Molecular Cloning and Characterization of a Novel Gene, CORS26 , Encoding a Putative Secretory Protein and Its Possible Involvement in Skeletal Development*

We cloned a novel mouse cDNA, CORS26 (collagenous repeat-containing sequence of 26 -kDa protein), encoding a secretory protein by suppression subtractive hybridization between transforming growth factor- b 1-treated and untreated C3H10T1/2 cells. The deduced amino acid sequence of CORS26 consists of 246 amino acids with a secretory signal peptide and contains a collagenous region (Gly- X - Y repeats) at the NH 2 termi- nus and a complement factor C1q globular domain at the COOH terminus. CORS26 is structurally similar to C1q and to adipocyte-specific protein Acrp30. Transfection analysis suggested that CORS26 is a secretory protein. Northern blot analysis revealed that CORS26 mRNA was present at high levels in rib growth plate cartilage and at moderate levels in kidney of adult mice. CORS26 mRNA was not detected in NIH3T3 cells, BALB/3T3 cells, C3H10T1/2 cells, or osteoblastic MC3T3-E1 cells by reverse transcription-polymerase chain reaction analysis. In situ hybridization of mouse embryos between 13 and 15 days postcoitus revealed relatively high levels of CORS26 mRNA in condensed prechondrocytic cells of cartilage primordia and developing cartilages. How-ever, CORS26 mRNA were undetectable in mature chondrocytes.

In skeletal development in vertebrates, the formation of chondrocytes from undifferentiated mesenchymal cells is one of the important processes, but the molecular mechanisms are not well understood. Identifying the genes underlying the induction of chondrocyte differentiation will provide powerful tools for understanding skeletal development. The induction of chondrogenesis has been extensively studied in vitro using primary cells and clonal cell lines from a variety of sources (1)(2)(3)(4). The mouse embryonic fibroblast cell line, C3H10T1/2, are multipotential cells and have been induced to undergo differentiation into myocytes, adipocytes, osteoblasts, and chondrocytes under specific culture conditions and treatments (5)(6)(7)(8). The frequency of chondrogenic conversion in C3H10T1/2 cells was much lower and irregular compared with other types of conversion (5,8), but it was recently reported that the induction of chondrogenesis and the formation of spheroids in C3H10T1/2 cells preferentially occurred when treated with transforming growth factor (TGF) 1 -␤1 (9), bone morphogenetic protein-2 (10), or a combination of osteoinductive bone proteins (11) in high-density micromass cultures. The formation of the spheroids resembled the condensation of mesenchymal cells seen in precartilage. Thus, C3H10T1/2 cells in high-density micromass cultures are suited for studying the molecular mechanisms involved in skeletal development.
In the present study, to help clarify the mechanism for skeletal development, mRNAs expressed in TGF-␤1-treated C3H10T1/2 cells were subtracted with those in untreated C3H10T1/2 cells using the suppression subtractive hybridization (SSH) technique (12), and we isolated a novel gene, CORS26 (collagenous repeat-containing sequence of 26-kDa protein). Sequence analysis revealed that CORS26 possesses a collagenous structure at the NH 2 terminus and a complement factor C1q globular domain at the COOH terminus. Due to the structural similarity between CORS26 and subunits of complement factor C1q, this novel protein is thought to be a member of the C1q-related protein family. The presence of the signal peptide, plus the hydrophilic nature of CORS26, suggests that CORS26 is a secretory protein. Indeed, the CORS26 protein was secreted from COS-7 cells by transient transfection analysis. CORS26 mRNA is specifically expressed in cartilage and kidney in the adult mouse. Moreover, the expression of this novel gene was observed in cartilage primordium or developing cartilage in embryonal mouse tissues in vivo. Thus, a possible secretory protein encoded by the CORS26 gene may be one of the important signaling molecules produced by prechondrocytic mesenchymal cells or early chondrocytes during skeletal development.

EXPERIMENTAL PROCEDURES
Cell Lines and Cell Culture-The mouse cell line, C3H10T1/2, was obtained from the RIKEN Cell Bank (Tsukuba, Japan), and micromass cultures were performed as described previously (9). In brief, trypsinized cells were suspended in Ham's F-12 medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Inc.), penicillin (50 units/ml), and streptomycin (50 g/ * This work was supported by a Grant-in-aid from the Ministry of Education, Culture and Science of Japan (No. 11470396). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF246265.
§ To whom correspondence should be addressed: Dept. of Radiology and Radiation Oncology, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-2967; Fax: 81-6-6879-2970; E-mail: tmaeda@radiol.dent.osaka-u.ac.jp. ml) at a concentration of 10 7 cells/ml, and then a 10-l drop of this cell suspension was placed in the center of a 24-well dish at 37°C in a humidified atmosphere containing 5% CO 2 . The cells were allowed to adhere to the dish for 3 h, and the culture was flooded with 1 ml of medium. After 24-h incubation, 1 ng/ml TGF-␤1 was added to the culture medium. Human TGF-␤1 was purchased from R & D Systems (Minneapolis, MN). The mouse cell lines, NIH3T3 and MC3T3-E1, were also obtained from the RIKEN Cell Bank, and BALB/3T3 cells were from Cancer Cell Repository of Tohoku University (Sendai, Japan).
Construction of the Subtractive cDNA Library by SSH-Messenger RNAs obtained from C3H10T1/2 cells were isolated using the Fast-Track mRNA isolation kit (Invitrogen, San Diego, CA). SSH was performed using the PCR-Select cDNA subtraction kit (CLONTECH, Palo Alto, CA) according to the manufacturer's protocol. Tester cDNA was synthesized from mRNAs of C3H10T1/2 cells treated with TGF-␤1 for 6 h, and driver cDNA was from mRNAs of untreated C3H10T1/2 cells. Products from the secondary PCR were cloned into pT7Blue T vector (Invitrogen), and transformed into competent Escherichia coli JM109 cells.
Dot-blot Differential Screening-Cloned cDNA inserts were initially evaluated for differential expression by DNA dot-blot analysis. White colonies from a subtractive cDNA library were picked randomly and boiled for 5 min in 20 l of H 2 O and then centrifuged. DNA in the supernatant was amplified by PCR using universal vector primer M13 and M13R. The PCR products were purified by QIAquick PCR purification kit (Qiagen, GmbH, Hilden, Germany) and dot-blotted on Hybond-N ϩ nylon membranes (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). The membranes were hybridized with [ 32 P]dCTP-labeled tester cDNA as positive and [ 32 P]dCTP-labeled driver cDNA as negative probes, respectively. The nucleotide sequences of candidate positive clones were determined by sequencing, and cDNA inserts were used as probes in Northern blot analysis confirming differential gene expression.
DNA Sequencing and Sequence Analysis-Sequencing of cDNA inserts in the vector were performed by the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (PerkinElmer Life Sciences) with the M13 vector primers on an ABI 373 automated sequencer. For long cDNA inserts, synthetic oligonucleotides were used as primers. The cDNA sequence was analyzed using sequence analysis software, DNA-SIS (Hitachi Software Engineering, Yokohama, Japan). Data base homology searches for the cDNA sequence and deduced protein sequences were performed by BLAST programs with the DNA and protein data bases at the National Center for Biotechnology Information. Analysis of the primary structure of predicted proteins were performed by the PRO-SITE motif search program.
Northern Blot Analysis-Total RNA was isolated from C3H10T1/2 cells before and after 6-h treatment with TGF-␤1 or mouse rib growth plate cartilage using a RNeasy kit (Qiagen). Rib growth plate cartilages were obtained from 10-day-old mice. Twenty micrograms each of total RNA was denatured using glyoxal and electrophoresed in 1% agarose gel, then transferred to Hybond-N ϩ nylon membranes (Amersham Pharmacia Biotech) and UV-cross-linked onto the membrane. For analysis of CORS26 expression in various normal adult mouse tissues, the Multiple Tissue Northern blot (CLONTECH) was purchased. The blotting membrane was incubated in prehybridization solution of 50% formamide, 5ϫ standard saline citrate (SSC), 5ϫ Denhardt's solution, 0.1% SDS, and 50 g/ml salmon sperm DNA at 43°C for 3 h, and then hybridization was performed using a 1.6-kb cDNA fragment of CORS26 labeled with [ 32 P]dCTP by random priming (TaKaRa, Shiga, Japan) at 43°C for 16 h. The membrane was washed in 2ϫ SSC containing 0.1% SDS at room temperature for 15 min, 0.5ϫ SSC containing 0.1% SDS at 55°C for 60 min, and 0.1ϫ SSC at room temperature for 10 min, respectively. The membranes were autoradiographed with Hyper-film (Amersham Pharmacia Biotech) at Ϫ80°C. After autoradiography, the membrane was stripped and rehybridized with mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe for internal loading control.
Rapid Amplification of cDNA Ends-Rapid amplification of cDNA ends (RACE) was performed in both directions using the SMART cDNA amplification kit (CLONTECH) from mRNA of C3H10T1/2 cells treated with TGF-␤1. For the 3Ј-RACE method, 5Ј-GGGCCAAAGGTGAGAAA-GGA-3Ј and 5Ј-GCCCCCGTATCAGGTGTGTA-3Ј were designed as gene-specific primers, and for the 5Ј-RACE method, 5Ј-GGCCCCAAA-TCTCCCAGTCAT-3Ј and 5Ј-GGTCGCCTTTGTCTCCTTTCT-3Ј were designed as gene-specific primers. The nested PCR products were subcloned into the pT7Blue T vector and sequenced.
Amplification of Full-length CORS26 -The full-length coding sequence of CORS26 was amplified by LA Taq polymerase (TaKaRa) using primers 5Ј-CTGTCAAGCTTCCCTGCGAGACTCTT-3Ј and 5Ј-GCAAGCCAGATGGGAGAAAAGTTTAT-3Ј from TGF-␤1-treated C3H10T1/2 cDNA and cloned directly into pGEM-T Easy (Promega, Madison, WI). The amplification conditions were 80°C for 5 min, followed by 30 cycles at 94°C for 30 s, 65°C for 40 s, and 72°C for 2.5 min and a final incubation at 72°C for 5 min. This construct was sequenced for accuracy and was used for in vitro transcription and translation.
In Vitro Transcription and Translation-The TNT T7 quick coupled transcription/translation system (Promega) was used to transcribe and translate the full-length CORS26 cDNA construct in the presence of [ 35 S]methionine (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Five microliters of the products were electrophoresed on a 12.5% SDS-polyacrylamide gel. The gel was treated with Enlightning (PerkinElmer Life Sciences), dried and autoradiographed.
RT-PCR Analysis-Total RNAs isolated from various cell lines were digested with RNase-free DNase I (Promega) at 37°C for 15 min. Then the denatured 1 g of total RNA was reverse-transcribed in 20 l of a reaction mixture containing 5ϫ RT buffer, 10 mM dithiothreitol, 0.5 mM amount of each dNTP, 500 ng of oligo(dT) 15  Aliquots (10 l) of the PCR products were electrophoresed in 1.5% agarose gel, and the gel was stained with ethidium bromide and photographed under UV light. The amplified 300-bp PCR products were subcloned into pGEM-T Easy vector.
In Situ Hybridization-One microgram of recombinant pGEM-T Easy vector containing the 300-bp fragment located at the coding region (nucleotides 305-604) of CORS26 was used as a template. Plasmids were linearized with SpeI to prepare the antisense riboprobe and NcoI to prepare the sense riboprobe. In vitro transcription was performed with 35 S-UTP (Amersham Pharmacia Biotech) using T7 and SP6 RNA polymerase (Promega). Unincorporated labels were removed by ethanol precipitation, and the counts/min were determined on a scintillation counter.
Embryos were obtained from ICR mice. In situ hybridizations were carried out as described previously (14) with modifications. In brief, mouse embryos were fixed with 4% paraformaldehyde and embedded in paraffin for sectioning. The 7-m-thick sections were pretreated with proteinase K and HCl and acetylated. Hybridization was performed with riboprobes (1 ϫ 10 5 cpm per slide) at 53°C overnight. After hybridization, the sections were washed with 5ϫ SSC containing 10 mM dithiothreitol at 50°C for 30 min, SF solution (2ϫ SSC, 50% formamide, and 20 mM dithiothreitol) at 65°C for 30 min, NTE buffer (0.5 M NaCl, 10 mM Tris-Cl, and 1 mM EDTA) at 37°C for 30 min, treated with 20 g/ml RNase A at 37°C for 30 min and then washed with 2ϫ SSC and 0.1ϫ SSC at room temperature. The sections were dehydrated in a graded series of ethanol, air-dried, coated with NTB-2 emulsion (Eastman Kodak Co.) and exposed for 2-3 weeks. Microphotographs were taken using both light-and dark-field optics.
Transient Transfection with FLAG-tagged CORS26 and Immunoblotting-A CORS26 cDNA was tagged with a FLAG epitope at the COOH terminus by PCR using oligonucleotides 5Ј-TTTGCCGAGCCA-TGCTCGGGAGGC-3Ј and 5Ј-TCACTTGTCATCGTCGTCCTTGTAGT-CCTTAGTTTCAAAGAGCAGAAA-3Ј. The sequence for a FLAG tag is underlined. The amplification conditions were 80°C for 5 min, followed by 25 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min and a final incubation at 72°C for 5 min. The PCR product was cloned into pGEM-T Easy vector, and the insert was excised with EcoRI and recloned into the EcoRI site of pcDNA3.1(ϩ) vector (Invitrogen). The orientation and nucleotide sequence of pcDNA3.1/CORS26-FLAG was confirmed by an automated ABI 373 sequencer. The pcDNA3.1/CORS26-FLAG construct was transiently transfected into COS-7 cells (RIKEN Cell Bank) using the SuperFect Transfection Reagent (Qiagen) according to the manufacturer's instruction. A pcDNA3.1(ϩ) vector was transfected in parallel as a negative control. Transfections were performed on cells seeded into six-well tissue culture plates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS 24 h before use at a density of 3 ϫ 10 5 cells/well. Cells were typically transfected with 2 g of DNA, incubated with the DNA suspension for 3 h, and replenished with fresh medium. Then, 48 h after transfection, conditioned medium was collected, and cells were lysed with TNE buffer (10 mM Tris-HCl (pH 7.8), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40). Protein from the conditioned medium was concentrated for 1 h with a Centricon-10 concentrator (Millipore, Bedford, MA). The proteins from the medium and cell extracts were separated on a 12.5% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane in a Trans-Blot semidry electrophoretic transfer cell (Bio-Rad) and immunoblotted with anti-FLAG M2 monoclonal antibody (Sigma) diluted 1:500. Immunocomplexes were detected with a secondary antibody conjugated to peroxidase (Dako, Glostrup, Denmark) and visualized with ECL reagent (Amersham Pharmacia Biotech).
Cell Growth Analysis Using CORS26 Stably Transfected Cells-A DNA fragment containing a coding region of CORS26 was inserted into the EcoRI-EcoRV sites of pcDNA3.1(ϩ) vector. C3H10T1/2 cells were seeded into 35-mm culture dishes in DMEM supplemented with 10% FBS at a density of 3 ϫ 10 4 cells per dish. Twenty-four hours later, the cells were transfected with 2.5 g of either control pcDNA3.1 vector or pcDNA3.1/CORS26 using the SuperFect Transfection Reagent (Qiagen) according to the manufacturer's instruction. The cells were incubated for 48 h and then trypsinized and seeded at a 1:20 ratio in 100-mm culture dishes in DMEM containing 10% FBS. Sixteen hours later, the cells were switched to a selective medium containing 500 g/ml G418 (Promega). After 4 weeks of culture in the selective medium, clonal isolates were expanded, and expression of CORS26 mRNA was verified by RT-PCR as described above. To analyze the growth of C3H10T1/2 cells transfected with pcDNA3.1/CORS26, the transfectants were seeded in 24-well culture plates at a density of 1 ϫ 10 4 cells/well and cultured with DMEM supplemented with 10% FBS. The number of cells were counted with a hemocytometer at the indicated days.

Molecular Cloning and Sequence Analysis of the CORS26
cDNA-To identify genes specifically expressed during skeletal development, the SSH technique was utilized using the mRNA extracted from C3H10T1/2 cells with or without TGF-␤1 treatment, and we selected several clones specifically up-regulated by TGF-␤1 treatment.
We determined the sequence of the isolated partial cDNA clones and carried out homology searches in the GenBank TM using BLAST2. Among all clones that had no significant homology with any known genes in the nucleotide sequence data bases, a 380-bp cDNA clone (clone 129) showed a marked induction by TGF-␤1 treatment (Fig. 1). We, therefore, focused further analysis on this clone. To obtain a full-length sequence, we carried out the 5Ј-and 3Ј-RACE using a TGF-␤1-treated C3H10T1/2-derived cDNA. The full nucleic acid sequence obtained (1,879 bp) and the deduced amino acid sequence are shown in Fig. 2 an isoelectric point of 6.2. Indeed, a protein of ϳ26 kDa was generated by coupled in vitro transcription and translation of the CORS26 cDNA in rabbit reticulocyte lysates (Fig. 3A), suggesting that the predicted initiation sequence serves as the start site of the translation. Hydropathy analysis (16) revealed that the predicted protein was predominantly hydrophilic, and the signal peptide of 22 amino acid residues and the cleavage site were also predicted at the amino-terminal end on the basis of the rules of von Heijne (17) (Fig. 3B). No transmembrane spanning region was predicted. To investigate whether CORS26 protein was secreted from cells, FLAG-tagged CORS26 was transiently transfected into COS-7 cells and analyzed by Western blotting using an anti-FLAG antibody. These findings demonstrated that COS-7 cells synthesized the CORS26 protein, and the protein was secreted into the medium (Fig. 4). Amino acid sequence analysis of the predicted CORS26 protein using the BLASTp search program revealed that the COOH-terminal globular region of CORS26 was 34, 32, and 30% identical to precerebellin (18), complement protein C1qrelated factor (CRF) (19), and adipocyte complement-related protein of 30 kDa (Acrp30, equivalent to AdipoQ) (20,21), respectively. A unique feature of this protein is that the NH 2terminal part contained uninterrupted collagen-like Gly-X-Y repeats (23 repeats) immediately downstream of a short noncollagenous sequence (at amino acids positions 45-113). NH 2terminal Gly-X-Y repeats are known to be found in the complement protein C1q A, B, and C chains (22)(23)(24), Acrp30, chipmunks hibernation-specific proteins (HP-20, HP-25, and HP-27) (25) and CRF (19). In the COOH-terminal half, CORS26 shares homology with the globular domain of the C1q subunits, precerebellin, Acrp30, CRF, and collagens VIII and X (26) (Fig. 5).
It was also notable that there was a proline-rich region in the NH 2 -terminal part (at amino acid positions 55-62). The pre-dicted protein also contained other putative functional sites, phosphorylation sites for protein kinase C (at amino acid positions 152-154) and casein kinase II (at amino acid positions 77-80 and 138 -141) and several N-myristoylation sites.
Expression of CORS26 mRNA in the Adult Mouse Tissues-We examined the expression of CORS26 mRNA in various adult mouse tissues by Northern blot analysis (Fig. 6). CORS26 cDNA hybridized to double transcripts of 2.3 and 2.0 kb. The CORS26 mRNA was expressed in rib growth plate cartilage and kidney. CORS26 mRNA was not detected in other tissues. In growth plate cartilage, the hybridization signal at 2.0 kb was stronger than the signal at 2.3 kb, while, in kidney, the signal at 2.3 kb was stronger than the signal at 2.0 kb. The 2.0-kb size of the transcripts agrees approximately with the length of the cloned CORS26 cDNA.
In Situ Hybridization of CORS26 in the Embryonal Mouse Tissues-To examine the expression of CORS26 during embryonic development in vivo, we performed in situ hybridization using embryonal mouse serial sections at 13-15 days p.c. CORS26 transcripts were localized in regions of cartilage primordium of occipital bone and that of the vertebral body of a 13-day-p.c. embryo (Fig. 8, A-C and F-H). In Meckel's cartilage, high levels of CORS26 mRNAs were seen in a 15-day-p.c. embryo (Fig. 8, D and I). In the cartilage primordium of digital bone of a 14-day-p.c. embryo, CORS26 was expressed in prechondrocytes, but not in mature chondrocytes (Fig. 8, E and J). These experiments showed that CORS26 mRNA was present at relatively high concentrations in precartilaginous primordia and developing cartilages. No specific signal was detected above background levels by sense riboprobes as controls.
Growth of CORS26 Transfected Cells in Vitro-We generated C3H10T1/2 cells stably transfected with the mammalian expression vector containing CORS26. The expression of CORS26 mRNA in the transfectant was verified by RT-PCR analysis (data not shown). The growth of CORS26 transfectants was significantly enhanced when compared with that of control cells. Moreover, saturation densities of CORS26 transfectants were higher than that of control cells (Fig. 9).

DISCUSSION
During skeletal development, condensation of multipotential mesenchymal cells to differentiate toward the various cell types is an important process. One such process is the formation of chondrocytes from undifferentiated mesenchymal cells. It was recently demonstrated that the mouse embryonic mesenchymal cell line, C3H10T1/2, when cultured at high density,  1 and 3) or pcDNA3.1 vector (lanes 2 and 4). The proteins (20 g) were separated on 12.5% SDSpolyacrylamide gel and immunoblotted with an anti-FLAG M2 antibody.
is induced to undergo chondrogenic differentiation by TGF-␤1. C3H10T1/2 cells cultured under this condition form a threedimensional spheroid structure, and the morphology of the cells in the spheroid resembled that of the cells seen in precartilage condensations (9). The formation of the spheroid in vitro mimic the condensation event of chondrogenesis in vivo.
In the present study, we demonstrated isolation of a novel gene, CORS26, encoding a secretory protein of 246 amino acids using a suppression subtractive hybridization technique between TGF-␤1-treated and untreated C3H10T1/2 cells cultured at high density. Sequence analysis reveals that CORS26 protein has a hydrophobic signal peptide at the NH 2 terminus and lacks a putative transmembrane domain. The presence of a putative signal peptide suggests that CORS26 enters the secretory pathway. Actually, CORS26 was secreted from COS-7 cells after transient transfection of a FLAG-tagged CORS26 expression vector. The deduced amino acid sequence of CORS26 displays structural similarity to several C1q related proteins, such as C1q A, B, and C chains, Acrp30, and CRF, containing the collagenous repeats (Gly-X-Y) at the NH 2 terminus and the globular domain at the COOH terminus. This suggested that CORS26 may belong to the C1q family proteins.
C1q family proteins are known to homo-or hetero-oligomerize via the collagenous structures, suggesting that CORS26 might form oligomers with itself or other proteins. The COOH-terminal region of the protein contains three potential phosphorylation sites. Consequently, this molecule is a potential target for protein phosphorylation via protein kinase C and casein kinase II. In addition, a cysteine residue in the globular region of the C1q B and C chains plays an important role in the formation of disulfide bonds with IgG (27) and the stabilization of the triplex strands in the collagenous domain (24). These cysteine residues are replaced by other residues in CORS26 similar to Acrp30 and CRF.
Recently, it was reported that the crystal structure of Acrp30, a member of the C1q family of proteins, showed homology with that of the tumor necrosis factor (TNF) family proteins. Moreover, TNFs (28) and C1q proteins have similar gene structure in the globular domains (29). These similarities suggest an evolutionary link between the C1q-like proteins and TNFs and establish a C1q/TNF molecular superfamily. It has been reported that TNFs have monospecific receptors (TNFRs) (30). Since CORS26 and C1q share significant similarities in the structure of the collagenous domain, which has been shown to be important for ligand-receptor interaction (13), it is possible that CORS26 might signal through the TNFRs or TNFRlike receptors.
Both Northern blot analysis and RT-PCR analysis showed a unique pattern of gene expression of CORS26 in various tissues and cell lines. CORS26 mRNA expression was found in rib Aliquots of the PCR products were electrophoresed in 2% agarose gel, and the gel was stained with ethidium bromide. A 300-bp CORS26specific band was not seen in the mouse cell lines examined. PCR for GAPDH expression was also performed as a quality control. The molecular mass marker is X174/HaeIII. growth plate cartilage and early stages of chondrogenic differentiation of C3H10T1/2 cells from which the CORS26 cDNA was isolated. Although the expression level was lower than that in cartilage, CORS26 was also expressed in kidney of adult mice. Indeed, DNA sequence homology searches in the Gen-Bank TM Mouse EST data bases using the BLAST search program revealed that several mouse EST sequences isolated from whole embryo or adult kidney libraries were homologous or identical to the 5Ј and 3Ј ends of the CORS26 cDNA. These findings from the EST data base are consistent with the tissue distribution of the CORS26 gene. However, CORS26 mRNA was not detected in fibroblastic cell lines and osteoblastic cell lines. The expression of CORS26 in kidney raises the question about its possible role in this tissue, but at present, details are unclear.
The basic form of the skeleton are first recognizable when mesenchymal cells aggregate into regions of high cell density called condensations. They subsequently differentiate into cartilage and bone and continue to grow by cell proliferation, maturation, and matrix deposition. In situ hybridization of mouse embryos 13-15 days p.c. showed that CORS26 was highly expressed in precartilaginous primordia and Meckel's cartilage. In each case, CORS26 transcripts appeared to be localized in condensed prechondrocytic mesenchymal cells or in less mature chondrocytes, whereas the levels of CORS26 mRNA were lower or undetectable in the mature chondrocytes. Furthermore, to test the role of the CORS26 gene in vitro, we generated C3H10T1/2 cells stably transfected with pcDNA3.1/CORS26. Since CORS26 mRNA is expressed in condensed prechondrocytic mesenchymal cells in vivo, we examined the effect of the CORS26 gene on cell proliferation. Overexpression of CORS26 enhanced growth of C3H10T1/2 cells and increased saturation densities in vitro. This finding suggested that secreted CORS26 protein acted as a growth factor based on its mitogenic activity on C3H10T1/2 cells.
Although the function of CORS26 in vivo is not clear, one possible function is the local regulation of mesenchymal condensation as secreted autocrine/paracrine factors during an early stage of skeletal development. Further analysis of CORS26 will lead to a better understanding of the molecular mechanism in skeletal development.