Collagen/Annexin V Interactions Regulate Chondrocyte Mineralization
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m708456200
ISSN1083-351X
Autores Tópico(s)Protease and Inhibitor Mechanisms
ResumoPhysiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca2+ influx resulting in an increased intracellular Ca2+ concentration, [Ca2+]i, and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca2+]i, alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca2+ channel activities) decreased [Ca2+]i, alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization. Physiological mineralization in growth plate cartilage is highly regulated and restricted to terminally differentiated chondrocytes. Because mineralization occurs in the extracellular matrix, we asked whether major extracellular matrix components (collagens) of growth plate cartilage are directly involved in regulating the mineralization process. Our findings show that types II and X collagen interacted with cell surface-expressed annexin V. These interactions led to a stimulation of annexin V-mediated Ca2+ influx resulting in an increased intracellular Ca2+ concentration, [Ca2+]i, and ultimately increased alkaline phosphatase activity and mineralization of growth plate chondrocytes. Consequently, stimulation of these interactions (ascorbate to stimulate collagen synthesis, culturing cells on type II collagen-coated dishes, or overexpression of full-length annexin V) resulted in increase of [Ca2+]i, alkaline phosphatase activity, and mineralization of growth plate chondrocytes, whereas inhibition of these interactions (3,4-dehydro-l-proline to inhibit collagen secretion, K-201, a specific annexin channel blocker, overexpression of N terminus-deleted mutant annexin V that does not bind to type II collagen and shows reduced Ca2+ channel activities) decreased [Ca2+]i, alkaline phosphatase activity, and mineralization. In conclusion, the interactions between collagen and annexin V regulate mineralization of growth plate cartilage. Because annexin V is up-regulated during pathological mineralization events of articular cartilage, it is possible that these interactions also regulate pathological mineralization. The extracellular matrix not only plays a major role in maintaining the function of a tissue but it also interacts and communicates directly with the cell via cell receptor/matrix interactions. These interactions play crucial roles in cell adhesion, migration, proliferation, and differentiation. Various types of collagens are the main organic components of extracellular matrices of many tissues. For example, in bone the main organic extracellular matrix component is type I collagen, whereas the main collagenous component in growth plate cartilage is type II collagen (1Hausler G. Helmreich M. Marlovits S. Egerbacher M. Calcif. Tiss. Inter. 2002; 71: 212-218Crossref PubMed Scopus (37) Google Scholar). Type II collagen is highly expressed in the proliferative and prehypertrophic zones of growth plate cartilage. Just before mineralization starts, type II collagen synthesis is reduced and the hypertrophic growth plate chondrocytes produce type X collagen (2Mwale F. Tchetina E. Wu C.W. Poole A.R. J. Bone Miner. Res. 2002; 17: 275-283Crossref PubMed Scopus (80) Google Scholar, 3Kirsch T. von der Mark K. Bone Miner. 1992; 18: 107-117Abstract Full Text PDF PubMed Scopus (65) Google Scholar). Several cell surface receptors that mediate cell/matrix interactions have been identified in growth plate chondrocytes. Among these receptors are integrin receptors and non-integrin receptors, like annexin V and CD44. Several integrins have been identified to be expressed by growth plate chondrocytes. Among these integrins are α1, α2, α5, α6, αV, β1, and β5. Most of these integrins are expressed in all growth plate zones, whereas some integrins are restricted to certain zones. For example, β5 integrin is restricted to the hypertrophic zone (1Hausler G. Helmreich M. Marlovits S. Egerbacher M. Calcif. Tiss. Inter. 2002; 71: 212-218Crossref PubMed Scopus (37) Google Scholar, 4Wang W. Kirsch T. J. Biol. Chem. 2006; 281: 30848-30856Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The main collagen receptors in growth plate cartilage are α1β1 and α2β1 integrins (5Durr J. Goodman S. Potocnik A. von der Mark H. von der Mark K. Exp. Cell Res. 1993; 207: 235-244Crossref PubMed Scopus (169) Google Scholar). Annexin V was identified as another protein binding to types II and X collagen, and it has been demonstrated that chondrocytes attach to type II collagen using annexin V (6Reid D.L. Aydelotte M.B. Mollenhauer J. J. Orthop. Res. 2000; 18: 364-373Crossref PubMed Scopus (39) Google Scholar). It was originally isolated from the chondrocyte membrane fractions as a type II collagen-binding protein (7Mollenhauer J. Bee J.A. Lizarbe M.A. von der Mark K. J. Cell Biol. 1984; 98: 1572-1578Crossref PubMed Scopus (92) Google Scholar, 8Kirsch T. Pfaeffle M. FEBS Lett. 1992; 310: 143-147Crossref PubMed Scopus (70) Google Scholar). We and others have shown that annexin V, among other annexins, is highly expressed by hypertrophic and terminally differentiated growth plate chondrocytes (9Kirsch T. Swoboda B. Nah H.-D. Osteoarthritis Cartilage. 2000; 8: 294-302Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 10Mollenhauer J. Mok M.T. King K.B. Gupta M. Chubinskaya S. Koepp H. Cole A. J. Histochem. Cytochem. 1999; 47: 209-220Crossref PubMed Scopus (45) Google Scholar). Annexins are normally located in the cytoplasm or at the inner plasma membrane surface. However, several annexins have been detected extracellularly or surface-exposed. In addition, several annexins have been shown to act as receptors for a variety of molecules, including extracellular matrix proteins, fetuin A, vitamin D, and other cell surface receptors (11Chung C.Y. Erickson H.P. J. Cell Biol. 1994; 126: 539-548Crossref PubMed Scopus (205) Google Scholar, 12Chen N.X. O'Neill K.D. Chen X. Duan D. Wang E. Sturek M.S. Edwards J.M. Moe S.M. Amer. J. Physiol. 2007; 292: F599-F606Crossref PubMed Scopus (55) Google Scholar, 13Baran D.T. Quail J.M. Ray R. Leszyk J. Honeyman T. J. Cell Biochem. 2000; 78: 34-46Crossref PubMed Scopus (81) Google Scholar, 14Siever D.A. Erickson H.P. Int. J. Biochem. Cell Biol. 1997; 29: 1219-1223Crossref PubMed Scopus (103) Google Scholar, 15Takagi H. Asano Y. Yamakawa N. Matsumoto I. Kimata K. J. Cell Sci. 2002; 115: 3309-3318Crossref PubMed Google Scholar). We provided evidence that annexin V mediates Ca2+ influx into growth plate chondrocytes and that this annexin-mediated Ca2+ influx regulates terminal differentiation and mineralization events (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar, 17Wang W. Xu J. Kirsch T. Exp. Cell Res. 2005; 305: 156-165Crossref PubMed Scopus (27) Google Scholar). In addition, Ca2+ influx studies into liposomes have shown that binding of type II or type X collagen to annexin V stimulated its Ca2+ channel activities, resulting in an increased Ca2+ influx into liposomes (18Kirsch T. Harrison G. Golub E.E. Nah H.-D. J. Biol. Chem. 2000; 275: 35577-35583Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). However, it is not known whether collagen/annexin V interactions occur under physiological conditions and whether these interactions play a role in the differentiation events of growth plate chondrocytes. To determine whether collagen/annexin V interactions occur physiologically, we determined whether annexin V is surface-exposed on growth plate chondrocytes using flow cytometric analysis and whether cell surface-exposed annexin V interacts with type II or X collagen using co-immunoprecipitation analysis. To determine the function of collagen/annexin V interactions in growth plate chondrocytes, we interfered with collagen synthesis and secretion and annexin V expression and function (collagen binding abilities and channel function). We analyzed how these interferences affect cytosolic Ca2+ concentration, [Ca2+]i, as a regulator of mineralization and terminal differentiation events (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar), tissue nonspecific alkaline phosphatase (APase) 2The abbreviation used is: APase, alkaline phosphatase. 2The abbreviation used is: APase, alkaline phosphatase. activity, an enzyme required for mineralization, and the degree of mineralization of growth plate chondrocytes. Reagents—The preparation and specificity of rabbit polyclonal antibodies specific for chicken annexin V were described previously (9Kirsch T. Swoboda B. Nah H.-D. Osteoarthritis Cartilage. 2000; 8: 294-302Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Monoclonal antibodies specific for type II and type X collagen were obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City) and have been described by Mills and Daniel (19Mills D.K. Daniel J.C. Connect. Tissue Res. 1993; 30: 37-57Crossref PubMed Scopus (9) Google Scholar) and Schmid and Linsenmayer (20Schmid T.M. Linsenmayer T.F. J. Cell Biol. 1985; 100: 598-605Crossref PubMed Scopus (350) Google Scholar). Antibodies specific for chicken β1 integrin were purchased from Chemicon International Inc. (Temecula, CA). 3,4-Dehydro-l-proline and ascorbate were purchased from Sigma-Aldrich. Native chicken type II collagen was obtained from Chondrex, Inc. (Redmond, WA). 1,4-Benzothiazepine derivative K-201 is cell-permeable and a specific annexin channel blocker and was obtained from Toshizo Tanaka, Japan Tobacco Inc., Osaka, Japan (21Kaneko N. Ago H. Matsuda R. Inagaki E. Miyano M. J. Mol. Biol. 1997; 274: 16-20Crossref PubMed Scopus (57) Google Scholar). Phospholipids were obtained from Avanti Polar Lipids (Birmingham, AL). Fura-2 and fura-2AM were obtained from Molecular Probes/Invitrogen. Chondrocyte Culture—Chondrocytes were isolated from the hypertrophic zone of day 19 embryonic chick tibia growth plate cartilage as described previously (22Kirsch T. Nah H.D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (190) Google Scholar). Cells were grown in monolayer cultures in Dulbecco's modified Eagle's medium (Invitrogen) containing 5% fetal calf serum (HyClone, Logan, UT), 2 mm l-glutamine (Invitrogen), and 50 units/ml penicillin and streptomycin (Invitrogen) (complete medium) on tissue culture dishes or tissue culture dishes coated with type II collagen. For collagen coating, a 1:100 dilution of chicken type II collagen solution (Chondrex Inc.) was added to each well of 6-well tissue culture plates and incubated at 4 °C. After overnight incubations, excess solution was removed and freshly isolated chondrocytes were plated onto the collagen-coated wells. After cells reached confluency the various treatments were started. Cells were treated with 50 μg/ml ascorbate and 0.5 mm 3,4-dehydro-l-proline or 20 μm K-201 for various time points. The same concentrations of 3,4-dehydro-l-proline and K-201 were used in previous studies to inhibit collagen synthesis in fibroblasts, growth plate chondrocytes, and osteoblasts or annexin V channel activities in growth plate chondrocytes (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar, 23Franceschi R.T. Iyer B.S. Cui Y. J. Bone Miner. Res. 1994; 9: 843-854Crossref PubMed Scopus (388) Google Scholar, 24Sullivan T.A. Uschmann B. Hough R. Leboy P.S. J. Biol. Chem. 1994; 269: 22500-22506Abstract Full Text PDF PubMed Google Scholar, 25Abe T. Abe Y. Aida Y. Hara Y. Maeda K. J. Cell Physiol. 2001; 189: 144-151Crossref PubMed Scopus (51) Google Scholar). These concentrations were shown not to be toxic to growth plate chondrocytes (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar, 25Abe T. Abe Y. Aida Y. Hara Y. Maeda K. J. Cell Physiol. 2001; 189: 144-151Crossref PubMed Scopus (51) Google Scholar). The medium was changed every other day. For annexin overexpression studies, growth plate chondrocytes were incubated after 3 days with high titer retroviral stocks of replication-competent, non-transforming Rous sarcoma virus-based expression vector (RCAS-BP) or RCAS-BP containing full-length annexin V or N terminus-deleted mutant annexin V cDNA in a small volume (5 × 106 colony-forming units/106 cells in less than 1 ml of medium) for 4 h. Thereafter, cells were cultured in complete medium in the presence of 50 μg/ml ascorbate for up to 8 days. The degree of overexpression was detected by immunoblotting using antibodies specific for annexin V or antibodies specific for c-Myc. The construction and production of chicken retrovirus RCAS-BP was performed as described previously (26Wang W. Xu J. Kirsch T. J. Biol. Chem. 2003; 278: 3762-3769Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Fluorescence-activated Cell Sorter Analysis—Freshly isolated chondrocytes were cultured for 48 h and then trypsinized. After blocking with 4% heat-inactivated goat serum for 30 min, cells were stained with a rabbit anti-annexin V IgG and mouse anti-β1 integrin for 2 h on ice, followed by incubation with the respective secondary antibodies (Alexa Fluor 633 and Alexa Fluor 488; Molecular Probes/Invitrogen). A fluorescence-activated cell sorter (LSR1; BD Biosciences) was used to quantify bound antibody. Cytosolic Calcium Concentration [Ca2+]i Measurements—Chondrocytes were trypsinized, and then 2 × 106 cells were incubated with 4 μm fura-2 AM (Molecular Probes/Invitrogen) for 15 min at 37 °C. [Ca2+]i was measured as described previously (27Kirsch T. Swoboda B. von der Mark K. Differentiation. 1992; 52: 89-100Crossref PubMed Scopus (57) Google Scholar). Briefly, cells were resuspended in a buffer containing 140 mm NaCl, 5 mm KCl, 1 mm CaCl2, 20 mm HEPES, 1 mm NaH2PO4, 5.5 mm glucose, pH 7.4. This suspension was transferred into a cuvette (magnetically stirred and thermostated at 37 °C). Fluorescence was measured in a fluorimeter (Photon Technology; excitation and emission wavelengths were 340 and 505 nm, respectively). The fluorescence maximum (FMax) was determined by addition of ionomycin (2 pmol/liter; Calbiochem), and the fluorescence minimum (FMin) was determined in the presence of 1 mm EGTA/10 mm Tris, pH 7.4. [Ca2+]i was calculated according to the following equation: [Ca2+]i = Kd × [(F – FMin)/(FMax – F)], with Kd = 224 nm (28Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Measurement of APase Activity and Protein Content and Alizarin Red S Staining—APase activity was measured using p-nitrophenyl phosphate (Sigma-Aldrich) as a substrate as described previously (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar). Protein content was analyzed by the BCA protein assay from Pierce. To determine the degree of mineralization, chondrocytes were stained with alizarin red S as described previously (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar). To quantify the alizarin red S staining, alizarin red S-stained cultures were incubated with 100 mm cetylpyridinium chloride for 1 h to release calcium-bound alizarin red S into solution. 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/mg of protein in each culture. Immunoprecipitation—Growth plate chondrocytes were cultured for 48 h in the presence of 50 μg/ml ascorbate. Cells were then extracted with a buffer containing 50 mm Tris/HCl, pH 7.4, 1% Nonidet P-40, 0.1% Triton X-100, 150 mm NaCl, 5 mm EDTA, and proteinase inhibitor mixture. 1 mg of total protein from the cell extract was incubated with 3 μg of monoclonal mouse anti-chicken type II or chicken type X collagen IgG fractions for overnight at 4 °C. After incubation, 60 μl of 50% slurry of protein A beads (Pharmacia) were added. After 1 h of incubation at 4 °C, beads were harvested and washed four times with extraction buffer. 50 μl of SDS loading buffer was added, and the samples were boiled at 95 °C for 5 min. Supernatants were analyzed by SDS-PAGE and immunoblotting. Measurement of Ca2+ Influx into Fura-2-loaded Liposomes and Annexin V/Collagen Liposome Binding Studies—Large thin-walled liposomes containing phosphatidylserine and phosphatidyl-ethanolamine in a ratio of 9:1 were prepared using a dehydration/hydration method and were loaded with fura-2 as described previously (29Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (85) Google Scholar). Ca2+ influx into fura-2-loaded liposomes was measured in the absence or presence of recombinant full-length annexin V or N terminus-deleted mutant annexin V (200 nm) as described previously (29Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (85) Google Scholar). Recombinant full-length and N terminus-deleted mutated annexin V were prepared using the pGEX expression vector (Pharmacia) as described previously (29Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (85) Google Scholar). To test the binding of type II or type X collagen to liposomes in the absence or presence of full-length annexin V or N terminus-deleted mutant annexin V, 200 nm of these annexins were incubated with liposomes in the presence of 400 μm Ca2+. Liposomes containing annexin or without annexin were then incubated with type II or type X collagen (10 μg) for 1 h at room temperature. Liposomes were quantitatively pelleted by centrifugation at 200,000 × g for 15 min and washed twice, and aliquots of the liposome suspension were dotted onto nitrocellulose membranes and immunostained with antibodies specific for annexin V, type II collagen, or type X collagen as described previously (29Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (85) Google Scholar). The optical density of the color reaction was determined using a densitometer, and the optical density for staining of liposomes containing full-length annexin V and type II or type X collagen and immunostained with antibodies specific for type II or type X collagen was set as 1. Cell Viability Assay—Cell viability was determined using the Cell Counting Kit-8 (CCK-8) from Dojindo Molecular Technologies, Inc. (Gaithersburg, MD) following the manufacturer's instructions. Briefly, the CCK-8 solution was added to each culture dish and incubated for 4 h at 37 °C. After incubation the absorption was measured at 450 nm. The absorption value for the untreated cells was set as 100%. SDS-PAGE and Immunoblotting—To determine the secretion and synthesis of types II and X collagen the cell layer was extracted with 0.1% Triton X-100, 1 m NaCl, and 5 mm EDTA in 50 mm Tris-HCl, pH 7.4, including protease inhibitor mixture from Sigma-Aldrich and 1 mm phenylmethylsulfonyl fluoride. Cell extracts (30 μg of total protein) were subjected to SDS-PAGE and immunoblotted with primary antibodies specific for type II or type X collagen as described previously (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar). Statistical Analysis—Numerical data are presented as means ± S.D. (n ≥ 3), and statistical significance between the groups was identified by using the two-tailed Student t test (p values are reported in the figure legends). To establish the role of the interactions between collagen and annexin V in terminal differentiation and mineralization events of growth plate chondrocytes, we treated chondrocytes isolated from the hypertrophic zone of day 19 embryonic chick tibia growth plate cartilage with ascorbate in the absence or presence of 3,4-dehydro-l-proline. Ascorbate is required for collagen synthesis, whereas 3,4-dehydro-l-proline reduces the hydroxylation of prolyl residues and the secretion of collagen from the cell (30Kerwar S.S. Felix A.M. J. Biol. Chem. 1976; 251: 503-509Abstract Full Text PDF PubMed Google Scholar). We used the same concentration of 3,4-dehydro-l-proline (0.5 mm) as used in previous studies to inhibit collagen synthesis in fibroblasts, sternal growth plate chondrocytes, and osteoblasts (24Sullivan T.A. Uschmann B. Hough R. Leboy P.S. J. Biol. Chem. 1994; 269: 22500-22506Abstract Full Text PDF PubMed Google Scholar, 25Abe T. Abe Y. Aida Y. Hara Y. Maeda K. J. Cell Physiol. 2001; 189: 144-151Crossref PubMed Scopus (51) Google Scholar, 31Xiao G. Cui Y. Ducy P. Karsenty G. Franceschi R.T. Mol. Endocrinol. 1997; 11: 1103-1113Crossref PubMed Scopus (152) Google Scholar). Fig. 1A shows that a treatment over 6 days with 3,4-dehydro-l-proline in the absence or presence of ascorbate did not affect cell viability compared with the cell viability of untreated or ascorbate-treated cells. The amounts of type II and X collagen were markedly increased in cell extracts from ascorbate-treated growth plate chondrocytes compared with the amounts of these collagens in extracts from untreated cells (Fig. 1B, II, X). We only determined the amounts of collagens in cell and cell layer extracts and not the collagen amounts released in the medium because only the collagen remaining in the cell layer interacts with cell receptors, including annexin V. The expression of annexin V was not affected by the 72-h ascorbate treatment. 3,4-Dehydro-l-proline treatment did not affect annexin V expression in growth plate chondrocyte cultures in the absence or presence of ascorbate (Fig. 1B, AnV). 3,4-Dehydro-l-proline, however, markedly reduced the amounts of types II and X collagen in total cell extracts from ascorbate-treated growth plate chondrocytes (Fig. 1B, II, X). To determine whether types II and X collagen, which bind to annexin V and stimulate its Ca2+ channel activities in an artificial liposome system (18Kirsch T. Harrison G. Golub E.E. Nah H.-D. J. Biol. Chem. 2000; 275: 35577-35583Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), affect annexin Ca2+ channel activities and ultimately mineralization events in growth plate chondrocytes, we determined first whether the inhibition of collagen secretion by 3,4-dehydro-l-proline in ascorbate-treated growth plate chondrocytes affected their [Ca2+]i and their degree of mineralization. Ascorbate increased [Ca2+]i compared with [Ca2+]i of untreated growth plate chondrocytes (Fig. 2A). 3,4-Dehydro-l-proline (0.5 mm) led to a decrease of [Ca2+]i in ascorbate-treated cultures to levels similar to those of untreated cells (Fig. 2A). 3,4-Dehydro-l-proline led to a slight, but statistically not significant, increase of [Ca2+]i compared with the levels of untreated cells (Fig. 2A). Ascorbate treatment resulted in a marked increase of APase activity and degree of mineralization compared with the levels of APase activity and mineralization in untreated cultures, which showed only very little signs of mineralization (Fig. 2, B–D). APase activity was measured in cell extracts, because most APase activity is retained on either the outer cell surface or in matrix vesicles, which are present in the cell layer (22Kirsch T. Nah H.D. Shapiro I.M. Pacifici M. J. Cell Biol. 1997; 137: 1149-1160Crossref PubMed Scopus (190) Google Scholar). APase activity was decreased in ascorbate-treated and untreated growth plate chondrocyte cultures in the presence of 3,4-dehydro-l-proline (Fig. 2B). 3,4-Dehydro-l-proline also resulted in a notable decrease of mineralization of ascorbate-treated growth plate chondrocyte cultures (Fig. 2, C and D). These findings reveal that interfering with collagen secretion by 3,4-dehydro-l-proline affects [Ca2+]i, APase activity, and the degree of mineralization in ascorbate-treated growth plate chondrocytes. To further determine whether collagen/annexin V interactions mediate these effects, we determined whether annexin V is cell surface-expressed and interacts with types II and X collagen on the cell surface. Annexins are cytosolic proteins, which have no signal peptide for protein secretion. However, several studies have shown the secretion of annexins to the cell surface (14Siever D.A. Erickson H.P. Int. J. Biochem. Cell Biol. 1997; 29: 1219-1223Crossref PubMed Scopus (103) Google Scholar, 32Castro-Caldas M. Duarte C.B. Carvalho A.P. Lopes M.C. Mol. Cell. Biochem. 2002; 237: 31-38Crossref PubMed Scopus (15) Google Scholar). Flow cytometric analysis of growth plate chondrocytes revealed that the cells express β1 integrin (Fig. 3A, c: lower and upper right quadrants) and annexin V (c: upper left and upper right quadrants) on their surface. Furthermore, we performed co-immunoprecipitation experiments in which total cell extracts from growth plate chondrocytes treated for 48 h with ascorbate were immunoprecipitated with antibodies specific for type II or type X collagen and the immunoprecipitates were immunoblotted with antibodies specific for annexin V. Annexin V co-immunoprecipitated with type II collagen or type X collagen as shown by the immunoblot of type II collagen or type X collagen immunoprecipitates with antibodies specific for annexin V (Fig. 3B). Immunoblotting of the type II collagen immunoprecipitate with antibodies specific for type II collagen revealed a band for type II collagen, whereas immunoblotting of the type X collagen immunoprecipitate with antibodies specific for type X collagen revealed a band for type X collagen (Fig. 3B). These findings reveal that type II and type X collagens interact with cell surface-exposed annexin V. To further establish the role of collagen/annexin V interactions in growth plate chondrocyte mineralization, we cultured growth plate chondrocytes on tissue culture dishes or type II collagen-coated tissue culture dishes in the absence or presence of K-201, a specific annexin V Ca2+ channel blocker (33Kaneko N. Matsuda R. Toda M. Shimamoto K. Biochim. Biophys. Acta. 1997; 1330: 1-7Crossref PubMed Scopus (53) Google Scholar). K-201 was used in a concentration of 20 μm, which has been previously shown to be an effective concentration to inhibit annexin VCa2+ channel activities without affecting cell viability (16Wang W. Kirsch T. J. Cell Biol. 2002; 157: 1061-1069Crossref PubMed Scopus (109) Google Scholar). Cells cultured on type II collagen-coated dishes showed an increase of [Ca2+]i compared with [Ca2+]i of growth plate chondrocytes cultured on tissue culture dishes (Fig. 4A). APase activity and the degree of mineralization were also stimulated by culturing growth plate chondrocytes on type II collagen-coated dishes compared with the APase activity and degree of mineralization of cells cultured on tissue culture dishes (Fig. 4, B and C). The increase of [Ca2+]i, APase activity, and the degree of mineralization of growth plate chondrocytes cultured on type II collagen-coated dishes was inhibited by K-201 (Fig. 4, A–C). K-201 treatment had no effect on [Ca2+]i, APase activity, and mineralization of growth plate chondrocytes cultured on tissue culture dishes (Fig. 4, A–C). In the final set of experiments we determined whether a N terminus-deleted mutant annexin V, which has been previously shown to have reduced Ca2+ channel activities compared with full-length annexin V (34Berendes R. Burger A. Voges D. Demange P. Huber R. FEBS Lett. 1993; 317: 131-134Crossref PubMed Scopus (51) Google Scholar), had an effect on APase activity and the degree of mineralization of ascorbate-treated growth plate chondrocytes cultures. We prepared full-length annexin V (amino acids 1–321) and N terminus-deleted mutant annexin V (amino acids 13–321) using the pGEX expression vector as described previously (29Kirsch T. Nah H.-D. Demuth D.R. Harrison G. Golub E.E. Adams S.L. Pacifici M. Biochemistry. 1997; 36: 3359-3367Crossref PubMed Scopus (85) Google Scholar). We determined whether full-length or N terminus-deleted mutant annexin V mediates Ca2+ influx into fura-2-loaded phosphatidylserine-enriched liposomes. The N terminus-deleted mutant annexin V mediated Ca2+ influx into liposomes to a lesser degree than full-length annexin V (Fig. 5A). Next we tested whether binding of type II or X collagen to liposomes is affected by the N-terminal domain of annexin V. Liposomes were incubated with type II or X collagen in the absence or presence of full-length annexin V or N terminus-deleted mutant annexin V, and liposome-bound type II or X collagen was detected with antibodies specific for type II or type X collagen. In the absence of annexin V, native type II or type X collagen did not bind t
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