WO2004013326A1

WO2004013326A1 - Cartilage differentiation inhibiting gene

Info

Publication number: WO2004013326A1
Application number: PCT/JP2003/009939
Authority: WO
Inventors: Shuji Muramatsu; Akio Matsuda; Goichi Honda
Original assignee: Asahi Kasei Pharma Corporation
Priority date: 2002-08-05
Filing date: 2003-08-05
Publication date: 2004-02-12
Also published as: AU2003250547A1

Abstract

The present invention provides a protein inhibiting type II collagen expression useful in diagnosis, treatment and prevention of diseases associated with cartilage impairments. Using the plasmid CPE43, the cDNA encoding a protein that can inhibit type II collagen expression is cloned from the cDNA library constructed from mouse cell line ATDC5 and human lung fibroblast, and the DNA sequence and the deduced amino acid sequence are determined. The protein, the DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector are useful in screening for a substance inhibiting or promoting the type II collagen expression.

Description

DESCRIPTION

CARTILAGE DIFFERENTIATION INHIBITING GENE

Technical Field

The present invention relates to a protein that can inhibit type II collagen expression, a DNA sequence encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein. The present invention also relates to use of the protein, DNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with cartilage disorders.

The present invention also relates to a method for screening a substance that can inhibit or suppress the chondrocyte proliferation and differentiation by using the protein, DNA, recombinant vector and transformant.

Background Art

A cartilage is a connective tissue consisting of a chondrocyte and a surrounding extracellular matrix, and is present in a joint, spinal disc, costicartilage, auricle, extemal acoustic meatus, pubic symphysis, guttural operculum and the like. The main components of the extracellular matrix are collagen and aggrecan (cartilage proteoglycan). The collagen and aggrecan are produced by a chondrocyte. A collagen fiber is known to be involved in the rigidity against a tension and a shear stress on a cartilage, while an aggrecan is known to be involved in the swelling nature characteristic of a cartilage tissue.

A cartilage tissue is present predominantly and abundantly in the skeleton of a fetus. However, the amount of cartilage tissue decreases gradually in response to the postnatal development and growth, and moreover, it remains in only limited regions. In a matured body, for example, the cartilage tissue remains only as an articular cartilage that covers mainly an epiphyseal surface.

The chondrocytes of an epiphyseal cartilage are differentiated into a static chondrocyte, proliferating chondrocyte, pre-hypertrophic chondrocyte and hypertrophic chondrocyte in this order in the direction from the epiphyseal region to the diaphseal region, and the cell populations on syntropic differentiation stages are aligned in layers very orderly. Each cartilage layer expresses and produces a molecule specific thereto corresponding to its degree of the differentiation. A proliferating chondrocyte expresses as its differentiation indexes a cartilage-specific type II collagen and a proteoglycan that is a complex of saccharides and proteins, especially an aggrecan. A hypertrophic chondrocyte expresses type X collagen rather than type II.

A chondrocyte is differentiated from a mesenchymal cell (pluripotent undifferentiated mesenchymal cell). More recent studies revealed factors which regulate the proliferation and differentiation of a chondrocyte (see for example, Crombrugghe B. et al.: Current Opinion in Cell Biology, 13, p.721-727 (2001 )). A Sox (sry-type HMG box) gene group is a transcriptional control gene group having a domain highly homologous to an HMG (high mobility group) box of a sex determining gene Sry (sex determining region Y), and 20 types or more of the Sox genes have currently been identified. Among these genes, Sox9 was reported to serve as a cause for a camptomelic dysplasia when it is mutated in a human (Foster J. W. et al.: Nature, 372, p.525-530 (1994)) and also to be expressed predominantly in a cartilage tissue during the development process of a mouse (Wright E. M. et al.: Nature Genet., 9, p.15-20 (1995), and an experiment using an ES cell having a Sox9 gene mutation revealed that a Sox9^_/" cells were blocked in their differentiation to become chondrocyte tissue and can not express an extracellular gene specific to a chondrocyte such as type II collagen (Bi W. et al., Nature Genet., 22, p.85-89 (1999)), suggesting a role as a transcription factor which is important for chondrogenesis. However, the function of the Sox9 is not limited to a chondrocyte, and the activation of type II collagen gene is reported to require some unknown partner gene in addition to the Sox9 (Kamachi Y. et al.: Mol. Cell. Biol. 19, p.107-120 (1999)), still reflecting a poorly elucidated nature. Similarly, the Sox9 expression control mechanism has not been made clear yet. Other factors that have been reported to be involved in the proliferation and differentiation of a chondrocyte are bone morphogenetic protein (BMP), fibroblast growth factor (FGF), insulin-like growth factor (IGF), parathyroid hormone related peptide (PTHrP), sonic hedgehog, Indian hedgehog and the like. Any of these factors is a common basic controlling factor playing an important role during the formation of various tissue and organ in addition to cartilages and, specific differentiation inducing factors for these factors have not been identified yet.

While a well-vasculized bone has a relatively potent regeneration ability, the articular cartilage, which covers the surface of the bones, has an extremely poor regeneration ability. A damage on the surface of an articular cartilage leads readily to osteoarthritis, which is very difficult to be recovered by means of the cartilage. The ethiology of a cartilage disease will be more fully understood and the development of therapeutic means will be more promoted if the molecular mechanisms of the chondrocyte proliferation and differentiation is clarified.

However, the mechanisms of the chondrocyte proliferation and differentiation are still left mostly unknown, and their entire nature is not understood. It is thus desired to identify a novel molecule involved in the chondrocyte proliferation and differentiation and to further clarify the mechanisms.

Disclosure of Invention

The object of the present invention is to identify a new gene and protein that can control the proliferation and differentiation of a chondrocyte, and to provide a method for using them in medicaments, diagnostics and therapy.

Thus, the present invention provides a novel protein that can inhibit type II collagen expression, a DNA encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, and antibody directed against the protein or a peptide fragment thereof, a process for producing the antibody, and a pharmaceutical composition containing the protein or the gene.

The present invention also provides, utilizing a protein, DNA, recombinant vector and transformant described above, a method for screening for a substance that can inhibit or promote type II collagen expression, a kit for the screening, a substance that can inhibit or promote type II collagen expression obtainable by the screening method or the screening kit, a process for producing such a substance, a pharmaceutical composition containing a substance that can inhibit or promote type II collagen expression. A mouse EC (embryonal carcinoma)-derived cloned cell line ATDC5 can not only allow cartilage differentiation to be induced very efficiently but also fulfills the requirements essential for a cartilage stem cell because of its high self replication ability (Atsumi T. et al.: Cell Diff. Dev., 30, p.109-116 (1990), Shukunami C. et al.: J. Cell Biol., 133, p.457-468 (1996)). Recently, it has become possible to utilize this cell to analyze the cartilage differentiation controlling mechanisms. An undifferentiated ATDC5 cell cultured in the presence of an insulin results in the formation of a cell aggregation region in the culture system, as is observed in a limb bud mesenchyme cell. An undifferentiated cell characteristic of the aggregation region may be referred to as a pro-chondrocyte, and will be differentiated into a proliferating chondrocyte in this region. On such a stage, the expressed collagen type is changed dramatically from type I to type II. A chondrocyte expressing type II collagen proliferates slowly to form a cartilage nodule. Then it will further be differentiated into a hypertrophic chondrocyte expressing type X collagen, finally resulting in a calcification of an extracellular matrix. As mentioned above, all differentiation stages of a cartilage can be simulated using the ATDC5 cells.

On the other hand, there are reports with regard to the control of the expression of type II collagen which is a molecule specific to a cartilage which teaches that a regulatory sequence (enhancer) required to express type II collagen specifically in a cartilage is present in the first intron of a type II collagen gene and is conserved between mammalian type II collagen genes (see for example, Lefebvre V. et al.: Mol. Cell. Biol. 16, p.4512-4523 (1996), Lefebvre V. et al.: Mol. Cell. Biol. 17, p.2336-2346 (1997), Bell, D.M. et al.: Nature Genet., 16, p.174-178 (1997), Zhou G. et al.: J. Biol. Chem. 272, p.14989-14997 (1998)).

The inventors have intensively studied under the technical circumstance described above to solve the problems mentioned above, and as a result and have succeeded in constructing a full-length cDNA library by using the oligo-capping method; establishing a gene function assay system by an expression cloning method using ATDC5 cells; and isolating a new DNA (cDNA) encoding a protein having a function of inhibiting type II collagen expression by using the assay system. This new DNA is proven to inhibit type II collagen expression by its expression in ATDC5 cells. These results show that this new DNA is a molecule involved in the type II collagen expression, whereby establishing the invention.

Thus, the present invention relates to: (1) A purified protein selected from the group consisting of:

(a) a protein which consists of an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,

40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78; and

(b) a protein that inhibits type II collagen expression and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78.

(2) A purified protein that inhibits type II collagen expression and comprises an amino acid sequence having at least 95% identity to the protein according to the above-mentioned (1) over the entire length thereof.

(3) A protein having that inhibits the differentiation of a chondrocyte which is a protein according to the above-mentioned (1) or (2).

(4) An isolated polynucleotide which comprises a nucleotide sequence encoding a protein selected from the group consisting of:

(a) a protein which consists of an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78; and

(5) An isolated polynucleotide comprising a nucleotides sequence selected from the group consisting of:

(a) a nucleotide sequence represented by any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77;

(b) a nucleotide sequence encoding a protein that inhibits type II collagen expression and hybridizing with a polynucleotide having the nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions; and

(c) a nucleotide sequence encoding a protein that inhibits type II collagen expression and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in nucleotide represented by any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77.

(6) An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence represented by the coding region of any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77;

(c) a nucleotide sequence encoding a protein that inhibits type II collagen expression and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition, which is any of the coding region represented by any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77.

(7) An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that inhibits type II collagen expression and has at least 95% identity to the polynucleotide according to any of the above-mentioned (4) to (6) over the entire length thereof.

(8) A polynucleotide encoding a protein that inhibits the differentiation of a chondrocyte consisting of the nucleotide sequence according to any one of the above-mentioned (4) to (7).

(9) A purified protein encoded by the polynucleotide according to any one of the above-mentioned (4) to (7).

(10) A recombinant vector which comprises a polynucleotide according to any one of the above-mentioned (4) to (7).

(11) A gene therapeutic agent that comprises as an active ingredient the recombinant vector according to the above-mentioned (9).

(12) A transformant which comprises the recombinant vector according to the above-mentioned (10).

(13) A membrane of the transformant according to the above-mentioned (12), having the protein according to the above-mentioned 1 or 2 which is a membrane protein.

(14) A process for producing a protein according to the above-mentioned (1 ), (2), or (9) comprising the steps of:

(a) culturing the transformant according to above-mentioned (12) under conditions providing expression of the protein according to above-mentioned (1 ),

(2) or (9); and

(b) recovering the protein from the culture.

(15) A process for diagnosing a disease or susceptibility to the disease related to expression or activity of the protein of the above-mentioned (1), (2) or (9) in a subject comprising the step of:

(a) determining the presence or absence of a mutation in a gene encoding the protein in the genome of the subject; and/or

(b) analyzing the amount of expression of the gene in a sample derived from the subject. Preferably the presence of the disease is diagnosed when the amount of the protein expressed is 2-fold or higher, or half or lower than normal.

(16) A method for screening for a compound that inhibits or promotes type II collagen expression that comprises the steps of:

(a) introducing a gene encoding the protein that inhibits type II collagen expression according to the (1 ), (2) or (9) and a gene that encodes a signal capable of detecting type II collagen expression into a host to form a transformant;

(b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds; (c) measuring a signal capable of detecting type II collagen expression; and

(d) selecting a candidate compound capable of changing signal amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression. This method is accomplished preferably by isolating or identifying as an inhibitor compound a compound that increases the signal 1.2-fold or higher than normal and isolating or identifying as an promoter compound a compound that decreases the signal 0.8-fold or less than normal.

(17) A method for screening for a compound that inhibits or promotes type II collagen expression that comprises the steps of:

(a) introducing a gene encoding the protein that inhibits type II collagen expression according to the above-mentioned (1), (2) or (9) into a host to form a transformant;

(b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds;

(c) measuring type II collagen expression; and

(d) selecting a candidate compound capable of changing type II collagen expression amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression. (18) A compound having an activity inhibiting or promoting type II collagen expression, selected by the screening method according to the above-mentioned

(15) or (16).

(19) A process for producing a pharmaceutical composition that comprises the steps of: (a) introducing a gene encoding the protein that inhibits type II collagen expression according to the above-mentioned (1), (2) or (9) and a gene encoding a signal capable of detecting type II collagen expression into a host to form a transformant; (b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds;

(c) measuring a signal capable of detecting type II collagen expression;

(d) selecting a candidate compound capable of changing signal amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression; and

(e) preparing a pharmaceutical composition containing the compound selected in the step (d).

In the present invention, this method is accomplished preferably by isolating or identifying as an inhibitor compound a compound that increases the signal 1.2-fold or higher than normal and isolating or identifying as an activator compound a compound that decreases the signal 0.8-fold or less than normal.

(20) A process for producing a pharmaceutical composition that comprises the steps of: (a) introducing a gene encoding the protein that inhibits type II collagen expression according to the above-mentioned (1 ), (2) or (9) into a host to form a transformant;

(b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds; (c) measuring type II collagen expression;

(d) selecting a candidate compound capable of changing type II collagen expression amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression; and (e) preparing a pharmaceutical composition containing the compound selected in the step (d).

(21) A kit for screening for a compound that inhibits or promotes type II collagen expression that comprises: (a) a transformant containing an introduced gene encoding the protein that inhibits type II collagen expression according to the above-mentioned (1 ), (2) or (9) and an introduced gene encoding a signal capable of detecting type II collagen expression; and (b) reagents for measuring the signal.

(22) A monoclonal or polyclonal antibody or a fragment thereof that recognizes the protein according to the above-mentioned (1), (2) or (9).

(23) A process for producing the monoclonal or polyclonal antibody or a fragment thereof according to the above-mentioned (22) that comprises administering the protein according to the above mentioned (1), (2) or (9) as an antigen or epitope-bearing fragment to a non-human animal.

(24) An antisense oligonucleotide that has a complementary sequence to a part of the polynucleotide according to any one of the above-mentioned (4) to (7) which inhibits the expression of a protein that inhibits the type II collagen expression.

(25) A ribozyme or deoxyribozyme which promotes the type II collagen expression by the cleavage of an RNA encoding the protein according to the above-mentioned (1), (2) or (9).

(26) A double-stranded RNA having a sequence corresponding to a part of the polynucleotide sequence according to any one of the above-mentioned (3) to (7), that suppresses the expression of a protein that inhibits type II collagen expression.

(27) A process of treating a disease comprising administering a compound screened by the method according to the above-mentioned (16) or (17) and/or monoclonal or polyclonal antibody or a fragment thereof according to the above-mentioned (22) or (23) and/or anti-sense oligonucleotide according above-mentioned (24) and/or ribozyme or deoxyribozyme according to the above-mentioned (25) and/or double-stranded RNA according to the above-mentioned (26) in amount effective for preventing and/or treating of the cartilage disease to a subject.

(28) A pharmaceutical composition produced by the method according to the above-mentioned (19) or (20) for inhibiting or promoting the type II collagen expression.

(29) The pharmaceutical composition according to the above-mentioned (28) for the prevention and/or the treatment of a cartilage disease.

(30) The pharmaceutical composition according to the above-mentioned (28) for the prevention and the treatment of osteoarthritis, cartilage defect or rheumatoid arthritis.

(31) A method for preventing and treating a cartilage disease comprising administering a pharmaceutical composition produced by the method according to the above-mentioned (19) or (20) to a patient suffering from a disease associated with a cartilage. (32) A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to the above-mentioned (22) as an active ingredient.

(33) A pharmaceutical composition which comprises an antisense oligonucleotide according to the above-mentioned (24) as an active ingredient. (34) A pharmaceutical composition which comprises the ribozyme or deoxyribozyme according to the above-mentioned (25) as an active ingredient. (35) A pharmaceutical composition comprising as an active ingredient the double-stranded RNA according to the above-mentioned (26) or a vector capable of expressing the double-stranded RNA. (36) The pharmaceutical composition according to the above-mentioned (32), (33), (34) or (35)for the treating and/or preventing a cartilage-related disease. (37) A computer-readable medium on which a data set comprising at least one nucleotide sequence represented by SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77 or a nucleotide sequence of a coding region thereof, and/or a data set comprising at least one amino acid sequence represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are stored.

(38) A method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on the medium according to the above-mentioned (37) with data of other nucleotide sequences and/or amino acid sequences.

(39) An insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77 are fixed. (40) An insoluble substrate to which polypeptides comprising all or a part of the amino acid sequence selected from the amino acid sequences represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are fixed.

Best Mode for Carrying Out the Invention

The basic feature of the present invention shall be explained below while explaining first the process that led to the invention. To obtain a new gene having a function of inhibiting type II collagen expression, the inventors conducted experiments that are explained below.

First, using the oligo-capping method, a full-length cDNA was produced from mRNA prepared from ATDC5 cells (purchased from RIKEN GENE BANK) or normal human lung fibroblasts (purchased from Sanko Junyaku Co., Ltd.), and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864). Next, the cDNA library was transformed into E. coli cells, and plasmid preparation was carried out per clone. Then, a reporter plasmid containing a promoter sequence and an enhancer sequence of a type II collagen gene upstream of the DNA encoding luciferase, i.e., a reporter plasmid that can investigate the expression of the type II collagen gene and the above full-length cDNA plasmid were co-transfected into ATDC5 cells. Subsequently, an insulin-like growth factor-l (IGF-I), fibroblast growth factor-basic (bFGF) were added at the final concentration of 50 ng/ml, whereby inducing the type II collagen gene. After 48 hours of culture, luciferase activity was measured, and the plasmid with significantly reduced luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA) was selected (the selected plasmid showed a 0.6-fold or less reduction in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was determined. The protein encoded by the cDNA thus obtained shows that this protein is a molecule involved in type II collagen expression.

The present invention is described in detail below. The present invention provides a protein as follows in relation with the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78: (a) comprises one of the above amino acid sequences; (b) is a peptide having one of the above amino acid sequences;

(c) inhibits type II collagen expression and consists of an amino acid sequence having one or more, favorably several, amino acid deletion, substitution or addition in the above amino acid sequences; (d) comprises an amino acid sequence, which has at least 95% identity, preferably at least 97 to 99% identity, to an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 over the entire length thereof.

"Identity" in this specification as known in the art is a relationship between two or more protein sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein or nucleotide sequences, as determined by the match between protein or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" can readily be calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. "Homology" can be determined by using the BLAST (Basic Local Alignment Search Tool) by Altschul et al. program (for example, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215, p.403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25, p.3389-3402 (1997)). When using a software such as BLAST, it is preferred to use a default value. A major initial condition employed generally in a BLAST search may for example be, but not limited to, the following conditions.

An amino acid substitution matrix is a matrix obtained by converting the analogy of each pair of the 20 amino acids into a numerical value, and one employed generally is a BLOSUM62 default matrix. The theory of this amino acid substitution matrix is detailed in Altschul S. R, J. Mol. Biol., 219:555-565 (1991), while the application to the comparison of DNA sequences is detailed in States D. J., Gish W., Altschul S. R, Methods, 3:66-70 (1991). An optimum gap cost in this procedure has empirically been determined, and the BLOSUM62 preferably employs the parameters of Existence 11 , Extension 1. An expectative value (EXPECT) is a threshold with regard to the statistical significance of the matching to a database sequence, and its default value is 10.

For example, a protein having an identity of 95% or more to the amino acid sequence described in SEQ ID NO. 2 may contain up to 5 variations of the amino acids per 100 amino acids in the amino acid sequence described in SEQ ID NO. 2. In other words, a protein having an identity of 95% or more to a control amino acid sequence may undergo deletion or substitution of up to 5% of the entire amino acids in the control sequence, or up to 5% of the amino acids in the entire amino acid sequence of the control sequence may be inserted into the control sequence. Such a variation in the control sequence may be present on the amino terminal or carboxyl terminal of the control amino acid sequence, or may be present in any position between the both terminals, or may occur serially one or more times in the control sequence. The Examples described below demonstrate that the protein consisting of an amino acid sequence of the above SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 can inhibit type collagen expression.

The present invention also provides a polynucleotide of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77 or a polynucleotide containing a coding region (CDS) shown in these sequences. (Unless otherwise specified, these polynucleotides are, as used herein, referred to as "polynucleotide of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77". The nucleotide sequences represented by SEQ ID NOs. are handled similarly.) In relation with this, the present invention further provides an isolated polynucleotide as follows: (a) polynucleotides having the above-described sequences;

(b) polynucleotides comprising a nucleotide sequence that has at least 95% identity, preferably at least 97-99% identity to one of the above sequences and encoding a protein that inhibits type II collagen expression; (c) polynucleotides having a nucleotide sequence encoding a protein that inhibits type II collagen expression and has at least 95% identity, preferably at least 97-99% identity, to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. Polynucleotide that are identical or substantially identical to nucleotide sequences contained in the above nucleotide sequences may be used as hybridization proves to isolate full-length cDNA and genomic clones encoding the protein of the present invention, or cDNA or genomic clones of other genes that have a high sequence similarity to the above sequences, or may be used as primers for nucleic acid amplification reactions. Typically, these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences. The probes or primers will generally comprise at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.

The polynucleotide of the present invention may be either in the form of a DNA (such as cDNA, a genomic DNA obtained by cloning or synthetically produced), or may be in the form of RNA (such as mRNA). The polynucleotide may be single-stranded or double-stranded. The double-stranded polynucleotides may be double-stranded DNA, double-stranded RNA or DNA:RNA hybrid. The single-strand polynucleotide may be a sense strand (also known as a coding strand) or an antisense strand (also known as a non-coding strand).

Those skilled in the art can prepare a protein having the same type II collagen expression inhibiting activity as the protein having an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, by means of appropriate substitution of an amino acid in the protein using known methods. One such method involves using conventional mutagenesis procedures for the DNA encoding the protein. Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd). Mutation of amino acids in proteins may also occur in nature. Thus, the present invention also includes a mutated protein that can inhibit type II collagen expression and that has at least one amino acid deletion, substitution or addition relative to the protein of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, and the DNA encoding the protein. The number of mutations of amino acids is not limited, preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3.

The substitutions of amino acids are preferably conservative substitutions, specific examples of which are substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (aspargine, glutaine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).

Based on the DNA (e.g., SEQ ID NO: 1) encoding a protein consisting of an amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 or a fragment thereof, those skilled in the art can routinely isolate a DNA with a high sequence similarity to these nucleotide sequences by using hybridization techniques and the like, and obtain proteins having the same type II collagen expression inhibiting activity as the protein having amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. Thus, the present invention also includes a protein that inhibits type II collagen expression and has a high identity to the protein of the amino acid sequence of the above SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78. "High identity" refers to an amino acid sequence having an identity of at least 95%, preferably at least 97 to 99% over the entire length of an amino acid sequence expressed by the above SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78.

A type II collagen expression inhibiting effect mentioned in the present invention means a direct or indirect inhibition of the type II collagen expression upon transducing a gene into an appropriate cell and allowing the gene to overexpress a protein encoded thereby. The type II collagen expression may be measured for example by means of a reporter assay employing a reporter plasmid that can investigate the type II collagen gene expression constructed by using type II collagen gene promoter sequence and regulatory (enhancer) sequence. A type II collagen expression inhibiting effect means an ability of reducing the type II collagen expression when compared with a control group (cell showing no such overexpression of the protein). The reduction in the type II collagen expression is preferably 0.8-fold or lower, more preferably 0.6-fold or lower.

A gene encoding a protein that inhibits the type II collagen expression can be examined by cloning a polynucleotide (for example cDNA) encoding a protein to be expressed into an appropriate expression vector, co-transfecting the expression vector with a reporter plasmid that can investigate the type II collagen expression into an appropriate cell, stimulating the cell with a factor known to induce the type II collagen expression, incubating for a certain period and then measuring the reporter activity. Such an appropriate expression vector is well known to those skilled in the art, and may for example be pME18S-FL3, pcDNA3.1 (Invitrogen) and the like. A reporter gene may be any of those whose expression can readily be detected by those skilled in the art, including genes encoding luciferase, chloramphenicol acetyl transferase and β-galactosidase. A gene encoding luciferase is employed most preferably. A reporter plasmid that can investigate type II collagen expression may for example be CPE43 herein described. An appropriate cell may for example be ATDC5 cell. Cell culture and gene transfection to a cell can be optimized by those skilled in the art in a customary manner.

In a preferred method, the cells are inoculated to a 96-well plate for cell culture at the density of 7500 cells/well in a culture medium (1 :1 mixture of HAM F-12 medium and D-MEM (Dulbecco's Modified Eagle medium), supplemented with 10 μg/ml human transferring and 0.3 nmol/l sodium selenite) in the presence of 5% FBS (Fetal Bovine Serum), and incubated in the presence of 5% CO₂ at 37°C for 24 hours, and then individual wells were co-transfected with a CPE43 reporter plasmid and an expression vector using FuGENE6 (Roche). Subsequently, a factor known to induce the type II collagen expression such as FGF, IGF, BMP and the like or a factor known to induce the chondrocyte proliferation or differentiation is added to the culture medium. After incubation at 37°C for 48 hours, a long term luciferase assay system PICK-A-GENE LT2.0 (Toyo Ink Mfg. Co., Ltd.) is employed to measure the luciferase activity, whereby determining the type II collagen expression. The luciferase activity measurement can be accomplished for example by using Wallac ARVOTMST 1420 MULTILABEL COUNTER supplied from Perkin Elmer. The method of gene transfection by FuGENEδ and the method of luciferase activity measurement by PICK-A-GENE LT2.0 are in accordance with the respective protocols attached. The method of gene transfection to a 96-well plate using FuGENE6 employs 0.3 to 0.5 μl, preferably 0.4 μl of FuGENE6, 50 to 100 ng, preferably 100 ng of the CPE43 reporter plasmid and 50 to 100 ng, preferably 100 ng of the expression vector. The concentration of FGF, IGF, BMP and the like is preferably 10 to 100 ng/ml. The ability of inhibiting the type II collagen expression means an ability of reducing the activity of the reporter (luciferase activity) when compared with a control experiment (cell transfected only with a blank vector). The reduction in the reporter activity as an index is preferably 0.8-fold or lower, more preferably 0.6-fold or lower. The proteins of the present invention may be natural proteins derived from any human or mammal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques. The protein may or may not be subjected to post-translational modification such as sugar chain addition or physphorylation. A protein encoded by the gene of the present invention may for example be a secreted protein (proliferation factor, cytokine, hormone and the like), protein modifying enzyme (protein kinase, protein dephosphorylating enzyme, protease and the like), signal transmitting molecule (protein interaction molecule and the like), intranuclear protein (intranuclear receptor, transcription factor and the like) and membrane protein. The membrane protein may for example be a receptor, cell adhesion molecule, ion channel, transporter and the like. When the protein is a membrane protein, it serves as a more useful research tool for a pharmaceutical compound since a compound selected by the screening described below is assumed to migrate readily into the cell or transmit a signal into the cell.

The present invention also provides a polynucleotide encoding the above protein of the present invention. Specific examples of nucleotide sequences encoding a protein consisting of an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 include nucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77. The DNA includes cDNA, genomic DNA, and chemically synthesized DNA. In accordance with the degeneracy of the genetic code, at least one nucleotide in the nucleotide sequence encoding a protein consisting of an amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 can be substituted with other nucleotides without altering the amino acid sequence of the protein produced from the gene. Therefore, the DNA sequence of the invention also includes nucleotide sequences altered by substitution based on the degeneracy of the genetic code. Such DNA sequences can be synthesized using known methods.

The DNA of the present invention includes a DNA that encodes a protein that can inhibit type II collagen expression and hybridizes under stringent conditions with the DNA consisting of the nucleotide sequence complementary with the nucleotide sequence represented by any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77. Stringent conditions are apparent to those skilled in the art and can be easily attained in accordance with various laboratory manuals such as one by T. Maniatis et al. (Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory 1982, 1989). That is, "stringent conditions" refer to overnight incubation at 37°C in a hybridization solution (5 x SCC (0.75 M NaCl, 75 mM trisodium citrate), 5 x Denhardt's solution, 0.5% SDS, 100 μg/ml denatured, sheared salmon sperm DNA), containing 30% formamide, followed by washing three times in 2 x SSC, 0.1 % SDS for 10 minutes at room temperature, then followed by washing two times in 1 x SSC, 0.1 % SDS for 10 minutes at 37°C (low stringency). Preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 40% formamide, followed by washing three times in 2 x SSC, 0.1 % SDS for 10 minutes at room temperature, then followed by washing two times in 0.2 x SSC, 0.1 % SDS for 10 minutes at 42°C (moderate stringency). Most preferred stringent conditions are overnight incubation at 42°C in a hybridization solution containing 50% formamide, followed by washing three times in 2 x SSC, 0.1 % SDS for 10 minutes at room temperature, followed by washing two times in 0.2 x SSC, 0.1 % SDS for 10 minutes at 50°C (high stringency). The DNA thus obtained must encode a protein that can inhibit type II collagen expression.

The present invention also includes a polynucleotide comprising a nucleotide that encodes a protein that can inhibit type II collagen expression and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (4), (5) or (6). Typically these nucleotide sequences are 95% identical, preferably 97% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (4), (5) or (6) over the entire length thereof.

The protein of the present invention has an ability of inhibiting type II collagen expression in ATDC5 cells. A chondrocyte is differentiated from an aggregated mesenchymal stem cell. The mesenchymal stem cell is differentiated to other cells than a chondrocyte, and it can be judged whether it is differentiated to a chondrocyte or not on the basis of the presence or absence of a type II collagen expressed specifically in the chondrocyte. It is well known that the type II collagen is a molecule expressed specifically in a chondrocyte (for example, see Horton W. et al.: Proc. Natl. Acad. Sci. USA 84, p.8864-8868 (1987)). Accordingly, it is considered that the differentiation from mesenchymal stem cell to a chondrocyte can be inhibited by inhibiting the expression of type II collagen. On the other hand, ATDC5 cells are known to be a cell line that can simulate all stages of the chondrocyte differentiation (see for example Atsumi T. et al.: Cell Diff. Dev., 30, p.109-116 (1990), Shukunami C. et al.: J. Cell Biol., 133, p.457-468 (1996)). An ATDC5 cell has the characteristics of a cartilage progenitor cell, and is differentiated to a chondrocyte upon stimulation by a proliferation factor. An undifferentiated ATDC5 cell does not express a type II collagen, but a culture in the presence of insulin until confluent leads to the formation of a specific cell aggregation region, from which a chondrocyte emerges to form a cartilage node. The formation of the cartilage node is reported to be accompanied with the type II collagen expression (Shukunami C, et al.: J. Cell Biol., 133, p.457-468 (1996)). Thus, the present invention is a protein according to above item (1 ) or (2) having an ability of inhibiting the differentiation of a chondrocyte. The present invention is also a nucleotide sequence encoding a protein that inhibits the differentiation of a chondrocyte consisting of a nucleotide sequence according to any of above items (4) to (8).

The above DNA of the present invention can be used to produce the above protein using recombinant DNA techniques. The DNA and peptide of the present invention can be obtained by: (A) cloning the DNA encoding the protein of the present invention; (B) inserting the DNA encoding the entire coding region of the protein or a part thereof into an expression vector to construct a recombinant vector;

(C) transforming hosts with the recombinant vector thus constructed; and,

(D) culturing the obtained hosts to express the protein or its analogue, and then purifying it by column chromatography or the like. General procedures necessary to handle DNA and recombinant hosts

(e.g., E. coli) at the above steps are well known to those skilled in the art, and can easily be carried out in accordance with various laboratory manuals such as one by T. Maniatis et al. All the enzymes, reagents, etc., used in these procedures are commercially available, and unless otherwise stated, such commercially available products can be used according to the use conditions specified by the manufacturer's instructions to attain completely its objects. The above steps (A) to (D) can be further illustrated in more details as follows. Techniques for cloning the DNA encoding the protein of the present invention at the step (A) include, in addition to the methods described in the specification of the application, PCR amplification using a synthetic DNA having a part of the nucleotide sequence of the present invention (e.g., SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77) as a primer, or selection of the DNA inserted into a suitable vector by hybridization with a labeled DNA fragment encoding a partial or full region of the protein of the present invention or a labeled synthetic DNA. Another technique involves direct amplification from total RNAs or mRNA fractions prepared from cells or tissues, using the reverse transcriptase polymerase chain reaction (RT-PCR method).

As a DNA inserted into a suitable vector, for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used. Techniques for hybridization are normally used in the art, and can easily be carried out in accordance with various laboratory manuals such as by T. Maniatis et al. Depending on the intended purpose, the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker. The DNA thus obtained may be a gene having a nucleotide sequence of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77 or a polynucleotide of above items (4) to (8). The DNA to be inserted into an expression vector at the above step (B) may be a full-length cDNA or a DNA fragment encoding the above full-length protein, or a DNA fragment constructed so that it expresses a part thereof. Thus, the present invention provides a recombinant vector, which comprises the above DNA. The expression vector to express the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.

Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses. However, the vectors should be selected to be compatible as hosts.

Suitable combinations of host-expression vector system are selected depending on the desired expression product.

When microorganisms are used as hosts, plasmid vectors compatible with these microorganisms are generally used as replicable expression vectors for recombinant DNAs.

For example, the plasmids pBR322 and pBR327 can be used to transform E. coli. Plasmid vectors normally contain an origin of replication, a promoter and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the transformant transformed with the recombinant DNA. Examples of such promoters include lac promoter and trp promoter. Examples of such marker gene include an ampicillin resistance gene, and a tetracycline resistance gene. Examples of suitable expression vectors include the plasmids pUC18 and pUC19 in addition to pBR322, pBR327.

In order to express the DNA of the present invention in yeast, for example, YEp24 can be used as a replicable expression vector. The plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene. Examples of promoters in expression vectors for yeast cells include promoters derived from genes for 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.

Examples of promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin. Examples of suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83. Examples of promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter. Examples of expression vectors suitable for insect cells include vaculovirus.

Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as a promoter to be placed upstream of the DNA of the present invention, a polyadenylation site and a transcription termination sequence. Such functional sequences, which can be used to express the DNA of the present invention in animal cells, can be obtained from viruses and viral substances. Examples of such functional sequences include an SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SRα promoter can preferably be used. As promoters to be placed inherently upstream of the gene encoding the protein of the present invention, any promoters can be used so long as they are suitable for use in the above host-vector systems. Examples of origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors that can be integrated into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed. Examples of suitable expression vectors include the plasmid pSV-dhfr (ATCC 37146), pBPV-1 (9-1 ) (ATCC 37111), pcDNA3.1 (INVITROGEN) and pME18S-FL3.

The present invention also provides a transformant, which comprises the above recombinant vector. Microorganisms or cells transformed with the recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes and genes encoding dihydrofolate reductase.

As hosts for use at the above step (C), any of prokaryotes (e.g., E. coli), microorganisms (e.g., yeast and fungi) as well as insect and animal cells can be used so long as such hosts are compatible with the expression vectors used. Examples of such microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coli JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E. coli, such as Salmonella typhimurium or Serratia marcesans; and various strains belonging to the genus Pseudomonas.

Examples of such yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of such fungi include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550). As insect cells, for example, Spodoptera frugiperda (Sf cells), High Five

TM cells derived from eggs of Trichoplusia ni, etc., can be used when the virus is AcNPV. Examples of such animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, Chinese hamster ovary (CHO) cell and ATDC5 cells. Among them, CHO cells and HEK 293 cells are preferred. When animal cells are used as hosts, combinations of expression vectors and host cells to be used vary with experimental intentions. According to such combinations, two types of expression (i.e., transient expression and constitutive expression) can be included. "Transformation" of hosts at the above step (C) refers to introducing DNA into microorganisms or cells used as hosts by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form. Those skilled in the art can carry out transformation by known methods (see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)"). For example, in the case of animal cells, DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, Iipofection, etc. For obtaining the cells which stably express the protein of the present invention using animal cells, there is a method in that selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated. For example, transformants can be selected using the above selectable marker as an indication of successful transformation. In addition, the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention. When a Dihydroforate reductase (DHFR) gene is used as a selectable marker, one can culture animal cells while gradually increasing the concentration of methotrexate (MTX) and select the resistant strains, thereby amplifying the DNA encoding the protein of the present invention in the cells together with the DHFR gene to obtain animal cell strains having higher levels of expression.

The above transformant can be cultured under conditions that permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced. Thus, the present invention also provides a process for producing a protein, which comprises culturing a transformant comprising the isolated polynucleotide according to above items (4) to (8) under conditions providing expression of the encoded protein and recovering the protein from the culture, i.e. cells themselves or medium.

The above transformants can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4" YODOSHA CO., LTD.). For example, animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, or suspension culture in which cells attached to cell culture carriers (microcarriers) or productive cells themselves are suspended. Examples of media for use in the culture include media commonly used for animal cell culture, such as D-MEM and RPMI1640.

In order to separate and purify the protein of the present invention from the above culture, suitable combinations of separation and purification methods known per se can be used. Examples of such methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance liquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing. For example, the protein of the present invention can be recovered and purified from recombinant cell cultures by well known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.

The protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the scope of the present invention. For the expression of such fusion proteins, any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein. Examples of proteins to which a peptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His). The fusion protein of the protein of the present invention with another protein can be advantageously purified by affinity chromatography using a substance with an affinity for the fusion partner protein. For example, fusion proteins with GST can be purified by affinity chromatography using glutathione as a ligand.

When the protein of the present invention is a membrane protein, a transformant containing introduced DNA encoding the above-described protein of the present invention can express a protein on its membrane. A membrane prepared from such a transformant and having the protein of the present invention is also within the scope of the present invention. The membrane of a cell referred to in this specification includes a plasma membrane, and all membranes of cell organelles. Preparation of the membrane of a cell can be conducted by methods known to those skilled in the art. For example, cells are recovered from a culture obtained by culturing a transformant, suspended in a suitable buffer, then, cells are broken using a homogenizer or by adding glass beads and using Vortex. The broken solution is subjected to centrifugal separation to remove unbroken cells and the like, the resulted supernatant is subjected to ultracentrifuge under suitable conditions, and the resulted precipitate is suspended in a buffer, to prepare a membrane fraction. The conditions for ultracentrifuge can be appropriately set depending on the kind of the membrane, and the like.

According to the present invention, a protein inhibiting the activity of the protein of the present invention is also provided. Examples of the protein include other proteins such as an antibody or a protein that bond to the active center of the protein of the present invention, and the like, to prevent manifestation of activity.

The present invention relates to an antibody that reacts to the protein of the present invention or a fragment thereof, and to production of such an antibody. Further preferably, the present invention relates to an antibody specifically reacting to the protein of the present invention or a fragment thereof, and to production of such an antibody. The term "specifically" here referred to means that there is little cross-reactivity, more preferably, there is no cross-reactivity.

The antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention, including polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies. Antibody fragments can be produced by known techniques. Examples of such antibody fragments includes, but not limited to,

F(ab')2 fragments, Fab' fragments, Fab fragments and Fv fragments. For example, a monoclonal or polyclonal antibody can be obtained by administering the protein according to above item (1) or (2) as an antigen or epitope-bearing fragments to a non-human animal. The antibody against the protein of the present invention can be produced using the protein of the present invention or a peptide thereof as an immunogen according to methods known per se for producing antibodies or antisera. Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken lgaku(Experimental Medicine)".

In the case of polyclonal antibodies, for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected. The polyclonal antibody can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized. In the case of monoclonal antibodies, for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting hybrid cells (i.e. hybridoma cells), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma cells can be implanted intraperitoneally into mice, and the ascitic fluid is recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the protein has been immobilized.

When the resulting antibody is used to administer it to humans, it is preferably used as a humanized antibody or human antibody in order to reduce its immunogenicity. The humanized antibody can be produced using transgenic mice or other mammals. For a general review of these humanized antibodies and human antibodies, see, for example, Morrison, S. L. et al. (Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)); Jones, P. T. et al. (Nature 321 :522-525 (1986)); Hiroshi Noguchi, (March of Medical Science), 167:457-462 (1993)); Takashi Matsumoto, (Chemistry and Biology 36:448-456 (1998)). Humanized chimeric antibodies can be produced by linking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a humanized antibody for a region other than a complementarity-determining region (CDR) from a mouse monoclonal antibody. In addition, human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems.

These antibodies can be used to isolate or to identify clones expressing the protein. In addition, these antibodies can be used to purify the protein of the invention from a cell extract or transformants producing the protein of the present invention. These antibodies can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems for detection of proteins of the present invention in cells and tissues. These assay system can be used for diagnostic purposes for detecting an amount of the protein of the present invention in a body sample in a tissue or a fluid in the blood of an animal, preferably human. For example, such antibodies can be used for diagnosing a disease characterized by an abnormality in a cartilage caused by an abnormality (expression) in the protein of the present invention, such as osteoarthritis and rheumatoid arthritis.

In order to provide a basis for diagnosis of a disease, a normal value about the expression of the protein of the present invention, that is, a standard value must be established. However, this is a well known technique to those skilled in the art. For example, there is a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention). The presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention. These antibodies can also be used as reagents for studying functions of the protein of the present invention. The antibody of the present invention can be used as a pharmaceutical composition as described below. When the antibody of the present invention is used as a pharmaceutical composition, it is necessary to use an antibody that can inhibit an action to inhibit the expression of type II collagen of the protein of the present invention (namely, neutralizing antibody).

The antibodies of the present invention can be purified and then administered to patients characterized by abnormal type II collagen expression and/or abnormal chondrocyte differentiation resulting from the expression abnormality of the protein of the present invention. Thus, in another aspect, the present invention provides a pharmaceutical composition that comprises the above antibody as an active ingredient, and therapy using the antibody of the present invention. The pharmaceutical compositions of the present invention comprises the above antibody as an active ingredient and can be further prepared with the other active ingredients for therapeutic use or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers or excipients and the like. The pharmaceutical composition containing the antibody of the present invention can be sterilized by filtration and lyophilized, and formulated into a dosage form as a stock in an administration vial or in a stabilized aqueous preparation. Administration to a patient can be intra-arterial administration, intravenous administration and subcutaneous administration, which are well known to those skilled in the art. The dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art. The antibodies of the present invention exhibit therapeutic activity by inhibiting abnormal type II collagen expression and/or abnormal chondrocyte differentiation mediated by the protein of the present invention. More typically, an antibody of the invention is useful as a pharmaceutical for treating or preventing a disease associated with a chondrocyte such as osteoarthritis, chondrodystrophy, fracture-induced chondrodystrophy, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans and the like.

The DNA of the present invention can also be used to isolate, identify and clone other proteins involved in chondrocyte differentiation regulation. For example, the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid system (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") that can associate with the protein of the present invention from the cDNA or genome DNA library. In a similar manner, it can be determined whether the protein of the invention can associate with other proteins. In another method, proteins that can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation (see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)") using antibodies directed against the protein of the present invention. In still another method, the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein in order to isolate a protein that can associate with the protein of the present invention.

According to the present invention, a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein in a subject comprising (a) determining the presence or absence of a mutation in a gene encoding the protein of the present invention in the genome of the subject; and/or (b) analyzing the amount of expression of the gene in a sample derived from the subject.

In the diagnosis process of the present invention, a disease or susceptibility to the disease can be diagnosed by detecting a mutation in a gene of the protein of the present invention having a function of promoting type II collagen expression. Further, such a disease may be diagnosed also by a method comprising analyzing at protein level or mRNA level the expression amount of the gene in a sample derived from a subject, and measuring abnormal decrease or increase of the expression amount.

Here, as the method of detecting a mutation in a gene encoding the protein of the present invention having a function promoting type II collagen expression, there is mentioned a method in which RT-PCR is conducted using as a primer a part of the nucleotide sequence in the gene, then, the sequence is determined by a usual nucleotide sequence determining method, to detect the presence or absence of a mutation. Alternatively, the presence or absence of a mutation can be checked also by a PCR-SSCP method (Genomics, 5: 874-879, 1989, "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)".

Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantification of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR, and methods such as RNase protection assay, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein in a sample derived from a host are well known to those skilled in the art. Such assay methods include radioimmunoassays, competitive binding assays, western blot analysis and ELISA assays. For checking expression amount at protein level, measurement can be effected utilizing the above-described antibody of the present invention.

Thought the extent of abnormality of the expression amount of a gene in a sample is not particularly restricted, the presence of diseases can be diagnosed when the expression amount of a protein is 2-fold or more or half or lower than normal, according to one embodiment of the present invention. According to another embodiment of the present invention, the presence of diseases can be diagnosed when the expression amount of a protein is 3-fold or more or one-third or lower than normal.

The DNA of the present invention is useful for gene diagnosis regarding damage, mutation, reduction, increase or overexpression of a DNA or mRNA encoding a protein of the present invention or a partial peptide thereof since it can be used to detect an abnormality in such a DNA or mRNA.

When a mutation is detected in a gene encoding the protein of the present invention in the genome of a subject, there is a possibility for the mutation to cause a disease related to abnormal control of type II collagen expression.

Further, when, as a result of the expression amount of the protein in a sample derived from a subject, the expression amount shows a different value from the normal value, abnormality of the expression amount of the novel protein of the present invention having an action of inhibiting type II collagen expression has a possibility to cagse a disease related to type II collagen expression.

The present invention also relates to a method of screening for a compound that inhibits or promotes type II collagen expression and/or chondrocyte differentiation using the proteins of the present invention. The above-described screening method comprises the steps of:

(a) introducing a gene encoding the protein that inhibits type II collagen expression of the present invention and a gene that encodes a signal capable of detecting type II collagen expression into a host to form a transformant;

(c) measuring a signal capable of detecting type II collagen expression; and (d) selecting a candidate compound capable of changing signal amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression.

An exemplary embodiment is accomplished by isolating or identifying as an inhibitor compound a compound that increases the detectable signal 1.2-fold or higher than normal and isolating or identifying as an promoter compound that decreases the detectable signal 0.8-fold or lower than normal.

Examples of genes encoding a signal capable of detecting type II collagen expression include reporter genes. Reporter genes are used instead of directly detecting the activation of transcription factors of interest, and the transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).

Any peptide or gene encoding the protein can be used so long as those skilled in the art can measure the activity or amount of the expression product

(including the amount of the produced mRNA) of the reporter genes. For example, enzymatic activity of chloramphenicol acetyltransferase, β-galactosidase, luciferase, etc., can be measured. Any reporter plasmids can be used to evaluate type II collagen expression so long as the reporter plasmids have a type II collagen gene promoter sequence and a regulatory sequence necessary for allowing the type II collagen to be expressed specifically in a cartilage inserted upstream of the reporter gene. For example, CPE43 described in the specification can be used. Other examples include reporter plasmids described in Murakami S. et al., Proc. Natl. Acad. Sci., USA, 96, p.1113-1118 (2002) (48-bp chondrocyte-specific Col2al enhancer construction).

Any hosts may be used so long as type II collagen expression can be detected in the hosts. Preferred host cells are mammalian cells such as ATDC5 cells. Transformation and culture of the cells can be carried out as described above.

In a specific embodiment, the method for screening for a compound which inhibits or promotes type II collagen expression comprises culturing the transformant for a certain period of time, adding a certain amount of a test substance, measuring the reporter activity expressed by the transformant after a certain period of time, and comparing the activity with that of a cell to which the test substance has not been added. The reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.). Examples of test substances include, but not limited to, low molecular weight compounds, high molecular weight compounds and peptides. Test compounds may be artificially synthesized compounds or naturally occurring compounds. Test compounds may be a single substance or mixtures. For example, libraries of compounds of lower molecular weights, libraries of compounds synthesized by combinatorial chemistry, natural substances containing cells, plants, animals and parts thereof, and extracted substances derived from these natural substances, and the like can also be used. When a compound containing a plurality of compounds is used as a test substance for screening, by further conducting screening for a test substance having a recognized activity of inhibiting or promoting the activity of type II collagen expression by the protein of the present invention, a single substance having this activity can be isolated. Further, isolation and purification of the intended compound from a mixture can be conducted by known methods, for example, filtration, extraction, washing, drying, concentration, crystallization, various chromatographies and the like appropriately in combination.

The screening method of the present invention can be conducted also by the following steps: (a) introducing a gene encoding the protein that inhibits type II collagen expression of the present invention into a host to form a transformant;

(c) measuring type II collagen expression; and (d) selecting a candidate compound capable of changing type II collagen expression amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression.

As the method of measuring type II collagen expression according to the above-described method, there are a method of measuring the protein amount of type II collagen by effecting ELISA assay or western blot assay using an antibody of type II collagen, and a method of measuring mRNA of type II collagen by northern hybridization, RT-PCR method and the like. The antibody may be produced by known methods, or commercially available products (for example, manufactured by FUNAKOSHI CO., LTD.) may also be used. In addition, the present invention provides a method for producing a pharmaceutical composition according to the following steps: (a) introducing a gene encoding the protein that inhibits type II collagen expression and a gene encoding a signal capable of detecting type II collagen expression into a host to form a transformant; (b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds;

(c) measuring a signal capable of detecting type II collagen expression;

The step (d) is accomplished preferably by isolating or identifying as an inhibitor compound that increases the signal 1.2-fold or higher than normal and isolating or identifying as a promoter compound a compound that decreases the signal 0.8-fold or less than normal.

In addition, the present invention provides a method for producing a pharmaceutical composition according to the following steps;

(a) introducing a gene encoding the protein that inhibits type II collagen expression into a host to form a transformant;

The protein of the invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by the following steps: (a) determining in the first instance the three-dimensional structure of the protein;

(b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist, antagonist or inhibitor;

(c) synthesizing candidate compounds that are predicted to bind or react with the deduced binding or reactive site; and, (d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitor.

According to the present invention, a compound selected by the above-described screening is provided. This compound has an activity of inhibiting or promoting type II collagen expression and/or chondrocyte differentiation. More in detail, this compound has an activity of inhibiting or promoting type II collagen expression and/or chondrocyte differentiation, by the protein of the present invention. A compound obtained by the above-described screening has an activity of inhibiting or promoting type II collagen expression, therefore, it is useful as a pharmaceutical composition for treating and/or preventing a disease derived from abnormal differentiation of a chondrocyte.

When a salt of a compound is wished to be obtained, if a compound is obtained in the form of salt, it may be itself purified, and if obtained in free form, it may be dissolved or suspended in a suitable solvent by a usual method, and a desired acid or base may be added to form a salt which is isolated and purified.

When the compounds or salts thereof obtained by the method of the invention are prepared as a pharmaceutical composition, they can be formulated for example by a standard method as described below. Thus, the above compounds or their pharmaceutically acceptable salt in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed. A form of formulation suitable for the mode of administration is selected. A composition suitable for oral administration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and suspension. A form useful for parenteral administration includes sterile solution, emulsion and suspension. The above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as corn, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate and magnesium stearate, talc, vegetable oil, alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol. Examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol. The present invention also provides a kit for screening for a compound that inhibits or promotes type II collagen expression. The kit comprises:

(a) a transformed cell containing an introduced gene encoding a protein that inhibits type II collagen expression and an introduced gene encoding a signal capable of detecting type II collagen expression, and

(b) reagents for measuring the above-mentioned signal, and it contains reagents necessary for screening for a compound that inhibit or promote type II collagen expression.

In another aspect, the present invention provides a diagnostic kit that comprises:

(a) a polynucleotide of the present invention having a nucleotide sequence represented by SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77; (b) a polynucleotide having the nucleotide sequence complementary to that of (a);

(c) a protein of the present invention having an amino acid sequence represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78, or a fragment thereof; and (d) an antibody to the protein of the present invention of (c).

A diagnostic kit comprising at least one of (a) to (d) is useful for diagnosing a disease or susceptibility to a disease such as osteoarthritis, rheumatoid arthritis, cartilage defect and the like.

As a result of the discovery of a novel protein having an ability of inhibiting type II collagen expression according to the report in the present specification, a novel pharmaceutical and a therapeutic method for treating or preventing a disease associated with a cartilage impairment is provided. In a further embodiment, the present invention relates to a method of using a compound that activates or inhibits the function of a protein having an ability of inhibiting type II collagen expression for the purpose of regulating the proliferation and the differentiation of a chondrocyte. The compound that activates or inhibits the type II collagen expression obtained by the screening method described above is useful as a pharmaceutical for treating or preventing a disease associated with a cartilage impairment. Such a disease may for example be osteoarthritis, chondrodystrophy, fracture-induced cartilage defect, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans, heterotopic ossification and heterotopic calcification.

Further, a gene encoding the protein of the present invention is useful also for gene therapy intending treatment of diseases such as osteoarthritis, chondrodystrophy, fracture-induced cartilage defect, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans, heterotopic ossification, heterotopic calcification and the like. The gene therapy means administration into a human body of a gene or a cell containing an incorporated gene. The protein of the present invention or DNA encoding the protein can be used also for the purpose of diagnosis. Namely, according to the present invention, a gene therapeutic agent containing a gene encoding the protein of the present invention is provided.

When a gene encoding the protein of the present invention is used for gene therapy, a method of RNA interference (RNAi) described later can also be applied. Namely, according to the present invention, there is also provided a vector for gene therapy which vector expresses a double stranded nucleic acid having a gene sequence encoding the protein of the present invention.

Though the form of a gene therapeutic agent is not particularly restricted, there are listed, for example, medical compositions containing an expression vector containing the gene of the present invention in a drug carrier composed of a physiological buffer, and the like. As the drug carrier, other suitable stabilizers (for example, nuclease inhibitor, and the like), chelating agents (for example, EDTA and the like), and/or other auxiliaries can also be contained. Alternatively, the gene therapeutic agent of the present invention may also be provided in the form of complex of a ribosome with an expression vector containing the gene of the present invention. The above-described gene therapeutic agent can also be applied, for example, utilizing a catheter. For example, it is also possible to directly inject the gene therapeutic agent of the present invention into a blood vessel of a patient, and the like. The dosage of the gene therapeutic agent of the present invention should be appropriately varied depending on the age, sex, body weight, conditions of the patient, and the route of administration and the like. In general, the amount of DNA of an active ingredient per administration for an adult is in the range from about 1 μg/kg to about 1000 mg/kg, preferably in the range from about 10 μg/kg to about 100 mg/kg. The frequency of administration is not particularly restricted.

The compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions according to conventional procedures. For example, tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions can be formulated. The formulations thus obtained are safe and of low toxicity, and therefore can be administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys). Administration to patients can be carried out by methods known in the art, such as intraarterial injection, intravenous injection and subcutaneous injection. The dosage and the mode of administration may vary depending on the weight and age of the patient, but those skilled in the art can appropriately select the administration mode and the suitable dosage depending thereon. When the compound can be encoded by a DNA, the DNA can be inserted into a vector for gene therapy, and then the gene therapy can be carried out. Thus, the present invention also provides a pharmaceutical composition that comprises a compound that inhibits or activates the type II collagen expression as an active ingredient.

A compound described above is useful as a pharmaceutical for treating or preventing a disease associated with a cartilage impairment. Thus, the present invention provides a pharmaceutical for treating or preventing a cartilage disease comprising a compound that inhibits or activates type II collagen expression. Specifically, the pharmaceutical is useful as a therapeutic or prophylactic agent for example against osteoarthritis, chondrodystrophy, fracture-induced cartilage defect, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans, heterotopic ossification and heterotopic calcification.

Even further, the present invention also provides use of the above-described compositions in production of the pharmaceutical compositions for treatment of osteoarthritis, chondrodystrophy, fracture-induced cartilage defect, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans, heterotopic ossification and heterotopic calcification and the like.

The present invention also provides an antisense oligonucleotide against a polynucleotide of any one of above-mentioned items (4) to (8). An antisense oligonucleotide is an oligonucleotide having sequence complementary to the target gene sequence. The use of antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions. In this case, the antisense oligonucleotide may be RNA or DNA. The DNA sequence of the present invention can be used to produce an antisense oligonucleotide that can hybridize with the mRNA transcribed from the gene encoding the protein of the present invention. It is known that an antisense oligonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., "Saibou Kougaku (Cell Engineering)" Vol. 13, No. 4 (1994)). The oligonucleotide containing an antisense coding sequence against a gene encoding the protein of the present invention can be introduced into a cell by standard methods. The oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.

The antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form (see e.g., Murakami and Makino, "Saibou Kougaku (Cell Engineering)" Vol. 13, No. 4, p.259-266 (1994); Akira Murakami, "Tanpakushitsu Kakusan Kouso (Protein Nucleic acid and enzyme)", Vol. 40, No. 10, p.1364-1370 (1995), Tsunenari Takeuchi et al., "Jikken Igaku (Experimental Medicine)", Vol. 14, No. 4, p.85-95 (1996)). Thus, the oligonucleotide may have modified sugar moieties or inter-sugar moieties. Examples of such modified forms include phosphothioates and other sulfur-containing species used in the art. According to several preferred embodiments of the present invention, at least one phosphodiester bond in the oligonucleotide is substituted with the structure that can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.

Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure. The antisense oligonucleotide may also contain at least some modified nucleotide forms. Thus, it may contain purine and pyrimidine derivatives other than naturally occurring purine and pyrimidine. Similarly, the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-alkyl and 2'-halogen substituted nucleotides. Examples of modifications in sugar moieties at their 2-posiiton include OH, SH, SCH₃, OCH₃, OCN or O(CH₂)_nCH₃ (wherein n is 1 to about 10), and other substituents having similar properties. All the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit the functions of the RNA.

The antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 15 to about 30 nucleotides, more preferably about 20 to about 25 nucleotides. The antisense oligonucleotide of the present invention can be produced by the well known solid phase synthesis technique. Devices for such synthesis are commercially available from some manufacturers including Applied Biosystems. Other oligonucleotides such as phosphothioates can also be produced by methods known in the art.

The antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention. Those skilled in the art can easily design an antisense oligonucleotides based on a given gene sequence (for example, Murakami and Makino, "Saibou Kougaku (Cell Engineering)" Vol. 13, No. 4, p.259-266 (1994); Akira Murakami, "Tanpakushitu Kakusan Kouso (Protein Nucleic acid and enzyme)", Vol. 40, No. 10, p.1364-1370 (1995), Tsunenari Takeuchi et al., "Jikken Igaku (Experimental Medicine)", Vol. 14, No. 4, p.85-95 (1996)). A recent study suggests that antisense oligonucleotides designed in a region containing 5' region of mRNA, preferably the translation initiation site, are most effective for the inhibition of the expression of a gene. The length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is desirable to confirm that there is no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search. The evaluation of whether the designed antisense molecule is functional or not can be determined by introducing the antisense oligonucleotide into a suitable cell and measuring the amount of the target mRNA (for example by northern blotting or RT-PCR), or the amount of the target protein (for example by western blotting or fluorescent antibody technique), to confirm the effect of expression inhibition by a method kwon to those skilled in the art. Another method including the triple helix technique involves forming a triple helix on the targeted intra-nuclear DNA, thereby regulating its gene expression, mainly at the transcription stage. The antisense oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention. Such RNA, DNA and oligonucleotide can be produced using known synthesizers.

An antisense oligonucleotide of the present invention may be introduced into the cells containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, lipofection, electroporation, and microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses. An antisense oligonucleotide expression vector can be prepared using a suitable retrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.

The DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique to inhibit type II collagen expression inhibition and/or chondrocyte differentiation inhibition mediated by the protein of the present invention.

The antisense oligonucleotide against the gene encoding the protein of the present invention is useful as a pharmaceutical to treat or prevent diseases associated with a chondrocyte such as osteoarthritis, chondrodystrophy, fracture-induced cartilage defect, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans and the like. Thus, the present invention also provides a pharmaceutical that comprises the above antisense oligonucleotide as an active ingredient. The antisense oligonucleotide of the invention can also be used to detect such diseases using northern hybridization or PCR.

The present invention also provides a ribozyme or deoxyribozyme that suppresses the inhibition of type II collagen expression and/or the inhibition of chondrocyte differentiation. A ribozyme and deoxyribozyme is an RNA that can recognize a nucleotide sequence of a nucleic acid and cleave the nucleic acid (see e.g., Hiroshi Yanagawa, Experimental Medicine Bioscience 12: New Age of RNA). The ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention). Based on the nucleotide sequence of the DNA encoding the protein of the present invention, the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed. Such ribozyme or deoxyribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction (or entire loss) of the expression of the protein of the present invention. The level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells. There are two types of ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme. In particular, hammerhead ribozymes or deoxyribozymes have been well studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes or deoxyribozymes solely on the nucleotide sequence information for the DNA encoding the protein of the present invention (see e.g., lida et al., "Saibou Kougaku (Cell Engineering)" Vol. 16 No. 3, p.438-445 (1997); Ohkawa and Taira, "Jikken Igaku (Experimental Medicine)", Vol. 12, No. 12, p.83-88 (1994)). It is known that the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II) forming a chain complementary to target RNA and an active site, and cleave the target RNA at the 3' end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites. In particular, the GUC (or GUA) has been found to have the highest activity (see e.g., Koizumi, M. et al., Nucl. Acids Res. 17:7059-7071 (1989); lida et al., "Saibou Kougaku (Cell Engineering)" Vol. 16, No. 3, p.438-445 (1997); Ohkawa and Taira, "Jikken Igaku (Experimental Medicine)", Vol. 12, No. 12, p.83-88 (1994)); Kawasaki and Taira, "Jikken Igaku (Experimental Medicine)", Vol. 18, No. 3, p.381-386 (2000)).

Therefore, the sequence GTC (or GTA) is searched out, and a ribozyme is designed to form several, up to 10 to 20 complementary pairs around that sequence. The suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in a literature by Ohkawa and Taira, ("Jikken Igaku (Experimental Medicine)", Vol. 12, No. 12, p.83-88 (1994)). The ribozyme can be prepared by method known in the art to synthesize RNA molecules.

Alternatively, the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymatically synthesize an RNA in vitro. Such ribozymes can be introduced into cells by gene transfer methods such as microinjections. Another method involves inserting a ribozyme-encoding DNA into a suitable expression vector and introducing the vector into cell strains, cells or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell, and examples of vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors). Such ribozymes can inhibit type II collagen expression inhibition and/or chondrocyte differentiation inhibition mediated by the protein of the present invention. An ribozyme or deoxyribozyme of the present invention is useful as a pharmaceutical for treating or preventing a disease associated with a chondrocyte such as osteoarthritis, chondrodystrophy, fracture-induced cartilage defect, trauma-induced damage of articular cartilage or articular disk, tuberculous arthritis, rheumatoid arthritis, disk hemiation, spondylosis deformans and the like. Thus, the present invention is a pharmaceutical containing as an active ingredient a ribozyme or deoxyribozyme described above. The present invention also provides a double-stranded nucleic acid that inhibits type II collagen expression.

A phenomenon, called RNA interference (RNAi), has been recently made clear that when a double-stranded RNA is introduced into a cell, mRNA corresponding to this sequence is specifically degraded to suppress gene expression. As a method of utilizing RNAi, there is mentioned, as one example, a method in which artificially synthesized small interfering RNA (siRNA) is introduced into a cell. siRNA is a double-stranded RNA having 19 to 25 base pairs, called trigger important for causing the RNAi phenomenon. With respect to a 19 to 25 base sequence in a suitable region in a sequence of a gene encoding the protein of the present invention, a sense RNA (RNA obtained by substituting a DNA sequence by an RNA sequence) and an antisense RNA (RNA having a sequence complimentary to sense RNA) are synthesized to produce siRNA, and this siRNA is introduced into a cell by, for example, a method of lipofection of FuGENE6 and the like, to utilize RNAi. Further, it as been recently gradually made clear that a method in which a

19 to 25 base sequence in a suitable region in a sequence of a gene encoding the protein of the present invention or a sequence complimentary to this in incorporated in a plasmid, and siRNA is transiently expressed in a cell is also an effective method, as the other method than introduction of synthetic siRNA. Specifically, pSilencer siRNA Expression Vector and the like manufactured by Ambion, for example, can be used (Takashi MORITA, et al., "Tanpakushitsu Kakusan Kouso (Protein Nucleic acid Enzyme)" Vol. 47, No. 14, p.1939 to p.1945 (2002), Asako SUGIMOTO, "Kagaku to Seibutsu (Chemistry and Biology)", Vol. 40, No. 11 , p.713 to 718).

The present invention further provides a method for obtaining a novel gene having a function. More particularly, the present invention provides a method for obtaining a novel gene that comprises using the oligo-capping method to construct a full-length cDNA library, and using a signal factor to detect the presence of a protein having such a function. An example of such a signal factor is a reporter gene.

Methods using a cDNA library containing a lot of non-full-length cDNAs are inefficient in obtaining many genes (cDNAs) having functions. Therefore libraries with a high ratio of the number of the full-length cDNA clones to the total number of the clones are necessary. "Full-length cDNA" refers to a complete DNA copy of mRNA from a gene. The cDNA libraries produced using the oligo-capping method contain full-length cDNAs in a ratio of 50 to 80%, namely, a 5 to 10-fold increase in full-length cDNA clones compared to the cDNA libraries produced by prior art methods (Sumio Sugano, the monthly magazine, BIO INDUSTRY, Vol. 16, NO. 11 , p.19-26). Generally, full-length cDNA clones are essential for protein expression in functional analysis of genes, and full-length cDNA clones themselves are very important materials for activity measurement. Thus, cloning of full-length cDNA is necessary for functional analysis of genes. Sequencing of the cDNA not only provides important information for establishing the primary sequence of the protein encoded by the cDNA, but also reveals the entire exon sequence of the gene. Thus, the full-length cDNA provides valuable information for identifying a gene, such as information for determining the primary sequence of a protein, exon-intron structure, the transcription initiation site of mRNA, the location of a promoter, etc.

The construction of full-length cDNA libraries by the oligo-capping method can be carried out, for example, according to the method described in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)" (1999). Thus, the oligo-capping method used herein involves substituting a cap structure with a synthetic oligo by using BAP, TAP and an RNA ligase, as described in Suzuki and Sugano, "Idenshi Kougaku Handbook "Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)".

A reporter gene that can be used as a signal factor indicating the presence of a protein having a function consists of an appropriate expression regulatory sequence part (one or more) to which a protein factor such as a transcription factor can be attached and a structural gene part enabling the measurement by activation for example by such a transcription factor. The structural gene part may be a gene that encodes any of peptides and proteins so long as it allows those skilled in the art to measure the activity or the amount of the expression product (including the amount of mRNA produced). For example, a polynucleotide encoding chloramphenicol acetyl transferase, β-galactosidase, luciferase and the like may be utilized by measuring the respective enzymatic activity. A reporter gene indicating the presence of a protein having a function may for example be a CPE43 reporter plasmid described in Examples in this specification as well as a reporter gene having a gene expression regulatory sequence for aggrecan, type XI collagen and the like that is a marker gene expressed specifically in a chondrocyte.

For example, when a gene having a function of inhibiting the expression of a type II collagen gene is desired, a CPE43 reporter plasmid that can monitor the expression of the type II collagen and a full-length cDNA clone prepared by an oligo-capping method are co-transfected to cells, and then a factor which induces the type II collagen expression is used to stimulate the cells, from which a plasmid inhibiting an increase of reporter activity is selected to accomplish the objectives described above. The introduction of the cDNA clone into the cells may be effected using a single clone or several clones may be introduced simultaneously. Such a method is exemplified in Examples in this specification in more detail. Nevertheless, by preparing a reporter gene appropriately, it is possible to obtain a gene encoding a protein having an ability of activating any of various physiologically active factors (for example, NF-κB, MAP kinase, neovascularization factor, various transcription factors), in addition to a gene encoding a protein having a type II collagen expression inhibiting effect.

The method for obtaining a novel gene according to the present invention uses an in vitro system or a cell-based system, preferably a cell-based system. Examples of such cells include cells of prokaryotes such as E. coli, microorganisms such as yeast and fungi, as well as insects and animals. Preferred examples include animal cells, in particular, ATDC5 cell.

Since the cDNA of the present invention is full-length, its 5' end sequence is the transcription initiation site of the corresponding mRNA. Therefore, the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA sequence with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and to determine its nucleotide sequence. Thus, by comparing the nucleotide sequence of the cDNA of the present invention with a genomic sequence, the promoter region of the gene located upstream of the cDNA can be identified. In addition, the promoter fragment of the gene thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene. In general, the DNA fragment spanning 2 kB (preferably 1 kb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid. The reporter plasmid can be used to screen for a compound that enhances or reduces the expression of the gene. For example, such a screening can be carried out by transforming a suitable host with the reporter plasmid, culturing the transformant for a certain period of time, adding a certain amount of a test substance, measuring the reporter activity expressed by the transformant after a certain period of time, and comparing the activity with that of a cell to which the test substance has not been added. These methods are also included in the scope of the present invention.

The present invention further relates to a computer-readable medium on which a sequence data set has been stored, the sequence data set comprising at least one nucleotide sequence represented by any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77 and/or at least one amino acid sequence represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78.

In another aspect, the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences. Thus, the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology. These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG. In this manner, a sequence in a database having a certain homology can be found easily. The computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commercially available floppy discs, tapes, chips, hard drives, compact disks and video discs. The data on the medium allows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises steps of providing a first nucleotide sequence containing the nucleotide sequence of the present invention for the computer-readable medium, and then comparing the first nucleotide sequence with at least one second nucleotide or polypeptide sequence to identify the homology. The present invention also relates to an insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from any of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 and 77 are fixed. A plurality of the various polynucleotides that are DNA probes are fixed on a specifically processed substrate such as a slide glass to form a DNA microarray and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes. The data obtained is analyzed and the gene expression is determined.

The present invention further relates to an insoluble substrate to which polypeptides comprising all or part of the amino acid sequences selected from amino acid sequences represented by any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 and 78 are fixed. By mixing organism-derived cell extract with the insoluble substrate on which these proteins are fixed, it is possible to isolate to identify cell-derived components such as proteins captured on the insoluble substrate that can be expected to be useful in diagnosis or drug development. EXAMPLES

The following examples further illustrate, but do not limit the invention. Various changes and modification are possible by those the skilled in the art which are included in the present invention.

EXAMPLE 1 : Construction of full-length cDNA library using oligo-capping method (1) Preparation of RNA from mouse ATDC5 cells

ATDC5, a cell strain cloned from mouse EC (embryonal carcinoma) (Atsumi, T. et al.: Cell Diff. Dev., 30:p.109-116 (1990)) was repeatedly subcultured to obtain fifty 10 cm dishes, and then the cells were recovered with a cell scraper. Then, total RNA was obtained from the recovered cells by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE). Then, poly A+ RNA was obtained from the total RNA by using an oligo-dT cellulose column. Specifically, the poly A⁺ RNA was obtained according to Maniatis et al., supra. The ATDC5 cells were cultured in a 1 :1 mixture of HAM F-12 medium

(SIGMA) and D-MEM (Dulbecco's Modified Eagle medium: SIGMA), supplemented with 5% fetal bovine serum (Invitrogen), 10 μg/ml human transferrin (SIGMA) and 0.3 nmol/l sodium selenite (Wako Pure Chemical Industries, Ltd.) in the presence of 5% CO₂ at 37°C. (2) Preparation of RNA from human lung fibroblast (Cryo NHLF)

Human lung fibroblasts (Cryo NHLF: purchased from Sanko Junyaku CO.,

Ltd.) were cultured according to the attached protocol. After repeating subculturing the cells to obtain fifty 10 cm dishes, the cells were recovered with a cell scraper. Thereafter, poly A+ RNA was obtained by a method similar to that of (1) above.

(3) Construction of full-length cDNA library by oligo-capping method

A full-length cDNA library was constructed from ATDC5 cells and poly A⁺ RNA of the above human lung fibroblasts by the oligo-capping method according to the method of Sugano et al. (e.g., Maruyama, K. and Sugano, S. Gene, 138:171-174 (1994); Suzuki, Y. et al., Gene, 200:149-156 (1997); Suzuki and Sugano, S. "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", the 3rd edition, an extra issue of "Jikken Igaku (Experimental Medicine)". (4) Preparation of plasmid DNA

The full-length cDNA library constructed as above was transformed into E. coli strain TOP 10 by electroporation, then spread on LB agar medium including

100 μg/ml ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96

Ultra Plasmid Kit (QIAGEN), the plasmids were recovered from the E. coli colonies grown on ampicillin-containing LB agar medium.

EXAMPLE 2: Cloning of DNA having ability of inhibiting type II collagen expression (1) Screening for cDNA encoding protein having ability of inhibiting type II collagen expression

ATDC5 cells were inoculated to a 96-well plate for cell culture at the density of 7500 cells/1 OOμl/well and incubated in the presence of 5% CO₂ at 37°C for a day. The culture medium employed was a 1 :1 mixture of HAM F-12 medium (SIGMA) and D-MEM (Dulbecco's Modified Eagle medium: SIGMA), supplemented with 5% fetal bovine serum (Invitrogen), 10 μg/ml human transferrin (SIGMA) and 0.3 nmol/l sodium selenite (Wako Pure Chemical Industries., Ltd.). Then, 100 ng of CPE43 reporter plasmid and 2 μl of the full-length cDNA plasmid prepared at the step (4) in the first Example 1 described above were co-transfected into individual wells using FuGENE6 (purchased from Roche). The transfection was conducted in accordance with the manufacturer's protocol attached. Subsequently, an insulin-like growth factor-l (IGF-I: purchased from SIGMA) and a fibroblast growth factor-basic (bFGF: purchased from SIGMA) were added to the culture medium each at the final concentration of 50 ng/ml. After incubation in the presence of 5% CO₂ at 37°C for 2 days, a long term luciferase assay system, PICK-A-GENE LT2.0 (Toyo Ink Mfg. Co., Ltd.) was employed to measure the reporter activity (luciferase activity) of the CPE43 reporter plasmid in accordance with the attached protocol. The luciferase activity measurement was conducted by using Wallac ARVOTMST 1420 MULTILABEL COUNTER supplied from Perkin Elmer.

The reporter plasmid CPE43 having type II collagen gene promoter sequence and enhancer sequence together with a DNA encoding a luciferase as a structural gene part was constructed as described below. The CPE43 was constructed with referring to a reporter plasmid constructed by Lefebvre et al. (Mol. Cell. Biol., 16, p.4512-4523 (1996)). First, for the purpose of cloning the sequence of the promoter region of a human type II collagen gene, the synthetic oligonucleotides, 5'-AGATCTGGTTACAGCCCAGCTGGG-3' (SEQ ID NO: 79) and 5'-AAGCTTAGCAGCGCTCTGCGTCTTC-3' (SEQ ID NO: 80) were designed as primers based on the nucleotide sequence of a human type II collagen gene (GenBank Accession M60299), which were used to conduct a PCR using a human genome as a template, and about 0.12 kb fragment thus amplified was isolated and digested with the restriction enzymes Bglll and Hindlll and then inserted into a firefly luciferase reporter vector pGL3-Basic Vector (Promega) between the Bglll site and Hindlll site using a T4 DNA ligase (GIBCO/BRL). The nucleotide sequence of the resultant clone was analyzed by a standard method. The PCR was conducted in a 50 μl reaction solution containing 5 μl of a 10-fold concentrated reaction buffer (buffer attached to TaKaRa Ex Taq, Takara Shuzo), 5 μl of a 2.5 mMdNTP mixture, each 2 μl of the primers described above (each 10 mM concentration), 0.5 μl of TaKaRa Ex Taq polymerase (Takara Shuzo) and 200 ng of a human Genomic DNA (CLONTECH) by incubating at 96°C for 2 minutes, followed by 30 cycles of 94°C for 1 minute, 56°C for 1 minute and 20 seconds, 72°C for 1 minute and 40 seconds using TaKaRa PCR Thermal Cycler MP (Takara Shuzo).

Then, an enhancer sequence of the type II collagen gene was produced using synthetic oligonucleotides. 2 strands of synthetic oligonucleotides, 5'-GATCCTGTGAATCGGGCTCTGTATGCGCTTGAGAAAAGCCCCATTCATGAG A-3' (SEQ ID NO: 81) and δ'-GATCTCTCATGAATGGGGCTTTTCTCAAGCGCATACAGAGCCCGATTCACA G-3' (SEQ ID NO: 82) were synthesized. Each of these synthetic oligonucleotides was dissolved in a sterilized water each at 1 μg/μl, and each 1 μl was mixed and made 20 μl with the sterilized water. This solution was heated at 95°C for 5 minutes, and then cooled gradually to room temperature to prepare a double-stranded oligonucleotide solution. This solution was subjected to a ligation using a T4 DNA ligase, digestion with the restriction enzymes BamHI and Bglll, and then an electrophoresis on an agarose gel. About 0.2 kb DNA fragment consisting of the double-stranded oligonucleotides ligated 4 times in the same direction (tandem) was cut out, and purified by Geneclean Spin Kit (BIO 101). This DNA fragment was inserted into the plasmid prepared above (having human type II collagen gene promoter region sequence inserted into pGL3-Basic Vector) at the Bglll site. A clone into which the synthetic oligonucleotides were inserted in such a manner that the restriction enzyme Bglll site occurs between the type II collagen promoter and the synthetic oligonucleotides was selected and employed as CPE43 reporter plasmid. (2) Nucleotide sequencing

The screening was carried out, and plasmids showing a 0.6-fold or less decrease in the luciferase activity compared to that of the control experiment (luciferase activity of the cell into which vacant vector pME18S-FL3 is introduced instead of full-length cDNA) were selected. One pass sequencing was carried out from the 5' end of the cloned cDNA (sequencing primer: 5'-CTTCTGCTCTAAAAGCTGCG-3' (SEQ ID NO: 83) and from the 3' end (sequencing primer: 5'-CGACCTGCAGCTCGAGCACA-3' (SEQ ID NO: 84)) so that as long nucleotide sequence as possible is determined. The sequencing was carried out using the reagent BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (Applied Biosystems) and the device ABI PRISM 377 sequencer or ABI PRISM 3100 sequencer according to the manufacturer's instructions attached to the kit. (3) Full-length sequencing

The full-length nucleotide sequences for the 39 new clones obtained by the above-described screening were determined (SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77, and the amino acid sequences of the protein coding region (open reading frames) were deduced (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78). For 24 clones of the above-described resulted clones, the results of measurement of a reporter activity (luciferase activity) of a CPE43 reporter plasmid reflecting type II collagen expression are shown in Table 1 below. In Table 1 , the value of activity is represented by a ratio when a value obtained in a control experiment (luciferase activity of a cell containing an introduced empty vector pME18S-FL3) is 1.

Table 1

Industrial Applicability

The present invention provides industrially highly useful proteins that can inhibit type II collagen gene expression as well as their genes. The proteins or genes of the present invention are useful in screening for a compound useful in treating or preventing a cartilage impairment-associated disease and also in the production of a diagnostic agent for such a disease.

Free text in Sequence Listing SEQ ID NO 79: Primer SEQ ID NO 80: Primer SEQ ID NO 81 : Enhancer SEQ ID NO 82: Enhancer SEQ ID NO 83: Sequence Primer

SEQ ID NO 84: Sequence Primer

Claims

1. A purified protein selected from the group consisting of:

^•40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78; and

2. A purified protein that inhibits type II collagen expression and comprises an amino acid sequence having at least 95% identity to the protein according to claim 1 over the entire length thereof.

3. An isolated polynucleotide which comprises a nucleotide sequence encoding a protein selected from the group consisting of:

4. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77; (b) a nucleotide sequence encoding a protein that inhibits type II collagen expression and hybridizing with a polynucleotide having the nucleotide sequence complementary to the nucleotide sequence of (a) under stringent conditions; and (c) a nucleotide sequence encoding a protein that inhibits type II collagen expression and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in nucleotide sequence represented by any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77.

5. An isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:

(a) a nucleotide sequence represented by the coding region of any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77;

(c) a nucleotide sequence encoding a protein that inhibits type II collagen expression and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition, which is any of the coding region represented by any of SEQ ID NOs: 1,3,5,7,9, 11, 13, 15, 17, 19,21,23,25,27,29,31,33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77.

6. An isolated polynucleotide comprising a nucleotide sequence encoding a protein that inhibits type II collagen expression and has at least 95% identity to the polynucleotide according to claim 3 over the entire length thereof.

7. An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that inhibits type II collagen expression and has at least 95% identity to the polynucleotide according to claim 4 or 5 over the entire length thereof.

8. A purified protein encoded by the polynucleotide according to any one of claims 3 to 7.

9. A recombinant vector which comprises a polynucleotide according to any one of claims 3 to 7.

10. A gene therapeutic agent that comprises as an active ingredient the recombinant vector according to claim 9.

11. A transformant which comprises the recombinant vector according to claim 9.

12. A membrane of the transformant according to claim 11 , having the protein according to claim 1 or 2 which is a membrane protein.

13. A process for producing a protein according to claim 1 , 2 or 8 comprising the steps of:

(a) culturing the transformant according to claim 11 under conditions providing expression of the protein according to claim 1 , 2 or 8; and

(b) recovering the protein from the culture.

14. A process for diagnosing a disease or susceptibility to the disease related to expression or activity of the protein of claim 1 , 2 or 8 in a subject comprising the steps of: (a) determining the presence or absence of a mutation in a gene encoding the protein in the genome of the subject; and/or

(b) analyzing the amount of expression of the gene in a sample derived from the subject.

15. A method for screening for a compound that inhibits or promotes type II collagen expression that comprises the steps of: (a) introducing a gene encoding the protein that inhibits type II collagen expression according to claim 1 , 2 or 8 and a gene that encodes a signal capable of detecting type II collagen expression into a host to form a transformant; (b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds;

(c) measuring a signal capable of detecting type II collagen expression; and

(d) selecting a candidate compound capable of changing signal amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression.

16. A method for screening for a compound that inhibit or promote type II collagen expression that comprises the steps of:

(a) introducing a gene encoding the protein that inhibits type II collagen expression according to claim 1 , 2 or 8 into a host to form a transformant;

17. A compound having an activity inhibiting or promoting type II collagen expression, selected by the screening method according to claim 15 or 16.

18 A process for producing a pharmaceutical composition that comprises the steps of: (a) introducing a gene encoding the protein that inhibits type II collagen expression according to claim 1 , 2 or 8 and a gene encoding a signal capable of detecting type II collagen expression into a host to form a transformant; (b) culturing the transformant under conditions that permits the expression of the gene in the presence or absence of one ore more candidate compounds; (c) measuring a signal capable of detecting type II collagen expression;

19. A process for producing a pharmaceutical composition that comprises the steps of:

(c) measuring type II collagen expression; (d) selecting a candidate compound capable of changing type II collagen expression amount, as compared with a case in the absence of the candidate compound, as a compound that inhibits or promotes type II collagen expression; and (e) preparing a pharmaceutical composition containing the compound selected in the step (d).

20. A kit for screening for a compound that inhibits or promotes type II collagen expression that comprises: (a) a transformant containing an introduced gene encoding the protein that inhibits type II collagen expression according to claim 1 , 2 or 8 and an introduced gene encoding a signal capable of detecting type II collagen expression; and (b) reagents for measuring the signal.

21. A monoclonal or polyclonal antibody or a fragment thereof that recognizes the protein according to claim 1 , 2 or 8.

22. A process for producing the monoclonal or polyclonal antibody or a fragment thereof according to claim 21 that comprises administering the protein according to claim 1 , 2 or 8 as an antigen or epitope-bearing fragment to a non-human animal.

23. An antisense oligonucleotide that has a complementary sequence to a part of the polynucleotide according to any one of claims 3 to 7 which inhibits the expression of a protein that inhibits the type II collagen expression.

24. A ribozyme or deoxyribozyme which promotes the type II collagen expression by the cleavage of an RNA encoding the protein according to claim 1 , 2 or 8.

25. A double-stranded RNA having a sequence corresponding to a part of the polynucleotide sequence according to any one of claim 3 to 7, that suppresses the expression of a protein that inhibits type II collagen expression.

26. A process of treating a disease comprising administering a compound screened by the method according to claim 15 or 16 and/or monoclonal or polyclonal antibody or a fragment thereof according to claim 21 or 22 and/or anti-sense oligonucleotide according claim 23 and/or ribozyme or deoxyribozyme according to claim 24 and/or double-stranded RNA according to claim 25 in amount effective for preventing and/or treating of the cartilage disease to a subject.

27. A pharmaceutical composition produced by the method according to claim 18 or 19 for inhibiting or promoting the type II collagen expression.

28. The pharmaceutical composition according to claim 27 for the treatment of a cartilage disease.

29. The pharmaceutical composition according to claim 27 for the prevention and/or the treatment of osteoarthritis, cartilage defect or rheumatoid arthritis.

30. A method for treating a cartilage disease comprising administering a pharmaceutical composition produced by the method according to claim 18 or 19 to a patient suffering from a disease associated with a cartilage.

31. A pharmaceutical composition which comprises the monoclonal or polyclonal antibody or a fragment thereof according to claim 21 as an active ingredient.

32. A pharmaceutical composition which comprises the antisense oligonucleotide according to claim 23 as an active ingredient.

33. A pharmaceutical composition which comprises the ribozyme or deoxyribozyme according to claim 24 as an active ingredient.

34. A pharmaceutical composition comprising as an active ingredient the double-stranded RNA according to claim25 or a vector capable of expressing the double-stranded RNA.

35. The pharmaceutical composition according to claim 31 , 32, 33 or 34 for the treating and/or preventing a cartilage-related disease.

36. A computer-readable medium on which a data set comprising at least one nucleotide sequence represented by SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, 51 , 53, 55, 57, 59, 61 , 63, 65, 67, 69, 71 , 73, 75 or 77 or a nucleotide sequence of a coding region thereof, and/or a data set comprising at least one amino acid sequence represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are stored.

37. A method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on a medium according to claim 36 with data of other nucleotide sequences and/or amino acid sequences.

38. An insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from any of SEQ ID NOs: 1, 3,5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 or 77 are fixed.

39. An insoluble substrate to which polypeptides comprising all or a part of the amino acid sequence selected from the amino acid sequences represented by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,

40.42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76 or 78 are fixed.