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• W2002113583 abstract "Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca2+ influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca2+, whereas K-201, a specific annexin Ca2+channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca2+ influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression ofbcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca2+influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes. Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca2+ influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca2+, whereas K-201, a specific annexin Ca2+channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca2+ influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression ofbcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca2+influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes. retinoic acid alkaline phosphatase activity retinoic acid receptor phosphate-buffered saline 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling Maturation of epiphyseal growth plate chondrocytes, which plays an important role during endochondral ossification, is accompanied by major changes of chondrocyte morphology, biosynthetic activities, and energy metabolism. These processes involve an ordered progression of various cell differentiation stages, including proliferation, hypertrophic differentiation, terminal differentiation, and ultimately programmed cell death (apoptosis) (1Hickok N.J. Haas A.R. Tuan R.S. Microsc. Res. Tech. 1998; 43: 174-190Crossref PubMed Scopus (53) Google Scholar, 2Gibson G. Microsc. Res. Tech. 1998; 43: 191-204Crossref PubMed Scopus (137) Google Scholar). During normal development these sequential events are under the strict control of local and systematic factors such as hormones and growth factors. If these processes, however, occur during pathological conditions, they can result in serious cartilage or bone defects. Evidence of endochondral ossification is also seen during osteophyte formation in osteoarthritic cartilage (3Hoyland J.A. Thomas J.T. Donn R. Marriott A. Ayadh S. Boot-Handford R.P. Grant M.E. Freemont A.J. Bone Miner. 1991; 15: 151-163Abstract Full Text PDF PubMed Scopus (109) Google Scholar, 4Aigner T. Reichenberger E. Bertling W. Kirsch T. Stoss H. von der Mark K. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1993; 63: 205-211Crossref PubMed Scopus (125) Google Scholar). Terminal differentiation of growth plate chondrocytes is an essential process, which primes the cartilage skeleton for its subsequent invasion by osteoblasts and its replacement by a bone matrix. Despite the obvious importance of these terminal differentiation events still little is known about mechanisms regulating these processes. cbfa1, a member of the runt domain family of transcription factors, was originally discovered as a key transcription factor, which controls osteoblast differentiation. In cbfa1-null mice no endochondral and intramembranous bone formation occurs due to an arrest in osteoblast differentiation (5Mundlos S. Otto F. Mundlos C. Mulliken J.B. Aylsworth A.S. Albright S. Lindhout D. Cole W.G. Henn W. Knoll J.H. Owen M.J. Mertelsmann R. Zabel B.U. Olsen B.R. Cell. 1997; 89: 773-779Abstract Full Text Full Text PDF PubMed Scopus (1285) Google Scholar, 6Otto F. Thornell A.P. Crompton T. Denzel A. Gilmour K.C. Rosewell I.R. Stamp G.W. Beddington R.S. Mundlos S. Olsen B.R. Selby P.B. Owen M.J. Cell. 1997; 89: 765-771Abstract Full Text Full Text PDF PubMed Scopus (2430) Google Scholar, 7Ducy P. Zhang R. Geoffroy V. Ridall A.L. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3668) Google Scholar, 8Komori T. Yagi H. Nomura S. Yamaguchi A. Sasaki K. Deguchi K. Shimizu Y. Bronson R.T. Gao Y.H. Inada M. Sato M. Okamoto R. Kitamura Y. Yoshiki S. Kishimoto T. Cell. 1997; 89: 755-764Abstract Full Text Full Text PDF PubMed Scopus (3678) Google Scholar). Recent studies have indicated that cbfa1 also plays an important regulative role in terminal chondrocyte maturation. Transgenic mice, which overexpresscbfa1 in non-hypertrophic chondrocytes, display an acceleration of endochondral ossification. Overexpression ofcbfa1 in chondrocytes of cbfa1-null mice partially rescued the abnormalities of cbfa1-null mutant mice. In particular, it rescued hypertrophic chondrocyte differentiation in the humerus and femur (9Takeda S. Bonnamy J.P. Owen M.J. Ducy P. Karsenty G. Genes Dev. 2001; 15: 467-481Crossref PubMed Scopus (451) Google Scholar). Thus, cbfa1seems to play dual functions in endochondral bone formation; it plays a key role in osteoblast differentiation from mesenchymal precursor cells, and it has the ability to stimulate hypertrophic and terminal chondrocyte differentiation. Chondrocyte hypertrophy and terminal differentiation are accompanied by an increase in cytosolic calcium, [Ca2+]i(10Iannotti J.P. Brighton C.T. J. Orthop. Res. 1989; 7: 511-518Crossref PubMed Scopus (34) Google Scholar, 11Gunter T.E. Zuscik M.J. Puzas J.E. Gunter K.K. Rosier R.N. Cell Calcium. 1990; 11: 445-457Crossref PubMed Scopus (37) Google Scholar, 12Kirsch T. Swoboda B. von der Mark K. Differentiation. 1992; 52: 89-100Crossref PubMed Scopus (57) Google Scholar). Calcium is recognized as an important regulator of many cellular processes, and it controls a diverse range of cell functions, including adhesion, motility, gene expression, cell differentiation, and proliferation. For example, the amplitude and duration of calcium signals control differential activation of different transcription factors in B lymphocytes (13Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1564) Google Scholar). Calcium has been shown to play several roles in vesiculation and the formation of vesicles. For example, Iannotti et al. (14Iannotti J.P. Naidu S. Noguchi Y. Hunt R.M. Brighton C.T. Clin. Orthop. Relat. Res. 1994; 306: 222-229PubMed Google Scholar) have shown a correlation between increasing [Ca2+]i and the release of matrix vesicles (14Iannotti J.P. Naidu S. Noguchi Y. Hunt R.M. Brighton C.T. Clin. Orthop. Relat. Res. 1994; 306: 222-229PubMed Google Scholar). Matrix vesicles are small membrane-enclosed particles, which are released from the plasma membrane of growth plate chondrocytes and which initiate the mineralization process (15Anderson H.C. Clin. Orthop. Relat. Res. 1995; 314: 266-280PubMed Google Scholar). We have previously shown that RA,1 which stimulates terminal differentiation and mineralization of hypertrophic chondrocytes, induces Ca2+ influx into growth plate chondrocytes causing the release of mineralization-competent matrix vesicles (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (113) Google Scholar). Annexins II, V, and VI, which are highly expressed in hypertrophic and mineralizing growth plate cartilage, are major components of matrix vesicles (17Kirsch T. Swoboda B. Nah H.-D. Osteoarthritis Cartilage. 2000; 8: 294-302Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 18Pfander D. Swoboda B. Kirsch T. Am. J. Pathol. 2001; 159: 1777-1783Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 19Kirsch T. Nah H.D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (194) Google Scholar). Annexins II, V, and VI belong to a family of Ca2+- and phospholipid-binding proteins. In addition, annexins II, V, and VI have been shown to form Ca2+channels in phospholipid bilayers or in liposomes (20Gerke V. Moss S.E. Physiol. Rev. 2002; 82: 331-371Crossref PubMed Scopus (1645) Google Scholar). They also form Ca2+ channels in matrix vesicles enabling Ca2+influx into these particles as a possible initial step for the formation of the first mineral phase within the vesicle lumen (21Kirsch T. Harrison G. Golub E.E. Nah H.-D. J. Biol. Chem. 2000; 275: 35577-35583Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Careful studies have provided evidence that apoptosis is the final fate of terminally differentiated growth plate chondrocytes. Chondrocyte apoptosis in the growth plate is centered at the site of the transition of cartilage to bone (2Gibson G. Microsc. Res. Tech. 1998; 43: 191-204Crossref PubMed Scopus (137) Google Scholar, 22Hatori M. Klatte K.J. Teixeira C.C. Shapiro I.M. J. Bone Miner. Res. 1995; 10: 1960-1968Crossref PubMed Scopus (176) Google Scholar). These apoptotic chondrocytes show characteristic hallmarks of apoptosis, including condensed nuclei, DNA fragmentation, activation of caspase cascade, and phosphatidylserine externalization. Previous studies have indicated that elevation of [Ca2+]i is involved in the induction of apoptosis. For example, apoptosis of cultured human endothelial cells was inhibited by chelating extracellular calcium with EGTA or by inhibiting the calcium influx by calcium channel blockers. It has been suggested that elevated [Ca2+]i leads to activation of proteases, lipases, and nucleases. All these actions can contribute to cell death (23Waring P. Beaver J. Exp. Cell Res. 1996; 227: 264-276Crossref PubMed Scopus (66) Google Scholar, 24Escargueilblanc I. Meilhac O. Pieraggi M.T. Arnal J.F. Salvayre R. Negresalvayre A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 331-339Crossref PubMed Scopus (135) Google Scholar, 25Srinivasan S. Stevens M.J. Sheng H.B. Hall K.E. Wiley J.W. J. Clin. Invest. 1998; 102: 1454-1462Crossref PubMed Scopus (63) Google Scholar). We have provided evidence that RA promotes annexin channel formation in growth plate chondrocytes and that annexin-mediated Ca2+influx into these cells controls Ca2+ homeostasis (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (113) Google Scholar). To determine the role of annexin-mediated alteration of Ca2+homeostasis in terminal differentiation, mineralization, and apoptosis of growth plate chondrocytes, we cotreated growth plate chondrocytes isolated from the hypertrophic zone of day 19 embryonic chicken growth plate cartilage with RA and K-201, a specific annexin channel blocker, or RA and BAPTA-AM, a cytosolic Ca2+ chelator, and analyzed the rate of terminal differentiation, mineralization, and apoptosis in these cells. Chondrocytes were isolated from the hypertrophic zone of day 19 embryonic chick tibia growth plate cartilage as described previously (19Kirsch T. Nah H.D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (194) Google Scholar). Briefly, sliced growth plate cartilage was digested with 0.25% trypsin and 0.05% collagenase for 5 h at 37 °C. Cells were plated at a density of 3 × 106 in 10-cm tissue culture dishes and grown in monolayer cultures in Dulbecco's modified Eagle's medium (Invitrogen) containing 5% fetal calf serum (Hyclone, Logan, UT), 2 mml-glutamine (Invitrogen), and 50 units/ml penicillin and streptomycin (complete medium). After cultures reached confluency, chondrocytes were cultured in the presence of 1.5 mmphosphate and in the absence or presence of (a) 35 nm RA (Sigma-Aldrich), (b) 35 nm RA and 2 μm 1,4-benzothiazepine derivative K-201 (JTV519) (provided by Drs. Noboro Kareko, Dokkyo University, Tochigo, Japan and Toshizo Tanaka, Japan Tobacco Inc., Osaka, Japan), and (c) 35 nm RA and 10 μm BAPTA-AM (Molecular Probes Inc., Eugene, OR). Total RNA was isolated from untreated, RA-treated, RA/K-201-treated, and RA/BAPTA-treated chondrocyte cultures after 1-, 3-, 5-day treatments using RNeasy Mini Kit (Qiagen, Stanford, CA). 1 μg of RNA was reverse-transcribed using Ominiscript RT Kit (Qiagen). A 1:100 dilution of the resulting cDNA was used as the template to quantify the relative content of mRNA by real time PCR (ABI PRISM 7700 sequence detection system) using respective primers and SYBR Green. The following primers for real time PCR were designed using Primer Express software. Annexin II: forward primer, 5′-CATGCCTATCTGCTCTTCGTT-3′; reverse primer, 5′-AGCCACCACACCGTCCATAA-3′; annexin V: forward primer, 5′-AGAGACATCAGGCCATTTTCAGA-3′; reverse primer, 5′-CTGCCATCAGGATCTCTATTTGC-3′; annexin VI: forward primer, 5′-GCGGCTGATTGTAAGCTTGAT-3′; reverse primer, 5′-GTCGGTGGTCCAGCACTTA-3′; type I collagen (α1(I)): forward primer, 5′-CAGCCGCTTCACCTACAGC-3′; reverse primer, 5′-TTTTGTATTCAATCACTGTCTTGCC-3′; type II collagen: forward primer, 5′-GGCAATAGCAGGTTCACGTAC-3′; reverse primer, 5′-CGATAACAGTCTTGCCCCACTT-3′; type X collagen: forward primer, 5′-AGTGCTGTCATTGATCTCATGGA-3′; reverse primer, 5′-TCAGAGGAATAGAGACCATTGGATT-3′; cbfa1: forward primer, 5′-CGCGGAGCTGCGAAAT-3′; reverse primer, 5′-ACGAATCGCAGGTCATTGAAT-3′; APase: forward primer, 5′-CCCTGACATCGAGGTGATCCT-3′; reverse primer, 5′-GGTACTCCACATCGCTGGTGTT-3′; osteocalcin: forward primer, 5′-TCGCGGCGCTGCTCACATTCA-3′; reverse primer, 5′-TGGCGGTGGGAGATGAAGGCTTTA-3′; bcl-2: forward primer, 5′-GGTGACCCGAAGCATCAAA-3′; reverse primer, 5′-AGCGACACGAAAACCCAAAC-3′. PCR reactions were performed with the TaqMan PCR master mix kit (Applied Biosystems) using 1 cycle at 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The 18 S RNA was amplified at the same time and used as an internal control. The cycle threshold values for 18 S RNA and that of the samples were measured and calculated by computer software. Relative transcript levels were calculated asx = 2−ΔΔ Ct , in which ΔΔCt = ΔE − ΔC and ΔE = Ct exp −Ct 18 S; ΔC =Ct ctl − Ct 18 S. Matrix vesicles were isolated from chondrocyte cultures after a 6-day treatment by enzymatic digestion and ultracentrifugation as described previously (19Kirsch T. Nah H.D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (194) Google Scholar). APase activity was measured using p-nitrophenyl phosphate (Sigma-Aldrich) as a substrate as described previously (19Kirsch T. Nah H.D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (194) Google Scholar). Protein content was analyzed by the BCA protein assay from Pierce. To determine the amounts of annexin II, V, and VI in matrix vesicles, vesicle fractions (total protein of 30 μg) were subjected to SDS-PAGE and immunoblotted with primary antibodies specific for annexins II, V, and VI. Samples were dissolved in 4× NuPAGE SDS sample buffer (Invitrogen). Prior to electrophoresis, the reducing reagent was added to the sample solution, denatured at 70 °C for 10 min, and analyzed by electrophoresis in 10% Bis-Tris gels following the NuPAGE electrophoresis protocols. Samples were electroblotted onto nitrocellulose filters after electrophoresis. After blocking with a solution of low fat milk protein, blotted proteins were immunostained with primary antibodies followed by peroxidase-conjugated secondary antibody, and the signal was detected by enhanced chemiluminescene (Pierce). To determine the degree of mineralization chondrocyte cultures were stained with alizarin red S after a 6-day treatment as described previously (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (113) Google Scholar). Briefly, chondrocyte cultures were fixed with 70% ethanol and then stained with 0.5% alizarin red S solution, pH 4.0, for 5 min at room temperature. To quantify the intensity of alizarin red S staining, alizarin red S-stained cultures were incubated with 100 mmcetylpyridinium chloride for 1 h to solubilize and release calcium-bound alizarin red S into solution (26Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (86) Google Scholar). The absorbance of the released alizarin red S staining was measured at 570 nm using a spectrophotometer. Data were expressed as units of alizarin red S released per mg of protein in each culture. Caspase-3 activity was determined using the ApoAlert caspase fluorescent assay kit (Clontech) following the manufacturer's protocol. Briefly, after a 6-day treatment chondrocyte cultures were washed twice with ice-cold phosphate-buffered saline (PBS), scraped into tubes, and centrifuged at 1500 rpm for 10 min. Cell pellets were washed one more time with ice-cold PBS and centrifuged again. Then air-dried cell pellets were resuspended in 60 μl of chilled cell lysis buffer and incubated on ice for 10 min. Cellular debris was removed by centrifugation, and 50 μl of 2 × reaction buffer/dithiothreitol mixture and 5 μl of 1 mm caspase-3 substrate (DEVD-7-amino-4-trifluoromethylcoumarin) were added to 50 μl of each sample and incubated for 1 h at 37 °C. Caspase-3 activity was measured in a fluorimeter (Photon Technology Instruments) using the excitation wavelength of 400 nm and the emission wavelength of 505 nm. Caspase-3 activity was quantitated using 7-amino-4-trifluoromethylcoumarin standard and normalized to the protein content in each culture. Apoptotic chondrocytes in day 6 chondrocyte cultures were detected using ApopTag in situ apoptosis detection kit to label apoptotic cells by modifying genomic DNA utilizing terminal deoxynucleotidyltransferase (TdT) (Intergen Co., Purchase, NY). Briefly, chondrocytes were washed twice with PBS and fixed with 1% paraformaldehyde/PBS solution (pH 7.4) for 10 min. Then fixed chondrocytes were incubated with 1% Triton/PBS solution, followed by incubation with a proteinase K solution (20 μg/ml) for 10 min at room temperature. Samples were then incubated in 3% hydrogen peroxide/PBS for 5 min at room temperature to quench endogenous peroxidases, followed by rinsing with PBS and incubation with equilibration buffer. Samples were incubated with a reaction mixture containing terminal deoxynucleotidyltransferase enzyme and digoxigenin-labeled dNTPs at 37 °C in a humidified chamber. After 1 h, the reaction was stopped, and digoxigenin-labeled nucleotides were detected by peroxidase-conjugated anti-digoxigenin antibodies in a humidified chamber for 30 min at room temperature. The signal was detected using 3,3′-diaminobenzidine as a color substrate. Sections were counterstained with methylene green, mounted, and viewed under an Olympus microscope. To gain insights into the extent of apoptosis in the various chondrocyte cultures, the percentage of stained cells was determined. 500 chondrocytes were counted in 10 randomly chosen areas of three different cultures. Data were expressed as the mean ± S.D. of the percentage of total cells that show TUNEL staining. Numerical data are presented as mean ± S.D. (n > 4), and statistical significance between groups was identified using the two-tailed Student's t test (p values are reported in the figure legends). Treatment of hypertrophic growth plate chondrocytes with RA induced terminal differentiation of these cells, as indicated by up-regulation of terminal differentiation marker genes, includingcbfa1 (Fig. 1 A), APase (Fig. 1 B), and osteocalcin (Fig. 1 C), compared with the expression levels in untreated cells. An approximately 9-fold increase in cbfa1 gene expression was detected after a 3-day treatment, whereas APase gene expression was up-regulated by ∼16-fold after a 5-day treatment, and osteocalcin gene expression increased ∼16-fold after a 3-day treatment and ∼14-fold after a 5-day treatment. Furthermore, alizarin red S staining revealed that RA-treated cultures were heavily mineralized after a 6-day treatment, whereas untreated cultures showed only little signs of mineralization (Fig. 2). Previously, we have shown that RA treatment led to a 3-fold increase in [Ca2+]i of growth plate chondrocytes compared with the concentration of untreated cells. In addition, we provided evidence that most of this increase was mediated by Ca2+influx through annexin channels (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (113) Google Scholar). Thus, it is possible that annexin-mediated Ca2+ influx into growth plate chondrocytes regulates terminal differentiation events of these cells. To test this hypothesis, we cotreated cells with RA and the annexin-specific Ca2+ channel blocker K-201 or antibodies specific for annexin V. Blocking annexin channel activities with K-201 led to a significant reduction of cbfa1, APase, and osteocalcin gene expression (Fig. 1), and mineralization of RA-treated chondrocyte cultures (Fig. 2). We have previously shown that antibodies specific for annexin V blocked its Ca2+ channel activities (26Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (86) Google Scholar). Cotreatment of RA-treated cultures with antibodies specific for annexin V also significantly reduced the rate of mineralization compared with the degree of mineralization in RA-treated cultures (Fig. 2).Figure 2Extent of matrix mineralization in chondrocyte cultures treated with RA , RA and K-201, or RA and antibodies specific for annexin V. Hypertrophic growth plate chondrocytes were treated with RA, RA/K-201, or RA/anti-annexin V IgGs for 6 days. A, alizarin red S staining of untreated, RA-treated, RA/K-201-treated, and RA/anti-annexin V IgG-treated cultures. Note the intense staining in RA-treated cultures, while untreated, RA/K-201-, and RA/anti-annexin V IgG-treated cultures showed little staining. B, to quantitate alizarin red S staining, alizarin red S-stained cultures were incubated with 100 mmcetylpyridium chloride for 1 h. The alizarin red staining released into the solution was collected, diluted when necessary, and read as units of alizarin red released (1 unit is equivalent to 1 unit of absorbance density at 570 nm) per mg of protein. Data were obtained from four different experiments, and values are mean ± S.D. (*,p ≤ 0.01; RA-treated versusRA/K-201-treated cultures).View Large Image Figure ViewerDownload Hi-res image Download (PPT) RA treatment also up-regulated annexin II, V, and VI gene expression. K-201 significantly reduced the up-regulation of annexin II, V, and VI gene expression in RA-treated cultures to levels similar to untreated cultures (Fig. 3, A,B, and C). Type I collagen gene expression was up-regulated in RA-treated cultures (Fig. 4 A), whereas type II collagen gene expression was down-regulated in these cultures (Fig. 4 B). Cultures cotreated with RA and K-201 showed levels of type I and II collagen gene expression similar to levels in untreated cultures (Fig. 4, A and B). Type X collagen gene expression was not affected by RA or RA/K-201 treatment (Fig. 4 C).Figure 4Quantitative real time PCR analysis of type I (A), II (B), and X (C) collagen gene expression in untreated, RA-treated, and RA/K-201-treated growth plate chondrocytes. Total RNA was isolated from day 1, 3, and 5 untreated, RA-, and RA/K-201-treated chondrocytes. Gene expressions of type I collagen (A), type II collagen (B), and type X collagen (C) were detected by quantitative real time PCR. Data were obtained from triplicated PCR reactions of three different cultures, and values are mean ± S.D. (*, p ≤ 0.01; **, p≤ 0.05; RA versus untreated or RA/K-201 treatment).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have demonstrated that RA treatment led to the release of mineralization-competent APase and annexin II-, V-, and VI-containing matrix vesicles, whereas untreated chondrocytes released vesicles that contain no or little APase activity and annexins II, V, and VI (16; see also Fig. 5). Cotreatment of chondrocytes with RA and K-201 significantly reduced the amounts of APase activity (Fig. 5 A) and annexins II, V, and VI (Fig. 5 B) in matrix vesicle fractions. Thus, annexin-mediated Ca2+influx into growth plate chondrocytes regulates expression of terminal differentiation marker genes, the release of APase- and annexin II-, V-, and VI-containing matrix vesicles, and subsequent mineralization. It is now well established that the final fate of terminally differentiated chondrocytes is apoptosis (2Gibson G. Microsc. Res. Tech. 1998; 43: 191-204Crossref PubMed Scopus (137) Google Scholar). Therefore, we addressed the question of whether RA triggers the whole cascade of terminal differentiation events including apoptosis and whether annexin-mediated Ca2+ influx into chondrocytes is also involved in the regulation of apoptotic changes. To determine the degree of apoptosis in the various treated chondrocyte cultures we measuredbcl-2 gene expression and caspase-3 activity and performed TUNEL labeling. A 5-day treatment with RA led to a significant decrease in gene expression of bcl-2, an anti-apoptotic protein (27Amling M. Neff L. Tanaka S. Inoue D. Kuida K. Weir E. Philbrick W.M. Broadus A.E. Baron R. J. Cell Biol. 1997; 136: 205-213Crossref PubMed Scopus (275) Google Scholar) (Fig. 6). In contrast, caspase-3 activity, an active cell death protease involved in the execution phase of apoptosis (28Porter A.G. Janicke R.U. Cell Death Differ. 1999; 6: 99-104Crossref PubMed Scopus (2917) Google Scholar), was more than 5-fold elevated in cultures treated for 6 days with RA compared with untreated cells (Fig. 7). Cotreatment of cultures with RA and the cytosolic Ca2+ chelator BAPTA-AM abolished the decrease in bcl-2 gene expression (Fig. 6) and the increase in caspase-3 activity (Fig. 7), suggesting that cytosolic calcium is directly involved in the regulation of apoptotic events. Interestingly,bcl-2 gene expression was also higher in RA/K-201-treated cells than in RA-treated cells (Fig. 6), whereas caspase-3 activity was lower (Fig. 7). In addition, TUNEL labeling revealed that in RA-treated cultures more than 10% of cells were TUNEL-positive, whereas only ∼2% were TUNEL-positive in untreated and RA/BAPTA-treated cells, and ∼4% of cells were TUNEL-positive in RA/K-201-treated cultures (Fig. 8).Figure 7Caspase-3 activity in untreated, RA -, RA/K-201-, and RA/BAPTA-treated growth plate chondrocytes. After a 6-day treatment, caspase-3 activities in untreated, RA-, RA/K-201-, and RA/BAPTA-treated chondrocytes were determined using the ApoAlert caspase fluorescent assay kit as described under “Experimental Procedures.” Caspase-3 activity was calibrated with AFC calibration curve and normalized to the protein content in each culture. Data were obtained from four different experiments, and values are mean ± S.D. (*, p ≤ 0.01; RA versus RA/K-201 treatment, RA versus RA/BAPTA treatment).View Large" @default.
• W2002113583 created "2016-06-24" @default.
• W2002113583 creator A5029527559 @default.
• W2002113583 creator A5046597133 @default.
• W2002113583 creator A5086648115 @default.
• W2002113583 date "2003-02-01" @default.
• W2002113583 modified "2023-10-08" @default.
• W2002113583 title "Annexin-mediated Ca2+ Influx Regulates Growth Plate Chondrocyte Maturation and Apoptosis" @default.
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