PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CARTILAGE DISEASES

Abstract

A pharmaceutical composition for preventing or treating cartilage diseases, and pharmaceutical preparation that includes the pharmaceutical composition as an active ingredient are provided. The pharmaceutical composition includes, as an active ingredient, at least one of an integrin beta-like 1 (ITGBL1) protein, ITGBL1 DNA or RNA encoding the ITGBL1 protein.

Claims

1. A method for treating cartilage diseases, comprising administering a pharmaceutically effective amount of an integrin beta-like 1 (ITGBL1) protein, ITGBL1 DNA or RNA encoding the ITGBL1 protein, a recombinant vector comprising the ITGBL1 DNA sequences, or recombinant cells transformed with the recombinant vector to a subject in need of treatment of the cartilage diseases.

2. The method of claim 1, wherein the cartilage diseases comprise degenerative arthritis, posttraumatic arthritis, or osteochondritis dissecans.

3. The method of claim 1, wherein the ITGBL1 protein has an amino acid sequence of SEQ ID NO: 25.

4. The method of claim 1, wherein the DNA encoding the ITGBL1 protein has a nucleotide sequence of SEQ ID NO: 26.

5. The method of claim 1, wherein the recombinant vector is a viral vector or a nonviral vector.

6. The method of claim 5, wherein the viral vector is selected from the group consisting of an adenovirus vector, an adeno-associated virus vector, a helper-dependent adenovirus vector, and a retroviral vector.

7. The method of claim 1, wherein the recombinant cells are mammalian cells.

8. The method of claim 1, wherein the ITGBL1 or the ITGBL1 protein inhibits an integrin-extracellular matrix (ECM) interaction.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] These and/or other aspects, features, and advantages of the invention will become apparent and more readily appreciated from the following description of example embodiments, taken in conjunction with the accompanying drawings of which:

[0058] FIG. 1A illustrates an experiment to verify expression of integrin beta-like 1 (ITGBL1) in cartilage tissues during a Xenopus laevis embryogenesis, and FIGS. 1B and 1C illustrate results indicating whether ITGBL1 is expressed in chondrocytes of Xenopus laevis embryos according to an example embodiment. FIG. 1D illustrates results indicating that ITGBL1 is expressed in cartilage tissues based on a comparison between an expression pattern of Col2a1 that is a cartilage tissue marker gene and an expression pattern of ITGBL1, and FIG. 1E illustrates results indicating that ITGBL1 is strongly expressed at a cartilage tissue formation time (stage 30).

[0059] FIGS. 2A and 2C illustrate results indicating that cartilage tissues were reduced in size and cartilage tissue formation is significantly suppressed when expression of ITGBL1 in chondrocytes of Xenopus laevis embryos is inhibited, and that cartilage is formed normally and cartilage increases in size when the expression of ITGBL1 is increased, according to an example embodiment, and FIGS. 2B and 2D are graphs illustrating sizes of cartilages of FIGS. 2A and 2C, respectively. FIG. 2E illustrates results indicating that expression of Sox9 and Col2a1 that are markers of cartilage tissues is increased in embryos in which the expression of the ITGBL1 is increased.

[0060] FIGS. 3A and 3B illustrate results indicating that expression of ITGBL1 increases during a chondrocyte differentiation of each bone marrow-derived mesenchymal stem cell (BM-MSC) and that expression of ITGBL1 decreases during a bone tissue formation. FIGS. 3C and 3D illustrate results that a formation of a cartilage tissue is inhibited when expression of an ITGBL1 protein decreases in human chondrocytes. FIG. 3E illustrates results indicating that expression of Col2a1 and Sox9 that are genes promoting chondrogenesis gradually increases in response to a gradual increase in expression of ITGBL1 in a mouse chondrocyte line, and FIG. 3F illustrates results of a quantitative analysis of the expression of FIG. 3E using quantitative polymerase chain reaction (qPCR). FIG. 3G illustrates results indicating that a cartilage differentiation is promoted in response to an increase in expression of ITGBL1 of limb-bud mesenchyme that is a part of an arm and leg of a mouse embryo and differentiates into chondrocytes. FIG. 3H illustrates a result of a measurement of an amount of glycosaminoglycan (GAG) in the chondrocytes of FIG. 3G indicating that a cartilage formation is promoted by overexpression of ITGBL1.

[0061] FIGS. 4A through 4F illustrate results indicating that when expression of an ITGBL1 protein is increased in chondrocytes of a mouse in which an inflammation is caused by treatment with IL-1β, expression of Sox9 and Col2a1 that are chondrogenic factors is recovered and expression of MMP3 and MMP13 that are inflammatory factors is reduced, according to an example embodiment. FIGS. 4G and 4H illustrate results indicating that arthritis is not developed when expression of ITGBL1 is increased by injecting ITGBL1-containing adenovirus into knee joint cavities of mice with arthritis induced by damaging medial meniscus cartilage of knee joints of mice.

[0062] FIGS. 5A and 5C illustrate results indicating that an amount of focal adhesion complexes formed in a site in which integrin and a cell matrix bind significantly increases, by suppressing expression of ITGBL1 using ITGBL1-siRNA in a PC3 cell line, and FIG. 5B is a graph illustrating an intensity and the number of FAK puncta that is a marker of the focal adhesion complexes of FIG. 5A. FIG. 5D is a graph illustrating an intensity and the number of integrin-beta 1 puncta that is another marker of the focal adhesion complexes of FIG. 5C.

[0063] FIG. 5E illustrates results indicating that an amount of activated integrin-beta 1 increases when expression of ITGBL1 is inhibited and that the amount of activated integrin-beta 1 decreases when the expression of ITGBL1 is increased, and FIG. 5F illustrates results indicating, using fluorescent staining, that an amount of activated integrin-beta 1 increases when expression of ITGBL1 is reduced. FIG. 5G illustrates results indicating that integrin-beta 1 and ITGBL1 protein bind to each other, using a co-immunoprecipitation experiment.

[0064] FIG. 6A illustrates results indicating that when expression of ITGBL1 is increased (Itgbl1-O.E.), PC3 cells do not properly adhere onto a surface coated with fibronectin, but that when activation of integrins is increased by Mn.sup.2+ ions, the PC3 cells properly adhere onto the surface, and FIG. 6B illustrates a result of a measurement of a cell area of FIG. 6A. FIGS. 6C and 6D illustrate results indicating that when expression of ITGBL1 increases in BM-MSCs, a cell adhesion is inhibited. FIGS. 6E and 6F illustrate results of experiments of FIG. 6A performed in human chondrocytes, and the results of FIGS. 6E and 6F indicate that when expression of ITGBL1 is increased in human chondrocytes, a cell adhesion is inhibited and when activation of integrins is increased by Mn.sup.2+ ions, cells properly adhere onto the surface again.

[0065] FIG. 7A illustrates results indicating that expression of Sox9 and Col2a1 increases when expression of ITGBL1 increases in chondrocytes, but that the expression of Sox9 and Col2a1 decreases again when activation of integrins is increased through treatment with Mn.sup.2+ or DTT, and FIG. 7B illustrates a result of a quantitative analysis of the results of FIG. 7A using qPCR. FIG. 7C illustrates results indicating that a size of cartilage is increased by overexpression of ITGBL1 in a process of forming a cartilage tissue using an ATDC5 chondrocyte line. The size of cartilage is reduced by adding Mn.sup.2+ or DTT which activate integrins. FIGS. 7D and 7 E illustrate analysis of the results of FIG. 7C based on a size of cartilage and an amount of GAG included in cartilage, respectively. FIG. 7F illustrates results indicating that expression of Sox9 increases when expression of integrin alpha and beta subunits of a table is inhibited. FIG. 7G illustrates results indicating that expression of Sox9 further increases when expression of integrin alpha and beta subunits is reduced in a state in which ITGBL1 is overexpressed.

[0066] FIGS. 8A through 8C illustrate results indicating that expression of MMP3 and MMP13 that promote decomposition of cartilage is increased in chondrocytes treated with 29-kDa fibronectin fragments (29-kDa Fn-fs) known to cause a cartilage degeneration of a patient with arthritis, but that the expression of MMP3 and MMP13 decreases again when expression of ITGBL1 is increased, and that the expression of MMP3 and MMP13 is increased again when integrins are activated through treatment with Mn.sup.2+ or DTT. FIGS. 8D and 8E illustrate results of a measurement of an adhesion level of 29-kDa Fn-fs to chondrocytes.

[0067] FIGS. 9A through 9C illustrate results indicating that expression of Mmp3 and Mmp13 increased by ITGBL1 depletion is not inhibited by treating subtype-specific integrin inhibitors Bio1211 (integrin-α4β1 inhibitor), obtustatin (integrin-α1β1 inhibitor), or ATN-161 (integrin-α5β1 inhibitor), but the expression of MMP3 and MMP13 is inhibited when a various types of integrin inhibitors are treated together, and that ATN-161 among integrin inhibitors is most effective. FIGS. 9D and 9E illustrate results indicating that articular cartilage deteriorates when expression of ITGBL1 is inhibited by injecting ITGBL1-shRN-containing adenovirus into knee joint cavities of mice, and that knee cartilage is recovered by injecting ATN-161 that is an integrin-beta 1 inhibitor into knee joint capsule in which the expression of ITGBL1 is inhibited.

[0068] FIG. 10 illustrates an example of a mechanism of promoting chondrogenesis and inhibiting an inflammation of cartilage diseases by inhibiting activation of integrins by expression of ITGBL1.

DETAILED DESCRIPTION

[0069] Hereinafter, the present disclosure will be described in more detail with reference to examples. The following examples are given for the purpose of illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

Reference Example 1. Culture of Xenopus laevis Embryos

[0070] Female Xenopus laevis were cultured in a 16° C. incubator (JSR, JSBI-150C), ovulation was induced by injecting 800 units of human chorionic gonadotropin (hCG, DS HCG Inj.) into the female Xenopus laevis. When the female Xenopus laevis began to lay eggs by hormones, the eggs were artificially squeezed and fertilized in vitro with male testes, followed by waiting for 30 minutes. Whether the eggs were fertilized was checked with naked eyes, the eggs were dejellied using 3% (w/v) cysteine (pH 7.8, Sigma:C7880), and Xenopus laevis embryos were cultured in 1/3× Marc's Modified Ringer's (MMR).

Reference Example 2. Cell Culture

[0071] Human PC3 cells, HEK293T cells, or human bone marrow-derived mesenchymal stem cells (hBMSCs) were cultured in 1% L-glutamine, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, an RPMI 1640 medium, a Dulbecco's modified Eagle medium (DMEM) and an α-MEM. To assess chondrogenesis of hBMSCs, pellet, micromass, or Transwell culture systems were employed.

[0072] Articular chondrocytes were isolated from femoral condyles and tibial plateaus of postnatal day 5 mice. Cartilage tissues were digested with 0.2% collagenase type II. Chondrocytes were maintained in a DMEM containing 10% FBS, penicillin and streptomycin.

[0073] Human chondrocytes were purchased from Cell Application, Inc. (San Diego, Calif., USA), and hBMSCs were purchased from the ATCC.

[0074] Mesenchymal cells obtained from embryos of ICR mice were digested with 1% trypsin and 0.2% collagenase type II and maintained to induce chondrogenesis and hypertrophic maturation. A total of 2×10.sup.7 cells/ml was suspended in a DMEM/F-12 medium (2:3) containing 10% (v/v) FBS. The cells were spotted as 20 μl drops on culture dishes and maintained for 6 days to induce chondrogenesis.

Example 1. Confirmation of Expression of ITGBL1 in Chondrocytes

[0075] 1-1. Confirmation of Expression Site of ITGBL1

[0076] To analyze genes expressed in pharyngeal arches of Xenopus laevis embryos, the following experiment was conducted.

[0077] The pharyngeal arches of Xenopus laevis embryos at stage 37 in which a face of an embryo is developed were dissected, and each of the dissected pharyngeal arches were divided into three parts in a cephalocaudal direction, and divided into two parts in a dorsoventral direction. RNA was extracted using a Trizol reagent (Sigma), an RNA-seq library was prepared from the extracted RNA using an Illumina TruSeq RNA Library Prep Kit, and a sequence analysis was commissioned by the Genome Sequencing Service Center (GSSC) at Stanford, U.S.A.

[0078] A result of the above analysis is shown in FIG. 1A, and analysis results are shown in Table 1 below.

[0079] Also, to determine whether ITGBL1 is expressed in chondrocytes of Xenopus laevis embryos, the following experiment was conducted.

[0080] An RNA probe having a base sequence complementary to ITGBL1 mRNA was prepared using an Ambion Megascript T7 RNA Transcription kit (Promega, P2077), and craniofacial tissue slices of Xenopus laevis embryos at stage 37 that were dissected as described above and fixed, were treated with a prehybridization buffer (formamide 1.13 g/ml, Biosesang, F1014 in 2×SSC; 0.3 M NaCl-Biosesang, 30 mM sodium citrate Biosesang, C1029) at 65° C. for 12 hours. The treated craniofacial tissue slices were washed twice with a 2×SSC solution at 65° C. for 30 minutes, were treated with RNase, were washed twice with a 0.2×SSC solution for 30 minutes, were treated with an anti-digoxigenin antibody (Sigma) for 12 hours and were washed three times with TBST. A color reaction was induced using BM purple (Sigma).

[0081] Sequences of primers to prepare the RNA probe are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Name Sequence (5′ .fwdarw. 3′) SEQ ID NO: ITGBL1 5′ augcacgcuggagccuu 1 ITGBL1 3′ aggauauucgcuuccaagcca 2

[0082] Analysis results are shown in FIGS. 1B and 1C.

[0083] As shown in FIGS. 1B and 1C, it is confirmed that ITGBL1 is expressed in chondrocytes of Xenopus laevis embryos.

[0084] 1-2. Comparison between Expression Pattern of Col2a1 and Expression Pattern of ITGBL1

[0085] Fixed Xenopus laevis embryos at stage 37 were cross-sectioned at a thickness of 100 μm using a Vibratome (Leica, VT 1000S). An RNA probe having a base sequence complementary to ITGBL1 mRNA, and an RNA probe having a base sequence complementary to Col2a1 mRNA were prepared, and craniofacial tissue slices of the fixed Xenopus laevis embryos at stage 37 were treated with a prehybridization buffer (formamide 1.13 g/ml, Biosesang, F1014) (w/v) at 65° C. for 12 hours. Each of the prepared RNA probes were diluted in a prehybridization solution, and the craniofacial tissue slices were treated at 65° C. for 1 day. The treated craniofacial tissue slices were washed twice with a 2×SSC solution (0.3 M NaCl-Biosesang, 20 mM sodium citrate-Biosesang, C1029) at 65° C. for 30 minutes, were treated with RNase, were washed twice with a 0.2×SSC solution for 30 minutes, were treated with an anti-digoxigenin antibody (Sigma) for 12 hours, and were washed three times with TBST. A color reaction was induced using BM purple (Sigma). In another method, a DAB staining, that is, an immunostaining of treating a slice with a thickness of 100 μm with a Col2a1 antibody (DSHB, II-II6B3) to confirm an cartilage-specific matrix expression, was conducted.

[0086] As shown in FIG. 1D, it is confirmed that ITGBL1 is expressed in cartilage tissues because an expression pattern of Col2a1 and an expression pattern of ITGBL1 are similar to each other.

[0087] 1-3. Confirmation of ITGBL1 Expression Time

[0088] RNA was extracted from fertilized Xenopus laevis embryos at stages 20, 24, 27, 30, 33, 35 and 41 using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). Reverse transcription polymerase chain reaction (RT-PCR; BIO RAD, T100 Thermal Cycler) was performed on the synthesized cDNA using Taq polymerase (Coregen, CE-500U).

[0089] As shown in FIG. 1E, it is confirmed that ITGBL1 is most strongly expressed at stage 30 that is a period in which cartilage tissues are formed.

Example 2. ITGBL1's Function of Promoting Formation of Cartilage Tissues

[0090] 2-1. Case of Inhibiting ITGBL1 Expression

[0091] To confirm a function of ITGBL1 to promote a formation of cartilage tissues, expression of ITGBL1 was inhibited in the following manner.

[0092] To use splice-blocking antisense morpholino oligonucleotides to inhibit expression of ITGBL1 in chondrocytes of Xenopus laevis embryos, a preparation of splice-blocking antisense morpholino oligonucleotides was requested to Gene Tools, LLC, and a sequence thereof is shown in Table 3 below.

TABLE-US-00003 TABLE 3 Name Sequence (5′ .fwdarw. 3′) SEQ ID NO: ITGBL1 MO AGTAGGGAAGATATACAGACCTGCA 3

[0093] The prepared splice-blocking antisense morpholino oligonucleotides were injected into dorsal-ventral axis of Xenopus laevis embryos at 2-cell stage, to inhibit the ITGBL1 expression in chondrocytes. Embryos into which the splice-blocking antisense morpholino oligonucleotides were injected were cultured up to embryo stage 45, and fixed with MEMFA (4% Formaldehyde, Biosesang, F1012). A specific staining of cartilage was performed using Alcian blue (1% Alcian Blue 8GX, Georgiachem, AB1082) and tissues other than the cartilage were removed using trypsin, to remove a nonspecific staining using a 10% (v/v) methanol solution. The cartilage was observed using an Olympus (SZX16-ILLB) microscope.

[0094] As a control group, an experiment was conducted in the same manner as described above, except that morpholino oligonucleotides that inhibit expression of ITGBL1 in chondrocytes of Xenopus laevis embryos were not injected.

[0095] Results of the experiment are shown in FIGS. 2A and 2B.

[0096] As shown in FIGS. 2A and 2B, it is confirmed that when the expression of the ITGBL1 in the chondrocytes of the Xenopus laevis embryos is inhibited, a size and shape of cartilage are reduced abnormally in comparison to the control group in which expression of ITGBL1 is not inhibited.

[0097] 2-2. Case of Increasing ITGBL1 Expression

[0098] To confirm a function of ITGBL1 to promote a formation of cartilage tissues, expression of ITGBL1 was increased in the following manner.

[0099] ITGBL1 cDNA (SEQ ID NO: 4) obtained from facial chondrocytes of Xenopus laevis embryos was applied to an mMESSAGE mMACHINE SP6 kit (Ambion, AM1340), to synthesize ITGBL1 mRNA. The ITGBL1 mRNA was injected into Xenopus laevis embryos in the same manner as in Example 2-1, to increase expression of ITGBL1 in chondrocytes. An Alcian blue staining was performed in the same manner as in Example 2-1, and expression of Sox9 and Col2a1 was confirmed through whole-mount in situ hybridization (WISH).

[0100] As a control group, an experiment was conducted in the same manner as described above, except that ITGBL1 mRNA that increases expression of ITGBL1 in chondrocytes of Xenopus laevis embryos was not injected.

[0101] Results of the experiments are shown in FIGS. 2C, 2D and 2E.

[0102] As shown in FIGS. 2C and 2D, it is confirmed that when the expression of the ITGBL1 is increased by injecting the ITGBL1 mRNA into the facial chondrocytes of the Xenopus laevis embryos, a size of cartilage is increased 1.2 times to 1.3 times in comparison to the control group in which the expression of the ITGBL1 is not increased. As shown in FIG. 2E, it is confirmed that expression of Sox9 and Col2a1 that are markers of cartilage tissues is increased in comparison to the control group.

Example 3. Effect of Promoting Chondrogenesis by ITGBL1 Protein in Human and Mouse Chondrocytes

[0103] 3-1. Comparison of ITGBL1 Expression Between Chondrocytes and Bone Tissues During a Differentiation of BM-MSCs

[0104] To determine whether chondrogenesis is promoted by an ITGBL1 protein during a differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs), an experiment was conducted in the following manner hBMSCs (ATCC) were cultured in an α-minimal essential medium (α-MEM; Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics). After forming a cell mass using a micromass method, the cell mass was treated with a chondrogenic inducer (TGF-β, dexamethasome, ascorbate-2-phosphate) together with the culture solution, to induce a formation of cartilage tissues. The formation of the cartilage tissues was induced for 12 days, and RNA was extracted using a PureLink RNA Mini Kit (Invitrogen, 12183018A), to synthesize cDNA using a GoScript Reverse Transcriptase (Promega, A5004A). Quantitative Reverse Transcription (qRT)-PCR was performed on the synthesized cDNA using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). All reactions were performed on 96-well plates, and a mean value was used to calculate mRNA expression. Results of the experiment are shown in FIGS. 3A and 3B.

[0105] As shown in FIG. 3A, it is confirmed that expression of ITGBL1 increases during a differentiation into chondrocytes of hBMSCs. As shown in FIG. 3B, it is confirmed that expression of ITGBL1 decreases during a formation of bone tissues of the hBMSCs.

[0106] 3-2. Case of Inhibiting ITGBL1 Expression During Differentiation into Chondrocytes of hBMSCs and Case of Increasing ITGBL1 Expression in Chondrocytes

[0107] To determine whether chondrogenesis is promoted by an ITGBL1 protein in human, an experiment was conducted in the following manner hBMSCs (ATCC) were cultured in an α-minimal essential medium (α-MEM; Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and ITGBL1 siRNA (Genolution, SEQ ID NO: 5 and 6) were transfected into the hBMSCs using a method of a protocol provided by a manufacturer. After forming a cell mass using a micromass method, the cell mass was treated with a chondrogenic inducer (TGF-β, dexamethasome, ascorbate-2-phosphate) together with the culture solution, to induce a formation of cartilage tissues. After the formation of the cartilage tissues was induced for 7 days, the cartilage tissues were fixed through a treatment with 4% (w/v) paraformaldehyde. The fixed cartilage tissues were added to an OCT solution (Cell Path, KMA-0.00-00A) and were cut at a thickness of 15 μm using a cryotome (Bright, OTF5000), and an immunofluorescent staining was performed to confirm expression of Col2a1. To determine whether chondrogenesis is promoted, an Alcian blue staining was performed in the same manner as in Example 2-1.

[0108] In the case of inhibiting expression of ITGBL1, ITGBL1 siRNA was not transfected in a control group. To increase ITGBL1 expression in chondrocytes, human chondrocytes (Cell Application, Inc. San Diego, Calif., USA) were cultured in an α-MEM (Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and ITGBL1 cDNA (SEQ ID NO: 7, ITGBL1 protein SEQ ID NO: 8) were transfected into the human chondrocytes using a method of a protocol provided by a manufacturer. The cell mass was treated with a chondrogenic inducer (TGF-β, dexamethasome, ascorbate-2-phosphate) together with the culture solution, to induce a formation of cartilage tissues.

[0109] After the formation of the cartilage tissues was induced for 7 days, total RNA was extracted from chondrocytes in which ITGBL1 DNA was transfected, using a PureLink RBA Mini Kit (Invitrogen, 12183018A), cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004), and qRT-PCR was performed using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). All reactions were performed on 96-well plates, and a mean value was used to calculate mRNA expression. Sequences of ITGBL1 siRNA and primers used in the qRT-PCR are shown in Table 4 below.

TABLE-US-00004 TABLE 4 Name Sequence (5′ .fwdarw. 3′) Tm (° C.) SEQ ID NO: Human Sense: GAGCUGUCUAUGACCGAUAUU N/A 5, 6 ITGBL1- Antisense: UAUCGGUCAUAGACAGCUCUU siRNA Mouse ATGCATCCTCCAGGCTTCAGGAACTTCTT N/A 7 ITGBL1 GTTGCTGGTGTCCTCCCTTCTCTTCATTGG cDNA GCTGTCAGCTGCTCCTCAAAGCTTCTTAC CATCTCTGAGAAGCCTGTCGGGCGCCCCC TGCAGGCTGTCCCGGGCAGAGTCCGAAC GCAGATGTCGTGCACCTGGGCAGCCCCC AGGGAGCGCTCTGTGCCATGACCGTGGC CGGTGCGAGTGTGGGGTCTGCATCTGTCA CGTGACCGAACCTGGCACCTACTTCGGTC CACTGTGTGAGTGCCATGAGTGGATATG CGAGACCTACGACGGGAAAACCTGTGCA GGCCACGGTAATTGTGACTGCGGCAAGT GCAAGTGTGATGTGGGATGGTCTGGGGA AGCTTGTCAGTACCCAACCAAGTGTGAC CTGACCAAAAAAATCAGCAACCAGATGT GCAAGAACTCCCAAGATGTCATCTGCTCC AATGCAGGTACATGTCACTGTGGCAGGT GTAAGTGTGATAATTCAGATGGACATGG ACTCATTTATGGTAAATTTTGTGAATGTG ATGATAGAGAATGCATAGATGATGAAAC AGAAGAAGTATGTGGAGGCCATGGGAAG TGTTACTGTGGAAACTGTTACTGTGAGGC TGGTTGGCATGGCGATAAATGCGAGTTC CAGTGTGACATCACCCCATGGGAAAGCA AGCGAAGATGCACATCTCCAGATGGCAA AGTCTGTAGCAACAGAGGAACATGTGTA TGTGGTGAATGTTCTTGCCATGATGTTGA TCCAACTGGGGACTGGGGAGACATTCAT GGGGACACGTGTGAGTGTGATGAAAGGG ACTGCAGAGCTGTTTATGATCGATACTCT GATGATTTCTGTTCAGGTCATGGGCAGTG TAACTGTGGAAGATGTGACTGCAGAGCA GGCTGGTATGGGAAGAAATGTGAGCACC CAAGGAATTGCCCATTGTCAGCTGAGGA GAGCACCAAGAAGTGCCAGGGTAGTTCT GATCTTCCTTGCTCTGGAAGGGGCAGATG CGAATGTGGCAGATGCACTTGTTACCCTC CTGGGGACAGCAGAGTCTATGGCAAGAC CTGTGAGTGTGATGACCGGCGCTGCGAG GACCTGGATGGTGTGGTCTGCGGAGGCC ATGGCATGTGCTCCTGTGGTCGCTGTGTT TGTGAGAAAGGATGGTTTGGTAAGCTCT GCCAACACCTGCGGAAGTGTAATATGAC AGAAGAACAAAGCAGGAGTCTGTGTGAG TCAGCAGATGGCACATTGTGCTCAGGGA AGGGTTCTTGTCATTGTGGAAAGTGCATT TGTTCTGGAGAAGAGTGGTATATTTCAGG GGAGTTTTGTGACTGTGATGACAGAGAC TGTGACAAACACGATGGTCTCATTTGCAC AGGGAATGGAATCTGTAGCTGTGGAAAC TGTGAATGCTGGGATGGATGGAATGGAA ATGCATGTGAAATCTGGCTTGGTACCGA ATATCCTTAA Mouse MHPPGFRNFLLLVSSLLFIGLSAAPQSFLPS N/A 8 ITGBL1 LRSLSGAPCRLSRAESERRCRAPGQPPGSA Protein LCHDRGRCECGVCICHVTEPGTYFGPLCEC HEWICETYDGKTCAGHGNCDCGKCKCDV GWSGEACQYPTKCDLTKKISNQMCKNSQD VICSNAGTCHCGRCKCDNSDGHGLIYGKF CECDDRECIDDETEEVCGGHGKCYCGNCY CEAGWHGDKCEFQCDITPWESKRRCTSPD GKVCSNRGTCVCGECSCHDVDPTGDWGDI HGDTCECDERDCRAVYDRYSDDFCSGHGQ CNCGRCDCRAGWYGKKCEHPRNCPLSAEE STKKCQGSSDLPCSGRGRCECGRCTCYPPG DSRVYGKTCECDDRRCEDLDGVVCGGHG MCSCGRCVCEKGWFGKLCQHLRKCNMTE EQSRSLCESADGTLCSGKGSCHCGKCICSG EEWYISGEFCDCDDRDCDKHDGLICTGNGI CSCGNCECWDGWNGNACEIWLGTEYP SOX9-F AAGGAGAGCGAGGAGGACAAGTTC 62 9 SOX9-B TGTTCTTGCTGGAGCCGTTG 57.4 10 MMMP3-F GATGCGCAAGCCCAGGTGTG 64.13 11 MMP3-B GCCAATTTCATGAGCAGCAACGA 59.57 12 MMP13-F AGGAGCATGGCGACTTCTACCC 62.88 13 MMP13-B TTTGTCTGGCGTTTTTGGATGTTT 56.23 14 Co12a1-F CAGTTGGGAGTAATGCAAG 58 15 Co12a1-B GCCTTGAGCAGTTCACCTTC 58 16

[0110] Results of the experiment are shown in FIGS. 3C, 3D and 3E.

[0111] As shown in FIGS. 3C and 3D, it is confirmed that when expression of an ITGBL1 protein is inhibited, a formation of cartilage tissues is suppressed.

[0112] As shown in FIGS. 3E and 3F, it is confirmed that expression of Sox9 and Col2a1 that are chondrogenic factors gradually increases when expression of an ITGBL1 protein gradually increases and that a formation of cartilage tissues is promoted.

[0113] Also, limb bud mesenchymes differentiating from arms and legs of mouse (limb bud) embryos into chondrocytes were isolated. An adenovirus vector (Ad-ITGBL1; Vector Biolabs, Malvern, Pa. 19355 USA, customized by a company) that contains ITGBL1 was transduced, to increase expression of ITGBL1 and induce a cartilage differentiation. It is confirmed that the cartilage differentiation is promoted using an Alcian blue staining scheme. Results of the experiment are shown in FIGS. 3G and 3H.

[0114] As shown in FIGS. 3G and 3H, in chondrocytes in which expression of ITGBL1 is increased, an amount of glycosaminoglycan (GAG) is increased and chondrogenesis is promoted.

Example 4. Effect of ITGBL1 Protein to Promote Chondrogenesis in Arthritis-Induced Mice

[0115] To determine whether an ITGBL1 protein promotes chondrogenesis in arthritis-induced mice, an experiment was conducted in the following manner.

[0116] Chondrocytes isolated from knee cartilage of postnatal day 5 mice were cultured for 2 days, and were treated with 5 ng/ml of IL-1β (GenScript, 201-LB) for 72 hours in order to induce an inflammation. An adenovirus vector Ad-ITGBL1 was transduced into the chondrocytes in which the inflammation was induced, and a change in expression of a chondrogenic factor (Sox9 and Col2a1) was analyzed by qRT-PCR in the same manner as in Example 3. Sequences of primers used in the qRT-PCR are shown in Table 5 below.

[0117] As a control group, an experiment was conducted in the same manner as described above, except that an adenovirus vector was not transduced and a treatment with IL-1β was not performed. The control group is indicated by “None” in FIGS. 4B through 4F.

TABLE-US-00005 TABLE 5 Tm SEQ Name Sequence (5′ .fwdarw. 3′) (° C.) ID NO: Co12a1-F CACACTGGTAAGTGGGGCAAGA 55 21 Co12a1-B GGATTGTGTTGTTTCAGGGTTCG 55 22 SOX9-F CATCAGCAGCACCGCACCCA 58 23 SOX9-B CGGGTGATGGGCGGGTAGGA 58 24

[0118] Results of the experiment are shown in FIGS. 4A through 4D.

[0119] As shown in FIG. 4A, when an inflammation is induced by treating chondrocytes isolated from mouse knee cartilage with IL-1β, expression of ITGBL1 is significantly reduced. As shown in FIGS. 4B through 4D, when expression of ITGBL1 is increased, expression of a chondrogenic factor (Sox9 and Col2a1) is increased.

[0120] Also, osteoarthritis (OA) was induced in a joint of a mouse by a destabilization of medial meniscus (DMM) surgery as a meniscectomy used to induce arthritis by removing medial meniscuses at knee joints of mice, and an adenovirus vector Ad-ITGBL1 was transduced into the joint of the mouse, to increase expression of an ITGBL1 protein. After 6 weeks, the joint of the mouse was isolated and stained with Safranin 0, and expression levels of Col2a1 and Sox9 were compared using immunohistochemistry. Results of the experiment are shown in FIGS. 4G and 4H.

[0121] As a control group, an experiment was conducted in the same manner as described above, except that a meniscectomy was not performed. The control group is indicated by “Sham” in FIGS. 4G and 4H.

[0122] As shown in FIGS. 4G and 4H, it is confirmed that when expression of an ITGBL1 protein is increased in a joint of an arthritis-induced mouse, an arthritis score (Osteoarthritis Research Society International (OARSI)) is reduced and expression of Col2a1 and Sox9 is increased, thereby inhibiting arthritis.

Example 5. Inflammation Inhibitory Effect of ITGBL1 Protein in Arthritis-Induced Mice

[0123] To confirm an inflammation inhibitory effect of an ITGBL1 protein in arthritis-induced mice, an experiment was conducted in the following manner.

[0124] As described above in Example 4, mouse chondrocytes were treated with 5 ng/ml of IL-1β for 72 hours in order to induce an inflammation. An adenovirus vector containing ITGBL1 DNA (Ad-ITGBL1) was transduced into the mouse chondrocytes in which the inflammation was induced, and a change in expression of MMP3 and MMP13 that are inflammatory factors was analyzed using qRT-PCR in the same manner in Example 3. Sequences of primers used in the qRT-PCR are shown in Table 6 below.

[0125] As control groups, an experiment was conducted in the same manner as described above, except that an adenovirus vector into which ITGBL1 was not inserted was transduced, as indicated by “Mock” and that an adenovirus vector was not transduced and a treatment with IL-1β was not performed as indicated by “None”.

TABLE-US-00006 TABLE 6 Tm SEQ Name Sequence (5′ .fwdarw. 3′) (° C.) ID NO: MMP3-F TCCTGATGTTGGTGGCTTCAG 58 17 MMP3-B TGTCTTGGCAAATCCGGTGTA 58 18 MMP13-F ACCACATCGAACTTCGA 58 19 MMP13-B CGACCATACAGATACTG 58 20

[0126] Results of the experiment are shown in FIGS. 4E and 4F.

[0127] As shown in FIGS. 4E and 4F, it is confirmed that when expression of an ITGBL1 protein is increased in mouse chondrocytes in which arthritis is induced by a treatment with IL-1β, expression of an inflammatory factor (MMP3 and MMP13) is significantly inhibited.

[0128] Also, osteoarthritis (OA) was induced in a joint of a mouse by a destabilization of medial meniscus (DMM) surgery as a meniscectomy, and an adenovirus vector Ad-ITGBL1 was transduced into the joint of the mouse, to increase expression of an ITGBL1 protein. After 6 weeks, the joint of the mouse was dissected, and expression of MMP3 and MMP13 were examined by performing an immunohistochemistry staining. Results thereof are shown in FIGS. 4G and 4H.

[0129] As a control group, an experiment was conducted in the same manner as described above, except that a meniscectomy was not performed, as indicated by “Sham” in FIGS. 4G and 4H.

[0130] As shown in FIGS. 4G and 4H, it is confirmed that when expression of an ITGBL1 protein is increased in a joint of an arthritis-induced mouse, expression of MMP3 and MMP13 that are inflammatory factors is reduced and osteoarthritis does not develop as severe as control.

Example 6. Effect of ITGBL1 Protein to Inhibit Integrin Activation

[0131] 6-1. Analysis of Change in Focal Adhesion Complexes Based on Inhibition of ITGBL1 Expression

[0132] To confirm a change in focal adhesion complexes by an ITGBL1 protein, an experiment was conducted in the following manner.

[0133] PC3 cells were cultured in an RPMI 1640 medium (gibco 22400-099, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 6-well plate and were transfected with ITGBL1 siRNA (Genolution 1) or ITGBL1 DNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer. The transfected cells were detached from the bottom using 0.25% Trypsin EDTA (gibco, 25200-072), were attached onto a fibronectin-coated coverslip for 4 hours, and were fixed in 4% (w/v) paraformaldehyde. To find a change in focal adhesion complexes, an immunofluorescent staining analysis was performed using anti-FAK (abcam, ab40794) and anti-01 integrin (DSHB, AIIB2). Results of the experiment are shown in FIGS. 5A through 5D.

[0134] As shown in FIGS. 5A through 5D, it is confirmed that an amount of focal adhesion complexes generated in a binding site of a cell matrix and integrin significantly increases when expression of ITGBL1 is inhibited in a PC3 cell line using ITGBL1-siRNA.

[0135] 6-2. Analysis of Correlation Between ITGBL1 and Integrin Activation

[0136] To confirm a correlation between an ITGBL1 protein and integrin activation, the following experiment was conducted.

[0137] PC3 cells were cultured in an RPMI 1640 medium (gibco 22400-099, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 6-well plate and were transfected with ITGBL1 siRNA (Genolution 1) or ITGBL1 DNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer. The transfected cells were detached from the bottom using 4 mM EDTA, an immunofluorescent staining was performed using an antibody (MILLIPORE, MAB2079Z) that binds to β1-integrin activated in a fluorescence-activated cell sorting (FACS) buffer (1×PBS, 2% FBS), and an analysis was performed using BD LSRFortessa™.

[0138] As shown in FIGS. 5E and 5F, it is confirmed that an amount of activated integrin increases when expression of ITGBL1 is inhibited, and that the amount of activated integrin decreases when expression of ITGBL1 is increased.

[0139] To confirm an interaction between ITGBL1 and integrin-β1, a co-immunoprecipitation experiment was conducted.

[0140] An ITGBL1-HA vector and an ITGB1-flag (integrin-β1) vector were transfected into HEK 293T cells, to extract cell proteins. The extracted cell proteins were subjected to the co-immunoprecipitation experiment using paramagnetic beads (Dynabeads, ThermoFisher) with an HA antibody. Results of the experiment are shown in FIG. 5G.

[0141] As shown in FIG. 5G, it is confirmed that ITGBL1 binds to ITGB1 in a state in which calcium ions are present.

[0142] 6-3. Confirmation of Cell Adhesion and Integrin Activity Based on Increase in ITGBL1 Expression

[0143] PC3 cells were cultured in an RPMI 1640 medium (gibco 22400-099, containing 10% (v/v) FBS and 1% (v/v) antibiotics), chondrocytes were cultured in an α-MEM (Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 6-well plate and were transfected with ITGBL1 siRNA (Genolution 1) or ITGBL1 DNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer. The transfected cells were detached from the bottom using 0.25% Trypsin EDTA (gibco, 25200-072), were attached onto a fibronectin-coated coverslip for 4 hours by treating the cells on the media based on a concentration of Mn.sup.2+, and were fixed in 4% (w/v) paraformaldehyde. The cells were observed using an Olympus IX73 microscope.

[0144] It is confirmed that when expression of ITGBL1 is increased in all of PC3 cells (FIGS. 6A and 6B), human mesenchymal stem cells (FIGS. 6C and 6D) and human chondrocytes (FIGS. 6E and 6F), a cell adhesion is inhibited, but that when integrin activation is increased by a treatment with Mn.sup.2+, the cell adhesion is increased again. Thus, it is demonstrated that the ITGBL1 protein inhibits the integrin activation to inhibit the cell adhesion.

Example 7. Analysis of Correlation Between Integrin Activation Inhibition Function of ITGBL1 Protein and Control of Expression of Chondrogenic Factor of Chondrocytes

[0145] 7-1. Case of Increasing ITGBL1 Expression

[0146] To analyze an influence of an integrin activation inhibition function of ITGBL1 on chondrogenesis during the chondrogenesis, the following experiment was conducted. Chondrocytes were cultured in an α-MEM (Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 6-well plate and were transfected with ITGBL1 DNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer. The transfected chondrocytes were treated with Mn.sup.2+ or DTT and incubated at 37° C. RNA was extracted from the cells using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). RT-PCR (BIO RAD, T100 Thermal Cycler) was performed on the synthesized cDNA using Taq polymerase (Coregen, CE-500U). Also, qRT-PCR was performed using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). All reactions were performed on 96-well plates, and a mean value was used to calculate mRNA expression. Results of the experiment are shown in FIGS. 7A and 7B.

[0147] As shown in FIGS. 7A and 7B, it is confirmed that when expression of ITGBL1 is increased in chondrocytes, expression of Sox9 and Col2a1 increases, but that when integrin activation is increased by a treatment with Mn.sup.2+ or DTT, the expression of Sox9 and Col2a1 decreases.

[0148] Thus, it is demonstrated that the integrin activation inhibition function of ITGBL1 promotes expression of a chondrogenic gene.

[0149] Also, to analyze an influence of the integrin inactivation function of ITGBL1 on the chondrogenesis, a micromass culture experiment was conducted in the following manner Chondrocytes (mouse chondrocytes; Sigma, 402-051) were cultured in a DMEM/F-12 medium (gibco, 10565-018, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and were transfected with ITGBL1 DNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer. A cell mass was formed by the transfected chondrocytes using a micromass method, and was treated with Mn.sup.2+ or DTT that activate integrins, and with a chondrogenic inducer (TGF-β, dexamethasone, ascorbate-2-phosphate) together with the culture solution, to induce a formation of cartilage tissues. The formation of the cartilage tissues was induced for 7 days, and the cartilage tissues were treated with 4% (w/v) paraformaldehyde and fixed. The fixed cartilage tissues were added to an OCT solution (Cell Path, KMA-0.00-00A) and were sectioned at a thickness of 15 μm using a cryotome (Bright, OTF5000), and an Alcian blue staining was performed, to measure an amount of glycosaminoglycan (GAG). Results of the experiment are shown in FIGS. 7C through 7E.

[0150] As shown in FIGS. 7C through 7E, it is confirmed that a size of cartilage increased by overexpression of ITGBL1 is reduced due to an addition of Mn.sup.2+ or DTT, and accordingly an amount of GAG and a size of cartilage are reduced.

[0151] Thus, it is demonstrated that the integrin inactivation function of ITGBL1 promotes the formation of the cartilage tissues.

[0152] 7-2. Analysis of Expression of Chondrogenic Gene in Case of Increasing ITGBL1 Expression while Inhibiting Expression of Alpha and Beta Subunits of Integrins

[0153] To confirm a correlation between integrins and ITGBL1 during chondrogenesis, the following experiment was conducted. Chondrocytes were cultured in an α-MEM (Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 60-mm plate and were transfected with integrin 13-1 siRNA, integrin α-1 siRNA, integrin α-3 siRNA, integrin α-5 siRNA and integrin α-10 siRNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer, as shown in tables of FIGS. 7F and 7G, to inhibit expression of the above integrins. Also, to express ITGBL1 and inhibit expression of integrins at the same time, the expression of the integrins was inhibited and ITGBL1 DNA was transfected, and a culture was performed at 37° C. RNA extracted from the cells using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). RT-PCR (BIO RAD, T100 Thermal Cycler) was performed on the synthesized cDNA using Taq polymerase (Coregen, CE-500U). Also, qRT-PCR was performed using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). All reactions were performed on 96-well plates, and a mean value was used to calculate mRNA expression.

[0154] As shown in FIGS. 7F and 7G, it is confirmed that when expression of alpha and beta subunits of integrins is inhibited, expression of Sox9 increases, and that when the expression of the alpha and beta subunits of the integrins is reduced in a state in which expression of ITGBL1 is increased, the expression of Sox9 further increases.

[0155] Based on the results of the experiments conducted in Examples 6 and 7, it is found that the ITGBL1 protein inhibits integrin activation and that the integrin inactivation function of ITGBL1 promotes expression of chondrogenic factors of chondrocytes and a formation of cartilage tissues.

Example 8. Analysis of Correlation Among Integrin Inactivation Function of ITGBL1, Inflammatory Response and Cartilage Degeneration Inhibitory Effect

[0156] 8-1. Analysis of Expression of Cartilage Degeneration Factor in Case of Increasing ITGBL1 Expression and Activating Integrins

[0157] Chondrocytes were cultured in an α-MEM (Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 60-mm plate and were treated with 29-kDa Fn-fs that are fragmented fibronectin known to promote cartilage destruction. The cells were transfected with an ITGBL1 vector using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer, were treated with Mn.sup.2+ or DTT used to activate integrins, and were incubated at 37° C. RNA was extracted from the cells using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNA was synthesized using GoScript Reverse Transcriptase (Promega, A5004A). RT-PCR (BIO RAD, T100 Thermal Cycler) was performed on the synthesized cDNA using Taq polymerase (Coregen, CE-500U). Also, qRT-PCR was performed using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). All reactions were performed on 96-well plates, and a mean value was used to calculate mRNA expression. Results of the experiment are shown in FIGS. 8A through 8C.

[0158] As shown in FIGS. 8A through 8C, increased expressions of MMP3 and MMP13 by the 29-kDa Fn-fs treatment were reduced when expression of ITGBL1 is increased. Also, when integrins are activated by a treatment with Mn.sup.2+ or DTT again, expression of MMP3 and MMP13 increases. Thus, it is found that an integrin inactivation function of ITGBL1 reduces expression of MMP3 and MMP13 which are known to cause cartilage destruction and inflammation.

[0159] Also, to determine whether an ITGBL1 protein inhibits a binding between chondrocytes and 29-kDa Fn-fs that promote cartilage destruction, the following experiment was conducted. 2×10.sup.5 cells were seeded on a 60-mm plate and were transfected with ITGBL1 siRNA or DNA using a jetPRIME (Polyplus, 114-15) based on a method of a protocol provided by a manufacturer. 29-kDa Fn-fs (sigma, F9911) were conjugated to the Alexa-488 (Thermo Fisher, A10235) according to the manufacturer's protocol. The Alexa-488 conjugated 29-kDa Fn-fs were treated to the transfected chondrocytes, and the chondrocytes were fixed in 4% paraformaldehyde. The fixed cells were observed using a confocal microscope (Zeiss, LSM880). Results of the experiment are shown in FIGS. 8D and 8E.

[0160] As shown in FIGS. 8D and 8E, when expression of ITGBL1 is increased, the binding between the chondrocytes and the 29-kDa Fn-fs is significantly reduced. When treatment with Mn.sup.2+ or DTT is performed, the binding is increased again. Thus, ITGBL1 may inhibit integrin activation and the binding between the chondrocytes and the 29-kDa Fn-fs, and suppress expression of a cartilage degeneration factor.

[0161] 8-2. Analysis of Change in Cartilage Degeneration Factor in Case of Inhibiting ITGBL1 Expression and Integrin Activation

[0162] To confirm a correlation between an integrin inactivation function of an ITGBL1 protein and expression of a cartilage degeneration factor, the following experiment was conducted. Chondrocytes were cultured in an α-MEM (Welgene, LM008-01, containing 10% (v/v) FBS and 1% (v/v) antibiotics), and 2×10.sup.5 cells were seeded on a 60-cm plate and were transfected with ITGBL1-siRNAusing a jetPRIME (Polyplus, 114-15) according to the manufacturer's protocol. Bio1211 (TOCRIS, 3910, integrin-α4β1 inhibitor), obtustatin (TOCRIS, 4664, integrin-α1β1 inhibitor), or ATN-161 (TOCRIS, 6058, integrin-α5β1 inhibitor) were added to the α-MEM to inhibit the activation of integrin subtypes, and incubated at 37° C. Total RNAs were extracted from the cells using a PureLink RNA Mini Kit (Invitrogen, 12183018A), and cDNAs were synthesized using GoScript Reverse Transcriptase (Promega, A5004A). RT-PCR (BIO RAD, T100 Thermal Cycler) was performed on the synthesized cDNA using Taq polymerase (Coregen, CE-500U). Also, qRT-PCR was performed using a QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, Calif., USA). All reactions were performed on 96-well plates, and a mean value was used to calculate mRNA expression. Results of the experiment are shown in FIGS. 9A through 9C.

[0163] As shown in FIGS. 9A through 9C, it is confirmed that when expression of ITGBL1 is inhibited, expression of MMP3 and MMP13 is increased, that when the cells are treated with various types of integrin inhibitors together, the expression of MMP3 and MMP13 is inhibited again, and that ATN-161 among the integrin inhibitors is most effective.

[0164] Also, inflammatory responses and cartilage degeneration of ITGBL1 depleted knee joints were analyzed by conducting the following experiment.

[0165] Expression of ITGBL1 was inhibited by injecting an ITGBL1-shRNA-containing adenovirus vector (Ad-ITGBL1 shRNA) into a knee joint cavity. Then, ATN-161, an integrin-α5β1 inhibitor, was injected into the knee joint cavity in which expression of ITGBL1 was inhibited by injecting the ITGBL1-shRNA-containing adenovirus vector (Ad-ITGBL1 shRNA), knee joint tissues were excised, and a Safranin 0 staining and immunohistochemistry were performed, so that expression of Col2a1 and Sox9 was observed.

[0166] As a control group, an experiment was conducted in the same manner as described above, except that an adenovirus vector (Ad-C) that does not include ITGBL1-shRNA is transduced. Results of experiment are shown in FIGS. 9D and 9E.

[0167] As shown in FIGS. 9D and 9E, it is confirmed that depletion of ITGBL1 by intra-articular injection of ITGBL1-shRNA-containing adenovirus vector caused OA-like cartilage degeneration in mouse. Also, it is confirmed that when ATN-161, an integrin-α5β1 inhibitor, is injected into an ITGBL1-depleted mouse knee-joint, the knee cartilage degeneration is recovered.

[0168] According to example embodiments, a pharmaceutical composition for preventing or treating cartilage diseases includes, as an active ingredient, at least one of an ITGBL1 protein, ITGBL1 DNA or RNA encoding the ITGBL1 protein. An inhibitor of integrin activation, and a pharmaceutical preparation that includes the pharmaceutical composition as an active ingredient are provided. The pharmaceutical composition that includes at least one of the ITGBL1 protein, ITGBL1 DNA or RNA encoding the ITGBL1 protein as an active ingredient may have an effect of promoting chondrogenesis and inhibiting an inflammation based on a function of an ITGBL1 protein to inhibit integrin activation, and thus it is possible to use the pharmaceutical composition as an effective therapeutic agent for cartilage diseases.

[0169] A number of example embodiments have been described above. Nevertheless, it should be understood that various modifications may be made to these example embodiments. For example, suitable results may be achieved if the described techniques are performed in a different order and/or if components in a described system, architecture, device, or circuit are combined in a different manner and/or replaced or supplemented by other components or their equivalents. Accordingly, other implementations are within the scope of the following claims.

PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING CARTILAGE DISEASES

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A61K35/32

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A61K38/1777

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A61K35/761

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C07K14/70546

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A61P19/02

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C12N2710/10343

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A61K38/17

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A61K35/32

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A61P19/02

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Abstract

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