 4D; a substance that inhibits the binding of plexin B2 and Traf2; and a substance that inhibits the binding of plexin B2 and c-Met. In addition, the present invention provides a method for screening a drug for preventing or treating cartilage/bone/joint diseases, the method comprising a step for selecting a substance to be tested that inhibits the signal transduction of semaphorin 4D-plexin B2.

Description

軟骨・骨・関節疾患の予防または治療用医薬組成物および軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法Pharmaceutical composition for prevention or treatment of cartilage / bone / joint disease and screening method for drug for prevention or treatment of cartilage / bone / joint disease
　本発明は、軟骨・骨・関節疾患の予防または治療用医薬組成物および軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法に関するものである。 The present invention relates to a pharmaceutical composition for preventing or treating cartilage / bone / joint disease and a method for screening a drug for preventing or treating cartilage / bone / joint disease.
　関節軟骨の破壊は、関節リウマチ（RA：rheumatoid arthritis）や変形性関節症（OA：osteoarthritis）などの慢性の軟骨・骨・関節疾患の最も一般的な特徴である。関節と軟骨の微小環境では、免疫細胞がいくつかの古典的な炎症誘発性サイトカインを産生することにより、関節軟骨の破壊に重要な役割を果たすことが知られている。炎症誘発性サイトカインには、腫瘍壊死因子-α（TNFα）、インターロイキン-1β（IL-1β）、インターロイキン6（IL-6）などが含まれ、これらはマトリックスメタロプロテイナーゼ（MMP）およびアグリカナーゼの発現を直接的および／または間接的に促進する。しかし、マクロファージ、T細胞およびB細胞を含む免疫細胞が、軟骨・骨・関節疾患の発症時に関節軟骨の軟骨細胞とどのように相互作用するかについては、未だ不明な点が多い。 Destruction of articular cartilage is the most common feature of chronic cartilage, bone and joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). In the articular and cartilage microenvironment, immune cells are known to play an important role in the destruction of articular cartilage by producing some classical pro-inflammatory cytokines. Inflammatory-inducing cytokines include tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), interleukin 6 (IL-6), etc., which are matrix metalloproteinases (MMPs) and aggrecanases. Promotes expression directly and / or indirectly. However, there are still many unclear points about how immune cells, including macrophages, T cells and B cells, interact with the chondrocytes of articular cartilage at the onset of cartilage, bone and joint disease.
　特許文献１には、セマフォリン4DとプレキシンB1との結合阻害物質を有効成分とする骨疾患の予防・治療剤が記載されており、骨疾患は骨折、骨欠損、骨粗しょう症、骨軟化症、骨減少症、腰背痛、骨ページェット病、硬直性脊椎炎、関節リウマチおよび変形性関節症から選択されることが記載されている。しかし、特許文献１はセマフォリン4DとプレキシンB2のシグナル伝達について言及していない。 Patent Document 1 describes a prophylactic / therapeutic agent for bone diseases containing a binding inhibitor of semaphorin 4D and plexin B1 as an active ingredient, and bone diseases include bone fracture, bone defect, osteoporosis, and bone softening. , Osteoporosis, lumbar backache, bone paget disease, rigid spondylitis, rheumatoid arthritis and osteoarthritis. However, Patent Document 1 does not refer to signal transduction of semaphorin 4D and plexin B2.
WO2012/157237WO2012 / 157237
　本発明は、関節軟骨の破壊を引き起こす炎症性サイトカインを見出し、当該炎症性サイトカインを標的とする軟骨・骨・関節疾患の予防または治療用医薬組成物および当該炎症性サイトカインを標的とする軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法を提供することを課題とする。 The present invention has found an inflammatory cytokine that causes destruction of articular cartilage, a pharmaceutical composition for preventing or treating cartilage / bone / joint disease targeting the inflammatory cytokine, and a cartilage / bone targeting the inflammatory cytokine. -It is an object to provide a screening method for a drug for preventing or treating joint diseases.
　本発明は、上記の課題を解決するために以下の各発明を包含する。
［１］セマフォリン４Ｄ－プレキシンＢ２のシグナル伝達を阻害する物質を有効成分として含有する軟骨・骨・関節疾患の予防または治療用医薬組成物。
［２］前記物質が、プレキシンＢ２とセマフォリン４Ｄの結合を阻害する物質、ｃ－Ｍｅｔとセマフォリン４Ｄの結合を阻害する物質、プレキシンＢ２とＴｒａｆ２の結合を阻害する物質またはプレキシンＢ２とｃ－Ｍｅｔの結合を阻害する物質である、前記［１］に記載の医薬組成物。
［３］前記物質が抗プレキシンＢ２抗体またはプレキシンＢ２の発現を阻害する核酸またはプレキシンＢ２の機能を阻害する物質である、前記［１］に記載の医薬組成物。
［４］前記物質がｃ－Ｍｅｔ阻害剤である、前記［１］に記載の医薬組成物。
［５］前記物質がＩκＢζまたはＣＥＢＰδの発現または機能を阻害する物質である、前記［１］に記載の医薬組成物。
［６］前記軟骨・骨・関節疾患が、関節リウマチ、変形性関節症または骨粗しょう症である、前記［１］～［５］のいずれかに記載の医薬組成物。
［７］セマフォリン４Ｄ－プレキシンＢ２のシグナル伝達を阻害する被験物質を選択する工程を含む、軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法。
［８］セマフォリン４Ｄにより発現誘導される遺伝子の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とセマフォリン４Ｄと被験物質を接触させる工程と、レポーター遺伝子の発現量を測定する工程と、被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。
［９］ＩκＢζまたはＣＥＢＰδが認識する発現制御領域の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とＩκＢζまたはＣＥＢＰδと被験物質を接触させる工程と、レポーター遺伝子の発現量を測定する工程と、被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。
［１０］プレキシンＢ２とセマフォリン４Ｄと被験物質を接触させる工程と、プレキシンＢ２とセマフォリン４Ｄとの結合を測定する工程と、被験物質を添加していない対照と比較してプレキシンＢ２とセマフォリン４Ｄとの結合を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。
［１１］プレキシンＢ２とｃ－Ｍｅｔと被験物質を接触させる工程と、プレキシンＢ２とｃ－Ｍｅｔとの結合を測定する工程と、被験物質を添加していない対照と比較してプレキシンＢ２とｃ－Ｍｅｔとの結合を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。
［１２］プレキシンＢ２とＴｒａｆ２と被験物質を接触させる工程と、プレキシンＢ２とＴｒａｆ２との結合を測定する工程と、被験物質を添加していない対照と比較してプレキシンＢ２とＴｒａｆ２との結合を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。
［１３］ＩκＢζおよび／またはＣＥＢＰδを発現する細胞と被験物質を接触させる工程と、ＩκＢζおよび／またはＣＥＢＰδの発現量を測定する工程と、被験物質を接触させていない対照と比較してＩκＢζおよび／またはＣＥＢＰδの発現量を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。
［１４］前記軟骨・骨・関節疾患が、関節リウマチ、変形性関節症または骨粗しょう症である、前記［７］～［１３］のいずれかに記載のスクリーニング方法。 The present invention includes the following inventions in order to solve the above problems.
[1] A pharmaceutical composition for preventing or treating cartilage, bone, and joint diseases, which contains a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient.
[2] The substance is a substance that inhibits the binding between plexin B2 and semaphorin 4D, a substance that inhibits the binding between c-Met and semaphorin 4D, a substance that inhibits the binding between plexin B2 and Traf2, or a substance that inhibits the binding between plexin B2 and c-. The pharmaceutical composition according to the above [1], which is a substance that inhibits the binding of Met.
[3] The pharmaceutical composition according to the above [1], wherein the substance is a nucleic acid that inhibits the expression of an anti-plexin B2 antibody or plexin B2, or a substance that inhibits the function of plexin B2.
[4] The pharmaceutical composition according to the above [1], wherein the substance is a c-Met inhibitor.
[5] The pharmaceutical composition according to the above [1], wherein the substance is a substance that inhibits the expression or function of IκBζ or CEBPδ.
[6] The pharmaceutical composition according to any one of the above [1] to [5], wherein the cartilage / bone / joint disease is rheumatoid arthritis, osteoarthritis or osteoarthritis.
[7] A method for screening a drug for preventing or treating cartilage, bone, or joint disease, which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2.
[8] The step of contacting semaphorin 4D with the test substance and the expression level of the reporter gene are measured by contacting the cells of a cultured cell or a genetically modified animal in which a reporter gene is inserted downstream of the gene whose expression is induced by semaphorin 4D. The screening method according to [7] above, comprising the step of selecting a test substance that reduces the expression level of the reporter gene as compared with a control that has not been contacted with the test substance.
[9] A step of contacting a cultured cell or a gene-modified animal cell in which a reporter gene is inserted downstream of an expression control region recognized by IκBζ or CEBPδ with a test substance, and measuring the expression level of the reporter gene. The screening method according to [7] above, comprising the step of selecting a test substance that reduces the expression level of the reporter gene as compared with a control that has not been contacted with the test substance.
[10] A step of contacting plexin B2 with semaphorin 4D and a test substance, a step of measuring the binding between plexin B2 and semaphorin 4D, and a step of measuring the binding between plexin B2 and semaphorin 4D, and plexin B2 and semaphorin as compared with a control to which no test substance was added. The screening method according to the above [7], which comprises a step of selecting a test substance that reduces binding to 4D.
[11] A step of contacting plexin B2 and c-Met with a test substance, a step of measuring the binding between plexin B2 and c-Met, and a step of comparing plexin B2 and c- with a control to which no test substance was added. The screening method according to the above [7], which comprises a step of selecting a test substance that reduces binding to Met.
[12] The step of contacting plexin B2 and Traf2 with the test substance, the step of measuring the binding between plexin B2 and Traf2, and the step of measuring the binding between plexin B2 and Traf2 are reduced as compared with the control to which the test substance is not added. The screening method according to the above [7], which comprises a step of selecting a test substance to be subjected to.
[13] A step of contacting a cell expressing IκBζ and / or CEBPδ with a test substance, a step of measuring the expression level of IκBζ and / or CEBPδ, and a step of measuring the expression level of IκBζ and / or CEBPδ with IκBζ and / or a control not contacted with the test substance. Alternatively, the screening method according to the above [7], which comprises a step of selecting a test substance that reduces the expression level of CEBPδ.
[14] The screening method according to any one of [7] to [13] above, wherein the cartilage / bone / joint disease is rheumatoid arthritis, osteoarthritis or osteoarthritis.
　本発明により、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質を有効成分として含有する軟骨・骨・関節疾患の予防または治療用医薬組成物を提供することができる。また、本発明により、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する被験物質を選択する工程を含む、軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法を提供することができる。 INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a pharmaceutical composition for preventing or treating cartilage, bone and joint diseases containing a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient. The present invention also provides a method for screening a drug for preventing or treating cartilage, bone, and joint diseases, which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2.
LPS処理した骨髄由来マクロファージの培養上清が、関節軟骨表層細胞のマトリックスメタロプロテイナーゼ13（Mmp13）発現を誘導することを確認した結果を示す図である。It is a figure which shows the result of having confirmed that the culture supernatant of the bone marrow-derived macrophage treated with LPS induces the expression of matrix metalloproteinase 13 (Mmp13) of the articular cartilage surface cell. LPS処理した骨髄由来マクロファージの培養上清を50KDaまたは100KDaで分画し、どの画分にMmp13発現誘導因子が存在するかを確認した結果を示す図であり、（A）が50kDaで分画した場合の結果、（B）が100kDaで分画した場合の結果である。It is a figure which shows the result of fractionating the culture supernatant of the bone marrow-derived macrophage treated with LPS by 50KDa or 100KDa, and confirming which fraction the Mmp13 expression-inducing factor is present, and (A) is fractionated by 50kDa. As a result of the case, (B) is the result when fractionated by 100 kDa. 骨髄由来マクロファージが発現するセマフォリンファミリー遺伝子発現レベルを、LPS処理した場合とLPS処理していない場合について、マイクロアレイで分析した結果を示す図である。It is a figure which shows the result of having analyzed the semaphorin family gene expression level expressed by a bone marrow-derived macrophage by a microarray in the case of LPS treatment and the case of not LPS treatment. LPS処理した骨髄由来マクロファージがセマフォリン4D（Sema4D）をシェディングする酵素を発現することを確認した結果を示す図である。It is a figure which shows the result of having confirmed that the bone marrow-derived macrophage treated with LPS expresses the enzyme which sheds semaphorin 4D (Sema4D). LPS処理したBMDMおよびLPS処理していないBMDMから分泌される可溶型Sema4Dを免疫ブロッティングで検出した結果を示す図である。It is a figure which shows the result of having detected the soluble Sema4D secreted from LPS-treated BMDM and LPS-untreated BMDM by immunoblotting. セマフォリン4D（Sema4D）による関節軟骨表層細胞のMmp13発現誘導を確認した結果を示す図であり、（A）がSema4D濃度依存性を確認した結果、（B）がSema4D処理時間依存性を確認した結果、（C）が免疫ブロッティングの結果である。It is a figure showing the result of confirming the induction of Mmp13 expression in articular cartilage surface cells by semaphorin 4D (Sema4D), (A) confirmed the Sema4D concentration dependence, and (B) confirmed the Sema4D treatment time dependence. As a result, (C) is the result of immune blotting. セマフォリン4Dによる各種細胞のMmp13発現誘導を確認した結果を示す図であり、（A）が肋軟骨細胞の結果、（B）が関節軟骨深層細胞の結果、（C）が骨芽細胞の結果、（D）が骨髄由来マクロファージの結果である。It is a figure showing the result of confirming the induction of Mmp13 expression in various cells by semaphorin 4D. (A) is the result of costal chondrocyte, (B) is the result of deep articular cartilage cell, and (C) is the result of osteoblast. , (D) are the results of bone marrow-derived macrophages. セマフォリン4D処理した関節軟骨表層細胞における軟骨マトリックス分解酵素の発現レベルをRNA-Seq分析した結果を示す図であり、（A）がMmp13の結果、（B）がMmp3の結果、（C）がAdamts4の結果、（D）がAdamts5の結果である。It is a figure which shows the result of RNA-Seq analysis of the expression level of the cartilage matrix degrading enzyme in the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of Mmp13, (B) is the result of Mmp3, (C) is. As a result of Adamts4, (D) is the result of Adamts5. 器官培養したマウス大腿骨頭軟骨の組織切片をサフラニンOおよびファストグリーンで染色し、顕微鏡で観察した結果であり、（A）がコントロールの結果、（B）がセマフォリン4Dを含む培地で培養した結果、（C）がインターロイキン-1βを含む培地で培養した結果である。Tissue sections of organ-cultured mouse femoral head cartilage were stained with safranin O and fast green and observed under a microscope. (A) is the result of control, and (B) is the result of culturing in a medium containing semaphorin 4D. , (C) is the result of culturing in a medium containing interleukin-1β. 器官培養したマウス大腿骨頭軟骨から培地へのアグリカン放出量を分析した結果を示す図である。It is a figure which shows the result of having analyzed the amount of agrecan release from the organ-cultured mouse femoral head cartilage to the culture medium. コラーゲン-LPS誘発関節炎を誘発した野生型（WT）マウスおよびSema4dノックアウトマウスの関節組織の組織切片をヘマトキシリン・エオジンで染色し、顕微鏡で観察した結果であり、（A）が野生型マウスの結果、（B）がSema4dノックアウトマウスの結果である。Tissue sections of the joint tissue of wild-type (WT) mice and Sema4d knockout mice induced with collagen-LPS-induced arthritis were stained with hematoxylin and eosin and observed under a microscope, and (A) is the result of wild-type mice. (B) is the result of Sema4d knockout mouse. コラーゲン-LPS誘発関節炎を誘発した野生型（WT）マウスおよびSema4dノックアウトマウスにおける関節炎の臨床スコアの経時変化を示す図である。It is a figure which shows the time course of the clinical score of arthritis in the wild type (WT) mouse and the Sema4d knockout mouse which induced collagen-LPS-induced arthritis. コラーゲン-LPS誘発関節炎を誘発した野生型（WT）マウスおよびSema4dノックアウトマウスの足（paws）の組織標本におけるびらんスコアの結果を示す図である。It is a figure which shows the result of the erosion score in the tissue specimen of the foot (paws) of the wild type (WT) mouse and the Sema4d knockout mouse which induced collagen-LPS-induced arthritis. 関節軟骨表層細胞におけるSema4D受容体の発現レベルをRNA-Seqで評価した。The expression level of Sema4D receptor in articular cartilage surface cells was evaluated by RNA-Seq. セマフォリン4D処理した関節軟骨表層細胞におけるプレキシン-B1またはプレキシン-B2のノックダウン効果を確認した結果を示す図である。It is a figure which shows the result of having confirmed the knockdown effect of plexin-B1 or plexin-B2 in the articular cartilage superficial cell treated with semaphorin 4D. プレキシン-B2ノックアウトマウスの関節軟骨表層細胞に対するセマフォリン4Dの効果を確認した結果を示す図であり、（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。It is a figure which shows the result of having confirmed the effect of semaphorin 4D on the articular cartilage superficial cell of the plexin-B2 knockout mouse, (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. 関節軟骨表層細胞におけるセマフォリン4D依存性Mmp13発現誘導に対する各種阻害剤の効果を確認した結果を示す図であり、（A）がMEK阻害剤PD98059、RAS阻害剤サリラシブおよびc-Met阻害剤K252aの結果、（B）がMEK阻害剤U0126およびc-Met阻害剤SU11274の結果、（C）がRhoキナーゼ阻害剤RKI-1447およびErbB2阻害剤AG825の結果、（D）がRhoキナーゼ阻害剤Y-27632およびAkt阻害剤MK-2206の結果である。It is a figure showing the result of confirming the effect of various inhibitors on the induction of semaphorin 4D-dependent Mmp13 expression in the superficial cells of articular cartilage. As a result, (B) is the result of MEK inhibitor U0126 and c-Met inhibitor SU11274, (C) is the result of Rho kinase inhibitor RKI-1447 and ErbB2 inhibitor AG825, and (D) is the result of Rho kinase inhibitor Y-27632. And the result of the Akt inhibitor MK-2206. セマフォリン4D処理した関節軟骨表層細胞におけるc-Metのノックダウン効果を確認した結果を示す図であり、（A）がc-Metの発現量の結果、（B）がMmp13の発現量の結果である。It is a figure which shows the result of having confirmed the knockdown effect of c-Met in the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of the expression level of c-Met, and (B) is the result of the expression level of Mmp13. Is. セマフォリン4Dまたは肝細胞増殖因子（HGF）で処理した関節軟骨表層細胞の細胞溶解液における抗c-Met抗体免疫沈降物を抗リン酸化チロシン抗体、細胞溶解液を抗リン酸化Erkl/2抗体を用いた免疫ブロッティングによって分析した結果を示す図である。Anti-c-Met antibody in cytolytic fluid of articular cartilage surface cells treated with semaphorin 4D or hepatocellular proliferation factor (HGF) Anti-phosphorylated tyrosine antibody for immunoprecipitates, anti-phosphorylated Erkl / 2 antibody for cytolytic fluid It is a figure which shows the result of analysis by the immunoblotting used. Plexin-B2発現ベクターおよびFLAG-TRAF2発現ベクターのいずれか一方または両方を導入したHEK293T細胞の細胞溶解液における抗プレキシン-B2抗体免疫沈降物を、抗Plexin-B2抗体および抗FLAG抗体を用いた免疫ブロッティングによって分析した結果を示す図である。Immunization of anti-Plexin-B2 antibody immunoprecipitates in cytolytic fluid of HEK293T cells into which either or both of the Plexin-B2 expression vector and FLAG-TRAF2 expression vector have been introduced, using anti-Plexin-B2 antibody and anti-FLAG antibody. It is a figure which shows the result of analysis by blotting. セマフォリン4Dまたはインターロイキン-1β（陽性コントロール）で処理した関節軟骨表層細胞の細胞溶解液を細胞質画分と核画分に分画し、各分画を抗NF-κB（p65）抗体および抗ヒストンH3抗体用いた免疫ブロッティングによって分析した結果を示す図である。Cytolysis of articular cartilage surface cells treated with semaphorin 4D or interleukin-1β (positive control) was fractionated into cytoplasmic and nuclear fractions, each fraction being anti-NF-κB (p65) antibody and anti. It is a figure which shows the result of the analysis by the immune blotting using the histone H3 antibody. セマフォリン4Dまたはインターロイキン-1β（陽性コントロール）で処理した関節軟骨表層細胞を抗NF-κB（p65）抗体と蛍光標識二次抗体を用いて免疫蛍光染色し、蛍光顕微鏡で観察した結果を示す図である。Articular cartilage surface cells treated with semaphorin 4D or interleukin-1β (positive control) were immunofluorescently stained with anti-NF-κB (p65) antibody and fluorescently labeled secondary antibody, and the results observed with a fluorescence microscope are shown. It is a figure. 関節軟骨表層細胞に、NF-κB responsible elementsを含むルシフェラーゼレポータープラスミドを導入し、セマフォリン4Dまたはインターロイキン-1β（陽性コントロール）で処理してルシフェラーゼレポーターアッセイを行った結果を示す図である。It is a figure which shows the result of having performed the luciferase reporter assay which introduced the luciferase reporter plasmid containing NF-κB respondable elements into the articular cartilage surface cell, and treated with semaphorin 4D or interleukin-1β (positive control). 関節軟骨表層細胞におけるセマフォリン4D依存性Mmp13発現誘導に対するNF-κB阻害剤の効果を確認した結果を示す図である。It is a figure which shows the result of confirming the effect of the NF-κB inhibitor on the induction of semaphorin 4D-dependent Mmp13 expression in the articular cartilage surface cell. Traf2ノックアウトマウスの関節軟骨表層細胞に対するセマフォリン4Dの効果を確認した結果を示す図であり、（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。It is a figure which shows the result of having confirmed the effect of semaphorin 4D on the articular cartilage superficial cell of the Traf2 knockout mouse, (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. セマフォリン4Dまたはセマフォリン4DとMEK阻害剤で処理した関節軟骨表層細胞を抗NF-κB（p65）抗体と蛍光標識二次抗体を用いて免疫蛍光染色し、蛍光顕微鏡で観察した結果を示す図である。Figure showing the results of immunofluorescent staining of articular cartilage surface cells treated with semaphorin 4D or semaphorin 4D and MEK inhibitor using anti-NF-κB (p65) antibody and fluorescently labeled secondary antibody, and observed under a fluorescence microscope. Is. セマフォリン4Dで処理した関節軟骨表層細胞におけるIL-6の発現量および培地に分泌されたIL-6量を分析した結果を示す図である。It is a figure which shows the result of having analyzed the expression level of IL-6 in the articular cartilage superficial cell treated with semaphorin 4D, and the amount of IL-6 secreted into the culture medium. セマフォリン4Dで処理した関節軟骨表層細胞から培地に分泌されたIL-6、TNFαおよびIL-1β量を分析した結果を示す図であり、（A）がIL-6の結果、（B）がTNFαの結果、（C）がIL-1βの結果である。It is a figure which shows the result of having analyzed the amount of IL-6, TNFα and IL-1β secreted into the medium from the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of IL-6, (B) is As a result of TNFα, (C) is the result of IL-1β. セマフォリン4Dで処理した関節軟骨表層細胞におけるタンパク質合成阻害の効果を確認した結果を示す図であり、（A）がMmp13の発現量の結果、（B）がIL-6の発現量の結果である。It is a figure which shows the result of having confirmed the effect of the protein synthesis inhibition in the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of IL-6. be. セマフォリン4Dまたはインターロイキン-6で処理した関節軟骨表層細胞におけるMmp13の発現量を分析した結果を示す図である。It is a figure which shows the result of having analyzed the expression level of Mmp13 in the articular cartilage superficial cell treated with semaphorin 4D or interleukin-6. 関節軟骨表層細胞におけるセマフォリン4D依存性Mmp13発現誘導に対する抗IL-6中和抗体の効果を確認した結果を示す図である。It is a figure which shows the result of having confirmed the effect of the anti-IL-6 neutralizing antibody on the induction of semaphorin 4D-dependent Mmp13 expression in the articular cartilage surface cell. 低濃度のセマフォリン4Dおよび／または低濃度のインターロイキン-6で処理した関節軟骨表層細胞におけるマトリックスメタロプロテイナーゼの発現量を確認した結果を示す図であり、（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。It is a figure which shows the result of having confirmed the expression level of the matrix metalloproteinase in the articular cartilage superficial cell treated with the low concentration of semaphorin 4D and / or the low concentration of interleukin-6, and (A) is the result of the expression level of Mmp13. , (B) are the results of the expression level of Mmp3. セマフォリン4Dで処理した関節軟骨表層細胞におけるNfkbizおよびCebpdの発現量を分析した結果を示す図であり、（A）がNfkbizの発現量の結果、（B）がCebpdの発現量の結果である。It is a figure which shows the result of having analyzed the expression level of Nfkbiz and Cebpd in the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of the expression level of Nfkbiz, and (B) is the result of the expression level of Cebpd. .. セマフォリン4Dで処理した関節軟骨表層細胞の細胞溶解液を、抗IκBζ抗体および抗C/EBPδ抗体を用いた免疫ブロッティングによって分析した結果を示す図である。It is a figure which shows the result of having analyzed the cytolysis of the articular cartilage superficial cell treated with semaphorin 4D by the immunoblotting using the anti-IκBζ antibody and the anti-C / EBPδ antibody. IκBζおよび／またはC/EBPδを過剰発現するSFZ細胞を作製し、Mmp13の発現量を分析した結果を示す図である。It is a figure which shows the result of having prepared the SFZ cell which overexpresses IκBζ and / or C / EBPδ, and analyzed the expression level of Mmp13. Nfkbizノックアウトマウスの関節軟骨表層細胞に対するセマフォリン4Dの効果を確認した結果を示す図であり、（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。It is a figure which shows the result of having confirmed the effect of semaphorin 4D on the articular cartilage superficial cell of Nfkbiz knockout mouse, (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. セマフォリン4Dで処理した関節軟骨表層細胞におけるNfkbizおよびCebpdの発現に対するMEK阻害剤の効果を確認した結果を示す図であり、（A）がNfkbizの発現量の結果、（B）がCebpdの発現量の結果である。It is a figure showing the result of confirming the effect of the MEK inhibitor on the expression of Nfkbiz and Cebpd in the articular cartilage surface cells treated with semaphorin 4D. It is the result of quantity. セマフォリン4Dで処理した関節軟骨表層細胞におけるNfkbizおよびCebpdの発現に対するNF-κB阻害剤の効果を確認した結果を示す図であり、（A）がNfkbizの発現量の結果、（B）がCebpdの発現量の結果である。It is a figure which shows the result of having confirmed the effect of the NF-κB inhibitor on the expression of Nfkbiz and Cebpd in the articular cartilage superficial cell treated with semaphorin 4D, (A) is the result of the expression level of Nfkbiz, and (B) is Cebpd. Is the result of the expression level of. セマフォリン4Dを含む培地またはセマフォリン4Dとc-Met阻害剤を含む培地でマウス大腿骨頭軟骨を器官培養し、培地へのアグリカン放出量を分析した結果を示す図である。It is a figure which shows the result of having performed the organ culture of the mouse femoral head cartilage in the medium containing semaphorin 4D or the medium containing semaphorin 4D and a c-Met inhibitor, and analyzing the amount of aggrecan released into the medium. セマフォリン4Dを含む培地またはセマフォリン4DとNF-κB阻害剤を含む培地で器官培養したマウス大腿骨頭軟骨の組織切片をサフラニンOおよびファストグリーンで染色し、顕微鏡で観察した結果であり、（A）がコントロールの結果、（B）がセマフォリン4Dを含む培地で培養した結果、（C）がセマフォリン4DとNF-κB阻害剤を含む培地で培養した結果である。Tissue sections of mouse femoral head cartilage organ-cultured in a medium containing semaphorin 4D or a medium containing semaphorin 4D and an NF-κB inhibitor were stained with safranin O and fast green and observed under a microscope (A). ) Is the result of control, (B) is the result of culturing in a medium containing safranin 4D, and (C) is the result of culturing in a medium containing semaphorin 4D and an NF-κB inhibitor. 図４０の器官培養したマウス大腿骨頭軟骨から培地へのアグリカン放出量を分析した結果を示す図である。It is a figure which shows the result of having analyzed the amount of aggrecan release from the organ-cultured mouse femoral head cartilage into the culture medium of FIG. 40.
〔医薬組成物〕
　本発明は、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質を有効成分として含有する軟骨・骨・関節疾患の予防または治療用医薬組成物（以下「本発明の組成物」と記す）を提供する。本発明は、本発明者らが、炎症を誘導したマクロファージが関節軟骨の破壊に関与すること、炎症を誘導したマクロファージから分泌される可溶型セマフォリン4Dが軟骨細胞に作用してマトリックスメタロプロテイナーゼ（MMP）の発現を誘導すること、軟骨細胞におけるMMPの発現は可溶型セマフォリン4Dと軟骨細胞のプレキシン-B2との結合によるシグナル伝達により誘導されること、を見出したことに基づく発明である。さらに、本発明者らは、セマフォリン4D－プレキシンB2の結合は、TRAF2-NF-κBシグナル伝達経路とc-Met-Ras-Erk l/2シグナル伝達経路の両方の経路を活性化し、転写因子IκBζおよびCEBPδを誘導して作用を発揮することを見出した。また、セマフォリン4Dは、IL-6と協調的に作用することを明らかにした。 [Pharmaceutical composition]
The present invention provides a pharmaceutical composition for the prevention or treatment of cartilage, bone and joint diseases (hereinafter referred to as "the composition of the present invention") containing a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient. offer. In the present invention, the present inventors have stated that macrophages that induce inflammation are involved in the destruction of articular cartilage, and that soluble semaphorin 4D secreted from macrophages that induce inflammation acts on chondrocytes to form a matrix metalloproteinase. The invention is based on the finding that it induces the expression of (MMP) and that the expression of MMP in chondrocytes is induced by signal transduction by binding of soluble semaphorin 4D and chondrocyte plexin-B2. be. Furthermore, we found that semaphorin 4D-plexin B2 binding activates both the TRAF2-NF-κB signaling pathway and the c-Met-Ras-Erk l / 2 signaling pathway and is a transcription factor. It was found that it exerts its action by inducing IκBζ and CEBPδ. We also revealed that semaphorin 4D acts cooperatively with IL-6.
　本発明の治療用医薬組成物の対象疾患である軟骨・骨・関節疾患は、関節軟骨の破壊を伴う疾患であれば特に限定されない。具体的には、例えば、骨折、骨欠損、骨粗しょう症、骨軟化症、骨減少症、腰背痛、骨ページェット病、硬直性脊椎炎、関節リウマチ、変形性関節症、骨形成不全症、骨多孔症などが挙げられる。好ましくは、関節リウマチ、変形性関節症または骨粗しょう症である。 The cartilage / bone / joint disease which is the target disease of the therapeutic pharmaceutical composition of the present invention is not particularly limited as long as it is a disease accompanied by destruction of articular cartilage. Specifically, for example, fracture, bone defect, osteoporosis, osteomalacia, bone loss, lumbar backache, Paget's disease of bone, rigid spondylitis, rheumatoid arthritis, osteoarthritis, osteodysplasia. , Osteomalacia and the like. Rheumatoid arthritis, osteoarthritis or osteoporosis are preferred.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、プレキシンB2とセマフォリン4Dの結合を阻害する物質であってもよい。プレキシンB2とセマフォリン4Dの結合を阻害する物質としては、例えば、プレキシンB2と特異的に結合し、プレキシンB2とセマフォリン4Dの結合を阻害する抗体であってもよい。または、プレキシンB2とセマフォリン4Dの結合を阻害する低分子化合物やペプチドであってもよい。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between semaphorin B2 and semaphorin 4D. The substance that inhibits the binding between plexin B2 and semaphorin 4D may be, for example, an antibody that specifically binds to plexin B2 and inhibits the binding between plexin B2 and semaphorin 4D. Alternatively, it may be a small molecule compound or peptide that inhibits the binding of plexin B2 to semaphorin 4D.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、c-Metとセマフォリン4Dの結合を阻害する物質であってもよい。c-Metとセマフォリン4Dのの結合を阻害する物質としては、例えば、c-Metと特異的に結合し、c-Metとセマフォリン4Dの結合を阻害する抗体であってもよい。または、c-Metとセマフォリン4Dの結合を阻害する低分子化合物やペプチドであってもよい。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between c-Met and semaphorin 4D. The substance that inhibits the binding between c-Met and semaphorin 4D may be, for example, an antibody that specifically binds to c-Met and inhibits the binding between c-Met and semaphorin 4D. Alternatively, it may be a small molecule compound or peptide that inhibits the binding of c-Met to semaphorin 4D.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、プレキシンB2とTraf2の結合を阻害する物質であってもよい。プレキシンB2とTraf2の結合を阻害する物質としては、例えば、プレキシンB2と特異的に結合し、プレキシンB2とTraf2の結合を阻害する抗体であってもよい。または、Traf2と特異的に結合し、プレキシンB2とTraf2の結合を阻害する抗体であってもよい。または、プレキシンB2とTraf2の結合を阻害する低分子化合物やペプチドであってもよい。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between plexin B2 and Traf2. The substance that inhibits the binding between plexin B2 and Traf2 may be, for example, an antibody that specifically binds to plexin B2 and inhibits the binding between plexin B2 and Traf2. Alternatively, it may be an antibody that specifically binds to Traf2 and inhibits the binding of plexin B2 to Traf2. Alternatively, it may be a small molecule compound or peptide that inhibits the binding between plexin B2 and Traf2.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、プレキシンB2とc-Metの結合を阻害する物質であってもよい。プレキシンB2とc-Metの結合を阻害する物質としては、例えば、プレキシンB2と特異的に結合し、プレキシンB2とc-Metの結合を阻害する抗体であってもよい。または、c-Metと特異的に結合し、プレキシンB2とc-Metの結合を阻害する抗体であってもよい。または、プレキシンB2とc-Metの結合を阻害する低分子化合物やペプチドであってもよい。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the binding between plexin B2 and c-Met. The substance that inhibits the binding between plexin B2 and c-Met may be, for example, an antibody that specifically binds to plexin B2 and inhibits the binding between plexin B2 and c-Met. Alternatively, it may be an antibody that specifically binds to c-Met and inhibits the binding of plexin B2 to c-Met. Alternatively, it may be a small molecule compound or peptide that inhibits the binding of plexin B2 to c-Met.
　上記で例示した各抗体はポリクローナル抗体でもよく、モノクローナル抗体でもよい。抗体は、抗原結合能を有する抗体断片（例えば、Fab、Fab'、F(ab')2、Fv、scFv、ダイアボディ等）、抗体の可変部を結合させた低分子化抗体であってもよい。ポリクローナル抗体およびモノクローナル抗体は公知の方法で作製することができる。ポリクローナル抗体は、例えば、抗原をPBSに溶解し、所望により通常のアジュバント（例えばフロイント完全アジュバント）を適量混合したものを免疫原として哺乳動物（マウス、ラット、ウサギ、ヤギ、ウマ等）を免疫し、常法に従い免疫した動物から血液を採取して血清を分離し、ポリクローナル抗体画分を精製することにより作製することができる。免疫方法は特に限定されないが、例えば、１回または適当な間隔で複数回、皮下注射または腹腔内注射する方法が好ましい。モノクローナル抗体は、例えば、上記免疫された哺乳動物から得た免疫細胞（例えば脾細胞）とミエローマ細胞とを融合させてハイブリドーマを得、当該ハイブリドーマの培養物から抗体を採取することによって作製することができる。また、抗体遺伝子をハイブリドーマからクローニングし、適当なベクターに組み込んで、これを宿主細胞に導入し、遺伝子組換え技術を用いて組換え型のモノクローナル抗体を産生させることもできる。さらに、ファージディスプレイ法を用いてモノクローナル抗体を作製することもできる。抗体はヒト型キメラ抗体またはヒト化抗体であってもよい。 Each antibody exemplified above may be a polyclonal antibody or a monoclonal antibody. The antibody may be a low molecular weight antibody to which an antibody fragment having an antigen-binding ability (for example, Fab, Fab', F (ab') 2, Fv, scFv, diabody, etc.) or a variable portion of the antibody is bound. good. Polyclonal antibody and monoclonal antibody can be produced by a known method. Polyclonal antibody immunizes mammals (mouse, rat, rabbit, goat, horse, etc.) using, for example, an antigen dissolved in PBS and optionally mixed with an appropriate amount of a usual adjuvant (for example, Freund's complete adjuvant) as an immunogen. It can be prepared by collecting blood from an immunized animal according to a conventional method, separating the serum, and purifying the polyclonal antibody fraction. The immunization method is not particularly limited, but for example, a method of subcutaneous injection or intraperitoneal injection once or multiple times at appropriate intervals is preferable. The monoclonal antibody can be produced, for example, by fusing immune cells (for example, splenocytes) obtained from the immunized mammal and myeloma cells to obtain a hybridoma, and collecting the antibody from the culture of the hybridoma. can. It is also possible to clone an antibody gene from a hybridoma, incorporate it into an appropriate vector, introduce it into a host cell, and use gene recombination technology to produce a recombinant monoclonal antibody. Furthermore, monoclonal antibodies can also be prepared using the phage display method. The antibody may be a human chimeric antibody or a humanized antibody.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、プレキシンB2の発現を阻害する核酸であってもよい。プレキシンB2の発現を阻害する核酸としては、例えば、プレキシンB2遺伝子のsiRNA（short interfering RNA）、shRNA（short hairpin RNA）、アンチセンスオリゴヌクレオチド、アンチセンスmRNAなどが挙げられる。投与対象動物のプレキシンB2遺伝子の塩基配列は公知のデータベース（NCBI等）から取得することができる。siRNAは、約20塩基（例えば、約21～23塩基）またはそれ未満の長さの二本鎖RNAであり、このようなsiRNAを細胞に導入することにより、そのsiRNAの標的となる遺伝子（本発明においてはプレキシンB2遺伝子）の発現を抑制することができる。shRNAは、一本鎖RNAで部分的に回文状の塩基配列を含むことにより、分子内で二本鎖構造をとり、3'末端に突出部を有する短いヘアピン構造からからなる約20塩基対以上の分子のことをいう。そのようなshRNAは、細胞内に導入された後、細胞内で約20塩基（代表的には例えば、21塩基、22塩基、23塩基）の長さに分解され、siRNAと同様に標的となる遺伝子（本発明においてはプレキシンB2遺伝子）の発現を抑制することができる。siRNAおよびshRNAは、プレキシンB2の発現を抑制できるものであればどのような形態であってもよい。siRNAまたはshRNAは、標的遺伝子の塩基配列に基づいて、公知の方法により設計することができる。siRNAまたはshRNAは、人工的に化学合成することができる。また、例えばT7RNAポリメラーゼおよびT7プロモーターを用いて、鋳型DNAからアンチセンスおよびセンスのRNAをインビトロで合成することができる。アンチセンスオリゴヌクレオチドは、プレキシンB2遺伝子のDNA配列中の連続する5から100の塩基配列に対して相補的な、またはハイブリダイズするヌクレオチドであればよく、DNAまたはRNAのいずれであってもよい。また、機能に支障がない限り修飾されたものであってもよい。アンチセンスオリゴヌクレオチドは常法によって合成することができ、例えば、市販のDNA合成装置によって容易に合成することができる。アンチセンスmRNAは、プレキシンB2のmRNAの塩基配列に相補的な、またはハイブリダイズするRNAであればよい。アンチセンスmRNAは、人工的に化学合成することができる。また、例えばT7RNAポリメラーゼおよびT7プロモーターを用いて、鋳型DNAからアンチセンスおよびセンスのRNAをインビトロで合成することができる。合成の際には、ウリジンの代わりにシュードウリジンを用い、キャップ構造とポリＡシグナルを付与する。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a nucleic acid that inhibits the expression of plexin B2. Examples of the nucleic acid that inhibits the expression of plexin B2 include siRNA (short interfering RNA), shRNA (short hairpin RNA), antisense oligonucleotide, and antisense mRNA of the plexin B2 gene. The nucleotide sequence of the plexin B2 gene of the administration target animal can be obtained from a known database (NCBI, etc.). A siRNA is a double-stranded RNA having a length of about 20 bases (for example, about 21 to 23 bases) or less, and by introducing such siRNA into a cell, the gene targeted by the siRNA (book). In the invention, the expression of plexin B2 gene) can be suppressed. shRNA is a single-stranded RNA containing a partially palindromic base sequence, so that it has a double-stranded structure within the molecule and consists of a short hairpin structure with a protrusion at the 3'end of about 20 base pairs. Refers to the above molecules. After being introduced into a cell, such shRNA is decomposed into a length of about 20 bases (typically, for example, 21 bases, 22 bases, and 23 bases) in the cell and becomes a target like siRNA. The expression of a gene (plexin B2 gene in the present invention) can be suppressed. The siRNA and shRNA may be in any form as long as they can suppress the expression of plexin B2. The siRNA or shRNA can be designed by a known method based on the base sequence of the target gene. siRNA or shRNA can be artificially synthesized. Antisense and sense RNA can also be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and T7 promoter. The antisense oligonucleotide may be either DNA or RNA, as long as it is a nucleotide that is complementary or hybridizes to the consecutive 5 to 100 base sequences in the DNA sequence of the plexin B2 gene. Further, it may be modified as long as it does not interfere with the function. The antisense oligonucleotide can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer. The antisense mRNA may be an RNA that is complementary to or hybridizes to the base sequence of the mRNA of plexin B2. Antisense mRNA can be artificially chemically synthesized. Antisense and sense RNA can also be synthesized in vitro from template DNA using, for example, T7 RNA polymerase and T7 promoter. During synthesis, pseudouridine is used instead of uridine to impart a cap structure and poly-A signal.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、プレキシンB2の機能を阻害する物質であってもよい。プレキシンB2の機能としては、例えば、セマフォリン4Dとの結合能、Traf2との結合能、c-Metとの結合能などが挙げられるが、これらに限定されない。今後見出されるプレキシンB2の機能を阻害する物質も、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質に含まれる。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the function of plexin B2. Functions of plexin B2 include, but are not limited to, the ability to bind to semaphorin 4D, the ability to bind to Traf2, and the ability to bind to c-Met. Substances that inhibit the function of plexin B2, which will be discovered in the future, are also included in the substances that inhibit the signal transduction of semaphorin 4D-plexin B2.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、IκBζまたはCEBPδの発現を阻害する物質であってもよい。IκBζまたはCEBPδの発現を阻害する物質は、例えば、IκBζ遺伝子またはCEBPδ遺伝子のsiRNA、shRNA、アンチセンスオリゴヌクレオチド、アンチセンスmRNAなどが挙げられる。投与対象動物のIκBζ遺伝子の塩基配列、CEBPδ遺伝子の塩基配列は、公知のデータベース（NCBI等）から取得することができる。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the expression of IκBζ or CEBPδ. Examples of the substance that inhibits the expression of IκBζ or CEBPδ include siRNA, shRNA, antisense oligonucleotide, and antisense mRNA of the IκBζ gene or CEBPδ gene. The nucleotide sequence of the IκBζ gene and the nucleotide sequence of the CEBPδ gene of the administration target animal can be obtained from a known database (NCBI, etc.).
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、IκBζまたはCEBPδの機能を阻害する物質であってもよい。IκBζまたはCEBPδの機能としては、例えば、DNA結合能、転写活性化能、他の転写因子との結合能などがあげられる。したがって、IκBζまたはCEBPδの機能を阻害する物質は、例えば、IκBζまたはCEBPδが認識するDNAとの結合を阻害する物質、IκBζまたはCEBPδが認識する発現制御領域が関与する遺伝子の発現を低下させる物質などが挙げられる。 The substance that inhibits the signal transduction of semaphorin 4D-plexin B2 may be a substance that inhibits the function of IκBζ or CEBPδ. Functions of IκBζ or CEBPδ include, for example, DNA binding ability, transcriptional activation ability, and ability to bind to other transcription factors. Therefore, substances that inhibit the function of IκBζ or CEBPδ include, for example, substances that inhibit binding to DNA recognized by IκBζ or CEBPδ, substances that reduce the expression of genes involved in the expression control region recognized by IκBζ or CEBPδ, and the like. Can be mentioned.
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質は、c-Met阻害剤であってもよい。本発明者らは、マウス大腿骨頭軟骨を器官培養し、培地にセマフォリン4Dまたはセマフォリン4Dとc-Met阻害剤クリゾチニブを培地に添加して培養したところ、c-Met阻害剤を添加することにより、培地へのアグリカン放出量が有意に抑制されること、すなわち、軟骨破壊が抑制されることを確認している（実施例8参照）。したがって、c-Met阻害剤は軟骨・骨・関節疾患の予防または治療用医薬組成物の有効成分として有用である。c-Met阻害剤は特に限定されないが、例えばクリゾチニブ、テポチニブ、カプマチニブなどが挙げられる。 The substance that inhibits semaphorin 4D-plexin B2 signal transduction may be a c-Met inhibitor. The present inventors cultivated mouse femoral head cartilage, added semaphorin 4D or semaphorin 4D and the c-Met inhibitor crizotinib to the medium, and then added the c-Met inhibitor. It has been confirmed that the amount of aggrecan released into the medium is significantly suppressed, that is, cartilage destruction is suppressed (see Example 8). Therefore, c-Met inhibitors are useful as active ingredients in pharmaceutical compositions for the prevention or treatment of cartilage, bone and joint diseases. The c-Met inhibitor is not particularly limited, and examples thereof include crizotinib, tepotinib, and capmatinib.
　本発明の医薬組成物は、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質を有効成分とし、常套手段に従って製剤化することができる。例えば、経口投与のための製剤としては、固体または液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤などが挙げられる。これらの製剤は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤もしくは賦形剤を含有するものである。例えば、錠剤用の担体、賦形剤としては、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムなどが用いられる。非経口投与のための製剤としては、例えば、注射剤、坐剤などが用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤、関節内注射剤などの剤形を包含する。このような注射剤は、公知の方法に従って、例えば、上記有効成分を通常注射剤に用いられる無菌の水性もしくは油性液に溶解、懸濁または乳化することによって調製される。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液などが用いられ、適当な溶解補助剤、例えば、アルコール（例えば、エタノール等）、ポリアルコール（例えば、プロピレングリコール、ポリエチレングリコール等）、非イオン界面活性剤（例えば、ポリソルベート80、HCO-50等）などと併用してもよい。油性液としては、例えば、ゴマ油、大豆油などが用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコールなどを併用してもよい。直腸投与に用いられる坐剤は、上記有効成分を通常の坐薬用基剤に混合することによって調製される。 The pharmaceutical composition of the present invention contains a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient, and can be formulated according to conventional means. For example, formulations for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and the like. Examples include syrups, emulsions and suspending agents. These formulations are manufactured by known methods and contain carriers, diluents or excipients commonly used in the pharmaceutical field. For example, lactose, starch, sucrose, magnesium stearate and the like are used as carriers and excipients for tablets. For example, injections, suppositories, etc. are used as preparations for parenteral administration, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection, intra-articular injection. Including dosage forms such as. Such injections are prepared according to known methods, for example, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily solution normally used for injections. As the aqueous solution for injection, for example, a physiological saline solution, an isotonic solution containing glucose and other auxiliary agents, and the like are used, and appropriate solubilizing agents such as alcohol (for example, ethanol etc.) and polyalcohol (for example) are used. , Propylene glycol, polyethylene glycol, etc.), nonionic surfactant (eg, polysorbate 80, HCO-50, etc.) and the like. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. Suppositories used for rectal administration are prepared by mixing the active ingredient with a conventional suppository base.
　本発明の医薬組成物の有効成分が抗体またはペプチドである場合、薬学的に許容される担体とともに製剤化された注射剤または輸液として、非経口投与経路、例えば、静脈内、筋肉内、皮膚内、腹腔内、皮下または局所に投与することが好ましい。 When the active ingredient of the pharmaceutical composition of the present invention is an antibody or peptide, the parenteral route of administration, eg, intravenous, intramuscular, intradermal, as an injection or infusion formulated with a pharmaceutically acceptable carrier. , Preferably administered intraperitoneally, subcutaneously or topically.
　発明の医薬組成物の有効成分が核酸である場合、非ウイルスベクターまたはウイルスベクターの形態で投与することができる。非ウイルスベクターの形態で投与する場合、リポソームを用いて核酸分子を導入する方法（リポソーム法、HVJ-リポソーム法、カチオニックリポソーム法、リポフェクション法、リポフェクトアミン法など）、マイクロインジェクション法、遺伝子銃（Gene Gun）でキャリア（金属粒子）とともに核酸分子を細胞に移入する方法などを利用することができる。siRNAまたはshRNAをウイルスベクターの形態で投与する場合は、無毒化したレトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シンドビスウイルス、センダイウイルス、SV40などのDNAウイルスまたはRNAウイルスに、siRNAまたはshRNAを発現するDNAを導入し、細胞または組織にこの組換えウイルスを感染させることにより、細胞または組織内にsiRNAまたはshRNAを導入することができる。 When the active ingredient of the pharmaceutical composition of the invention is nucleic acid, it can be administered in the form of a non-viral vector or a viral vector. When administered in the form of a non-viral vector, a method of introducing a nucleic acid molecule using a liposome (liposome method, HVJ-liposome method, cationic liposome method, lipofection method, lipofectamine method, etc.), microinjection method, gene gun (GeneGun) can be used to transfer nucleic acid molecules into cells together with carriers (metal particles). When siRNA or shRNA is administered in the form of a viral vector, detoxified retrovirus, lentivirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, sindobis virus, Sendai virus, SV40 By introducing a DNA expressing siRNA or shRNA into a DNA virus or RNA virus such as, and infecting cells or tissues with this recombinant virus, siRNA or shRNA can be introduced into cells or tissues.
　このようにして得られる製剤は安全で低毒性であるので、例えば、ヒトや哺乳動物（例えば、ラット、マウス、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなど）に対して経口的にまたは非経口的に投与することができる。本発明の医薬組成物には、有効成分を0.001～50質量％、好ましくは0.01～10質量％、さらに好ましくは0.1～1質量％含有することができる。本発明の医薬組成物の投与量は、疾患の重篤度、患者の年齢、体重、性別、既往歴、有効成分の種類などを考慮して、適宜設定される。約65～70ｋｇの体重を有する平均的なヒトを対象とした場合、１日当たり0.02mg～4000mg程度が好ましく、0.1mg～200mg程度がより好ましい。１日当たりの総投与量は、単一投与量であっても分割投与量であってもよい。 The formulations thus obtained are safe and low toxicity, and therefore, for example, orally to humans and mammals (eg, rats, mice, rabbits, sheep, pigs, cows, cats, dogs, monkeys, etc.). Alternatively, it can be administered parenterally. The pharmaceutical composition of the present invention can contain 0.001 to 50% by mass, preferably 0.01 to 10% by mass, and more preferably 0.1 to 1% by mass of the active ingredient. The dose of the pharmaceutical composition of the present invention is appropriately set in consideration of the severity of the disease, the age, weight, sex, medical history, type of active ingredient, and the like of the patient. For an average human having a body weight of about 65 to 70 kg, about 0.02 mg to 4000 mg per day is preferable, and about 0.1 mg to 200 mg is more preferable. The total daily dose may be a single dose or a divided dose.
〔スクリーニング方法〕
　セマフォリン4D－プレキシンB2のシグナル伝達を阻害する被験物質を選択する工程を含む、軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法（以下「本発明のスクリーニング方法」と記す）を提供する。 [Screening method]
Provided is a method for screening a drug for preventing or treating cartilage, bone, or joint disease (hereinafter referred to as "the screening method of the present invention"), which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2. do.
　本発明のスクリーニング方法に供される被験物質は特に限定されず、例えば、天然化合物、有機化合物、無機化合物、核酸、核酸オリゴ、タンパク質、ペプチド等の単一物質、並びに化合物ライブラリー、核酸オリゴライブラリー、ペプチドライブラリー、遺伝子ライブラリーの発現産物、細胞抽出物、細胞培養上清、発酵微生物産生物、海洋生物抽出物、植物抽出物、原核細胞抽出物、真核単細胞抽出物、動物細胞抽出物等を挙げることができる。被験物質は、新規な物質であってもよいし、公知の物質であってもよい。これらの被験物質は塩を形成していてもよい。被験物質の塩としては、生理学的に許容される酸や塩基との塩が好ましい。 The test substance used in the screening method of the present invention is not particularly limited, and for example, a single substance such as a natural compound, an organic compound, an inorganic compound, a nucleic acid, a nucleic acid oligo, a protein, or a peptide, a compound library, or a nucleic acid oligolive. Rally, peptide library, gene library expression product, cell extract, cell culture supernatant, fermented microbial product, marine biological extract, plant extract, prokaryotic cell extract, eukaryotic single cell extract, animal cell extract Things can be mentioned. The test substance may be a novel substance or a known substance. These test substances may form salts. As the salt of the test substance, a salt with a physiologically acceptable acid or base is preferable.
　本発明のスクリーニング方法の第一の実施形態は、以下の工程（１）～（３）を含むものであればよい。
（１）プレキシンB2とセマフォリン4Dと被験物質を接触させる工程、
（２）プレキシンB2とセマフォリン4Dとの結合を測定する工程、および
（３）験物質を添加していない対照と比較してプレキシンB2とセマフォリン4Dとの結合を低下させる被験物質を選択する工程。 The first embodiment of the screening method of the present invention may include the following steps (1) to (3).
(1) Step of contacting plexin B2, semaphorin 4D and the test substance,
(2) A step of measuring the binding between plexin B2 and semaphorin 4D, and (3) selecting a test substance that reduces the binding between plexin B2 and semaphorin 4D as compared with a control to which no test substance is added. Process.
　工程（１）では、プレキシンB2とセマフォリン4Dと被験物質を接触させる。プレキシンB2とセマフォリン4Dと被験物質を接触させる系としては、例えば、溶液系または培養系が挙げられる。溶液系の場合、例えば、タンパク質溶液に適した緩衝液中で、プレキシンB2とセマフォリン4Dと被験物質を接触させる系を用いることができる。通常、被験物質を接触させない対照が設けられる。タンパク質溶液に適した緩衝液としては、例えば、pH6～9のトリス緩衝液、リン酸緩衝液、酢酸緩衝液、ホウ酸緩衝液、クエン酸緩衝液などが挙げられる。プレキシンB2およびセマフォリン4Dは、公知の遺伝子組み換え技術および組み換えタンパク質発現技術を用いて、組み換えタンパク質として作製することができる。セマフォリン4Dは可溶型セマフォリン4Dであってもよい。プレキシンB2のアミノ酸配列情報、プレキシンB2をコードする遺伝子の塩基配列情報、セマフォリン4Dのアミノ酸配列情報、セマフォリン4Dをコードする遺伝子の塩基配列情報は公知のデータベース（NCBI等）から取得することができる。例えば、ヒトおよびマウスの塩基配列およびアミノ酸配列のアクセッション番号は以下のとおりである。
プレキシンB2
ヒト　：NM_012401.3（塩基配列）、NP_036533.2（アミノ酸配列）
マウス：NM_001159521.2（塩基配列）、NP_001152993.1（アミノ酸配列）
セマフォリン4D
ヒト　：NM_006378.3（塩基配列）、NP_006369.3（アミノ酸配列）
マウス：NM_001281880.2（塩基配列）、NP_001268809.1（アミノ酸配列） In step (1), plexin B2, semaphorin 4D, and the test substance are brought into contact with each other. Examples of the system in which plexin B2 and semaphorin 4D are brought into contact with the test substance include a solution system or a culture system. In the case of a solution system, for example, a system in which plexin B2, semaphorin 4D and a test substance are brought into contact with each other in a buffer solution suitable for a protein solution can be used. Usually, a control is provided that is not in contact with the test substance. Examples of the buffer solution suitable for the protein solution include Tris buffer solution having a pH of 6 to 9, a phosphate buffer solution, an acetate buffer solution, a borate buffer solution, and a citrate buffer solution. Plexin B2 and semaphorin 4D can be produced as recombinant proteins using known gene recombination techniques and recombinant protein expression techniques. Semaphorin 4D may be soluble semaphorin 4D. The amino acid sequence information of plexin B2, the base sequence information of the gene encoding plexin B2, the amino acid sequence information of semaphorin 4D, and the base sequence information of the gene encoding semaphorin 4D can be obtained from a known database (NCBI, etc.). can. For example, the accession numbers of the human and mouse base sequences and amino acid sequences are as follows.
Plexin B2
Human: NM_012401.3 (base sequence), NP_036533.2 (amino acid sequence)
Mouse: NM_001159521.2 (base sequence), NP_001152993.1 (amino acid sequence)
Semaphorin 4D
Human: NM_006378.3 (base sequence), NP_006369.3 (amino acid sequence)
Mouse: NM_001281880.2 (base sequence), NP_001268809.1 (amino acid sequence)
　培養系の場合、例えば、プレキシンB2発現細胞を培養している培地にセマフォリン4Dまたは可溶型セマフォリン4Dと被験物質を添加する系、プレキシンB2を発現する培養細胞と可溶型セマフォリン4Dを分泌する細胞を共培養している培地に被験物質を添加する系などを用いることができる。通常、被験物質を添加しない対照が設けられる。プレキシンB2発現細胞としては、例えば、関節軟骨表層細胞、プレキシンB2発現ベクターを導入した培養細胞、軟骨細胞、骨芽細胞、肢芽細胞などが挙げられる。セマフォリン4Dを分泌する細胞としては、例えば、LPSで処理したマクロファージ、リンパ球、滑膜細胞、破骨細胞などが挙げられる。 In the case of a culture system, for example, a system in which semaphorin 4D or soluble semaphorin 4D and a test substance are added to a medium in which plexin B2-expressing cells are cultured, a system in which plexin B2-expressing cultured cells and soluble semaphorin 4D are added. It is possible to use a system in which the test substance is added to the medium in which the cells secreting the cells are co-cultured. Usually, a control without the addition of the test substance is provided. Examples of plexin B2-expressing cells include articular cartilage surface cells, cultured cells into which a plexin B2 expression vector has been introduced, chondrocytes, osteoblasts, and limb blast cells. Examples of cells that secrete semaphorin 4D include macrophages, lymphocytes, synovial cells, osteoclasts, etc. treated with LPS.
　工程（２）では、プレキシンB2とセマフォリン4Dとの結合を測定する。プレキシンB2とセマフォリン4Dとの結合を測定する方法は特に限定されず、公知の方法を適宜選択して使用することができる。例えば、ELISA法、蛍光偏光法、免疫染色法、質量分析法、構造解析、細胞を用いたHTS系によるスクリーニング法などを好適に用いることができる。ELISA法を用いる場合、可溶型セマフォリン4Dの組み換えタンパク質およびプレキシンB2の細胞外領域の組み替えタンパク質のいずれか一方を固相化し、そこに他方および被験物質を添加して反応させ、プレキシンB2とセマフォリン4Dの結合レベルを適当な一次抗体および二次抗体を用いて検出することができる。 In step (2), the binding between plexin B2 and semaphorin 4D is measured. The method for measuring the binding between plexin B2 and semaphorin 4D is not particularly limited, and a known method can be appropriately selected and used. For example, an ELISA method, a fluorescence polarization method, an immunostaining method, a mass spectrometry method, a structural analysis, a screening method using an HTS system using cells, or the like can be preferably used. When the ELISA method is used, either one of the recombinant protein of soluble semaphorin 4D and the recombinant protein of the extracellular region of plexin B2 is immobilized, and the other and the test substance are added thereto to react with plexin B2. The binding level of semaphorin 4D can be detected using the appropriate primary and secondary antibodies.
　工程（３）では、被験物質を添加していない対照と比較してプレキシンB2とセマフォリン4Dとの結合を低下させる被験物質を選択する。選択の基準は特に限定されないが、例えば、被験物質を添加していない対照の結合レベルと比較して、結合レベルを50％以下、40％以下、30％以下、20％以下、10％以下に低下させる被験物質を選択してもよい。 In step (3), a test substance that reduces the binding between plexin B2 and semaphorin 4D as compared with the control to which the test substance is not added is selected. Criteria for selection are not particularly limited, but for example, the binding level is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less as compared with the binding level of the control to which the test substance is not added. The test substance to be lowered may be selected.
　本発明のスクリーニング方法の第二の実施形態は、以下の工程（１）～（３）を含むものであればよい。
（１）プレキシンB2とTraf2と被験物質を接触させる工程、
（２）プレキシンB2とTraf2との結合を測定する工程、および
（３）験物質を添加していない対照と比較してプレキシンB2とTraf2との結合を低下させる被験物質を選択する工程。 The second embodiment of the screening method of the present invention may include the following steps (1) to (3).
(1) Step of contacting plexin B2 and Traf2 with a test substance,
(2) A step of measuring the binding between plexin B2 and Traf2, and (3) a step of selecting a test substance that reduces the binding between plexin B2 and Traf2 as compared with a control to which no test substance is added.
　工程（１）では、プレキシンB2とTraf2と被験物質を接触させる。プレキシンB2とTraf2と被験物質を接触させる系としては、例えば、溶液系または培養系が挙げられる。溶液系の場合、例えば、タンパク質溶液に適した緩衝液中で、プレキシンB2とTraf2と被験物質を接触させる系を用いることができる。培養系の場合、例えば、プレキシンB2発現細胞を培養している培地にTraf2と被験物質を添加する系を用いることができる。通常、被験物質を接触させない対照が設けられる。プレキシンB2およびTraf2は、公知の遺伝子組み換え技術および組み換えタンパク質発現技術を用いて、組み換えタンパク質として作製することができる。プレキシンB2の配列情報は上記の通りであり、Traf2のアミノ酸配列情報、Traf2をコードする遺伝子の塩基配列情報は公知のデータベース（NCBI等）から取得することができる。例えば、ヒトおよびマウスの塩基配列およびアミノ酸配列のアクセッション番号は以下のとおりである。
ヒト　：NM_021138.4（塩基配列）、NP_066961.2（アミノ酸配列）
マウス：NM_001290413.1（塩基配列）、NP_001277342.1（アミノ酸配列）
　プレキシンB2発現細胞としては、第一の実施形態で例示した細胞を用いることができる。 In step (1), plexin B2 and Traf2 are brought into contact with the test substance. Examples of the system in which plexin B2 and Traf2 are brought into contact with the test substance include a solution system and a culture system. In the case of a solution system, for example, a system in which plexin B2 and Traf2 are brought into contact with a test substance in a buffer solution suitable for a protein solution can be used. In the case of a culture system, for example, a system in which Traf2 and a test substance are added to a medium in which plexin B2-expressing cells are cultured can be used. Usually, a control is provided that is not in contact with the test substance. Plexins B2 and Traf2 can be produced as recombinant proteins using known gene recombination techniques and recombinant protein expression techniques. The sequence information of plexin B2 is as described above, and the amino acid sequence information of Traf2 and the base sequence information of the gene encoding Traf2 can be obtained from a known database (NCBI or the like). For example, the accession numbers of the human and mouse base sequences and amino acid sequences are as follows.
Human: NM_021138.4 (base sequence), NP_066961.2 (amino acid sequence)
Mouse: NM_001290413.1 (base sequence), NP_001277342.1 (amino acid sequence)
As the plexin B2-expressing cell, the cell exemplified in the first embodiment can be used.
　本発明のスクリーニング方法の第二の実施形態の工程（２）および（３）は、上記第一の実施形態においてセマフォリン4Dに代えてTraf2を用いること以外、上記第一の実施形態の工程（２）および（３）と同様に行うことができる。 The steps (2) and (3) of the second embodiment of the screening method of the present invention are the steps of the first embodiment except that Traf2 is used instead of semaphorin 4D in the first embodiment. It can be performed in the same manner as in 2) and (3).
　本発明のスクリーニング方法の第三の実施形態は、以下の工程（１）～（３）を含むものであればよい。
（１）プレキシンB2とc-Metと被験物質を接触させる工程、
（２）プレキシンB2とc-Metとの結合を測定する工程、および
（３）験物質を添加していない対照と比較してプレキシンB2とc-Metとの結合を低下させる被験物質を選択する工程。 The third embodiment of the screening method of the present invention may include the following steps (1) to (3).
(1) Step of contacting plexin B2, c-Met and the test substance,
(2) A step of measuring the binding between plexin B2 and c-Met, and (3) selecting a test substance that reduces the binding between plexin B2 and c-Met as compared with a control to which no test substance is added. Process.
　工程（１）では、プレキシンB2とc-Metと被験物質を接触させる。プレキシンB2とc-Metと被験物質を接触させる系としては、例えば、溶液系または培養系が挙げられる。溶液系の場合、例えば、タンパク質溶液に適した緩衝液中で、プレキシンB2とc-Metと被験物質を接触させる系を用いることができる。培養系の場合、例えば、プレキシンB2発現細胞を培養している培地にc-Metと被験物質を添加する系、c-Met発現細胞を培養している培地にプレキシンB2と被験物質を添加する系、プレキシンB2発現細胞とc-Met発現細胞を共培養している培地に被験物質を添加する系などを用いることができる。通常、被験物質を接触させない対照が設けられる。 In step (1), plexin B2, c-Met and the test substance are brought into contact with each other. Examples of the system in which the plexin B2 and c-Met are brought into contact with the test substance include a solution system and a culture system. In the case of a solution system, for example, a system in which plexin B2, c-Met and a test substance are brought into contact with each other in a buffer solution suitable for a protein solution can be used. In the case of a culture system, for example, a system in which c-Met and a test substance are added to a medium in which plexin B2-expressing cells are cultured, and a system in which plexin B2 and a test substance are added to a medium in which c-Met-expressing cells are cultured. , A system in which a test substance is added to a medium in which plexin B2-expressing cells and c-Met-expressing cells are co-cultured can be used. Usually, a control is provided that is not in contact with the test substance.
　セマフォリン4Dおよびc-Metは、公知の遺伝子組み換え技術および組み換えタンパク質発現技術を用いて、組み換えタンパク質として作製することができる。プレキシンB2の配列情報は上記の通りであり、c-Metのアミノ酸配列情報、c-Metをコードする遺伝子の塩基配列情報は公知のデータベース（NCBI等）から取得することができる。例えば、ヒトおよびマウスの塩基配列およびアミノ酸配列のアクセッション番号は以下のとおりである。
ヒト　：NM_001127500.3（塩基配列）、NP_001120972.1（アミノ酸配列）
マウス：NM_008591.2（塩基配列）、NP_032617.2（アミノ酸配列）
　プレキシンB2発現細胞としては、第一の実施形態で例示した細胞を用いることができる。c-Met発現細胞としては、関節軟骨表層細胞、c-Met発現ベクターを導入した培養細胞、軟骨細胞、骨芽細胞、肢芽細胞などが挙げられる。 Semaphorins 4D and c-Met can be produced as recombinant proteins using known gene recombination techniques and recombinant protein expression techniques. The sequence information of plexin B2 is as described above, and the amino acid sequence information of c-Met and the base sequence information of the gene encoding c-Met can be obtained from a known database (NCBI or the like). For example, the accession numbers of the human and mouse base sequences and amino acid sequences are as follows.
Human: NM_001127500.3 (base sequence), NP_001120972.1 (amino acid sequence)
Mouse: NM_008591.2 (base sequence), NP_032617.2 (amino acid sequence)
As the plexin B2-expressing cell, the cell exemplified in the first embodiment can be used. Examples of the c-Met expressing cell include articular cartilage surface layer cells, cultured cells into which a c-Met expression vector has been introduced, chondrocytes, osteoblasts, and limb blast cells.
　本発明のスクリーニング方法の第三の実施形態の工程（２）および（３）は、上記第一の実施形態においてセマフォリン4Dに代えてc-Metを用いること以外、上記第一の実施形態の工程（２）および（３）と同様に行うことができる。 The steps (2) and (3) of the third embodiment of the screening method of the present invention are the same as that of the first embodiment, except that c-Met is used instead of semaphorin 4D in the first embodiment. It can be carried out in the same manner as in steps (2) and (3).
　本発明のスクリーニング方法の第四の実施形態は、以下の工程（１）～（３）を含むものであればよい。
（１）IκBζおよび／またはCEBPδを発現する細胞と被験物質を接触させる工程、
（２）IκBζおよび／またはCEBPδの発現量を測定する工程、および
（３）被験物質を接触させていない対照と比較してIκBζおよび／またはCEBPδの発現量を低下させる被験物質を選択する工程。 The fourth embodiment of the screening method of the present invention may include the following steps (1) to (3).
(1) A step of contacting a test substance with a cell expressing IκBζ and / or CEBPδ,
(2) A step of measuring the expression level of IκBζ and / or CEBPδ, and (3) a step of selecting a test substance that reduces the expression level of IκBζ and / or CEBPδ as compared with a control not contacted with the test substance.
　工程（１）では、IκBζおよび／またはCEBPδを発現する細胞と被験物質を接触させる。例えば、IκBζおよび／またはCEBPδを発現する培養細胞の培地に被験物質を添加ることにより、細胞と被験物質を接触させることができる。通常、被験物質を接触させない対照が設けられる。IκBζおよび／またはCEBPδを発現する細胞としては、関節軟骨表層細胞、軟骨細胞、骨芽細胞、肢芽細胞などが挙げられる。 In step (1), the test substance is brought into contact with cells expressing IκBζ and / or CEBPδ. For example, the test substance can be brought into contact with the test substance by adding the test substance to the medium of the cultured cells expressing IκBζ and / or CEBPδ. Usually, a control is provided that is not in contact with the test substance. Examples of cells expressing IκBζ and / or CEBPδ include articular cartilage superficial cells, chondrocytes, osteoblasts, limb blast cells and the like.
　工程（２）では、IκBζおよび／またはCEBPδの発現量を測定する。発現量の測定は、タンパク質量を測定してもよく、mRNA量を測定してもよい。タンパク質量を測定する場合は、公知の方法で細胞からタンパク質を抽出し、公知のタンパク質量測定方法を用いて定量することができる。公知のタンパク質量測定方法としては、例えば、ウエスタンブロット法、EIA法、ELISA法、RIA法、タンパク質測定試薬を用いる方法などが挙げられる。mRNA量を測定する場合は、公知の方法で細胞からRNAを抽出し、公知のmRNA量測定方法を用いて定量することができる。公知のmRNA量測定方法としては、ノーザンブロット法、RT-PCR法、定量RT-PCR法、RNaseプロテクションアッセイなどが挙げられる。 In step (2), the expression levels of IκBζ and / or CEBPδ are measured. The expression level may be measured by measuring the amount of protein or mRNA. When measuring the amount of protein, the protein can be extracted from the cell by a known method and quantified by using a known method for measuring the amount of protein. Known methods for measuring the amount of protein include, for example, Western blotting, EIA method, ELISA method, RIA method, and a method using a protein measuring reagent. When measuring the amount of mRNA, RNA can be extracted from cells by a known method and quantified using a known method for measuring the amount of mRNA. Known methods for measuring the amount of mRNA include Northern blotting, RT-PCR, quantitative RT-PCR, RNase protection assay and the like.
　工程（３）では、被験物質を接触させていない対照と比較してIκBζおよび／またはCEBPδの発現量を低下させる被験物質を選択する。選択の基準は特に限定されないが、例えば、被験物質を添加していない対照の発現量と比較して、発現量を50％以下、40％以下、30％以下、20％以下、10％以下に低下させる被験物質を選択してもよい。 In step (3), a test substance that reduces the expression level of IκBζ and / or CEBPδ as compared with a control that has not been contacted with the test substance is selected. The criteria for selection are not particularly limited, but for example, the expression level is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less as compared with the expression level of the control to which the test substance is not added. The test substance to be lowered may be selected.
　本発明のスクリーニング方法の第五の実施形態は、以下の工程（１）～（３）を含むものであればよい。
（１）IκBζまたはCEBPδが認識する発現制御領域の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とIκBζまたはCEBPδと被験物質を接触させる工程、
（２）レポーター遺伝子の発現量を測定する工程、および
（３）被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する工程。 The fifth embodiment of the screening method of the present invention may include the following steps (1) to (3).
(1) A step of contacting a cultured cell or a genetically modified animal cell in which a reporter gene is inserted downstream of an expression control region recognized by IκBζ or CEBPδ with IκBζ or CEBPδ and a test substance.
(2) A step of measuring the expression level of the reporter gene, and (3) a step of selecting a test substance that reduces the expression level of the reporter gene as compared with a control not contacted with the test substance.
　工程（１）では、IκBζまたはCEBPδが認識する発現制御領域の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とIκBζまたはCEBPδと被験物質を接触させる。工程（１）で用いる培養細胞としては、株化培養細胞、関節表層軟骨細胞、軟骨細胞、骨芽細胞、肢芽細胞などが挙げられる。工程（１）で用いる遺伝子改変動物としては、マウス、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サルなどが挙げられる。IκBζまたはCEBPδが認識する発現制御領域の下流にレポーター遺伝子が挿入された培養細胞は、これら遺伝子発現制御領域を含むレポーター遺伝子を公知のトランスフェクション法、ウイルス導入法等を用いて遺伝子導入することにより作製することができる。IκBζまたはCEBPδが認識する発現制御領域の下流にレポーター遺伝子が挿入された遺伝子改変動物は、ゲノム編集法、相同組み替え法等でこれら遺伝子発現領域にレポーター遺伝子をノックイン（挿入）することにより作製することができる。 In step (1), IκBζ or CEBPδ and the test substance are brought into contact with cultured cells or genetically modified animal cells in which a reporter gene is inserted downstream of the expression control region recognized by IκBζ or CEBPδ. Examples of the cultured cells used in the step (1) include established cultured cells, articular superficial chondrocytes, chondrocytes, osteoblasts, and limb blasts. Examples of the genetically modified animal used in the step (1) include mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys and the like. In cultured cells in which a reporter gene is inserted downstream of the expression control region recognized by IκBζ or CEBPδ, the reporter gene containing these gene expression control regions is introduced by a known transfection method, virus transfer method, or the like. Can be made. Genetically modified animals in which a reporter gene is inserted downstream of the expression control region recognized by IκBζ or CEBPδ should be produced by knocking in (inserting) the reporter gene into these gene expression regions by genome editing, homologous recombination, etc. Can be done.
　レポーター遺伝子は、一般に用いられているものであれば特に限定されないが、安定かつ活性の定量が容易なものが好ましい。例えば、ルシフェラーゼ、β－ガラクトシダーゼ（lacZ）、β－グルクロニダーゼ（GUS）、クロラムフェニコールアセチルトランスフェラーゼ（CAT）、アルカリホスファターゼ（ALP）、ペルオキシダーゼ（POD）、緑色蛍光タンパク質（GFP）等をコードする遺伝子が挙げられる。 The reporter gene is not particularly limited as long as it is generally used, but a reporter gene that is stable and whose activity can be easily quantified is preferable. For example, genes encoding luciferase, β-galactosidase (lacZ), β-glucuronidase (GUS), chloramphenicol acetyltransferase (CAT), alkaline phosphatase (ALP), peroxidase (POD), green fluorescent protein (GFP), etc. Can be mentioned.
　工程（１）において培養細胞を用いる場合、例えば、培養細胞の培地にIκBζまたはCEBPδと被験物質を添加することにより、細胞とIκBζまたはCEBPδと被験物質を接触させることができる。工程（１）において遺伝子改変動物を用いる場合、例えば、当該遺伝子改変動物にIκBζまたはCEBPδと被験物質を投与することにより遺伝子改変動物の細胞とIκBζまたはCEBPδと被験物質を接触させることができる。投与経路は特に限定されないが、経口投与、静脈内投与、腹腔内投与等の全身投与、標的臓器や標的組織への局所投与などが挙げられる。通常、被験物質を接触させない対照が設けられる。 When the cultured cells are used in the step (1), for example, by adding IκBζ or CEBPδ and the test substance to the medium of the cultured cells, the cells can be brought into contact with the IκBζ or CEBPδ and the test substance. When a genetically modified animal is used in step (1), for example, by administering IκBζ or CEBPδ and the test substance to the genetically modified animal, the cells of the genetically modified animal can be brought into contact with IκBζ or CEBPδ and the test substance. The route of administration is not particularly limited, and examples thereof include systemic administration such as oral administration, intravenous administration, and intraperitoneal administration, and local administration to a target organ or target tissue. Usually, a control is provided that is not in contact with the test substance.
　工程（２）では、レポーター遺伝子の発現量を測定する。レポーター遺伝子の発現量は、用いるレポーター遺伝子に応じて、公知の測定方法を適宜選択して測定することができる。例えば、レポーター遺伝子がCAT遺伝子である場合には、該遺伝子産物によるクロラムフェニコールのアセチル化を検出することによって、レポーター遺伝子の発現レベルを測定することができる。例えば、レポーター遺伝子がlacZ遺伝子である場合には、該遺伝子発現産物の触媒作用による色素化合物の発色を検出することにより、ルシフェラーゼ遺伝子である場合には、該遺伝子産物の触媒作用によるルシフェリンの発光を検出することにより、レポーター遺伝子の発現レベルを測定することができる。 In step (2), the expression level of the reporter gene is measured. The expression level of the reporter gene can be measured by appropriately selecting a known measuring method according to the reporter gene to be used. For example, when the reporter gene is a CAT gene, the expression level of the reporter gene can be measured by detecting the acetylation of chloramphenicol by the gene product. For example, when the reporter gene is the lacZ gene, the color development of the dye compound due to the catalytic action of the gene expression product is detected, and when the reporter gene is the luciferase gene, the luminescence of luciferin due to the catalytic action of the gene product is emitted. By detecting, the expression level of the reporter gene can be measured.
　工程（３）では、被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する。選択の基準は特に限定されないが、例えば、被験物質を添加していない対照の発現量と比較して、発現量を50％以下、40％以下、30％以下、20％以下、10％以下に低下させる被験物質を選択してもよい。 In step (3), a test substance that reduces the expression level of the reporter gene as compared with a control that has not been contacted with the test substance is selected. The criteria for selection are not particularly limited, but for example, the expression level is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less as compared with the expression level of the control to which the test substance is not added. The test substance to be lowered may be selected.
　本発明のスクリーニング方法の第六の実施形態は、以下の工程（１）～（３）を含むものであればよい。
（１）セマフォリン4Dにより発現誘導される遺伝子の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とセマフォリン4Dと被験物質を接触させる工程、
（２）レポーター遺伝子の発現量を測定する工程、および
（３）被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する工程。 The sixth embodiment of the screening method of the present invention may include the following steps (1) to (3).
(1) A step of contacting semaphorin 4D with a test substance with cultured cells or genetically modified animal cells in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D.
(2) A step of measuring the expression level of the reporter gene, and (3) a step of selecting a test substance that reduces the expression level of the reporter gene as compared with a control that has not been contacted with the test substance.
　第六の実施形態の工程（１）では、セマフォリン4Dにより発現誘導される遺伝子の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とセマフォリン4Dと被験物質を接触させる。工程（１）で用いる培養細胞および遺伝子改変動物としては、第五の実施形態で例示した細胞および動物が挙げられる。セマフォリン4Dにより発現誘導される遺伝子としては、例えば、Mmp13、Mmp3、IL-6、Nfikbz、C/ebpdなどが挙げられる。セマフォリン4Dにより発現誘導される遺伝子の下流にレポーター遺伝子が挿入された培養細胞は、セマフォリン4Dにより発現誘導される遺伝子の下流にレポーター遺伝子を連結したコンストラクトを公知のトランスフェクション法、ウイルス導入法等を用いて遺伝子導入することにより作製することができる。セマフォリン4Dにより発現誘導される遺伝子の下流にレポーター遺伝子が挿入された遺伝子改変動物は、ゲノム編集法、相同組み替え法等でセマフォリン4Dにより発現誘導される遺伝子の下流にレポーター遺伝子をノックイン（挿入）することにより作製することができる。 In step (1) of the sixth embodiment, semaphorin 4D and the test substance are brought into contact with cultured cells or genetically modified animal cells in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D. Examples of the cultured cells and genetically modified animals used in the step (1) include the cells and animals exemplified in the fifth embodiment. Examples of the gene whose expression is induced by semaphorin 4D include Mmp13, Mmp3, IL-6, Nfikbz, C / ebpd and the like. In cultured cells in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D, a known transfection method or virus transfer method is used to construct a construct in which a reporter gene is linked downstream of a gene whose expression is induced by semaphorin 4D. It can be produced by introducing a gene using such as. A gene-modified animal in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D knocks in (inserts) a reporter gene downstream of a gene whose expression is induced by semaphorin 4D by genome editing, homologous recombination, etc. ) Can be produced.
　本発明のスクリーニング方法の第六の実施形態は、工程（１）で使用するレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞が異なる以外は、上記第五の実施形態と同様に実施することができる。 The sixth embodiment of the screening method of the present invention is carried out in the same manner as the fifth embodiment, except that the cultured cells into which the reporter gene used in the step (1) is inserted or the cells of the genetically modified animal are different. be able to.
　本発明には、以下の各発明が含まれる。
　軟骨・骨・関節疾患患者に、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質を投与する工程を含む、軟骨・骨・関節疾患の予防または治療方法。
　軟骨・骨・関節疾患の予防または治療に使用するための、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質。
　軟骨・骨・関節疾患の予防または治療用医薬組成物を製造するための、セマフォリン4D－プレキシンB2のシグナル伝達を阻害する物質の使用。 The present invention includes the following inventions.
A method for preventing or treating cartilage / bone / joint disease, which comprises a step of administering a substance that inhibits signal transduction of semaphorin 4D-plexin B2 to a patient with cartilage / bone / joint disease.
A substance that inhibits signal transduction of semaphorin 4D-plexin B2 for use in the prevention or treatment of cartilage, bone and joint diseases.
Use of substances that inhibit semaphorin 4D-plexin B2 signaling to produce pharmaceutical compositions for the prevention or treatment of cartilage, bone and joint diseases.
　以下、実施例により本発明を詳細に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.
〔実験方法〕
　すべての動物実験は、大阪大学研究所動物実験委員会によって承認され、規制ガイドラインに従って実施した。
（１）マウス
　C57BL/6J、ICRおよびDBA/1Jマウスは日本エスエルシーから購入した。Nfkbiz floxed（Nfkbizflox/flox）マウスは、Okumaら（Immunity 38, 450-460, doi:10.1016/j.immuni.2012.11.016 (2013)）に記載の方法で作製した。Traf2ノックアウト（Traf2-/-）マウス（以下「Traf2 KOマウス」記す）は、Piaoら（J Biol Chem 286, 17879-17888, doi:10.1074/jbc.M111.221853 (2011)）に記載の方法で作製した。CAG-CREトランスジェニックマウスは理化学研究所から入手した。Nfkbizノックアウト（Nfkbiz-/-）マウス（以下「Nfkbiz KOマウス」記す）を作製するために、Nfkbizflox/floxマウスをCAG-CREトランスジェニックマウスと交配し、Nfkbiz+/-マウスを得た。Nfkbiz+/-マウスをNfkbiz+/-マウスと交配し、Nfkbiz-/-マウスを作製した。 〔experimental method〕
All animal experiments were approved by the Animal Care and Use Committee of the Institute of Osaka University and conducted in accordance with regulatory guidelines.
(1) Mice C57BL / 6J, ICR and DBA / 1J mice were purchased from Japan SLC. Nfkbiz floxed (Nfkbiz flox / flox ) mice were produced by the method described in Okuma et al. (Immunity 38, 450-460, doi: 10.1016 / j.immuni.2012.11.016 (2013)). Traf2 knockout (Traf2 -/- ) mice (hereinafter referred to as "Traf2 KO mice") are prepared by the method described in Piao et al. (J Biol Chem 286, 17879-17888, doi: 10.1074 / jbc.M111.221853 (2011)). Made. CAG-CRE transgenic mice were obtained from RIKEN. In order to generate Nfkbiz knockout (Nfkbiz -/- ) mice (hereinafter referred to as "Nfkbiz KO mice"), Nfkbiz flox / flox mice were mated with CAG-CRE transgenic mice to obtain Nfkbiz +/- mice. Nfkbiz +/- mice were mated with Nfkbiz +/- mice to generate Nfkbiz -/- mice.
　Sema4dノックアウトマウス（以下「Sema4d KOマウス」記す）は、CRISPR/Cas9システムに基づくTAKE法（Technique for Animal Knockout system by Electroporation）を用いて作製した（Sci Rep 4, 6382, doi:10.1038/srep06382 (2014)）。Sema4d遺伝子の開始コドン周辺を標的とするガイドRNA（標的配列：5'- AGGATGTGTGCCCCCGTTAG -3'、配列番号1）を、Guide-it sgRNA In Vitro Transcription and Screening System（タカラバイオ）を用いてインビトロ転写し、Cas9タンパク質および停止コドンを導入するmRNAと共に、エレクトロポレーション法でC57BL/6Jマウスの受精卵細胞に導入した。Sema4d遺伝子のゲノムDNA配列解析を行い、Sema4d KOマウスの適切なゲノム編集を確認した。 Sema4d knockout mice (hereinafter referred to as "Sema4d KO mice") were produced using the TAKE method (Technique for Animal Knockout system by Electroporation) based on the CRISPR / Cas9 system (SciRep 4, 6382, doi: 10.1038 / srep06382 (2014). )). Guide RNA (target sequence: 5'-AGGATGTGCGCCCCGTTAG-3', SEQ ID NO: 1) targeting around the start codon of the Sema4d gene is transcribed in vitro using Guide-it sgRNA In Vitro Transcription and Screening System (Takara Bio). , Cas9 protein and mRNA that introduces a stop codon, were introduced into fertilized egg cells of C57BL / 6J mice by electroporation. Genomic DNA sequence analysis of the Sema4d gene was performed to confirm appropriate genome editing in Sema4d KO mice.
　Plxnb2flox/floxマウスは、2工程のTAKE法を使用して作製した。第1工程では、Plxnb2をコードするエクソン22の下流を標的とするガイドRNA（標的配列：5'- GATGAGGGGAGCCTTACCGG -3'、配列番号2）をインビトロ転写し、Cas9タンパク質、mRNAおよび一本鎖オリゴデオキシヌクレオチド（ssODN）（5'- GTCTCCATCTACTGCTACTGGTGAGTCCTGCCCCCGATAACTTCGTATAGCATACATTATACGAAGTTATGTAAGGCTCCCCTCATCCCTCCCGCATCAG -3'、配列番号3）と共に、エレクトロポレーション法でC57BL/6Jマウスの受精卵細胞に導入した。Plxnb2遺伝子のゲノムDNA配列解析を行い、Plxnb2 LoxPトランスジェニックマウスの適切なゲノム編集を確認した。第2工程では、Plxnb2遺伝子をコードするエクソン21の上流を標的とするガイドRNA（標的配列：5'- GGGACCCCATGCCAGCCGTA -3'、配列番号4）をインビトロ転写し、Cas9タンパク質、mRNAおよびssODN（5'- ACAAGCTCATCCACGCCCGGGTAAGAAGACCCCTACATAACTTCGTATAGCATACATTATACGAAGTTATGGCTGGCATGGGGTCCCGGGTCGGGCGGGG -3'、配列番号5）と共に、エレクトロポレーション法でPlxnb2 LoxPトランスジェニックマウスの受精卵細胞に導入した。2つのLoxP部位の導入は、DNA配列解析で確認した。Plxnb2flox/floxマウスをCAG-CREトランスジェニックマウスと交配し、Plxnb2+/-マウスを得た。Plxnb2+/-マウスをPlxnb2+/-マウスと交配し、Plxnb2ノックアウトマウス（以下「Plxnb2 KOマウス」記す）を作製した。CAG-CREトランスジェニックマウスとの交配によりこれらのエクソンが切除され、プレキシン-B2の膜貫通ドメインが欠失することを、DNA配列解析で確認した。 Plxnb2 flox / flox mice were generated using the two-step TAKE method. In the first step, a guide RNA (target sequence: 5'-GATGAGGGGAGCCTTACCGG -3', SEQ ID NO: 2) targeting downstream of exon 22 encoding Plxnb2 is transcribed in vitro, and Cas9 protein, mRNA and single-stranded oligodeoxy are transcribed. It was introduced into fertilized egg cells of C57BL / 6J mice by the electroporation method together with nucleotides (ssODN) (5'-GTCTCCATCTACTGCTACTGGTGAGTCCTGCCCCCGATAACTTCGTATAGCATACATTATACGAAGTTATGTAAGGCTCCCCTCATCCCTCCCGCATCAG -3', SEQ ID NO: 3). Genomic DNA sequence analysis of the Plxnb2 gene was performed to confirm appropriate genome editing of Plxnb2 LoxP transgenic mice. In the second step, a guide RNA (target sequence: 5'-GGGACCCCATGCCAGCCGTA -3', SEQ ID NO: 4) targeting the upstream of exon 21 encoding the Plxnb2 gene is transcribed in vitro, and Cas9 protein, mRNA and ssODN (5') are transcribed. --ACAAGCTCATCCACGCCCGGGTAAGAAGACCCCTACATAACTTCGTATAGCATACATTATACGAAGTTATGGCTGGCATGGGGTCCCGGGTCGGGCGGGG -3', SEQ ID NO: 5) was introduced into fertilized egg cells of Plxnb2 LoxP transgenic mice by the electroporation method. The introduction of two LoxP sites was confirmed by DNA sequence analysis. Plxnb2 flox / flox mice were mated with CAG-CRE transgenic mice to obtain Plxnb2 +/- mice. Plxnb2 +/- mice were mated with Plxnb2 +/- mice to produce Plxnb2 knockout mice (hereinafter referred to as "Plxnb2 KO mice"). DNA sequence analysis confirmed that these exons were excised by mating with CAG-CRE transgenic mice and that the transmembrane domain of plexin-B2 was deleted.
（２）関節炎誘発
　Sema4d KOマウスは、少なくとも5世代にわたってDBA/1Jバックグラウンドと戻し交配した。ニワトリII型コラーゲン（2 mg/ml、Chondrex）を等量の完全フロイントアジュバント（1 mg/ml、Chondrex）で乳化し、エマルジョンを調製した。7～8週齢のオスのSema4d KOマウスおよび野生型（WT）マウスの尾の付け根に0.1 mlのエマルジョンを皮内注射することにより免疫した（0日目）。追加免疫のために、50μgのLPS（Chondrex）を21日目に腹腔内注射した。関節炎の臨床スコアは、各足（paw）について0～4の5段階で判定した（0＝正常、1＝1本の指の紅斑と腫脹、2＝2本の指の紅斑と腫脹、または足首もしくは手首の紅斑と腫脹、3＝3本の指の紅斑と腫脹、または2本の指と足首もしくは手首の腫脹、4＝紅斑と足（paw）全体の腫脹）。各マウスの足（paws）を組織学的に評価した。足を4％PFA/PBSで固定した後、10％EDTAで4週間脱灰した。軟骨をパラフィン包埋し、厚さ5μmの切片を作製した。切片を脱パラフィンし、ヘマトキシリンおよびエオジン（HE）で染色した。関節の病変は、Maedaら（Nature Medicine 18, 405-U166, doi:10.1038/nm.2653 (2012)）の記載に従い評価および定量化した。 (2) Arthritis-induced Sema4d KO mice were backcrossed with DBA / 1J background for at least 5 generations. Chicken type II collagen (2 mg / ml, Chondrex) was emulsified with an equal amount of complete Freund's adjuvant (1 mg / ml, Chondrex) to prepare an emulsion. 7-8 week old male Sema4d KO and wild-type (WT) mice were immunized by intradermal injection of 0.1 ml emulsion at the base of the tail (day 0). For booster immunization, 50 μg LPS (Chondrex) was injected intraperitoneally on day 21. The clinical score for arthritis was determined on a scale of 0-4 for each paw (0 = normal, 1 = 1 finger erythema and swelling, 2 = 2 finger erythema and swelling, or ankle. Or wrist erythema and swelling, 3 = 3 finger erythema and swelling, or 2 finger and ankle or wrist swelling, 4 = erythema and paw whole swelling). The paws of each mouse were evaluated histologically. The feet were fixed with 4% PFA / PBS and then decalcified with 10% EDTA for 4 weeks. The cartilage was embedded in paraffin to prepare a section having a thickness of 5 μm. Sections were deparaffinized and stained with hematoxylin and eosin (HE). Joint lesions were evaluated and quantified according to the description of Maeda et al. (Nature Medicine 18, 405-U166, doi: 10.1038 / nm.2653 (2012)).
（３）細胞および細胞刺激
　HEK293細胞は、10％FCSおよびペニシリン/ストレプトマイシン/グルタミン（富士フイルム和光純薬）を添加したDMEM培地（富士フイルム和光純薬）で培養した。10％FCS、25％L929馴化培地、ピルビン酸ナトリウム、HEPES、2MEおよびペニシリン-ストレプトマイシンを添加したRPMI（富士フイルム和光純薬）中で脛骨および大腿骨の骨髄前駆細胞を分化させることにより、骨髄由来マクロファージ（bone marrow derived macrophages、以下「BMDM」と記す）を得た。マクロファージを炎症状態にするために、LPS（10 ng/ml）で6時間刺激した。関節軟骨表層（SFZ: superficial zone）細胞（以下「SFZ細胞」と記す）はYasuharaら（Lab Invest 91, 1739-1752, doi:10.1038/labinvest.2011.144 (2011)）に記載の方法で単離した。すなわち、4日齢のマウスまたはPlxnb2 KOマウス作製用の17.5日胚の膝関節から大腿骨の近位端および脛骨の遠位端を切断し、靭帯および腱をそれらの付着部位から注意深く除去した。0.25％トリプシンを含むハンクス平衡塩溶液（HBSS）で組織を1時間インキュベートし、続いて0.25％コラゲナーゼ（富士フイルム和光純薬）を含むDMEMで1.5時間消化した。解離した細胞をBioCoatTMフィブロネクチンプレート（Sigma-Aldrich）に播種し、20分間インキュベートした。非接着細胞をリン酸緩衝生理食塩水（PBS）で2回洗い流し、接着細胞をSFZ細胞として使用した。SFZ細胞は10％FBSおよびペニシリン/ストレプトマイシン/グルタミンを含むDMEM培地で維持した。 (3) Cells and cell stimulation HEK293 cells were cultured in DMEM medium (Fujifilm Wako Pure Chemical Industries) supplemented with 10% FCS and penicillin / streptomycin / glutamine (Fujifilm Wako Pure Chemical Industries, Ltd.). Bone marrow-derived by differentiating tibial and femoral bone marrow progenitor cells in RPMI (Fujifilm Wako Pure Drug) supplemented with 10% FCS, 25% L929 acclimatized medium, sodium pyruvate, HEPES, 2ME and penicillin-streptomycin. Macrophages (bone marrow derived macrophages, hereinafter referred to as "BMDM") were obtained. The macrophages were stimulated with LPS (10 ng / ml) for 6 hours to make them inflamed. Articular cartilage surface layer (SFZ: superficial zone) cells (hereinafter referred to as "SFZ cells") were isolated by the method described in Yasuhara et al. (Lab Invest 91, 1739-1752, doi: 10.1038 / labinvest.2011.144 (2011)). .. That is, the proximal end of the femur and the distal end of the tibia were cut from the knee joint of a 17.5 day embryo for the production of 4-day-old mice or Plxnb2 KO mice, and ligaments and tendons were carefully removed from their attachment sites. Tissues were incubated with Hanks Balanced Salt Solution (HBSS) containing 0.25% trypsin for 1 hour, followed by digestion with DMEM containing 0.25% collagenase (Wako Pure Chemical Industries, Ltd.) for 1.5 hours. Dissociated cells were seeded on BioCoat TM fibronectin plates (Sigma-Aldrich) and incubated for 20 minutes. Non-adherent cells were rinsed twice with phosphate buffered saline (PBS) and adherent cells were used as SFZ cells. SFZ cells were maintained in DMEM medium containing 10% FBS and penicillin / streptomycin / glutamine.
　関節軟骨深層（DZ: deep zone）細胞は、コラゲナーゼによる残存軟骨組織の追加消化によって分離した。DZ細胞は10％FBSおよびペニシリン/ストレプトマイシン/グルタミンを含むDMEM培地で維持した。初代骨芽細胞は、生後4日齢のマウスの頭蓋冠をコラゲナーゼで連続消化することにより取得した。分散液から骨芽細胞を回収し、10％FBSおよびペニシリン/ストレプトマイシン/グルタミンを添加したα-MEM培地で培養した。肋軟骨細胞は、4日齢のマウスの肋骨をコラゲナーゼ（富士フイルム和光純薬）で消化することにより単離した。肋軟骨細胞を分散液から収集し、10％FBSおよびペニシリン/ストレプトマイシン/グルタミンを含むDMEM培地で培養した。刺激アッセイのために、アッセイの2時間前に無血清DMEMに培地交換し、各刺激剤で処理した。 Deep zone (DZ: deep zone) cells were separated by additional digestion of residual cartilage tissue with collagenase. DZ cells were maintained in DMEM medium containing 10% FBS and penicillin / streptomycin / glutamine. Primary osteoblasts were obtained by continuous digestion of the calvaria of 4-day-old mice with collagenase. Osteoblasts were collected from the dispersion and cultured in α-MEM medium supplemented with 10% FBS and penicillin / streptomycin / glutamine. Costal chondrocytes were isolated by digesting the ribs of 4-day-old mice with collagenase (Wako Pure Chemical Industries, Ltd.). Costal chondrocytes were collected from the dispersion and cultured in DMEM medium containing 10% FBS and penicillin / streptomycin / glutamine. For the stimulus assay, medium was replaced with serum-free DMEM 2 hours prior to the assay and treated with each stimulant.
（４）プラスミド、トランスフェクションおよびレンチウイルス感染
　Plexin-B2(S)-Fc-HisおよびPlexin-B2(L)-Fc-His、pCMV-myc-Sema4D、pBabe-c-Met、およびpmPlexin-B2-VSVのプラスミドはAddgene社から購入した。マウスIκBζおよびC/EBPδの発現ベクターはGenScript社から購入した。IκBζおよびC/EBPδをpLVSIN-CMVPur Vector（タカラバイオ）にサブクローニングした。トランスフェクション試薬X-tremeGENE 9（Sigma-Aldrich）を用いて、各プラスミドを細胞へ導入した。ノックダウン実験では、Plxnb1を標的とするshRNA（Plxnb1 shRNA、TRCN0000078917、TRCN0000078916）、Plxnb2を標的とするshRNA（Plxnb2 shRNA、TRCN0000078853、TRCN0000078857）、c-Metを標的とするshRNA（c-Met shRNA、TRCN0000226122、TRCN0000235）がクローニングされているレンチウイルスpLKO.1プラスミド（Sigma-Aldrich）を購入した。空ベクターをネガティブコントロールとして使用した。レンチウイルス粒子の調製と感染は、製造元（Sigma-Aldrich）のプロトコルに従って行った。コントロールまたはshRNAを発現するレンチウイルスをSFZ細胞に感染させ、ピューロマイシン（2μg/ml）を含む培地で選択した。レンチウイルスのタンパク質導入には、Lenti-X 293T細胞（タカラバイオ）にLentiviral High Titer Packaging Mix（タカラバイオ）と共にpLVSIN-IκBζまたはpLVSIN-C/EBPδを導入した。レンチウイルス粒子の調製と感染は、製造元（タカラバイオ）のプロトコルに従って行った。 (4) plasmid, transfection and lentivirus infection Plexin-B2 (S) -Fc-His and Plexin-B2 (L) -Fc-His, pCMV-myc-Sema4D, pBabe-c-Met, and pmPlexin-B2- The VSV plasmid was purchased from Addgene. Expression vectors for mouse IκBζ and C / EBPδ were purchased from GenScript. IκBζ and C / EBPδ were subcloned into pLVSIN-CMVPur Vector (Takara Bio). Each plasmid was introduced into cells using the transfection reagent X-tremeGENE 9 (Sigma-Aldrich). In knockdown experiments, Plxnb1 targeting shRNAs (Plxnb1 shRNA, TRCN0000078917, TRCN0000078916), Plxnb2 targeting shRNAs (Plxnb2 shRNA, TRCN0000078853, TRCN0000078857), and c-Met targeting shRNAs (c-Met shRNA, TRCN0000226122). , TRCN0000235) was cloned into the lentivirus pLKO.1 plasmid (Sigma-Aldrich). An empty vector was used as a negative control. Lentivirus particles were prepared and infected according to the manufacturer's (Sigma-Aldrich) protocol. Lentiviruses expressing controls or shRNA were infected with SFZ cells and selected in media containing puromycin (2 μg / ml). For lentivirus protein introduction, pLVSIN-IκBζ or pLVSIN-C / EBPδ was introduced into Lenti-X 293T cells (Takara Bio) together with Lentiviral High Titer Packaging Mix (Takara Bio). Lentivirus particles were prepared and infected according to the manufacturer's (Takara Bio) protocol.
（５）化学物質およびその他の試薬
　LPS（L3024）はSigma-Aldrichから購入した。組換えヒトsCD100/Sema4D（310-29）はPeproTechから購入し、特に指定がない限り2μg/mlで使用した。組換えマウスIL-6（094-07001）および組換えマウスIL-1β（094-04681）は富士フイルム和光純薬から購入した。組換えマウスTNFα（315-01A）および組換えヒトHGF（100-39）はPeproTech社から購入した。抗マウスIL-6中和抗体（14-7061-81）はInvitrogenから購入した。
　各種阻害剤として、c-Met阻害剤：SU11274（富士フイルム和光純薬、1μM）、K252a（abcam、1μM）およびクリゾチニブ（Sigma、0.5 μM）、MEK阻害剤：PD98059（Cell Signaling Technology、10μM）およびU0126（Cell Signaling Technology、10μM）、RAS阻害剤：サリラシブ（abcam、10μM）、Rhoキナーゼ阻害剤：RKI-1447（Sigma-Aldrich、5μM）およびY-27632（Sigma-Aldrich、10μM）、ErbB2阻害剤：AG825（abcam、1μM）、Akt阻害剤：MK-2206（Selleck、1μM）、翻訳阻害剤：シクロヘキシミド（富士フイルム和光純薬、100μg/ml）およびNF-κB阻害剤BAY11-7082（Sigma、10μM）を使用した。 (5) Chemicals and other reagents LPS (L3024) was purchased from Sigma-Aldrich. Recombinant human sCD100 / Sema4D (310-29) was purchased from PeproTech and used at 2 μg / ml unless otherwise specified. Recombinant mouse IL-6 (094-07001) and recombinant mouse IL-1β (094-04681) were purchased from Wako Pure Chemical Industries, Ltd. Recombinant mouse TNFα (315-01A) and recombinant human HGF (100-39) were purchased from PeproTech. Anti-mouse IL-6 neutralizing antibody (14-7061-81) was purchased from Invitrogen.
As various inhibitors, c-Met inhibitors: SU11274 (Fujifilm Wako Pure Drug, 1 μM), K252a (abcam, 1 μM) and crizotinib (Sigma, 0.5 μM), MEK inhibitors: PD98059 (Cell Signaling Technology, 10 μM) and U0126 (Cell Signaling Technology, 10 μM), RAS Inhibitor: Salilacib (abcam, 10 μM), Rho Kinase Inhibitor: RKI-1447 (Sigma-Aldrich, 5 μM) and Y-27632 (Sigma-Aldrich, 10 μM), ErbB2 Inhibitor : AG825 (abcam, 1 μM), Akt inhibitor: MK-2206 (Selleck, 1 μM), Translation inhibitor: Cycloheximide (Fujifilm Wako Pure Drug, 100 μg / ml) and NF-κB inhibitor BAY11-7082 (Sigma, 10 μM) )It was used.
（６）RT-qPCR（Reverse transcriptase quantitative polymerase chain reaction）
　Nucleospin RNA Plus（Macherey-Nagel）を使用して、細胞からトータルRNAを単離した。トータルRNAを変性させた後、ReverTra Ace qPCR RT Master Mix（東洋紡）を使用してトータルRNAからcDNAを合成した。リアルタイムPCRは、THUNDERBIRD SYBR qPCR Mix（東洋紡）およびABI Step One PlusリアルタイムPCRシステム（Applied Biosystems）を使用して実施した。標的mRNA量はβ-アクチンmRNA量に対して標準化した。相対的mRNA発現レベルは、比較CT法を使用して計算した。使用したプライマーセットを表1に示す。 (6) RT-qPCR (Reverse transcriptase quantitative polymerase chain reaction)
Total RNA was isolated from cells using Nucleospin RNA Plus (Macherey-Nagel). After denaturing the total RNA, cDNA was synthesized from the total RNA using ReverTra Ace qPCR RT Master Mix (Toyobo). Real-time PCR was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) and ABI Step One Plus real-time PCR systems (Applied Biosystems). The target mRNA amount was standardized with respect to the β-actin mRNA amount. Relative mRNA expression levels were calculated using the comparative CT method. The primer set used is shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
（７）免疫ブロッティング、ELISAおよび免疫細胞化学
　全細胞溶解物の免疫ブロッティングでは、冷PBSで洗浄した細胞をSDSサンプルバッファーで直接溶解した。細胞胞溶解物を直ちに10分間煮沸した。馴化培地、免疫沈降および分画サンプルの免疫ブロッティングでは、馴化培地またはタンパク質抽出物をSDSサンプルバッファーで煮沸した。タンパク質サンプルをSDS-PAGEで分離し、PVDFメンブレンに転写した。メンブレンを一次抗体で免疫ブロットし、西洋ワサビペルオキシダーゼが結合した抗マウスIgG抗体（Jackson、1：5,000）、抗ウサギ抗体IgG抗体（Jackson、1：5,000）、抗ヤギ抗体IgG抗体（Jackson、1：5000）および抗ヒツジIgG抗体（Jackson、1：5000）と、ペルオキシダーゼ発光基質Immuno Star LD（富士フイルム和光純薬）を使用して可視化した。一次抗体として、抗MMP13抗体（ab39012、1：2000）および抗Myc抗体（ab9132、1：2000）をAbcamから購入し、抗c-Met抗体（82202、1：1000）、抗NF-κB（p65）抗体（8242、1：1000）、抗Erk1/2抗体（4696、1：2000）、抗P-Erk1/2抗体（9101、1：2000）、抗IκBζ抗体（76041、1：1000）およびC/EBPδ抗体（2318、1：1000）をCell Signaling Technologyから購入し、抗リン酸化チロシン抗体（GTX14167, 1:1000）をGeneTexから購入し、抗FLAG抗体（M185-3、1：2000）をMBLから購入し、抗β-アクチン抗体（017-24573、1：5000）および抗ヒストンH3抗体（MABI0301、1：1000）を富士フイルム和光純薬から購入し、抗α-チューブリン抗体（T6074、1：2000）をSigmaから購入し、抗Plexin-B2抗体（PA547880、1：400）をInvitrogenから購入し、抗Sema4D抗体（AF5235、1：1000）をR＆D Systemsから購入した。各サンプル中のタンパク質の量を評価するために、抗β-アクチン抗体を用いた免疫ブロッティングを実施した。 (7) Immunoblotting, ELISA and immunocytochemistry In immunoblotting of whole cell lysates, cells washed with cold PBS were directly lysed with SDS sample buffer. The cell vesicle lysate was immediately boiled for 10 minutes. For conditioned medium, immunoprecipitation and immunoblotting of fractionated samples, conditioned medium or protein extract was boiled in SDS sample buffer. Protein samples were separated by SDS-PAGE and transferred to PVDF membranes. The membrane was immunoblotted with a primary antibody, and anti-mouse IgG antibody (Jackson, 1: 5,000) to which Western wasabi peroxidase was bound, anti-rabbit antibody IgG antibody (Jackson, 1: 5,000), anti-goat antibody IgG antibody (Jackson, 1: 5,000). Visualization was performed using 5000) and an anti-sheep IgG antibody (Jackson, 1: 5000) and the peroxidase luminescent substrate Immuno Star LD (Fujifilm Wako Junyaku). Anti-MMP13 antibody (ab39012, 1: 2000) and anti-Myc antibody (ab9132, 1: 2000) were purchased from Abcam as primary antibodies, and anti-c-Met antibody (82202, 1: 1000) and anti-NF-κB (p65). ) Antibodies (8242, 1: 1000), anti-Erk1 / 2 antibodies (4696, 1: 2000), anti-P-Erk1 / 2 antibodies (9101, 1: 2000), anti-IκBζ antibodies (76041, 1: 1000) and C / EBPδ antibody (2318, 1: 1000) purchased from Cell Signaling Technology, anti-phosphorylated tyrosine antibody (GTX14167, 1: 1000) purchased from GeneTex, anti-FLAG antibody (M185-3, 1: 2000) MBL Anti-β-actin antibody (017-24573, 1: 5000) and anti-histon H3 antibody (MABI0301, 1: 1000) were purchased from Fujifilm Wako Pure Chemicals and anti-α-tubulin antibody (T6074, 1). : 2000) was purchased from Sigma, anti-Plexin-B2 antibody (PA547880, 1: 400) was purchased from Invitrogen, and anti-Sema4D antibody (AF5235, 1: 1000) was purchased from R & D Systems. Immune blotting with anti-β-actin antibody was performed to assess the amount of protein in each sample.
　細胞成分分画（subcellular fractionation）では、冷PBSで洗浄した細胞を氷上で10分間、溶解緩衝液（10 mM HEPES, pH7.5、1 mM MgCl2、10 mM KCl、0.1% TritonX-100、20% Glycerol、phosphatase inhibitors、protease inhibitors）で溶解した。次に、細胞溶解液を3,000 gで10分間、4℃で遠心分離し、上清（細胞質画分）を回収した。ペレットを溶解緩衝液で洗浄した後、高塩溶解緩衝液（10 mM HEPES, pH7.5、400 mM NaCl、1 mM EDTA、0.1% TritonX-100、20% Glycerol、phosphatase inhibitors、protease inhibitors）に再懸濁して氷上で20分間冷却し、12,000gで10分間、4℃で遠心分離して、上清（核画分）を回収した。タンパク質抽出物を免疫ブロッティングで分析した。 In the subcellular fractionation, cells washed with cold PBS are lysed buffer (10 mM HEPES, pH 7.5, 1 mM MgCl 2 , 10 mM KCl, 0.1% TritonX-100, 20) on ice for 10 minutes. % Glycerol, phosphatase inhibitors, protease inhibitors). Next, the cytolytic solution was centrifuged at 3,000 g for 10 minutes at 4 ° C., and the supernatant (cytoplasmic fraction) was collected. After washing the pellet with lysis buffer, rehydrate with high salt lysis buffer (10 mM HEPES, pH 7.5, 400 mM NaCl, 1 mM EDTA, 0.1% TritonX-100, 20% Glycerol, phosphatase inhibitors, protease inhibitors). It was suspended, cooled on ice for 20 minutes, centrifuged at 12,000 g for 10 minutes at 4 ° C, and the supernatant (nuclear fraction) was collected. Protein extracts were analyzed by immunoblotting.
　ELISAでは、培養上清を、IL-6 ELISAキット（Invitrogen、88-7064-88）、TNFαELISAキット（Invitrogen、88-7324-88）およびIL-1βELISAキット（Invitrogen、88-7013-88）で分析した。
　免疫細胞化学では、一次抗体に抗NF-κB（p65）抗体（8242、1：400）を使用し、二次抗体に抗ウサギIgG抗体Alexa Fluor 568（1：500）（A11036、Invitrogen）を使用した。蛍光染色用封入剤VECTASHIELD Hard Set Mounting Medium with DAPI（VECTOR LABORATORIES）を重層してカバーグラスを掛け、蛍光顕微鏡（LEICA、DM4B）で観察した。 In ELISA, culture supernatants are analyzed with IL-6 ELISA kit (Invitrogen, 88-7064-88), TNFα ELISA kit (Invitrogen, 88-7324-88) and IL-1β ELISA kit (Invitrogen, 88-7013-88). did.
In immunocytochemistry, anti-NF-κB (p65) antibody (8242, 1: 400) is used as the primary antibody, and anti-rabbit IgG antibody Alexa Fluor 568 (1: 500) (A11036, Invitrogen) is used as the secondary antibody. did. VECTASHIELD Hard Set Mounting Medium with DAPI (VECTOR LABORATORIES) was layered, covered with a cover glass, and observed with a fluorescence microscope (LEICA, DM4B).
（８）器官培養
　関節軟骨の器官培養は、Stantonら（Nat Protoc 6, 388-404, doi:10.1038/nprot.2010.179 (2011)）の記載に従って行った。すなわち、3週齢のICRマウスから大腿骨頭を単離し、Sema4D（20μg/ml）もしくはIL-1β（10ng/ml）を含む無血清DMEM培地、またはどちらも含まない無血清DMEM培地で3日間培養した。大腿骨頭軟骨を4％パラホルムアルデヒドで固定した後、10％EDTAで2週間脱灰した。軟骨をパラフィン包埋し、厚さ5μmの切片を作製した。切片を脱パラフィンし、PBSと蒸留水で洗浄した後、0.2％ファストグリーンで10分間、0.1％サフラニンOで20分間染色した。培地へのアグリカン放出は、ジメチルメチレンブルーアッセイで分析した。 (8) Organ culture Organ culture of articular cartilage was performed according to the description of Stanton et al. (Nat Protoc 6, 388-404, doi: 10.1038 / nprot.2010.179 (2011)). That is, the femoral head was isolated from 3-week-old ICR mice and cultured for 3 days in serum-free DMEM medium containing Sema4D (20 μg / ml) or IL-1β (10 ng / ml), or serum-free DMEM medium containing neither. did. The femoral head cartilage was fixed with 4% paraformaldehyde and then decalcified with 10% EDTA for 2 weeks. The cartilage was embedded in paraffin to prepare a section having a thickness of 5 μm. The sections were deparaffinized, washed with PBS and distilled water, and then stained with 0.2% fast green for 10 minutes and with 0.1% safranin O for 20 minutes. Aggrecan release into the medium was analyzed by the dimethylmethylene blue assay.
（９）馴化培地（conditioned media、以下「CM」と記す）の分画
　骨髄由来マクロファージの培地をアッセイの2時間前に無血清DMEMに交換し、その後LPS（10 ng/ml）を添加してまたは添加せずに6時間処理した。CMを回収し、0.22μmフィルターでろ過した。ろ過したCMを、製造元のプロトコルに従って、50kDaおよび100kDaのアミコンフィルター（Merck Millipore）で順次分画した。CMの50～100kDa画分を質量分析に供した。 (9) Fractionation of conditioned media (hereinafter referred to as "CM") Bone marrow-derived macrophage medium was replaced with serum-free DMEM 2 hours before the assay, and then LPS (10 ng / ml) was added. Alternatively, it was treated for 6 hours without addition. CM was collected and filtered through a 0.22 μm filter. The filtered CM was sequentially fractionated with 50 kDa and 100 kDa Amicon filters (Merck Millipore) according to the manufacturer's protocol. A 50-100 kDa fraction of CM was subjected to mass spectrometry.
（１０）免疫沈降
　冷PBSで洗浄した細胞を、氷冷NP-40バッファー（1％NP-40、50 mM Tris-HCl pH 7.4、150 mM NaCl、プロテアーゼ阻害剤カクテルおよびホスファターゼ阻害剤カクテル（富士フイルム和光純薬））で4℃、30分間溶解した。細胞溶解液を回収し、12,000×gで4℃、10分間遠心分離した。上清をDynabeads Protein G（Invitrogen）と4℃で1時間前培養し、ビーズを除去した。次に、上清を抗FLAG抗体（M185-3, 1:400）、抗Plexin-B2抗体（PA547880, 1:50）または抗c-Met抗体（1:50）（3127, Cell Signaling）と4℃で一晩インキュベートした後、Dynabeads Protein Gと4℃で1時間インキュベートした。ビーズをNP-40バッファーで5回洗浄した。結合したタンパク質をSDSサンプルバッファーで溶出した後、免疫ブロッティングを行った。プレキシン-B2結合タンパク質を同定するために、結合したタンパク質を、抗プレキシン-B2抗体（1：100）（abcam、ab193355）を使用した免疫沈降により精製した。結合したタンパク質をSDSサンプルバッファーで溶出した後、質量分析を行った。 (10) Immunoprecipitation Cold PBS-washed cells were subjected to ice-cold NP-40 buffer (1% NP-40, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, protease inhibitor cocktail and phosphatase inhibitor cocktail (Fuji Film). Wako Pure Chemical Industries)) was dissolved at 4 ° C for 30 minutes. Cytolysis was collected and centrifuged at 12,000 xg at 4 ° C for 10 minutes. The supernatant was precultured with Dynabeads Protein G (Invitrogen) at 4 ° C. for 1 hour to remove beads. Next, the supernatant is divided into anti-FLAG antibody (M185-3, 1: 400), anti-Plexin-B2 antibody (PA547880, 1:50) or anti-c-Met antibody (1:50) (3127, Cell Signaling) and 4 After incubating overnight at ° C, it was incubated with Dynabeads Protein G at 4 ° C for 1 hour. The beads were washed 5 times with NP-40 buffer. The bound protein was eluted with SDS sample buffer and then immunoblotting was performed. To identify the plexin-B2-binding protein, the bound protein was purified by immunoprecipitation using an anti-plexin-B2 antibody (1: 100) (abcam, ab193355). The bound protein was eluted with SDS sample buffer and then mass spectrometric analysis was performed.
（１１）質量分析
　CM画分またはプレキシン-B2結合画分をSDS-PAGEで分離し、各レーンをいくつかの断片にスライスした。各ゲルスライスのタンパク質をトリプシン消化した。トリプシン消化ペプチドをLC-ESI-MS/MS（Thermo Fisher Scientific、LTQ Orbitrap Velos + ETD）で分析した後、ショットガンプロテオミクス分析を行った（Murakamiら、J Immunol 202, 1942-1947, doi:10.4049/jimmunol.1801388 (2019)）。 (11) Mass spectrometry The CM fraction or plexin-B2 binding fraction was separated by SDS-PAGE, and each lane was sliced into several fragments. The protein in each gel slice was trypsin digested. Tryptic digestive peptides were analyzed by LC-ESI-MS / MS (Thermo Fisher Scientific, LTQ Orbitrap Velos + ETD) and then shotgun proteomics analysis (Murakami et al., J Immunol 202, 1942-1947, doi: 10.4049 / jimmunol.1801388 (2019)).
（１２）RNA-シーケシング（RNA-Seq）およびマイクロアレイ分析
　RNA-Seq用に、Nucleospin RNA Plusを使用してSFZ細胞からトータルRNAを単離した。ライブラリーの調製は、 TruSeq stranded mRNA sample prep kit（Illumina）を使用して、製造元の指示に従って行った。シーケンスは、Illumina NovaSeq 6000 プラットフォームで101塩基のシングルエンドモードで実行した。ベースコールにIllumina Casava1.8.2ソフトウェアを使用した。配列リードは、TopHat v2.0.13と、Bowtie2 ver.2.2.3およびSAMtools ver.0.1.19を組み合わせて用いて、マウス参照ゲノム配列（mm10）にマッピングした。FPKMs（fragments per kilobase of exon per million mapped fragments）は、Cufflinks version 2.2.1.を使用して計算した。スクリーニングされた遺伝子を評価するために、iDEP（http://bioinformatics.sdstate.edu/idep/）を使用したバイオインフォマティクス分析を行った。 (12) RNA-Seqing (RNA-Seq) and Microarray Analysis Total RNA was isolated from SFZ cells using Nucleospin RNA Plus for RNA-Seq. The library was prepared using the TruSeq stranded mRNA sample prep kit (Illumina) and according to the manufacturer's instructions. The sequence was run in single-ended mode with 101 bases on the Illumina NovaSeq 6000 platform. I used Illumina Casava 1.8.2 software for the base call. Sequence reads were mapped to the mouse reference genome sequence (mm10) using TopHat v2.0.13 in combination with Bowtie2 ver.2.2.3 and SAMtools ver.0.1.19. FPKMs (fragments per kilobase of exon per million mapped fragments) were calculated using Cufflinks version 2.2.1. Bioinformatics analysis using iDEP (http://bioinformatics.sdstate.edu/idep/) was performed to evaluate the screened genes.
　マイクロアレイ用に、Nucleospin RNA Plusを使用してBMDMからトータルRNAを単離した。マイクロアレイ分析は、Affymetrix Mouse Genome 430 2.0 Array（Affymetrix）を使用して、製造元のプロトコルに従って行った。 Total RNA was isolated from BMDM using Nucleospin RNA Plus for microarrays. Microarray analysis was performed using Affymetrix Mouse Genome 430 2.0 Array (Affymetrix) according to the manufacturer's protocol.
（１３）ルシフェラーゼレポーターアッセイ
　SFZ細胞に、NF-κB responsible elementsを含むルシフェラーゼレポータープラスミド（Clontech）を、トランスフェクション試薬X-tremeGENE 9（Sigma-Aldrich）を用いて導入した。トランスフェクションの24時間後、細胞をSema4D（2μg/ml）で12時間処理した。IL-1β（10 ng/ml）で処理した細胞を陽性コントロール、未処理細胞を陰性コントロールとした。Luciferase Assay System（Promega）を用いて製造元のプロトコルに従ってルシフェラーゼ活性を測定した。 (13) Luciferase Reporter Assay A luciferase reporter plasmid (Clontech) containing NF-κB responsible elements was introduced into SFZ cells using the transfection reagent X-tremeGENE 9 (Sigma-Aldrich). Twenty-four hours after transfection, cells were treated with Sema4D (2 μg / ml) for 12 hours. Cells treated with IL-1β (10 ng / ml) were designated as positive controls, and untreated cells were designated as negative controls. Luciferase activity was measured using the Luciferase Assay System (Promega) according to the manufacturer's protocol.
（１４）統計解析
　2群間の比較は、対応のない両側スチューデントt検定を使用して分析した。多群間の比較は、一元配置分散分析または二元配置分散分析に続くボンフェローニ事後検定を使用して分析した。p値が0.05未満である場合に有意差があるとみなした。すべてのデータは、平均±標準誤差（SEM）で表した。 (14) Statistical analysis The comparison between the two groups was analyzed using the unpaired two-sided Student's t-test. Multi-group comparisons were analyzed using a Bonferroni post-test following a one-way ANOVA or a two-way ANOVA. A significant difference was considered when the p-value was less than 0.05. All data are expressed as mean ± standard error (SEM).
〔参考例１：LPS処理したマクロファージは関節軟骨の破壊に関与する〕
　炎症を誘導したマクロファージが、関節軟骨の破壊に関与するか否かを確認するために、LPS処理したBMDMのCM、またはLPS処理していないBMDMのCMで、SFZ細胞を16時間培養し、マトリックスメタロプロテイナーゼ（以下「Mmp」と記す）13の発現レベルをRT-qPCRで分析した。対照としてSFZ細胞を通常の培地で培養した群（control）およびSFZ細胞の通常の培地にLPSを直接添加した群（LPS）を設けた。 [Reference Example 1: LPS-treated macrophages are involved in the destruction of articular cartilage]
To determine whether inflammation-inducing macrophages are involved in the destruction of articular cartilage, SFZ cells were cultured for 16 hours in LPS-treated BMDM CM or LPS-untreated BMDM CM for 16 hours and matrix. The expression level of metalloproteinase (hereinafter referred to as "Mmp") 13 was analyzed by RT-qPCR. As a control, a group in which SFZ cells were cultured in a normal medium (control) and a group in which LPS was directly added to the normal medium of SFZ cells (LPS) were provided.
　結果を図1に示した。LPS処理していないBMDMのCM（図中CM(-)）で培養したSFZ細胞はMmp13を発現しなかったが、LPS処理したBMDMのCM（図中CM(LPS)）で培養したSFZ細胞は顕著にMmp13発現を誘導した。 The results are shown in Fig. 1. SFZ cells cultured with LPS-treated BMDM CM (CM (-) in the figure) did not express Mmp13, but SFZ cells cultured with LPS-treated BMDM CM (CM (LPS) in the figure) did not express Mmp13. Remarkably induced Mmp13 expression.
〔実施例１：LPS処理したBMDMのCMに存在するMmp13発現誘導因子の特定〕
１－１　ショットガン質量分析
　LPS処理したBMDMから分泌される可溶性因子を特定するために、CMに存在するタンパク質のショットガン質量分析を行い、以下の表2に示す32種類の候補タンパク質を検出した。 [Example 1: Identification of Mmp13 expression-inducing factor present in CM of BMDM treated with LPS]
1-1 Shotgun Mass Spectrometry In order to identify soluble factors secreted from LPS-treated BMDM, shotgun mass spectrometry of proteins present in CM was performed, and 32 types of candidate proteins shown in Table 2 below were detected. ..
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
１－２　Mmp13発現誘導因子の分子量予測
　LPS処理したBMDMのCMを50kDaで分画し、CMの<50kDa画分、CMの>50kDa画分、分画していないCMでSFZ細胞を16時間培養し、Mmp13の発現レベルをRT-qPCRで分析した。また、LPS処理したBMDMのCMを100kDaで分画し、CMの<100kDa画分、CMの>100kDa画分、分画していないCMでSFZ細胞を16時間培養し、Mmp13の発現レベルをRT-qPCRで分析した（独立した実験を2回行った）。 1-2 Mmp13 Expression Inducing Factor Molecular Weight Prediction LPS-treated BMDM CM was fractionated by 50 kDa, and SFZ cells were cultured for 16 hours in CM <50 kDa fraction, CM> 50 kDa fraction, and unfractionated CM. Then, the expression level of Mmp13 was analyzed by RT-qPCR. In addition, LPS-treated BMDM CM was fractionated by 100 kDa, SFZ cells were cultured for 16 hours in CM <100 kDa fraction, CM> 100 kDa fraction, and unfractionated CM, and the expression level of Mmp13 was RT. -analyzed by PCR (two independent experiments).
　結果を図2に示した（A）が50kDaで分画した場合の結果、（B）が100kDaで分画した場合の結果である。（A）では>50kDa画分でMmp13が発現し、<50kDa画分ではMmp13がほとんど発現しなかった。（B）では>100kDa画分より<100kDa画分のほうがMmp13の発現レベルが顕著に高かった。この結果から、質量分析による32種類の候補タンパク質中、セマフォリン4D（Sema4D）に着目した。 The results shown in Fig. 2 are the results when (A) is fractionated by 50 kDa and (B) is the result when fractionated by 100 kDa. In (A), Mmp13 was expressed in the> 50 kDa fraction, and Mmp13 was hardly expressed in the <50 kDa fraction. In (B), the expression level of Mmp13 was significantly higher in the <100 kDa fraction than in the> 100 kDa fraction. From this result, we focused on semaphorin 4D (Sema4D) among 32 types of candidate proteins by mass spectrometry.
１－３　LPS処理したBMDMにおけるセマフォリンファミリー遺伝子発現レベルのマイクロアレイ分析
　LPS処理したBMDMおよびLPS処理していないBMDMからそれぞれトータルRNAを単離し、マイクロアレイ分析に供した。
　結果を図3に示した。BMDMはLPS処理の有無に関わらずSema4dを高発現することが示された。この結果から、LPS処理は、通常膜貫通タンパク質として存在するSema4Dのシェディングおよび可溶型Sema4Dの分泌を刺激すると考えられた。 1-3 Microarray analysis of semaphorin family gene expression levels in LPS-treated BMDM Total RNA was isolated from LPS-treated BMDM and LPS-untreated BMDM, respectively, and subjected to microarray analysis.
The results are shown in Fig. 3. BMDM was shown to highly express Sema4d with or without LPS treatment. From this result, LPS treatment was considered to stimulate the shedding of Sema4D, which normally exists as a transmembrane protein, and the secretion of soluble Sema4D.
１－４　LPS処理したBMDMにおけるSema4Dをシェディングする酵素の発現分析
　LPS処理したBMDMおよびLPS処理していないBMDMからそれぞれトータルRNAを単離し、Sema4Dをシェディングすることが知られているシェディング酵素（Adamts4、Mmp14、Adam17）の発現レベルをRT-qPCRで分析した（各n=2）。
　結果を図4に示した。（A）がAdamts4の結果、（B）がMmp14の結果、（C）がAdam17の結果である。BMDMをLPSで刺激することにより、Sema4Dのシェディング酵素であるAdamts4、Mmp14およびAdam17の発現を誘導することが確認された。 1-4 Expression analysis of Sema4D-sheding enzyme in LPS-treated BMDM A shedding enzyme known to shed Sema4D by isolating total RNA from LPS-treated BMDM and LPS-untreated BMDM, respectively. The expression levels of (Adamts4, Mmp14, Adam17) were analyzed by RT-qPCR (n = 2 each).
The results are shown in Fig. 4. (A) is the result of Adamts4, (B) is the result of Mmp14, and (C) is the result of Adam17. It was confirmed that stimulation of BMDM with LPS induces the expression of Adamts4, Mmp14 and Adam17, which are the shedding enzymes of Sema4D.
１－５　LPS処理したBMDMにおける可溶型Sema4Dの発現分析
　LPS処理したBMDMおよびLPS処理していないBMDMから分泌される可溶型Sema4Dを、免疫ブロッティングで検出した。
　結果を図5に示した。LPS処理は、BMDMからの可溶型Sema4Dの分泌を著しく増加させた。 1-5 Expression analysis of soluble Sema4D in LPS-treated BMDM Soluble Sema4D secreted from LPS-treated BMDM and LPS-untreated BMDM was detected by immunoblotting.
The results are shown in Fig. 5. LPS treatment significantly increased the secretion of soluble Sema4D from BMDM.
〔実施例２：軟骨細胞に対するSema4Dの効果〕
２－１　Sema4DによるSFZ細胞のMmp13発現誘導
　SFZ細胞の培地に0.5、1または2μg/mlの組換えSema4Dを添加して12時間培養し、Mmp13の発現量をRT-qPCRで分析した（n=3）。また、SFZ細胞の培地に2μg/mlの組換えSema4Dを添加して6、12または24時間培養し、Mmp13の発現量をRT-qPCRおよび免疫ブロッティングで分析した（n=3）。
　RT-qPCRの結果を図6に示した。（A）が3段階のSema4D濃度で培養したときのRT-qPCRの結果、（B）が3段階の培養時間で培養したときのRT-qPCRの結果、（C）が免疫ブロッティングの結果である。Sema4Dは、SFZ細胞において用量依存的および時間依存的にMmp13発現を誘導した（**P <0.01, ***P <0.001）。また、Sema4Dは、MMP13タンパク質を顕著に増加させた。 [Example 2: Effect of Sema4D on chondrocytes]
2-1 Induction of Mmp13 expression in SFZ cells by Sema4D 0.5, 1 or 2 μg / ml of recombinant Sema4D was added to the SFZ cell medium and cultured for 12 hours, and the expression level of Mmp13 was analyzed by RT-qPCR (n =). 3). In addition, 2 μg / ml recombinant Sema4D was added to the medium of SFZ cells and cultured for 6, 12 or 24 hours, and the expression level of Mmp13 was analyzed by RT-qPCR and immunobrotting (n = 3).
The results of RT-qPCR are shown in Fig. 6. (A) is the result of RT-qPCR when culturing at three stages of Sema4D concentration, (B) is the result of RT-qPCR when culturing at three stages of culture time, and (C) is the result of immune blotting. .. Sema4D induced dose- and time-dependent expression of Mmp13 in SFZ cells (** P <0.01, *** P <0.001). Sema4D also significantly increased the MMP13 protein.
２－２　Sema4Dによる肋軟骨細胞、関節軟骨深層細胞、骨芽細胞および骨髄由来マクロファージ（BMDM）のMmp13発現誘導
　各細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに、12時間培養し、Mmp13の発現量をRT-qPCRで分析した（n=2）。SFZ細胞を対照として用いた。
　結果を図7に示した。（A）が肋軟骨細胞（図中CC）の結果、（B）が関節軟骨深層細胞（図中DZ）の結果、（C）が骨芽細胞（図中OB）の結果、（D）がBMDMの結果である。Sema4Dは、SFZ細胞以外に、肋軟骨細胞および関節軟骨深層細胞においてもMmp13の発現を誘導した。骨芽細胞に対するMmp13の発現誘導は弱く、マクロファージに対してはMmp13の発現を誘導しなかった。 2-2 Induction of Mmp13 expression of costal chondrocytes, deep articular cartilage cells, osteoblasts and bone marrow-derived macrophages (BMDM) by Sema4D With or without addition of 2 μg / ml recombinant Sema4D to the medium of each cell , 12 hours culture, and the expression level of Mmp13 was analyzed by RT-qPCR (n = 2). SFZ cells were used as controls.
The results are shown in Fig. 7. (A) is the result of costal chondrocytes (CC in the figure), (B) is the result of deep articular cartilage cells (DZ in the figure), (C) is the result of osteoblasts (OB in the figure), and (D) is. It is the result of BMDM. Sema4D induced the expression of Mmp13 not only in SFZ cells but also in costal cartilage cells and deep articular cartilage cells. The induction of Mmp13 expression on osteoblasts was weak, and the expression of Mmp13 was not induced on macrophages.
２－３　SFZ細胞における軟骨マトリックス分解酵素の発現分析
　SFZ細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに、12時間培養し、RNAシーケンシング分析を行った（n=3）。
　軟骨マトリックス分解酵素の発現レベルを分析した結果を図8に示した。（A）がMmp13の結果、（B）がMmp3の結果、（C）がAdamts4の結果、（D）がAdamts5の結果である。Sema4Dは、SFZ細胞の軟骨マトリックス分解酵素の発現レベルを上昇させることが示された（***P <0.001, ****P <0.0001）。
　これらの結果は、Sema4Dが炎症性サイトカインとして機能し、SFZ細胞の軟骨マトリックス分解酵素の発現誘導を介して軟骨破壊に関与していることを示唆するものである。 2-3 Expression analysis of cartilage matrix-degrading enzyme in SFZ cells RNA-seqing analysis was performed by culturing for 12 hours with or without addition of 2 μg / ml recombinant Sema4D to the medium of SFZ cells (n). = 3).
The results of analysis of the expression level of cartilage matrix degrading enzyme are shown in Fig. 8. (A) is the result of Mmp13, (B) is the result of Mmp3, (C) is the result of Adamts4, and (D) is the result of Adamts5. Sema4D has been shown to increase the expression level of cartilage matrix degrading enzymes in SFZ cells (*** P <0.001, **** P <0.0001).
These results suggest that Sema4D functions as an inflammatory cytokine and is involved in cartilage destruction through induction of expression of cartilage matrix-degrading enzyme in SFZ cells.
〔実施例３：軟骨組織に対するSema4Dの効果〕
３－１　マウス関節軟骨の器官培養
　実験方法（８）に記載の方法で器官培養したマウス大腿骨頭軟骨の組織切片を、サフラニンOおよびファストグリーンで染色し、顕微鏡で観察した。IL-1βは陽性対照である。
　結果を図9に示した。（A）がコントロール（陰性対照）の結果、（B）がSema4Dを含む培地で培養した結果、（C）がIL-1βを含む培地で培養した結果である。スケールバーは200μmである。Sema4Dは、軟骨の表層および中間層においてサフラニンOによって染色されるプロテオグリカンを減少させた。 [Example 3: Effect of Sema4D on cartilage tissue]
3-1 Organ culture of mouse articular cartilage Tissue sections of mouse femoral head cartilage organ-cultured by the method described in Experimental Method (8) were stained with safranin O and fast green and observed under a microscope. IL-1β is a positive control.
The results are shown in Fig. 9. (A) is the result of control (negative control), (B) is the result of culturing in the medium containing Sema4D, and (C) is the result of culturing in the medium containing IL-1β. The scale bar is 200 μm. Sema4D reduced proteoglycans stained with safranin O in the superficial and intermediate layers of cartilage.
　培地へのアグリカン放出をジメチルメチレンブルーアッセイで測定した（n=6）。
　結果を図10に示した。Sema4Dは、軟骨からのアグリカンの放出を有意に増加させた（***P <0.001, ****P <0.0001）。 Aggrecan release into the medium was measured by the dimethylmethylene blue assay (n = 6).
The results are shown in Fig. 10. Sema4D significantly increased the release of agrecan from cartilage (*** P <0.001, **** P <0.0001).
３－２　Sema4KOマウスを用いた検討
　実験方法（２）に記載の方法で、Sema4d KOマウスおよび野生型（WT）マウスにコラーゲン-LPS誘発関節炎を誘発し、関節炎の臨床スコアを決定した。また、マウスの足（paws）の組織標本を作製し、顕微鏡で観察してびらんスコアを決定した。
　HE染色した組織標本の観察結果を図11に示した。（A）が野生型マウスの結果、（B）がSema4d KOマウスの結果である。軟骨破壊は、野生型マウスと比較してSema4d KOマウスにおいて減少していた。
　関節炎の臨床スコアのLPS投与後の経時変化を図12に示した。図中Day0はLPS投与日である。野生型マウスと比較してSema4d KOマウスのスコアは有意に低下した（*P <0.05, **P <0.01）。
　組織標本におけるびらんスコアの結果を図13に示した。野生型マウスと比較してSema4d KOマウスのスコアは有意に低下した（*P <0.05）。 3-2 Examination using Sema4KO mice Collagen-LPS-induced arthritis was induced in Sema4d KO mice and wild-type (WT) mice by the method described in Experimental Method (2), and the clinical score of arthritis was determined. In addition, tissue specimens of mouse paws were prepared and observed under a microscope to determine the erosion score.
The observation results of the HE-stained tissue specimen are shown in Fig. 11. (A) is the result of wild-type mouse, and (B) is the result of Sema4d KO mouse. Cartilage destruction was reduced in Sema4d KO mice compared to wild-type mice.
Figure 12 shows the change over time in the clinical score of arthritis after LPS administration. Day 0 in the figure is the LPS administration day. Sema4d KO mice scored significantly lower than wild-type mice (* P <0.05, ** P <0.01).
The results of the erosion score in the tissue specimen are shown in Fig. 13. Sema4d KO mice scored significantly lower than wild-type mice (* P <0.05).
〔実施例４：Sema4D依存性Mmp13発現誘導に関与する分子の探索〕
４－１　SFZ細胞におけるSema4D受容体の発現レベル
　SFZ細胞におけるSema4D受容体の発現レベルをRNA-Seqで評価した。
　結果を図14に示した。SFZ細胞において、プレキシン-B1（図中Plxnb1）、プレキシン-B3（図中Plxnb3）およびCD72（図中Cd72）と比較してプレキシン-B2（図中Plxnb2）の発現レベルか高いことが示された。 [Example 4: Search for molecules involved in Sema4D-dependent Mmp13 expression induction]
4-1 Expression level of Sema4D receptor in SFZ cells The expression level of Sema4D receptor in SFZ cells was evaluated by RNA-Seq.
The results are shown in FIG. It was shown that the expression level of Plexin-B2 (Plxnb2 in the figure) was higher than that of Plexin-B1 (Plxnb1 in the figure), Plexin-B3 (Plxnb3 in the figure) and CD72 (Cd72 in the figure) in SFZ cells. ..
４－２　プレキシン-B2ノックダウン
　SFZ細胞に、プレキシン-B1を標的とするshRNA（Plxnb1 shRNA）発現ベクター、プレキシン-B2を標的とするshRNA（Plxnb2 shRNA）発現ベクターまたは空ベクター（Control）をトランスフェクションした。それぞれ、Sema4Dで処理する群と処理しない群を設けた。各細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、Mmp13の発現量をRT-qPCRで分析した（n=3）。
　結果を図15に示した。プレキシン-B1をノックダウンしてもSema4D処理によりMmp13の発現が誘導されたが（図中shPlxnB1+Sema4D）、プレキシン-B2をノックダウンするとSema4D処理を行ってもMmp13の発現が誘導されなかった（図中shPlxnB2+Sema4D）。したがって、プレキシン-B2が主にSema4D依存性Mmp13発現誘導に関与していることが示された。 4-2 Plexin-B2 knockdown SFZ cells are transfected with a Plexin-B1 targeting shRNA (Plxnb1 shRNA) expression vector, a Plexin-B2 targeting shRNA (Plxnb2 shRNA) expression vector, or an empty vector (Control). did. A group treated with Sema4D and a group not treated with Sema4D were provided, respectively. The medium of each cell was cultured for 12 hours with or without 2 μg / ml recombinant Sema4D, and the expression level of Mmp13 was analyzed by RT-qPCR (n = 3).
The results are shown in FIG. Knockdown of plexin-B1 induced Mmp13 expression by Sema4D treatment (shPlxnB1 + Sema4D in the figure), but knockdown of plexin-B2 did not induce Mmp13 expression by Sema4D treatment (Sema4D treatment). In the figure shPlxnB2 + Sema4D). Therefore, it was shown that plexin-B2 is mainly involved in the induction of Sema4D-dependent Mmp13 expression.
４－３　プレキシン-B2 KOマウスのSFZ細胞に対するSema4Dの効果
　野生型マウスまたはPlxnb2 KO同腹児からSFZ細胞を調製した。それぞれ、Sema4Dで処理する群と処理しない群を設けた。各細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、Mmp13の発現量（n=3）およびMmp3の発現量（n=3）をRT-qPCRで分析した。
　結果を図16に示した。（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。プレキシン-B2の欠損により、Sema4D依存性のMmp13発現およびMmp3発現が有意に抑制されることが示された（****P <0.0001）。 4-3 Effect of Sema4D on SFZ cells in Plexin-B2 KO mice SFZ cells were prepared from wild-type mice or Plxnb2 KO littermates. A group treated with Sema4D and a group not treated with Sema4D were provided, respectively. Incubate for 12 hours with or without addition of 2 μg / ml recombinant Sema4D to the medium of each cell, and RT-qPCR the expression level of Mmp13 (n = 3) and the expression level of Mmp3 (n = 3). Analyzed in.
The results are shown in FIG. (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. It was shown that plexin-B2 deficiency significantly suppressed Sema4D-dependent Mmp13 and Mmp3 expression (**** P <0.0001).
４－４　各種阻害剤のSema4D依存性Mmp13発現誘導に対する効果の確認
　c-Met阻害剤としてSU11274（1μM）およびK252a（1μM）、MEK阻害剤としてPD98059（10μM）およびU0126（10μM）、RAS阻害剤としてサリラシブ（10μM）、Rhoキナーゼ阻害剤としてRKI-1447（5μM）およびY-27632（10μM）、ErbB2阻害剤としてAG825（1μM）、Akt阻害剤としてMK-2206（1μM）を使用した。SFZ細胞を、Sema4D（2μg/ml）またはSema4Dと各種阻害剤で12時間処理した。未処理のSFZ細胞をコントロールとした。Mmp13の発現量（各n=2）をRT-qPCRで分析した。
　結果を図17（A）～（D）に示した。c-Met阻害剤、RAS阻害剤およびMEK阻害剤はSema4D依存性Mmp13発現誘導を抑制した。一方、Rhoキナーゼ阻害剤、ErbB2阻害剤およびAkt阻害剤はSema4D依存性Mmp13発現誘導を抑制しなかった。 4-4 Confirmation of effects of various inhibitors on Sema4D-dependent Mmp13 expression induction c-Met inhibitors SU11274 (1 μM) and K252a (1 μM), MEK inhibitors PD98059 (10 μM) and U0126 (10 μM), RAS inhibitors Salilacib (10 μM) was used as a Rho kinase inhibitor, RKI-1447 (5 μM) and Y-27632 (10 μM) were used as Rho kinase inhibitors, AG825 (1 μM) was used as an ErbB2 inhibitor, and MK-2206 (1 μM) was used as an Akt inhibitor. SFZ cells were treated with Sema4D (2 μg / ml) or Sema4D and various inhibitors for 12 hours. Untreated SFZ cells were used as controls. The expression level of Mmp13 (n = 2 each) was analyzed by RT-qPCR.
The results are shown in FIGS. 17 (A) to 17 (D). c-Met inhibitors, RAS inhibitors and MEK inhibitors suppressed Sema4D-dependent Mmp13 expression induction. On the other hand, Rho-kinase inhibitors, ErbB2 inhibitors and Akt inhibitors did not suppress Sema4D-dependent Mmp13 expression induction.
４－５　c-Metノックダウン
　SFZ細胞に、c-Metを標的とするshRNA（c-Met shRNA）発現ベクターまたは空ベクター（Control）をトランスフェクションした。c-Met shRNA発現ベクターをトランスフェクションしたSFZ細胞から、2クローン（shcMet1およびshcMet2）を実験に使用した。各細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、c-Metの発現量（n=2）およびMmp13の発現量（n=2）をRT-qPCRで分析した。
　結果を図18に示した。（A）がc-Metの発現量の結果、（B）がMmp13の発現量の結果である。c-Metをノックダウンすると、Sema4D依存性のMmp13発現誘導が著しく阻害された。 4-5 c-Met knockdown SFZ cells were transfected with a c-Met-targeted shRNA (c-Met shRNA) expression vector or an empty vector (Control). Two clones (shcMet1 and shcMet2) from SFZ cells transfected with the c-Met shRNA expression vector were used in the experiment. Incubate for 12 hours with or without 2 μg / ml recombinant Sema4D in the medium of each cell, and RT the expression level of c-Met (n = 2) and the expression level of Mmp13 (n = 2). Analyzed by -qPCR.
The results are shown in FIG. (A) is the result of the expression level of c-Met, and (B) is the result of the expression level of Mmp13. Knockdown of c-Met markedly inhibited Sema4D-dependent induction of Mmp13 expression.
４－６　抗c-Met抗体を用いた免疫沈降
　SFZ細胞をSema4DまたはHGF（hepatocyte growth factor）で1分間または5分間処理した。コントロールには未処理のSFZ細胞を用いた。細胞溶解液を調製し、抗c-Met抗体を添加して免疫沈降物を回収した。免疫沈降タンパク質におけるリン酸化c-Metおよび細胞溶解液におけるリン酸化Erk l/2を、抗リン酸化チロシン抗体および抗リン酸化Erk l/2抗体を用いた免疫ブロッティングによって分析した。
　結果を図19に示した。Sema4Dはc-Metのリン酸化とSFZ細胞のc-Metの下流にあるErk l/2のリン酸化を刺激することが示された。 4-6 Immunoprecipitated SFZ cells with anti-c-Met antibody were treated with Sema4D or HGF (hepatocyte growth factor) for 1 or 5 minutes. Untreated SFZ cells were used as controls. Cytolysis was prepared, anti-c-Met antibody was added, and the immunoprecipitate was recovered. Phosphorylated c-Met in immunoprecipitated proteins and phosphorylated Erk l / 2 in cytolysis were analyzed by immunobrotting with anti-phosphorylated tyrosine and anti-phosphorylated Erk l / 2 antibodies.
The results are shown in FIG. Sema4D has been shown to stimulate c-Met phosphorylation and Erk l / 2 phosphorylation downstream of c-Met in SFZ cells.
　以上より、Semc4Dは、プレキシン-B2経路とc-Met-Ras-Erk l/2経路の両方を介して、SFZ細胞に対する効果を伝達することがわかった。 From the above, it was found that Semc4D transmits the effect on SFZ cells via both the plexin-B2 pathway and the c-Met-Ras-Erk l / 2 pathway.
〔実施例５：SFZ細胞におけるセマフォリン4D/プレキシン-B2シグナル伝達を媒介する分子の探索〕
　セマフォリン-プレキシンシステムは、主にRhoファミリーメンバーであるRhoAを介して、アクチン細胞骨格の再配列を調節することにより、細胞の形態と遊走を調節することが知られている（Perrot et al., J Biol Chem 277, 43115-43120 (2002)）。しかし、Rhoキナーゼ阻害剤はSema4D依存性Mmp13発現誘導を抑制しなかった（図17（c）、（d）参照）。この結果は、他のシグナル伝達経路がSFZ細胞のプレキシン-B2のシグナルを媒介してMmp13発現を調節することを示唆している。 [Example 5: Search for molecules that mediate semaphorin 4D / plexin-B2 signaling in SFZ cells]
The semaphorin-plexin system is known to regulate cell morphology and migration by regulating the rearrangement of the actin cytoskeleton, primarily via RhoA, a member of the Rho family (Perrot et al). ., J Biol Chem 277, 43115-43120 (2002)). However, Rho-kinase inhibitors did not suppress Sema4D-dependent Mmp13 expression induction (see FIGS. 17 (c) and 17 (d)). This result suggests that other signaling pathways mediate the signal of plexin-B2 in SFZ cells to regulate Mmp13 expression.
５－１　SFZ細胞におけるプレキシン-B2結合タンパク質のショットガン質量分析
　実験方法（１０）および（１１）に記載の方法でプレキシン-B2結合タンパク質のショットガン質量分析を行った。結果を表3に示した。NF-κBシグナル伝達経路の調節因子であるTNF受容体関連因子2（TRAF2）がプレキシン-B2に結合することを見出した。 Shotgun mass spectrometry of plexin-B2-binding protein in 5-1 SFZ cells Shotgun mass spectrometry of plexin-B2-binding protein was performed by the methods described in Experimental Methods (10) and (11). The results are shown in Table 3. We found that TNF receptor-related factor 2 (TRAF2), a regulator of the NF-κB signaling pathway, binds to plexin-B2.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
５－２　プレキシン-B2とTRAF2の会合の確認
　HEK293T細胞に、Plexin-B2発現ベクターおよびFLAG-TRAF2発現ベクターのいずれか一方または両方を導入した。発現ベクターを導入していないHEK293T細胞をコントロールとした。36時間細胞を培養した後細胞溶解液を調製した。細胞溶解液に抗Plexin-B2抗体または抗FLAG抗体を添加して免疫沈降物を回収した。抗Plexin-B2抗体による免疫沈降タンパク質、抗FLAG抗体による免疫沈降タンパク質、および後細胞溶解液を免疫ブロッティングに供し、抗Plexin-B2抗体および抗FLAG抗体で分析した。
　結果を図20に示した。Plexin-B2発現ベクターとFLAG-TRAF2発現ベクターを両方導入した細胞では、プレキシン-B2とTRAF2とが会合することが確認された。 5-2 Confirmation of association between plexin-B2 and TRAF2 HEK293T cells were introduced with either or both of the Plexin-B2 expression vector and the FLAG-TRAF2 expression vector. HEK293T cells into which an expression vector had not been introduced were used as controls. After culturing the cells for 36 hours, a cytolytic solution was prepared. Anti-Plexin-B2 antibody or anti-FLAG antibody was added to the cytolytic solution to recover the immunoprecipitate. Immunoprecipitated protein with anti-Plexin-B2 antibody, immunoprecipitated protein with anti-FLAG antibody, and post-cytolytic solution were subjected to immunoblotting and analyzed with anti-Plexin-B2 antibody and anti-FLAG antibody.
The results are shown in FIG. It was confirmed that Plexin-B2 and TRAF2 were associated in cells into which both the Plexin-B2 expression vector and the FLAG-TRAF2 expression vector were introduced.
５－３　Sema4DによるNF-κBの核移行誘導の確認
　SFZ細胞をSema4Dで1時間または2時間処理した。陽性コントロールとして、SFZ細胞をIL-1βで1時間処理した。未処理のSFZ細胞を陰性コントロールとした。細胞を破砕して細胞質画分と核画分に分画し、各画分を免疫ブロッティングに供し、抗NF-κB（p65）抗体および抗ヒストンH3抗体で分析した。また、上記の各細胞を固定し、一次抗体に抗NF-κB（p65）抗体、二次抗体に抗ウサギIgG抗体Alexa Fluor 568を用いて免疫蛍光染色し、蛍光顕微鏡で観察した。
　免疫ブロッティングの結果を図21に示した。免疫蛍光染色の結果を図22に示した。図22において、DAPIは核を示し、スケールバーは50μmである。これらの結果から、Sema4DはSFZ細胞において、NF-κBの細胞質から核への移行を誘導することが明らかになった。 5-3 Confirmation of induction of NF-κB nuclear translocation by Sema4D SFZ cells were treated with Sema4D for 1 or 2 hours. As a positive control, SFZ cells were treated with IL-1β for 1 hour. Untreated SFZ cells were used as the negative control. The cells were disrupted into cytoplasmic and nuclear fractions, each fraction was subjected to immunobrotting and analyzed with anti-NF-κB (p65) antibody and anti-histone H3 antibody. In addition, each of the above cells was immobilized, immunofluorescently stained with an anti-NF-κB (p65) antibody as the primary antibody, and an anti-rabbit IgG antibody Alexa Fluor 568 as the secondary antibody, and observed with a fluorescence microscope.
The results of immune blotting are shown in FIG. The results of immunofluorescent staining are shown in FIG. In Figure 22, DAPI shows the nucleus and the scale bar is 50 μm. From these results, it was clarified that Sema4D induces the translocation of NF-κB from the cytoplasm to the nucleus in SFZ cells.
５－４　ルシフェラーゼレポーターアッセイ
　実験方法（１３）に記載の方法でルシフェラーゼレポーターアッセイを行った。結果を図23に示した（各n=4）。Sema4DはSFZ細胞のNF-κB活性を有意に上昇させることが示された（****P <0.0001）。 5-4 Luciferase Reporter Assay A luciferase reporter assay was performed by the method described in Experimental Method (13). The results are shown in Fig. 23 (n = 4 each). Sema4D was shown to significantly increase NF-κB activity in SFZ cells (**** P <0.0001).
５－５　NF-κB阻害剤の効果の確認
　SFZ細胞の培地に、2μg/mlの組換えSema4DまたはSema4DとNF-κB阻害剤BAY11-7082（10μM）を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。Mmp13の発現量（n=2）をRT-qPCRで分析した。結果を図24に示した。NF-κB阻害剤はSFZ細胞におけるSema4D依存性Mmp13発現誘導を無効にした。 5-5 Confirmation of effect of NF-κB inhibitor 2 μg / ml of recombinant Sema4D or Sema4D and NF-κB inhibitor BAY11-7082 (10 μM) were added to the medium of SFZ cells and cultured for 12 hours. Untreated SFZ cells were used as controls. The expression level of Mmp13 (n = 2) was analyzed by RT-qPCR. The results are shown in FIG. NF-κB inhibitors abolished Sema4D-dependent Mmp13 expression induction in SFZ cells.
５－６　Traf2 KOマウスのSFZ細胞に対するSema4Dの効果
　野生型マウスまたはTraf2 KO同腹児からSFZ細胞を調製した。それぞれ、Sema4Dで処理する群と処理しない群を設けた。各細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、Mmp13の発現量（n=3）およびMmp3の発現量（n=3）をRT-qPCRで分析した。
　結果を図25に示した。（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。Traf2の欠損により、Sema4D依存性のMmp13発現およびMmp3発現が有意に抑制されることが示された（**P <0.01, ***P <0.001）。
　これらの結果から、Sema4DがSFZ細胞のプレキシン-B2およびTRAF2を介してNF-κBシグナル伝達を活性化し、Sema4DがNF-κBシグナル伝達を介してSFZ細胞に炎症作用を発現することが見出された。 Effect of Sema4D on SFZ cells in 5-6 Traf2 KO mice SFZ cells were prepared from wild-type mice or Traf2 KO littermates. A group treated with Sema4D and a group not treated with Sema4D were provided, respectively. Incubate for 12 hours with or without addition of 2 μg / ml recombinant Sema4D to the medium of each cell, and RT-qPCR the expression level of Mmp13 (n = 3) and the expression level of Mmp3 (n = 3). Analyzed in.
The results are shown in Fig. 25. (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. Traf2 deficiency was shown to significantly suppress Sema4D-dependent Mmp13 and Mmp3 expression (** P <0.01, *** P <0.001).
From these results, it was found that Sema4D activates NF-κB signaling via plexin-B2 and TRAF2 in SFZ cells, and Sema4D exerts an inflammatory effect on SFZ cells via NF-κB signaling. rice field.
５－７　MEK阻害剤の効果の確認
　Sema4DがTRAF2-NF-κBおよびc-Met-Ras-Erk l/2シグナル伝達経路を活性化することを示したため、MEK阻害剤を使用してSema4Dによって活性化されるNF-κB経路におけるc-Met-Ras-Erk l/2シグナル伝達経路の役割を調べた。
　SFZ細胞の培地に、2μg/mlの組換えSema4D、Sema4DとMEK阻害剤PD98059（10μM）またはSema4DとMEK阻害剤U0126（10μM）を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。上記の各細胞を固定し、一次抗体に抗NF-κB（p65）抗体、二次抗体に抗ウサギIgG抗体Alexa Fluor 568を用いて免疫蛍光染色し、蛍光顕微鏡で観察した。
　結果を図26に示した。DAPIは核を示し、スケールバーは50μmである。Sema4Dは、MEK阻害剤によってNF-κBの核への移行を誘導することが示された。この結果は、Sema4DによるNF-κBの活性化にはc-Met-Ras-Erk l/2シグナル伝達が必要であることを示唆するものである。 5-7 Confirmation of the effect of MEK inhibitor Since Sema4D was shown to activate the TRAF2-NF-κB and c-Met-Ras-Erk l / 2 signaling pathways, it was activated by Sema4D using a MEK inhibitor. We investigated the role of the c-Met-Ras-Erk l / 2 signaling pathway in the NF-κB pathway.
2 μg / ml of recombinant Sema4D, Sema4D and MEK inhibitor PD98059 (10 μM) or Sema4D and MEK inhibitor U0126 (10 μM) were added to the medium of SFZ cells and cultured for 12 hours. Untreated SFZ cells were used as controls. Each of the above cells was immobilized, immunofluorescently stained with anti-NF-κB (p65) antibody as the primary antibody and anti-rabbit IgG antibody Alexa Fluor 568 as the secondary antibody, and observed with a fluorescence microscope.
The results are shown in FIG. DAPI shows the nucleus and the scale bar is 50 μm. Sema4D has been shown to induce the translocation of NF-κB to the nucleus by a MEK inhibitor. This result suggests that c-Met-Ras-Erk l / 2 signaling is required for NF-κB activation by Sema4D.
〔実施例６：Sema4D依存性Mmp13発現誘導に関与する既知炎症性サイトカインの探索〕
　Sema4Dは、SFZ細胞においてNF-κBシグナル伝達を活性化し、炎症反応性遺伝子をアップレギュレートしたので、Sema4DがSFZ細胞に対して直接的または間接的に炎症作用を示すかどうか確認するために以下の実験を行った。 [Example 6: Search for known inflammatory cytokines involved in Sema4D-dependent Mmp13 expression induction]
Since Sema4D activated NF-κB signaling in SFZ cells and up-regulated inflammatory responsive genes, the following to determine whether Sema4D has a direct or indirect inflammatory effect on SFZ cells: Experiment was conducted.
６－１　Sema4D処理したSFZ細胞における既知炎症性サイトカイン発現
　SFZ細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養した。IL-6の発現量をRT-qPCRで分析し（n=3）、培養上清中のIL-6濃度をELISAで分析した（n=3）。
　結果を図27に示した。（A）がRT-qPCRの結果、（B）が培養上清のELISAの結果である。Sema4DはSFZ細胞のIL-6の発現を有意に誘導した（***P <0.001）。 Expression of known inflammatory cytokines in SFZ cells treated with 6-1 Sema4D 2 μg / ml of recombinant Sema4D was added to or without addition to the medium of SFZ cells and cultured for 12 hours. The expression level of IL-6 was analyzed by RT-qPCR (n = 3), and the IL-6 concentration in the culture supernatant was analyzed by ELISA (n = 3).
The results are shown in Fig. 27. (A) is the result of RT-qPCR, and (B) is the result of ELISA of the culture supernatant. Sema4D significantly induced the expression of IL-6 in SFZ cells (*** P <0.001).
　また、別途、SFZ細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、培養上清中のIL-6、TNFα、IL-1βの各濃度をELISAで分析した（n=2）。
　結果を図28に示した。（A）がIL-6の結果、（B）がTNFαの結果、（C）がIL-1βの結果である。Sema4DはSFZ細胞のTNFαおよびIL-1βの発現を誘導しなかった。 Separately, the cells were cultured for 12 hours with or without 2 μg / ml recombinant Sema4D added to the medium of SFZ cells, and the concentrations of IL-6, TNFα, and IL-1β in the culture supernatant were determined by ELISA. Analyzed in (n = 2).
The results are shown in FIG. (A) is the result of IL-6, (B) is the result of TNFα, and (C) is the result of IL-1β. Sema4D did not induce TNFα and IL-1β expression in SFZ cells.
６－２　タンパク質合成阻害の効果の確認
　SFZ細胞の培地に2μg/mlの組換えSema4DまたはSema4Dと翻訳阻害剤シクロヘキシミド（100μg/ml）を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。Mmp13の発現量およびIL-6の発現量をRT-qPCRで分析した（各n=2）。
　結果を図29に示した。（A）がMmp13の発現量の結果、（B）がIL-6の発現量の結果である。シクロヘキシミド（図中CHX）によりデノボタンパク質合成が阻害されると、Mmp13は発現誘導されなくなった。一方、IL-6の発現誘導は中程度に阻害された。 6-2 Confirmation of the effect of inhibiting protein synthesis To the medium of SFZ cells, 2 μg / ml of recombinant Sema4D or Sema4D and the translation inhibitor cycloheximide (100 μg / ml) were added and cultured for 12 hours. Untreated SFZ cells were used as controls. The expression level of Mmp13 and IL-6 was analyzed by RT-qPCR (n = 2 each).
The results are shown in Figure 29. (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of IL-6. When cyclohexidine (CHX in the figure) inhibited de novo protein synthesis, Mmp13 was no longer induced to be expressed. On the other hand, the induction of IL-6 expression was moderately inhibited.
６－３　Sema4D依存性Mmp13発現誘導におけるIL-6の関与
　SFZ細胞の培地にSema4D（2μg/ml）、IL-6（1ng/ml）またはIL-6（100ng/ml）を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。Mmp13の発現量をRT-qPCRで分析した（各n=2）。
　結果を図30に示した。IL-6（1ng/ml）処理した場合Mmp13の発現を誘導しなかったことから、SFZ細胞をSema4Dで処理した際に分泌されるIL-6の量（図27（B）参照）はSFZ細胞にMmp13の発現を誘導するのに十分でないことが分かった。 6-3 Involvement of IL-6 in inducing Sema4D-dependent Mmp13 expression 12 hours after adding Sema4D (2 μg / ml), IL-6 (1 ng / ml) or IL-6 (100 ng / ml) to the medium of SFZ cells. It was cultured. Untreated SFZ cells were used as controls. The expression level of Mmp13 was analyzed by RT-qPCR (n = 2 each).
The results are shown in FIG. Since Mmp13 expression was not induced when treated with IL-6 (1 ng / ml), the amount of IL-6 secreted when SFZ cells were treated with Sema4D (see Fig. 27 (B)) is SFZ cells. It was found that it was not sufficient to induce the expression of Mmp13.
　SFZ細胞の培地にSema4D（2μg/ml）、Sema4Dと抗IL-6抗体（1μg/ml）またはIL-6（10μg/ml）を添加して24時間培養した。未処理のSFZ細胞をコントロールとした。Mmp13の発現量をRT-qPCRで分析した（各n=2）。
　結果を図31に示した。抗IL-6抗体はSema4D依存性Mmp13発現誘導を多少抑制した。 Sema4D (2 μg / ml), Sema4D and anti-IL-6 antibody (1 μg / ml) or IL-6 (10 μg / ml) were added to the medium of SFZ cells and cultured for 24 hours. Untreated SFZ cells were used as controls. The expression level of Mmp13 was analyzed by RT-qPCR (n = 2 each).
The results are shown in Figure 31. The anti-IL-6 antibody slightly suppressed the induction of Sema4D-dependent Mmp13 expression.
　SFZ細胞の培地にSema4D（1μg/ml）、IL-6（1μg/ml）またはSema4DとIL-6を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。Mmp13の発現量およびMmp3の発現量をRT-qPCRで分析した（各n=3）。
　結果を図32に示した。（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。単独ではMmp13とMmp3の発現を上昇させない量のSema4DとIL-6の両方でSFZ細胞を処理すると、Mmp13とMmp3の発現を相乗的に上昇させた。
　以上より、Sema4Dは部分的にIL-6を介してMmp13とMmp3の発現を誘導する可能性が示唆された。 Sema4D (1 μg / ml), IL-6 (1 μg / ml) or Sema4D and IL-6 were added to the medium of SFZ cells and cultured for 12 hours. Untreated SFZ cells were used as controls. The expression level of Mmp13 and the expression level of Mmp3 were analyzed by RT-qPCR (n = 3 each).
The results are shown in Figure 32. (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. Treatment of SFZ cells with both Sema4D and IL-6 in amounts that did not increase Mmp13 and Mmp3 expression alone synergistically increased Mmp13 and Mmp3 expression.
These results suggest that Sema4D may induce the expression of Mmp13 and Mmp3 partially via IL-6.
〔実施例７：Sema4D依存性Mmp13発現誘導に関与する転写因子の探索〕
７－１　Sema4D処理SFZ細胞で高発現する転写因子遺伝子のRNA-Seq分析
　実施例６のデノボタンパク質合成阻害実験の結果（図29）は、Sema4D依存性Mmp13発現誘導に特定の転写因子が関与していることを示唆している。そこで、Sema4D処理したSFZ細胞において、未処理SFZ細胞と比較して10倍以上高発現している転写因子遺伝子をRNA-Seqで分析した。結果を表4に示した。Sema4DはSFZ細胞において、IκBζをコードするNfkbizとC/EBPδをコードするCebpdの発現を上昇させることが示された。 [Example 7: Search for transcription factors involved in Sema4D-dependent Mmp13 expression induction]
7-1 RNA-Seq analysis of transcription factor genes highly expressed in Sema4D-treated SFZ cells The results of the de novo protein synthesis inhibition experiment in Example 6 (Fig. 29) show that specific transcription factors are involved in the induction of Sema4D-dependent Mmp13 expression. It suggests that it is. Therefore, RNA-Seq was used to analyze transcription factor genes that were 10-fold or more highly expressed in Sema4D-treated SFZ cells as compared to untreated SFZ cells. The results are shown in Table 4. Sema4D has been shown to increase the expression of Nfkbiz, which encodes IκBζ, and Cebpd, which encodes C / EBPδ, in SFZ cells.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
７－２　Sema4D処理SFZ細胞におけるNfkbizおよびCebpdの発現レベル
　SFZ細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、NfkbizおよびCebpdの発現量をRT-qPCRで分析した（各n=3）。
　結果を図33に示した。（A）がNfkbizの発現量の結果、（B）がCebpdの発現量の結果である。Sema4D処理により、SFZ細胞におけるNfkbizおよびCebpd発現量が有意に上昇した（***P <0.001, ****P <0.0001）。 7-2 Expression level of Nfkbiz and Cebpd in Sema4D-treated SFZ cells The expression level of Nfkbiz and Cebpd was increased by culturing for 12 hours with or without addition of 2 μg / ml recombinant Sema4D to the medium of SFZ cells. Analyzed by qPCR (n = 3 each).
The results are shown in Figure 33. (A) is the result of the expression level of Nfkbiz, and (B) is the result of the expression level of Cebpd. Sema4D treatment significantly increased Nfkbiz and Cebpd expression in SFZ cells (*** P <0.001, **** P <0.0001).
７－３　Sema4D処理SFZ細胞におけるIκBζおよびC/EBPδの発現レベル
　SFZ細胞の培地に2μg/mlの組換えSema4Dを添加して12時間または24時間培養した。未処理のSFZ細胞をコントロールとした。細胞溶解液を調製し、抗IκBζ抗体および抗C/EBPδ抗体を用いて免疫ブロッティング分析を行った。
　結果を図34に示した。SFZ細胞をSema4Dで処理することにより、IκBζおよびC/EBPδの発現が誘導された。 7-3 Expression levels of IκBζ and C / EBPδ in Sema4D-treated SFZ cells 2 μg / ml of recombinant Sema4D was added to the medium of SFZ cells and cultured for 12 hours or 24 hours. Untreated SFZ cells were used as controls. Cytolytic solutions were prepared and immunoblotting analysis was performed using anti-IκBζ antibody and anti-C / EBPδ antibody.
The results are shown in Figure 34. Treatment of SFZ cells with Sema4D induced the expression of IκBζ and C / EBPδ.
７－４　レンチウイルスベクターによるIκBζおよびC/EBPδの過剰発現
　SFZ細胞に、IκBζ発現ベクターが導入されたレンチウイルスベクターおよび／またはC/EBPδ発現ベクターが導入されたレンチウイルスベクターを感染させ、IκBζを過剰発現するSFZ細胞、C/EBPδを過剰発現するSFZ細胞、IκBζとC/EBPδの両方を過剰発現するSFZ細胞を得た。空ベクターが導入されたレンチウイルスベクターを感染させた細胞をコントロールとした。感染後4日間培養し、Mmp13の発現量をRT-qPCRで分析した（各n=3）。
　結果を図35に示した。IκBζとC/EBPδをそれぞれ単独で過剰発現するSFZ細胞は、Mmp13の発現量が上昇した。また、IκBζとC/EBPδの両方を過剰発現するSFZ細胞では、両者は協調的な役割を行うことが示された。 7-4 Overexpression of IκBζ and C / EBPδ by lentiviral vector SFZ cells are infected with the lentiviral vector into which the IκBζ expression vector is introduced and / or the lentiviral vector into which the C / EBPδ expression vector is introduced, and IκBζ is produced. We obtained SFZ cells that overexpress, SFZ cells that overexpress C / EBPδ, and SFZ cells that overexpress both IκBζ and C / EBPδ. Cells infected with the lentiviral vector into which the empty vector was introduced were used as controls. After infection, the cells were cultured for 4 days, and the expression level of Mmp13 was analyzed by RT-qPCR (n = 3 each).
The results are shown in Figure 35. The expression level of Mmp13 was increased in SFZ cells that overexpressed IκBζ and C / EBPδ alone. It was also shown that in SFZ cells that overexpress both IκBζ and C / EBPδ, both play a coordinated role.
７－５　Nfkbiz KOマウスのSFZ細胞に対するSema4Dの効果
　野生型マウスまたはNfkbiz KO同腹児からSFZ細胞を調製した。それぞれ、Sema4Dで処理する群と処理しない群を設けた。各細胞の培地に2μg/mlの組換えSema4Dを添加して、または添加せずに12時間培養し、Mmp13の発現量（n=3）およびMmp3の発現量（n=3）をRT-qPCRで分析した。
　結果を図36に示した。（A）がMmp13の発現量の結果、（B）がMmp3の発現量の結果である。Nfkbiz KOマウスのSFZ細胞では、Sema4D処理によるMmp13およびMmp3の発現誘導を有意に阻害した（***P <0.001, ****P <0.0001）。 7-5 Effect of Sema4D on SFZ cells in Nfkbiz KO mice SFZ cells were prepared from wild-type mice or Nfkbiz KO littermates. A group treated with Sema4D and a group not treated with Sema4D were provided, respectively. Incubate for 12 hours with or without addition of 2 μg / ml recombinant Sema4D to the medium of each cell, and RT-qPCR the expression level of Mmp13 (n = 3) and the expression level of Mmp3 (n = 3). Analyzed in.
The results are shown in FIG. (A) is the result of the expression level of Mmp13, and (B) is the result of the expression level of Mmp3. In SFZ cells of Nfkbiz KO mice, the induction of Mmp13 and Mmp3 expression by Sema4D treatment was significantly inhibited (*** P <0.001, **** P <0.0001).
７－６　Sema4D誘導性のNfkbizおよびCebpdの発現に対するMEK阻害剤の効果
　SFZ細胞の培地にSema4D（2μg/ml）、Sema4DとMEK阻害剤PD98059（10μM）またはSema4DとMEK阻害剤U0126（10μM）を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。NfkbizおよびCebpdの発現量をRT-qPCRで分析した（各n=2）。
　結果を図37に示した。（A）がNfkbizの発現量の結果、（B）がCebpdの発現量の結果である。MEK阻害剤はema4D誘導性のNfkbizおよびCebpdの発現を顕著に阻害した。 7-6 Effect of MEK inhibitor on Sema4D-induced expression of Nfkbiz and Cebpd Sema4D (2 μg / ml), Sema4D and MEK inhibitor PD98059 (10 μM) or Sema4D and MEK inhibitor U0126 (10 μM) were added to the medium of SFZ cells. It was added and cultured for 12 hours. Untreated SFZ cells were used as controls. The expression levels of Nfkbiz and Cebpd were analyzed by RT-qPCR (n = 2 each).
The results are shown in Figure 37. (A) is the result of the expression level of Nfkbiz, and (B) is the result of the expression level of Cebpd. MEK inhibitors markedly inhibited ema4D-induced expression of Nfkbiz and Cebpd.
７－７　Sema4D誘導性のNfkbizおよびCebpd発現に対するNF-κB阻害剤の効果
　SFZ細胞の培地にSema4D（2μg/ml）またはSema4DとNF-κB阻害剤BAY11-7082（10μM）を添加して12時間培養した。未処理のSFZ細胞をコントロールとした。NfkbizおよびCebpdの発現量をRT-qPCRで分析した（各n=2）。
　結果を図38に示した。（A）がNfkbizの発現量の結果、（B）がCebpdの発現量の結果である。NF-κB阻害剤はema4D誘導性のNfkbizおよびCebpdの発現を顕著に阻害した。 7-7 Effect of NF-κB inhibitor on Sema4D-induced Nfkbiz and Cebpd expression 12 hours after addition of Sema4D (2 μg / ml) or Sema4D and NF-κB inhibitor BAY11-7082 (10 μM) to SFZ cell medium It was cultured. Untreated SFZ cells were used as controls. The expression levels of Nfkbiz and Cebpd were analyzed by RT-qPCR (n = 2 each).
The results are shown in Figure 38. (A) is the result of the expression level of Nfkbiz, and (B) is the result of the expression level of Cebpd. NF-κB inhibitors markedly inhibited ema4D-induced expression of Nfkbiz and Cebpd.
〔実施例８：c-Met阻害剤による関節軟骨からのアグリカン放出抑制〕
　実験方法（８）に記載の方法でマウス大腿骨頭軟骨を器官培養した。すなわち、3週齢のICRマウスから大腿骨頭を単離し、Sema4D（20μg/ml）またはSema4Dとc-Met阻害剤クリゾチニブ（0.5μM）を無血清DMEM培地に添加して3日間培養し、培地へのアグリカン放出量をジメチルメチレンブルーアッセイで分析した。コントロールはどちらも含まない無血清DMEM培地で3日間培養した。
　結果を図39に示した。c-Met阻害剤は、Sema4D処理により増加したアグリカン放出量を有意に抑制した（*P <0.05, ****P <0.0001）。この結果は、c-Met阻害剤はSema4Dによって生じる軟骨破壊の予防または治療に有効であることを示すものである。 [Example 8: Suppression of agrecan release from articular cartilage by a c-Met inhibitor]
Mouse femoral head cartilage was organ-cultured by the method described in Experimental Method (8). That is, the femoral head was isolated from 3-week-old ICR mice, Sema4D (20 μg / ml) or Sema4D and the c-Met inhibitor crizotinib (0.5 μM) were added to serum-free DMEM medium, cultured for 3 days, and then cultured into the medium. Aggrecan release was analyzed by dimethylmethylene blue assay. Controls were cultured in serum-free DMEM medium containing neither for 3 days.
The results are shown in Figure 39. The c-Met inhibitor significantly suppressed the aggrecan release increased by Sema4D treatment (* P <0.05, **** P <0.0001). This result indicates that c-Met inhibitors are effective in the prevention or treatment of cartilage destruction caused by Sema4D.
〔実施例９：NF-κB阻害剤による関節軟骨からのアグリカン放出抑制〕
　実験方法（８）に記載の方法でマウス大腿骨頭軟骨を器官培養した。すなわち、3週齢のICRマウスから大腿骨頭を単離し、Sema4D（20μg/ml）またはSema4DとNF-κB阻害剤BAY11-7082（10μM）を無血清DMEM培地に添加して3日間培養した。コントロールはどちらも含まない無血清DMEM培地で3日間培養した。大腿骨頭軟骨を4％パラホルムアルデヒドで固定した後、10％EDTAで2週間脱灰した。軟骨をパラフィン包埋し、厚さ5μmの切片を作製した。切片を脱パラフィンし、PBSと蒸留水で洗浄した後、0.2％ファストグリーンで10分間、0.1％サフラニンOで20分間染色した。培地へのプロテオグリカン放出は、ジメチルメチレンブルーアッセイで分析した。 [Example 9: Suppression of agrecan release from articular cartilage by an NF-κB inhibitor]
Mouse femoral head cartilage was organ-cultured by the method described in Experimental Method (8). That is, the femoral head was isolated from 3-week-old ICR mice, and Sema4D (20 μg / ml) or Sema4D and the NF-κB inhibitor BAY11-7082 (10 μM) were added to serum-free DMEM medium and cultured for 3 days. Controls were cultured in serum-free DMEM medium containing neither for 3 days. The femoral head cartilage was fixed with 4% paraformaldehyde and then decalcified with 10% EDTA for 2 weeks. The cartilage was embedded in paraffin to prepare a section having a thickness of 5 μm. The sections were deparaffinized, washed with PBS and distilled water, and then stained with 0.2% fast green for 10 minutes and with 0.1% safranin O for 20 minutes. Proteoglycan release into the medium was analyzed by the dimethylmethylene blue assay.
　関節軟骨切片の観察結果を図40に示した。（A）がコントロールの結果、（B）がSema4Dを含む培地で培養した結果、（C）がSema4DとBAY11-7082を含む培地で培養した結果である。スケールバーは200μmである。Sema4Dは軟骨の表層および中間層においてサフラニンOによって染色されるプロテオグリカンを減少させたが、BAY11-7082を加えることによりプロテオグリカンの減少が抑制されていることが観察された。
　ジメチルメチレンブルーアッセイの結果を図41に示した。NF-κB阻害剤は、Sema4D処理により増加したアグリカン放出量を有意に抑制した（**P <0.01）。これらの結果は、NF-κB阻害剤はSema4Dによって生じる軟骨破壊の予防または治療に有効であることを示すものである。 The observation results of the articular cartilage section are shown in Fig. 40. (A) is the result of control, (B) is the result of culturing in the medium containing Sema4D, and (C) is the result of culturing in the medium containing Sema4D and BAY11-7082. The scale bar is 200 μm. It was observed that Sema4D reduced the proteoglycan stained by safranin O in the superficial and intermediate layers of cartilage, but the addition of BAY11-7082 suppressed the reduction of proteoglycan.
The results of the dimethylmethylene blue assay are shown in Figure 41. The NF-κB inhibitor significantly suppressed the aggrecan release increased by Sema4D treatment (** P <0.01). These results indicate that NF-κB inhibitors are effective in the prevention or treatment of cartilage destruction caused by Sema4D.
　なお本発明は上述した各実施形態および実施例に限定されるものではなく、請求項に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献および特許文献の全てが、本明細書中において参考として援用される。 The present invention is not limited to the above-described embodiments and examples, and various modifications can be made within the scope of the claims, and the technical means disclosed in the different embodiments may be appropriately combined. The obtained embodiments are also included in the technical scope of the present invention. In addition, all of the academic and patent documents described in this specification are incorporated herein by reference.

Claims (14)

  1. 　セマフォリン４Ｄ－プレキシンＢ２のシグナル伝達を阻害する物質を有効成分として含有する軟骨・骨・関節疾患の予防または治療用医薬組成物。 A pharmaceutical composition for the prevention or treatment of cartilage, bone and joint diseases containing a substance that inhibits signal transduction of semaphorin 4D-plexin B2 as an active ingredient.
  2. 　前記物質が、プレキシンＢ２とセマフォリン４Ｄの結合を阻害する物質、ｃ－Ｍｅｔとセマフォリン４Ｄの結合を阻害する物質、プレキシンＢ２とＴｒａｆ２の結合を阻害する物質またはプレキシンＢ２とｃ－Ｍｅｔの結合を阻害する物質である、請求項１に記載の医薬組成物。 The substance is a substance that inhibits the binding between plexin B2 and semaphorin 4D, a substance that inhibits the binding between c-Met and semaphorin 4D, a substance that inhibits the binding between plexin B2 and Traf2, or a substance that inhibits the binding between plexin B2 and c-Met. The pharmaceutical composition according to claim 1, which is a substance that inhibits.
  3. 　前記物質が抗プレキシンＢ２抗体またはプレキシンＢ２の発現を阻害する核酸またはプレキシンＢ２の機能を阻害する物質である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the substance is a nucleic acid that inhibits the expression of an anti-plexin B2 antibody or plexin B2, or a substance that inhibits the function of plexin B2.
  4. 　前記物質がｃ－Ｍｅｔ阻害剤である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the substance is a c-Met inhibitor.
  5. 　前記物質がＩκＢζまたはＣＥＢＰδの発現または機能を阻害する物質である、請求項１に記載の医薬組成物。 The pharmaceutical composition according to claim 1, wherein the substance is a substance that inhibits the expression or function of IκBζ or CEBPδ.
  6. 　前記軟骨・骨・関節疾患が、関節リウマチ、変形性関節症または骨粗しょう症である、請求項１～５のいずれか１項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 5, wherein the cartilage / bone / joint disease is rheumatoid arthritis, osteoarthritis or osteoarthritis.
  7. 　セマフォリン４Ｄ－プレキシンＢ２のシグナル伝達を阻害する被験物質を選択する工程を含む、軟骨・骨・関節疾患の予防または治療用薬剤のスクリーニング方法。 A method for screening a drug for preventing or treating cartilage, bone, or joint disease, which comprises a step of selecting a test substance that inhibits signal transduction of semaphorin 4D-plexin B2.
  8. 　セマフォリン４Ｄにより発現誘導される遺伝子の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とセマフォリン４Ｄと被験物質を接触させる工程と、レポーター遺伝子の発現量を測定する工程と、被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する工程を含む、前記［７］に記載のスクリーニング方法。 A step of contacting a cultured cell or a gene-modified animal cell in which a reporter gene is inserted downstream of a gene whose expression is induced by semaphorin 4D with a test substance, a step of measuring the expression level of the reporter gene, and a step of measuring the expression level of the reporter gene. The screening method according to the above [7], which comprises a step of selecting a test substance that reduces the expression level of a reporter gene as compared with a control that has not been contacted with the test substance.
  9. 　ＩκＢζまたはＣＥＢＰδが認識する発現制御領域の下流にレポーター遺伝子が挿入された培養細胞または遺伝子改変動物の細胞とＩκＢζまたはＣＥＢＰδと被験物質を接触させる工程と、レポーター遺伝子の発現量を測定する工程と、被験物質を接触させていない対照と比較してレポーター遺伝子の発現量を低下させる被験物質を選択する工程を含む、請求項７に記載のスクリーニング方法。 A step of contacting a cultured cell or a gene-modified animal cell in which a reporter gene is inserted downstream of the expression control region recognized by IκBζ or CEBPδ with the test substance, a step of measuring the expression level of the reporter gene, and a step of measuring the expression level of the reporter gene. The screening method according to claim 7, comprising selecting a test substance that reduces the expression level of the reporter gene as compared to a control that has not been contacted with the test substance.
  10. 　プレキシンＢ２とセマフォリン４Ｄと被験物質を接触させる工程と、プレキシンＢ２とセマフォリン４Ｄとの結合を測定する工程と、被験物質を添加していない対照と比較してプレキシンＢ２とセマフォリン４Ｄとの結合を低下させる被験物質を選択する工程を含む、請求項７に記載のスクリーニング方法。 A step of contacting plexin B2 with semaphorin 4D and a test substance, a step of measuring the binding between plexin B2 and semaphorin 4D, and a step of plexin B2 and semaphorin 4D as compared with a control to which no test substance was added. The screening method according to claim 7, comprising the step of selecting a test substance that reduces binding.
  11. 　プレキシンＢ２とｃ－Ｍｅｔと被験物質を接触させる工程と、プレキシンＢ２とｃ－Ｍｅｔとの結合を測定する工程と、被験物質を添加していない対照と比較してプレキシンＢ２とｃ－Ｍｅｔとの結合を低下させる被験物質を選択する工程を含む、請求項７に記載のスクリーニング方法。 A step of contacting plexin B2 and c-Met with a test substance, a step of measuring the binding between plexin B2 and c-Met, and a step of plexin B2 and c-Met as compared with a control to which no test substance was added. The screening method according to claim 7, comprising the step of selecting a test substance that reduces binding.
  12. 　プレキシンＢ２とＴｒａｆ２と被験物質を接触させる工程と、プレキシンＢ２とＴｒａｆ２との結合を測定する工程と、被験物質を添加していない対照と比較してプレキシンＢ２とＴｒａｆ２との結合を低下させる被験物質を選択する工程を含む、請求項７に記載のスクリーニング方法。 A test substance that reduces the binding between plexin B2 and Traf2 as compared with a step of contacting plexin B2 and Traf2 with a test substance, a step of measuring the binding between plexin B2 and Traf2, and a control to which no test substance is added. 7. The screening method according to claim 7, comprising the step of selecting.
  13. 　ＩκＢζおよび／またはＣＥＢＰδを発現する細胞と被験物質を接触させる工程と、ＩκＢζおよび／またはＣＥＢＰδの発現量を測定する工程と、被験物質を接触させていない対照と比較してＩκＢζおよび／またはＣＥＢＰδの発現量を低下させる被験物質を選択する工程を含む、請求項７に記載のスクリーニング方法。 A step of contacting a cell expressing IκBζ and / or CEBPδ with a test substance, a step of measuring the expression level of IκBζ and / or CEBPδ, and a step of measuring the expression level of IκBζ and / or CEBPδ and the step of contacting IκBζ and / or CEBPδ with a control not contacted with the test substance. The screening method according to claim 7, comprising a step of selecting a test substance that reduces the expression level.
  14. 　前記軟骨・骨・関節疾患が、関節リウマチ、変形性関節症または骨粗しょう症である、請求項７～１３のいずれかに記載のスクリーニング方法。 The screening method according to any one of claims 7 to 13, wherein the cartilage / bone / joint disease is rheumatoid arthritis, osteoarthritis or osteoarthritis.
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