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• W2004193702 abstract "This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes. This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes. Endochondral ossification is an essential process for skeletal development, bone growth, and fracture healing and is implicated in pathological conditions such as osteoarthritis and ectopic ossification. During this process, chondrocytes first proliferate and then progressively differentiate into mature hypertrophic chondrocytes. Once fully matured, these hypertrophic cells mineralize the surrounding matrix and undergo apoptosis. This is followed by a local recruitment of blood vessels and osteoclasts, leading to progressive replacement of cartilage by bone. Thus, in this process of endochondral bone formation, proliferation, maturation, mineralization, and apoptosis of chondrocytes must be properly coordinated. To elucidate the molecular mechanisms of endochondral ossification, we have been attempting to isolate novel genes implicated in this process (1Koshizuka Y. Ikegawa S. Sano M. Nakamura K. Nakamura Y. Cytogenet. Cell Genet. 2001; 94: 163-168Crossref PubMed Google Scholar, 2Koshizuka Y. Ikegawa S. Sano M. Nakamura K. Nakamura Y. Genomics. 2001; 72: 252-259Crossref PubMed Scopus (18) Google Scholar, 3Ikegawa S. Sano M. Koshizuka Y. Nakamura Y. Cytogenet. Cell Genet. 2000; 90: 291-297Crossref PubMed Scopus (134) Google Scholar, 4Okawa A. Nakamura I. Goto S. Moriya H. Nakamura Y. Ikegawa S. Nat. Genet. 1998; 19: 271-273Crossref PubMed Scopus (342) Google Scholar). For this study, we took advantage of the naturally occurring mouse mutant ttw (tiptoe walking), which exhibits ectopic ossification in various soft tissues such as tendons, cartilage, and ligaments of the extremities and the spine (5Hosoda Y. Yoshimura Y. Higaki A. Ryumachi. 1981; 21: 157-164PubMed Google Scholar). We previously found that ttw is caused by a nonsense mutation of the nucleotide pyrophosphatase gene encoding an ectoenzyme generating phosphate and pyrophosphate (4Okawa A. Nakamura I. Goto S. Moriya H. Nakamura Y. Ikegawa S. Nat. Genet. 1998; 19: 271-273Crossref PubMed Scopus (342) Google Scholar). Based on the fact that a high phosphate diet accelerates ectopic ossification of ttw, using a differential display method, we identified nine mouse genes whose expression is regulated by a high phosphate diet (1Koshizuka Y. Ikegawa S. Sano M. Nakamura K. Nakamura Y. Cytogenet. Cell Genet. 2001; 94: 163-168Crossref PubMed Google Scholar). Six of the nine genes were novel; and among them, we isolated one, termed cystatin 10 (Cst10), 1The abbreviations used are: Cst10cystatin 10BACbacterial artificial chromosomeFITCfluorescein isothiocyanateRTreverse transcriptionCstCcystatin CPBSphosphate-buffered salinePIpropidium iodidepNAp-nitroanilideBMPbone morphogenetic proteinPTHrPparathyroid hormone-related protein.1The abbreviations used are: Cst10cystatin 10BACbacterial artificial chromosomeFITCfluorescein isothiocyanateRTreverse transcriptionCstCcystatin CPBSphosphate-buffered salinePIpropidium iodidepNAp-nitroanilideBMPbone morphogenetic proteinPTHrPparathyroid hormone-related protein. that is up-regulated by a high phosphate diet and is expressed exclusively in cartilage, suggesting its specific role in endochondral bone formation. cystatin 10 bacterial artificial chromosome fluorescein isothiocyanate reverse transcription cystatin C phosphate-buffered saline propidium iodide p-nitroanilide bone morphogenetic protein parathyroid hormone-related protein. cystatin 10 bacterial artificial chromosome fluorescein isothiocyanate reverse transcription cystatin C phosphate-buffered saline propidium iodide p-nitroanilide bone morphogenetic protein parathyroid hormone-related protein. In this study, we first characterized temporal and spatial expression patterns of Cst10, a novel member of the cystatin superfamily. The cystatin superfamily is known to inhibit the papain-like cysteine proteinases cathepsins B, H, and L by the formation of a tight reversible complex (6Barrett A.J. Fritz H. Grubb A. Isemura S. Jarvinen M. Katunuma N. Machleidt W. Muller-Esterl W. Sasaki M. Turk V. Biochem. J. 1986; 236: 312Crossref PubMed Scopus (277) Google Scholar). These cysteine proteinases are thought to be associated with terminal degradation of proteins in lysosomes, so the cystatin superfamily is ubiquitously expressed and exhibits various biological functions (7Reinheckel T. Deussing J. Roth W. Peters C. Biol. Chem. Hoppe-Seyler. 2001; 382: 735-741PubMed Google Scholar). However, the present study reveals that Cst10 is expressed exclusively in mature chondrocytes. In addition, overexpression of the Cst10 gene accelerates hypertrophic maturation, mineralization, and apoptosis of chondrocytes. These data suggest a crucial and specific role of Cst10 in the later stage of endochondral ossification, implying a physiological role distinct from those other members of the cystatin superfamily. Determination of the Genomic Structure of the Mouse Cst10 Gene—Bacterial artificial chromosome (BAC) clones containing the mouse Cst10 gene were isolated using a BAC PCR screening system (Genome Systems, St. Louis, MO) according to the manufacturer's protocol. The set of primers used for screening was Cst10/BAC/F (5′-TCCTGAGGATATATGTCAGGC-3′) and Cst10/BAC/R (5′-ATCTCTGTCTGAGGAAAGGAG-3′). To determine the size of introns of the Cst10 gene, interexon PCRs were carried out with primers designed according to the cDNA sequence we determined in this study. The BAC clones and PCR products were sequenced directly, and the exon-intron junctions were determined by comparing the genomic sequences obtained with the corresponding cDNA sequences. Chromosomal Localization—To determine the chromosomal localization of the mouse Cst10 gene, we performed fluorescence in situ hybridization as described previously (8Inazawa J. Saito H. Ariyama T. Abe T. Nakamura Y. Genomics. 1993; 17: 153-162Crossref PubMed Scopus (135) Google Scholar). A BAC clone containing the mouse Cst10 gene was labeled and hybridized to the mouse metaphase chromosome. Hybridization signals were rendered visible with fluorescein isothiocyanate (FITC)-avidin. Precise assignments of the signals were determined by visualization of the replicated G-bands. Animals—The ddY and ttw mice were purchased from Shizuoka Laboratories Animal Center (Shizuoka, Japan) and the Central Institute for Experimental Animals (Kanagawa, Japan), respectively. All animal experiments were performed according to the guidelines of the International Association for the Study of Pain (9Zimmermann M. Pain. 1983; 16: 109-110Abstract Full Text PDF PubMed Scopus (6755) Google Scholar). Cell Culture—Primary mesenchymal cells (osteoblasts, chondrocytes, and fibroblasts) were extracted from the calvariae, costal cartilage, and skin, respectively, of neonatal ddY mice as described previously (10Kawaguchi H. Manabe N. Miyaura C. Chikuda H. Nakamura K. Kuro-o M. J. Clin. Investig. 1999; 104: 229-237Crossref PubMed Scopus (166) Google Scholar, 11Shimoaka T. Ogasawara T. Yonamine A. Chikazu D. Kawano H. Nakamura K. Itoh N. Kawaguchi H. J. Biol. Chem. 2002; 277: 7493-7500Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Cells were cultured in α-modified minimal essential medium (Invitrogen) containing 5% fetal bovine serum (Invitrogen) at 37 °C. Mouse chondrogenic ATDC5 cells were obtained from the RIKEN Cell Bank (Saitama, Japan). The cells were cultured in medium consisting of a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Invitrogen) containing 5% fetal bovine serum, 10 μg/ml human transferrin (Roche Applied Science, Mannheim, Germany), and 3 × 10-8m sodium selenite (Sigma) as described previously (12Shukunami C. Ishizeki K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (250) Google Scholar). The inoculum density of the cells was 4 × 104 cells/well in 12-multiwell plates (Corning Inc., New York). For induction of chondrogenesis, the cells were cultured in medium supplemented with 10 μg/ml bovine insulin (Wako Pure Chemicals, Osaka, Japan). Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every other day. Expression of the Cst10 Transcript—Expression of the Cst10 mRNA was examined by semiquantitative reverse transcription (RT)-PCR, followed by Southern blotting using auricular cartilage from ttw mice, mesenchymal cells from neonatal ddY mice, and cultured mouse chondrogenic ATDC5 cells. For the experiments with ttw mice, the mice were divided into two groups according to the content of phosphate in the diet, i.e. high (0.87%) and low (0%) phosphate groups after weaning at 3 weeks of age. The animals were killed 0, 1, 3, 5, 7, 10, and 14 days after the start of the diet, and the auricular cartilage was resected en bloc. Total RNAs were extracted using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RT-PCR was done using the following set of primers: Cst10/3′/F (5′-TCC TGA GGA TAT ATG TCA AT-3′) and Cst10/3′/R (5′-GAA CAG TGG GCC TTT GAA AA-3′). The following amplification cycle was used: 2 min of initial denaturation at 94 °C, followed by 35 cycles at 94, 60, and 72 °C for 30 s each plus extension at 72 °C for 4 min. The primers and RT-PCR conditions used for type II and X collagens were as described previously (13Wang D. Canaff L. Davidson D. Corluka A. Liu H. Hendy G.N. Henderson J.E. J. Biol. Chem. 2001; 276: 33995-34005Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). PCR products were electrophoresed and detected by Southern hybridization. For quantification of type X collagen mRNA levels, the density of each band was measured by NIH Image Version 1.62 2Available at rsb.info.nih.gov/nih-image/download.html. and is expressed as the ratio to the density of glyceraldehyde-3-phosphate dehydrogenase. Immunoblot Analysis—Polyclonal antibody against the full-length Cst10 protein was raised in rabbits using a synthetic peptide of Cst10. For preparation of the whole cell lysate, adherent and detached cells were collected and resuspended in chilled lysis buffer (10 mm Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.1% SDS, 1 mm EDTA, 2 mm sodium orthovanadate, 10 mm sodium fluoride, and 10 mg/ml aprotinin). Collected cells were allowed to lyse by sonication on ice. The homogenate was centrifuged for 5 min in a microcentrifuge at 4 °C, and the supernatants were collected and boiled in SDS sample buffer. The culture medium was collected and centrifuged for 5 min in a microcentrifuge at 4 °C, and the supernatants were collected. The pellet obtained from the supernatants by centrifugation at 19,000 × g for 20 min was resuspended in SDS sample buffer. Fifty-μg portions of SDS sample buffer were loaded onto SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membrane (Amersham Biosciences). Mouse recombinant cystatin C (CstC) protein was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Protein bands on Western blots were visualized by chemiluminescent detection (ECL, Amersham Biosciences). Immunohistochemistry—The samples harvested from embryonic mice (18 days postcoitus) were demineralized in 10% EDTA for 1 week at 4 °C. The specimens were dehydrated with increasing concentrations of ethanol and then embedded in paraffin. Cst10 immunolocalization was examined in 4-μm-thick dewaxed paraffin sections. The sections were treated with phosphate-buffered saline (PBS) containing 0.3% hydrogen peroxide for 30 min at room temperature and then with PBS containing 1% bovine serum albumin (Sigma) for 60 min at room temperature. They were then incubated with polyclonal antibody against mouse Cst10 for 24 h at 4 °C and with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Dakopatts, Glostrup, Denmark) at a dilution of 1:500 for 1 h at room temperature. After washing with PBS, the sections were immersed in diaminobenzidine solution for 10 min at room temperature for visualization and counterstained with hematoxylin. Nonimmune rabbit serum at the same concentration was used as a negative control. For ultrastructural analysis, these sections were observed under a transmission electron microscope (H-7100, Hitachi, Tokyo) following the pre-embedding method described previously (14Hoshi K. Ejiri S. Ozawa H. J. Bone Miner. Res. 2001; 16: 289-298Crossref PubMed Scopus (55) Google Scholar). Briefly, embryonic mice (18 days postcoitus) were perfused through the left ventricle with a 2-ml dose of a mixture of 4% paraform-aldehyde and 0.1% glutaraldehyde in 0.08 m cacodylate buffer (pH 7.4). The tibiae were then removed and immersed in the same fixative for 2 h at 4 °C. Specimens were decalcified with 4.13% EDTA at 4 °C for 1 day and cryosectioned at a thickness of 10 μm by cryomicrotome. The cryosections were treated following methods similar to those used for immunohistochemical investigations, being post-fixed in 1% OsO4 in 0.1 m cacodylate buffer at 4 °C for 1 h. The specimens were dehydrated in a graded ethanol series and embedded in Poly/Bed 812 resin (Polysciences, Warrington, PA). Ultrathin sections stained with lead citrate were used for transmission electron microscopic observation. Establishment of ATDC5 Cells Stably Transfected with the Cst10 Gene—The entire sequence of Cst10 cDNA was amplified by PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA) and inserted into the mammalian expression vector pcDNA3.1 (Invitrogen) including the cytomegalovirus promoter with a hemagglutinin epitope tag at the C terminus (pCMV-Cst10). The subcloned cDNA fragment was confirmed by sequencing. ATDC5 cells (2 × 105) were plated in a 6-cm culture dish 24 h before transfection. pCMV-Cst10 (4 mg/6-cm culture dish) or the mock vector (pCMV) was transfected into ATDC5 cells by lipofection using SuperFect transfection reagent (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions. Two days later, cells were diluted 10-fold and incubated in a maintenance medium containing 400 μg/ml Geneticin (Invitrogen). After 2 weeks, we isolated drug-resistant colonies, each of which was derived from a single clone, and cultured them separately. To confirm the reproducibility of the effects of Cst10 overexpression, we performed two experiments with independent transfection procedures. For the first experiment, we established 18 stable clones from 18 different colonies of ATDC5 cells transfected with pCMV-Cst10 and selected three clones (clones 3, 8, and 12) with the highest expression of Cst10 (pCMV-Cst10/ATDC5) by RT-PCR analysis. Three mock vector-transfected clones (clones 1–3) that were confirmed by RT-PCR not to express Cst10 were also selected as negative controls (pCMV/ATDC5). For the second experiment, we independently transfected pCMV-Cst10 into ATDC5 cells as described above, randomly established four stable clones, and examined the relationship of expressions between Cst10 and collagens by RT-PCR. Alcian Blue and Alizarin Red Staining—pCMV-Cst10/ATDC5 and pCMV/ATDC5 cells were placed in 12-multiwell plates and cultured. Twenty-one days after induction by insulin, cells were rinsed with PBS and fixed with 95% methanol for 20 min. They were then stained overnight with 0.1% Alcian blue 8GS (Fluka, Buchs, Switzerland) in 0.1 m HCl. Twenty-eight days after induction by insulin, cultures were stained with 1% alizarin red S (pH 4.0) (Sigma) after fixation with 95% ethanol. Detection of Apoptosis and Activities of Caspases—Apoptosis of pCMV-Cst10/ATDC5 and pCMV/ATDC5 cells was examined by nuclear staining with Hoechst 33342, externalization of phosphatidylserine residues using FITC-labeled annexin V, mitochondrial membrane potential, and flow cytometric analysis. Annexin V binding assay was performed using an FITC-labeled annexin V apoptosis detection kit (Medical and Biological Laboratories, Nagoya, Japan) according to the manufacturer's protocol (15Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar, 16Koopman G. Reutelingsperger C.P. Kuijten G.A. Keehnen R.M. Pals S.T. van Oers M.H. Blood. 1994; 84: 1415-1420Crossref PubMed Google Scholar, 17Martin S.J. Reutelingsperger C.P. McGahon A.J. Rader J.A. van Schie R.C. LaFace D.M. Green D.R. J. Exp. Med. 1995; 182: 1545-1556Crossref PubMed Scopus (2538) Google Scholar). Briefly, ATDC5 cells were harvested 7 days after induction with insulin and washed with PBS. Cells were then incubated with binding buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 5 mm KCl, 1 mm MgCl2, and 1.8 mm CaCl2) containing 2 μl of FITC-labeled annexin V and 5 μg of propidium iodide (PI) for 15 min at room temperature in the dark. After incubation, they were viewed under a fluorescent microscope. PI was added to distinguish cells with membrane permeability due to the loss of membrane integrity, which is characteristic of necrotic cell death. To assess the mitochondrial membrane potential, a MitoCapture apoptosis detection kit (Medical and Biological Laboratories) was used (18Ogawa Y. Nishioka A. Kobayashi T. Kariya S. Hamasato S. Saibara T. Seguchi H. Yoshida S. Int. J. Mol. Med. 2001; 7: 603-607PubMed Google Scholar). ATDC5 cells harvested 7 days after induction were incubated with MitoCapture solution for 15 min at 37 °C and viewed under a fluorescent microscope using a band-pass filter (detects FITC and rhodamine). In healthy cells, MitoCapture accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. In apoptotic cells, MitoCapture cannot aggregate in the mitochondria due to the altered mitochondrial membrane potential and thus remains in the cytoplasm in its monomer form, fluorescing green. For flow cytometric analysis, ATDC5 cells were harvested 0, 1, 3, 5, 7, 10, and 14 days after induction by insulin and fixed with 75% ethanol and PBS at 4 °C for 1 h. After rinsing twice with PBS, cells were incubated for 30 min with 1 ml of PBS containing 1 mg of boiled RNase at 37 °C and then stained with 1 ml of PBS containing 10 μg of PI. A total of 2 × 104 cells were analyzed with a flow cytometer (FACSCaliber, BD Biosciences). To determine whether caspase-3 is activated in pCMV-Cst10/ATDC5, a PhiLux kit (Medical and Biological Laboratories) was used according to the manufacturer's protocol (19Komoriya A. Packard B.Z. Brown M.J. Wu M.L. Henkart P.A. J. Exp. Med. 2000; 191: 1819-1828Crossref PubMed Scopus (135) Google Scholar). Briefly, ATDC5 cells harvested 7 days after induction were incubated with 10 mm GDEVDGI and labeled with two molecules of rhodamine, which was selectively cut by caspase-3. After incubation, cells were viewed under a fluorescent microscope. Caspase-3 activity was determined with a caspase-3/CPP32 colorimetric protease assay kit (Medical and Biological Laboratories) according to the manufacturer's protocol. In brief, ATDC5 cells were harvested 7 days after induction with insulin and lysed in lysis buffer. Cell lysate (150 μg of protein in 50 μl of lysis buffer) was incubated with DEVD-p-nitroanilide (pNA) as a substrate for 1 h at 37 °C, and the amount of pNA generated was determined spectrophotometrically at 405 nm. Caspase-8 and caspase-9 activities were determined with caspase-8/FLICE and caspase-9/Mch6 colorimetric protease assay kits, respectively (Medical and Biological Laboratories), according to the manufacturer's protocols. IETD-p-nitroanilide for caspase-8 and LEHD-p-nitroanilide for caspase-9 were used as substrates. After incubation for 1 h at 37 °C, the amount of p-nitroanilide generated was determined spectrophotometrically at 405 nm (20Casciola-Rosen L. Nicholson D.W. Chong T. Rowan K.R. Thornberry N.A. Miller D.K. Rosen A. J. Exp. Med. 1996; 183: 1957-1964Crossref PubMed Scopus (575) Google Scholar, 21Datta R. Kojima H. Banach D. Bump N.J. Talanian R.V. Alnemri E.S. Weichselbaum R.R. Wong W.W. Kufe D.W. J. Biol. Chem. 1997; 272: 1965-1969Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 22Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Villancourt J.P. Chapman K.T. Nicholson D.W. J. Biol. Chem. 1997; 272: 17907-17911Abstract Full Text Full Text PDF PubMed Scopus (1834) Google Scholar). Statistical Analysis—Means of groups were compared by analysis of variance, and significance of differences was determined by post-hoc testing using Bonferroni's method. Characterization of the Cst10 Gene—In a previous study, using differential display analysis, we identified nine genes, including Cst10, whose expression is regulated in the auricular cartilage of ttw mice fed a high phosphate diet (1Koshizuka Y. Ikegawa S. Sano M. Nakamura K. Nakamura Y. Cytogenet. Cell Genet. 2001; 94: 163-168Crossref PubMed Google Scholar). The full-length cDNA sequence of the mouse Cst10 gene determined by 5′-rapid amplification of cDNA ends is 789 bp long and has an open reading frame of 447 bp, coding for 148 amino acids (DDBJ/GenBank™/EBI accession number AB036743). The mouse Cst10 gene consists of three exons spanning 4.2 kb on genomic DNA. All of the sequences of the exon-intron junction were confirmed to be the consensus sequences for splicing boundaries (AG/GT rule). The predicted amino acid sequence contains two intrachain disulfide bridges and a cystatin domain (consensus (GSTEQKRV)Q(LIVT)(VAF)(SAGQ)GX-(LIVMNK)X2(LIVMFY)X(LIVMFYA)(DENQKRHSIV)), indicating a novel member of the type II cystatin superfamily with 40.5 and 39.0% homologies to mouse and human CstC (Cst3), respectively, the closest cystatin family member (Fig. 1A). Its homologies to other mouse cystatins were around or less than 30%: 31.5% to Cst9, 31.4% to Cst7, 27.0% to CstEM, 28.6% to CstSC, and 26.7% to CstTE. To investigate the localization of the Cst10 gene in the mouse chromosome, >50 metaphase cells were examined by fluorescence in situ hybridization using a BAC clone containing the mouse Cst10 gene as a probe. Specific hybridization signals were identified on chromosome 2 in almost all cells, and no significant background was observed at any other chromosomal sites (Fig. 1B). Temporal and Spatial Expression of Cst10 in Vivo and in Vitro—We first examined the temporal expression pattern of Cst10 mRNA levels in the auricular cartilage of ttw mice whose endochondral ossification was enhanced with a high phosphate diet. Expression appeared 3 days after weaning and was upregulated by a high phosphate diet at 5 days and thereafter (Fig. 2A). Our previous study on the tissue distribution of Cst10 expression in a variety of mouse tissues showed that this gene is expressed exclusively in cartilage (1Koshizuka Y. Ikegawa S. Sano M. Nakamura K. Nakamura Y. Cytogenet. Cell Genet. 2001; 94: 163-168Crossref PubMed Google Scholar). We therefore examined the expression pattern of Cst10 using cell cultures. Among three cultured mesenchymal cells from neonatal ddY mice (primary osteoblasts from calvariae, chondrocytes from costal cartilage, and fibroblasts from skin), Cst10 expression was confirmed to be specific to chondrocytes (Fig. 2B). To characterize the expression pattern during differentiation of chondrocytes, we used the mouse chondrogenic cell line ATDC5, which can be induced to differentiate into mature chondrocytes in the presence of insulin (12Shukunami C. Ishizeki K. Atsumi T. Ohta Y. Suzuki F. Hiraki Y. J. Bone Miner. Res. 1997; 12: 1174-1188Crossref PubMed Scopus (250) Google Scholar). During induction of differentiation with insulin, expression of type II collagen remained unchanged throughout the culture period up to 14 days, whereas that of type X collagen, a marker for hypertrophic chondrocytes, appeared 7 days after induction (Fig. 2C). Expression of the Cst10 gene appeared at 3 days and increased thereafter, indicating that Cst10 expression is in synchrony with the maturation of chondrocytes. To examine the localization of Cst10 in cartilage, we first confirmed by Western blot analysis the specificity of a polyclonal antibody against Cst10 without cross-reactivity with CstC, the closest member of the cystatin superfamily (Fig. 2D). Using this antibody, we performed immunohistochemical analysis on the growth plates of embryonic ddY mice and found that Cst10 was expressed mainly in mature chondrocytes, including prehypertrophic and hypertrophic cells (Fig. 2E). Electron microscopic examination of a hypertrophic chondrocyte revealed that Cst10 was immunolocalized in the cytosolic areas, but was not found in the nucleus or within the lumen of the rough endoplasmic reticulum (Fig. 2F). These findings suggest that Cst10 is not transported into the Golgi-endoplasmic reticulum system, but acts as an intracellular protein in the cytosol. Overexpression of the Cst10 Gene Accelerates Maturation of ATDC5 Cells—To elucidate the function of Cst10 in chondrocytes, we established stable clones of ATDC5 cells overexpressing the Cst10 gene (pCMV-Cst10/ATDC5). We first compared by RT-PCR the differentiation of pCMV-Cst10/ATDC5 cells with that of control clones of ATDC5 cells transfected with the mock vector (pCMV/ATDC5) upon induction with insulin (Fig. 3A). In pCMV-Cst10/ATDC5 cells, Cst10 mRNA expression was clearly seen not only after, but also before induction (time 0), whereas in pCMV/ATDC5 cells, expression was faintly seen 7 days after induction and increased moderately thereafter. Western blot analysis revealed that the Cst10 protein was localized in the cell lysate, but not in the culture medium of pCMV-Cst10/ATDC5 cells (Fig. 3B), indicating that Cst10 is not a secreted protein. Expression of type II collagen, which is known to be produced by chondrocytes from their early phase of differentiation, was constitutively seen before and after induction, and expression was not different between pCMV-Cst10/ATDC5 and pCMV/ATDC5 cells (Fig. 3A). However, expression of type X collagen, a marker of hypertrophic chondrocytes, was observed earlier and was stronger in pCMV-Cst10/ATDC5 cells than in pCMV/ATDC5 cells. We also compared the cartilage nodule formation and mineralization between cultured pCMV-Cst10/ATDC5 and pCMV/ATDC5 cells using Alcian blue and alizarin red staining, respectively. No difference was seen in the Alcian blue staining between the two cells; however, alizarin red staining was stronger in cultured pCMV-Cst10/ATDC5 cells than in pCMV/ATDC5 cells (Fig. 3C). To confirm the reproducibility of the effects of Cst10 overexpression, we performed another experiment using ATDC5 cell clones that were independently transfected with pCMV-Cst10 and isolated. In this experiment, we randomly established four stable clones and compared the mRNA levels of Cst10 and type II and X collagens by RT-PCR before (time 0) and 5 days after induction (Fig. 3D). The Cst10 mRNA levels were not changed before or after induction in all clones. There was a good correlation between the Cst10 and type X collagen levels at 5 days, although the type II collagen levels were similar among the clones (Fig. 3D). These results indicate that overexpression of the Cst10 gene accelerates the later (but not earlier) stage of chondrocyte differentiation and mineralization. Overexpression of Cst10 Leads to Apoptosis of ATDC5 Cells—Staining with Hoechst 33342 revealed the existence of cells with fragmented and condensed nuclei with increased fluorescence, suggesting apoptotic cell death in pCMV-Cst10/ATDC5 cells, but not in pCMV/ATDC5 cel" @default.
• W2004193702 created "2016-06-24" @default.
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• W2004193702 date "2003-11-01" @default.
• W2004193702 modified "2023-10-10" @default.
• W2004193702 title "Cystatin 10, a Novel Chondrocyte-specific Protein, May Promote the Last Steps of the Chondrocyte Differentiation Pathway" @default.
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