1α,25(OH)2D3 Regulates Chondrocyte Matrix Vesicle Protein Kinase C (PKC) Directly via G-protein-dependent Mechanisms and Indirectly via Incorporation of PKC during Matrix Vesicle Biogenesis
2002; Elsevier BV; Volume: 277; Issue: 14 Linguagem: Inglês
10.1074/jbc.m110398200
ISSN1083-351X
AutoresZvi Schwartz, Victor L. Sylvia, Dennis Larsson, Ilka Nemere, David Casasola, David D. Dean, Barbara D. Boyan,
Tópico(s)Vitamin D Research Studies
ResumoMatrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1α,25(OH)2D3. Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1α,25(OH)2D3 in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1α,25(OH)2D3 during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1α,25(OH)2D3, PKCζ in matrix vesicles was inhibited, while PKCα in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCζ in matrix vesicles was increased, while PKCα in plasma membranes was unchanged. The effect of 1α,25(OH)2D3 was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1α,25(OH)2D3 membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1α,25(OH)2D3-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1α,25(OH)2D3 in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or Gi/Gs had no effect. However, inhibition of Gq blocked the effect of 1α,25(OH)2D3. The rapid effect of 1α,25(OH)2D3 also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1α,25(OH)2D3 at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid. Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1α,25(OH)2D3. Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1α,25(OH)2D3 in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1α,25(OH)2D3 during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1α,25(OH)2D3, PKCζ in matrix vesicles was inhibited, while PKCα in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCζ in matrix vesicles was increased, while PKCα in plasma membranes was unchanged. The effect of 1α,25(OH)2D3 was stereospecific and metabolite-specific. Monensin blocked the increase in matrix vesicle PKC after 24 h, suggesting the secosteroid-regulated packaging of PKC. In addition, the 1α,25(OH)2D3 membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1α,25(OH)2D3-dependent changes in PKC after both long and short treatment times. In contrast, antibodies to annexin II had no effect, and there was no evidence for the presence of the nuclear VDR on Western blots. To investigate the signaling pathways involved in regulating matrix vesicle PKC activity after biosynthesis, matrix vesicles were isolated and then treated for 9 min with 1α,25(OH)2D3 in the presence and absence of specific inhibitors. Inhibition of phosphatidylinositol-phospholipase C, phospholipase D, or Gi/Gs had no effect. However, inhibition of Gq blocked the effect of 1α,25(OH)2D3. The rapid effect of 1α,25(OH)2D3 also involved the 1,25-mVDR. Moreover, arachidonic acid was found to stimulate PKC when added directly to isolated matrix vesicles. These results indicate that matrix vesicle PKC is regulated by 1α,25(OH)2D3 at three levels: 1) during matrix vesicle biogenesis; 2) through direct action on the membrane; and 3) through production of other factors such as arachidonic acid. Costochondral growth plate chondrocytes metabolize 25(OH)D3 in a regulated manner, producing and secreting 1,25(OH)2D3 and 24,25(OH)2D3 (1.Schwartz Z. Brooks B.P. Swain L.D. Del Toro F. Norman A.W. Boyan B.D. Endocrinology. 1992; 130: 2495-2504Crossref PubMed Google Scholar, 2.Pedrozo H.A. Boyan B.D. Mazock J. Dean D.D. Gomez R. Schwartz Z. Calcif. Tissue Int. 1999; 64: 50-56Crossref PubMed Scopus (49) Google Scholar). The physiological importance of this is not yet well understood. 1α,25(OH)2D3 exerts direct effects on matrix vesicles isolated from the extracellular matrix of growth plate chondrocytes (3.Swain L.D. Schwartz Z. Caulfield K. Brooks B.P. Boyan B.D. Bone. 1993; 14: 609-617Crossref PubMed Scopus (69) Google Scholar), suggesting that the cells may use local production of the vitamin D metabolite as a mechanism for controlling events in the matrix. A number of observations support this hypothesis. Treatment of matrix vesicles with 1α,25(OH)2D3 causes increased alkaline phosphatase specific activity, which is associated with the onset of calcification (4.Schwartz Z. Knight G. Swain L.D. Boyan B.D. J. Biol. Chem. 1988; 263: 6023-6026Abstract Full Text PDF PubMed Google Scholar). In addition, phospholipase A2(PLA2) 1The abbreviations used are: PLA2phospholipase A2PKCprotein kinase CPLCphospholipase CPGE2prostaglandin E2PIphosphatidylinositolPLDphospholipase DmVDRmembrane vitamin D receptornVDRnuclear vitamin D receptorDOG1,2-dioctanoyl-sn-glycerolDAGdiacylglycerolMAPmitogen-activated protein 1The abbreviations used are: PLA2phospholipase A2PKCprotein kinase CPLCphospholipase CPGE2prostaglandin E2PIphosphatidylinositolPLDphospholipase DmVDRmembrane vitamin D receptornVDRnuclear vitamin D receptorDOG1,2-dioctanoyl-sn-glycerolDAGdiacylglycerolMAPmitogen-activated protein specific activity is increased (5.Schwartz Z. Boyan B.D. Endocrinology. 1988; 122: 2191-2198Crossref PubMed Scopus (143) Google Scholar), which may lead to a loss of membrane integrity and the release of proteinases capable of remodeling the matrix (6.Dean D.D. Schwartz Z. Muniz O.E. Gomez R. Swain L.D. Howell D.S. Boyan B.D. Calcif. Tissue Int. 1992; 50: 342-349Crossref PubMed Scopus (91) Google Scholar, 7.Einhorn T.A. Hirschman A. Kaplan C. Nashed R. Devlin V.J. Warman J. J. Orthop. Res. 1989; 7: 792-805Crossref PubMed Scopus (34) Google Scholar). One of the matrix metalloproteinases that are present in matrix vesicles, stromelysin-1 (MMP-3), has been shown to activate latent transforming growth factor β-1 (TGF-β1) in a 1α,25(OH)2D3-dependent manner (8.Maeda S. Dean D.D. Schwartz Z. Boyan B.D. J. Bone Miner. Res. 2001; 16: 1281-1290Crossref PubMed Scopus (75) Google Scholar). phospholipase A2 protein kinase C phospholipase C prostaglandin E2 phosphatidylinositol phospholipase D membrane vitamin D receptor nuclear vitamin D receptor 1,2-dioctanoyl-sn-glycerol diacylglycerol mitogen-activated protein phospholipase A2 protein kinase C phospholipase C prostaglandin E2 phosphatidylinositol phospholipase D membrane vitamin D receptor nuclear vitamin D receptor 1,2-dioctanoyl-sn-glycerol diacylglycerol mitogen-activated protein The mechanisms involved in the regulation of matrix vesicles by 1α,25(OH)2D3 are not known. 1α,25(OH)2D3 modulates proliferation and differentiation of growth plate chondrocytes via the nuclear vitamin D receptor (1,25-nVDR) and the concerted action of transcription factors and co-activators. However, matrix vesicles do not contain DNA or RNA, so genomic pathways are unlikely to play a role in mediating the direct effect of 1α,25(OH)2D3 on the organelles. Recent studies show that 1α,25(OH)2D3 also acts on cells through membrane-mediated mechanisms, resulting in rapid changes in calcium ion flux, phospholipid metabolism and kinase activation, including a rapid increase in protein kinase C (PKC) specific activity (9.Boyan B.D. Sylvia V.L. Dean D.D. Pedrozo H. Del Toro F. Nemere I. Posner G.H. Schwartz Z. Steroids. 1999; 64: 129-136Crossref PubMed Scopus (85) Google Scholar, 10.de Boland A.R. Boland R.L. Cell Signal. 1994; 6: 717-724Crossref PubMed Scopus (114) Google Scholar, 11.Morelli S. de Boland A.R. Boland R. Biochem. J. 1993; 289: 675-679Crossref PubMed Scopus (105) Google Scholar, 12.Facchinetti M.M. de Boland A.R. Cell Signal. 1999; 11: 39-44Crossref PubMed Scopus (17) Google Scholar, 13.Facchinetti M.M. Boland R. de Boland A.R. J. Lipid Res. 1998; 39: 197-204Abstract Full Text Full Text PDF PubMed Google Scholar). Many of the physiological responses of the chondrocytes to 1α,25(OH)2D3 are blocked by inhibiting PKC (14.Boyan, B. D., Sylvia, V. L., Dean, D. D., Del Toro, F., and Schwartz, Z. (2002) Crit. Rev. Oral Biol. Med., in pressGoogle Scholar). Moreover, both the PKC-dependent responses and the rapid increase in PKC itself are blocked with an antibody generated to a [3H]-1,25(OH)2D3-binding protein present in the basal lateral membranes of chick intestinal epithelium (Ab99) (15.Nemere I. Dormanen M.C. Hammond M.W. Okamura W.H. Norman A.W. J. Biol. Chem. 1994; 269: 23750-23756Abstract Full Text PDF PubMed Google Scholar, 16.Nemere I. Schwartz Z. Pedrozo H. Sylvia V.L. Dean D.D. Boyan B.D. J. Bone Miner. Res. 1998; 13: 1353-1359Crossref PubMed Scopus (187) Google Scholar, 17.Pedrozo H.A. Schwartz Z. Rimes S. Sylvia V.L. Nemere I. Posner G.H. Dean D.D. Boyan B.D. J. Bone Miner. Res. 1999; 14: 856-867Crossref PubMed Scopus (89) Google Scholar). The rapid effects of 1,25(OH)2D3 are stereospecific; only the 1α,25(OH)2D3 isomer elicits an increase in PKC or regulates the signaling pathways that lead to the increase in PKC (18.Sylvia V.L. Del Toro Jr., F. Hardin R.R. Dean D.D. Boyan B.D. Schwartz Z. J. Steroid Biochem. Mol. Biol. 2001; 78: 261-274Crossref PubMed Scopus (45) Google Scholar), indicating a 1,25(OH)2D3 membrane receptor (1,25-mVDR)-mediated mechanism is involved. The hypothesis that 1α,25(OH)2D3 regulates matrix vesicles via 1,25-mVDR-mediated changes in PKC is attractive. Ab99 recognizes a single protein band in Western blots of matrix vesicles with a Mr of 65,000, and matrix vesicles exhibit specific binding for [3H]-1,25(OH)2D3 (16.Nemere I. Schwartz Z. Pedrozo H. Sylvia V.L. Dean D.D. Boyan B.D. J. Bone Miner. Res. 1998; 13: 1353-1359Crossref PubMed Scopus (187) Google Scholar). Moreover, the effect of 1α,25(OH)2D3 on PKC is blocked by Ab99, just as it is in the cell. However, 1α,25(OH)2D3 stimulates PKC activity in growth zone chondrocytes and when incubated directly with chondrocyte plasma membranes, whereas it inhibits PKC activity when incubated directly with matrix vesicles (19.Sylvia V.L. Schwartz Z. Ellis E.B. Helm S.H. Gomez R. Dean D.D. Boyan B.D. J. Cell. Physiol. 1996; 167: 380-393Crossref PubMed Scopus (92) Google Scholar), even though the same receptor is involved. The purpose of the present study was to examine the mechanisms that regulate 1α,25(OH)2D3-dependent PKC activity in matrix vesicles. There are several reasons why regulation of matrix vesicle PKC might differ from that of the plasma membrane. First, there is a differential distribution of PKC isoforms between the two membrane fractions. PKCζ predominates in matrix vesicles, whereas PKCα predominates in plasma membranes (19.Sylvia V.L. Schwartz Z. Ellis E.B. Helm S.H. Gomez R. Dean D.D. Boyan B.D. J. Cell. Physiol. 1996; 167: 380-393Crossref PubMed Scopus (92) Google Scholar). The two membrane fractions differ in other ways as well, including phospholipid composition (20.Boyan B.D. Schwartz Z. Swain L.D. Carnes Jr., D.L. Zislis T. Bone. 1988; 9: 185-194Crossref PubMed Scopus (176) Google Scholar) and basal membrane fluidity (3.Swain L.D. Schwartz Z. Caulfield K. Brooks B.P. Boyan B.D. Bone. 1993; 14: 609-617Crossref PubMed Scopus (69) Google Scholar). Studies using chondrocytes from the resting zone of costochondral cartilage indicate that the responsive isoform in intact cells, as well as in isolated plasma membranes, is PKCα, whereas the responsive isoform in matrix vesicles is PKCζ. Whether this is also the case for growth zone cells is not known, however. PKC is regulated by 24R,25(OH)2D3 in resting zone cells, but 1α,25(OH)2D3 regulates activity in growth zone cells, and these two metabolites use two distinctly different mechanisms to regulate PKC activity in their target cells (9.Boyan B.D. Sylvia V.L. Dean D.D. Pedrozo H. Del Toro F. Nemere I. Posner G.H. Schwartz Z. Steroids. 1999; 64: 129-136Crossref PubMed Scopus (85) Google Scholar, 21.Schwartz Z. Sylvia V.L. Luna M.H. DeVeau P. Whetstone R. Dean D.D. Boyan B.D. Steroids. 2001; 66: 683-694Crossref PubMed Scopus (48) Google Scholar). Moreover, matrix vesicle composition, including phospholipids and enzyme activities, and regulation of matrix vesicle function differs between the two cell types (see Refs. 14.Boyan, B. D., Sylvia, V. L., Dean, D. D., Del Toro, F., and Schwartz, Z. (2002) Crit. Rev. Oral Biol. Med., in pressGoogle Scholar, 22.Boyan B.D. Dean D.D. Sylvia V.L. Schwartz Z. Feldman D. Glorieux F.H. Pike J.W. Vitamin D. Academic Press, San Diego, CA1997: 395-421Google Scholar for reviews). It is likely that 1α,25(OH)2D3 regulates matrix vesicle PKC during organelle biogenesis, as well as directly, once they are resident in the extracellular matrix. When growth plate chondrocytes are cultured with 1α,25(OH)2D3for 24 h, long enough for new gene expression and matrix vesicle synthesis, matrix vesicle PKC activity is increased (23.Sylvia V.L. Schwartz Z. Schuman L. Morgan R.T. Mackey S. Gomez R. Boyan B.D. J. Cell. Physiol. 1993; 157: 271-278Crossref PubMed Scopus (99) Google Scholar). While 1α,25(OH)2D3 has been shown to regulate the distribution of matrix proteinases in matrix vesicles (24.Dean D.D. Boyan B.D. Muniz O.E. Howell D.S. Schwartz Z. Calcif. Tissue Int. 1996; 59: 109-116Crossref PubMed Scopus (58) Google Scholar), it is not known if the increase in matrix vesicle PKC is due to preferential incorporation of specific isoforms of the enzyme. PKCα is sensitive to Ca2+ ions and to phospholipid, whereas PKCζ is insensitive to both co-factors (25.Newton A.C. J. Biol. Chem. 1995; 270: 28495-28498Abstract Full Text Full Text PDF PubMed Scopus (1468) Google Scholar), yet both Ca2+ ions and phospholipid are present at relatively high levels in the growth plate extracellular matrix (26.Boyan B.D. Schwartz Z. Howell D.S. Naski M. Ranly D.M. Sylvia V.L. Dean D.D. Coe F.L. Favus M.J. Disorders of Bone and Mineral Metabolism. 2nd Ed. Lippincott, Williams, and Wilkins, Inc., Philadelphia, PA2002Google Scholar), particularly in the growth zone. Thus, it is possible that other isoforms may be involved in the matrix vesicle response to 1α,25(OH)2D3. For example, in renal epithelial cells, 1α,25(OH)2D3 has been shown to increase PKCβ activity (27.Simboli-Campbell M. Gagnon A. Franks D.J. Welsh J. J. Biol. Chem. 1994; 269: 3257-3264Abstract Full Text PDF PubMed Google Scholar). Other aspects of the signaling pathway by which 1α,25(OH)2D3 modulates matrix vesicle PKC may differ as well. G-protein, specifically Gq but not Gi or Gs, mediates the effect of 1α,25(OH)2D3 on cellular PKC (21.Schwartz Z. Sylvia V.L. Luna M.H. DeVeau P. Whetstone R. Dean D.D. Boyan B.D. Steroids. 2001; 66: 683-694Crossref PubMed Scopus (48) Google Scholar); whether this is the case for matrix vesicle PKC is unknown. Phospholipid metabolism plays a major role in the mechanism of 1α,25(OH)2D3-dependent PKC activity in the intact cell, causing rapid increases in phospholipase A2 (PLA2) and phospholipase C (PLC) activity, release of arachidonic acid and diacylglycerol, and production of prostaglandin E2 (PGE2) (9.Boyan B.D. Sylvia V.L. Dean D.D. Pedrozo H. Del Toro F. Nemere I. Posner G.H. Schwartz Z. Steroids. 1999; 64: 129-136Crossref PubMed Scopus (85) Google Scholar). This may not be the case for matrix vesicles, however. Matrix vesicles possess an active phospholipid metabolism that is regulated independently from that of the cell (28.Schwartz Z. Schlader D.L. Swain L.D. Boyan B.D. Endocrinology. 1988; 123: 2878-2884Crossref PubMed Scopus (141) Google Scholar). Their phospholipid composition is distinct from that of the plasma membrane as well (29.Boyan B.D. Ritter N.M. Calcif. Tissue Int. 1984; 36: 332-337Crossref PubMed Scopus (21) Google Scholar, 30.Peress N.S. Anderson H.C. Sajdera S.W. Calcif. Tissue Res. 1974; 14: 275-281Crossref PubMed Scopus (131) Google Scholar, 31.Swain L.D. Schwartz Z. Boyan B.D. Bone Miner. 1992; 17: 192-196Abstract Full Text PDF PubMed Scopus (13) Google Scholar). Unlike the plasma membrane, which has a phospholipid composition higher in phosphatidylcholine, matrix vesicles contain higher levels of phosphatidylserine and phosphatidylinositol, as well as cardiolipin. The basal fluidity of the plasma membrane and matrix vesicles also differs (3.Swain L.D. Schwartz Z. Caulfield K. Brooks B.P. Boyan B.D. Bone. 1993; 14: 609-617Crossref PubMed Scopus (69) Google Scholar). Thus, it is likely that phospholipid metabolism may play a different role in the mechanism by which 1α,25(OH)2D3 modulates PKC activity in the extracellular organelle. This study tested the hypothesis that 1α,25(OH)2D3 regulates matrix vesicle PKC activity in multiple ways. The vitamin D metabolite first increases the amount of PKCζ incorporated during matrix vesicle biogenesis though genomic mechanisms. Once the matrix vesicles are released into the matrix, 1α,25(OH)2D3 acts directly on the matrix vesicle via the 1,25-mVDR, reducing PKCζ activity. The signaling pathways differ from those that participate in the increase in plasma membrane PKC activity. In addition, factors released from the cells by the action of 1α,25(OH)2D3 on the plasma membrane also modulate PKC activity in the extracellular organelle. We used two experimental models to examine the regulation of matrix vesicle PKC by 1α,25(OH)2D3. In the first set of experiments, we tested the hypothesis that 1α,25(OH)2D3 regulates the differential distribution of PKC isoforms during matrix vesicle biogenesis. Rat costochondral growth zone cartilage cells were treated with 1α,25(OH)2D3 for up to 24 h. Matrix vesicles and plasma membranes were then isolated from the cultures. The 1α,25(OH)2D3-dependent isoform in each membrane fraction was determined using isoform-specific antibodies, comparing the effect at 90 min to the effect at 24 h. We also examined the regulation of matrix vesicle production by 1α,25(OH)2D3 using monensin to block protein transport through the Golgi. While it is known that the 1,25-mVDR mediates the rapid increase in PKC at 9 min (16.Nemere I. Schwartz Z. Pedrozo H. Sylvia V.L. Dean D.D. Boyan B.D. J. Bone Miner. Res. 1998; 13: 1353-1359Crossref PubMed Scopus (187) Google Scholar), it is not known if the downstream genomic regulation of matrix vesicle PKC is also regulated via the 1,25-mVDR or any of the signaling pathways. The role of the 1,25-mVDR in the process was assessed using Ab99. We also examined whether the effect of 1α,25(OH)2D3on matrix vesicle PKC at 24 h is mediated by PLC, which was previously shown to mediate the 1,25-mVDR-dependent rapid increase in PKC activity in growth zone cells. For these experiments, cells were treated with 1α,25(OH)2D3 in the presence of the phosphatidylinositol-specific (PI-PLC) inhibitorU73122. The second model used matrix vesicles isolated from cultures not previously treated with 1α,25(OH)2D3 to examine the mechanism of the direct effect of the secosteroid. Matrix vesicles were incubated with 1α,25(OH)2D3 ± inhibitors of signal transduction pathways shown previously to mediate the activation of PKCα in a number of experimental systems. For these experiments, membrane fractions were incubated with the following:U73122 to inhibit PI-PLC activity; cholera toxin, pertussis toxin, and GDPβS to inhibit G-proteins; and wortmannin to inhibit phospholipase D (PLD). In addition, we examined the regulation of matrix vesicle PKC by agents shown previously to stimulate PKCα activity in growth zone cells. Matrix vesicles were treated directly with arachidonic acid, which is the product of PLA2 action, the arachidonic acid precursor, linolenic acid, and the arachidonic acid metabolite PGE2 as well as with diacylglycerol, the product of PLC action. The role of the 1,25-mVDR in the response of matrix vesicle PKC to 1α,25(OH)2D3 was assessed using Ab99. Specificity of the response was established using 1β,25(OH)2D3 and 24R,25(OH)2D3. The role of annexin II was assessed using antibodies to the C-terminal and N-terminal regions of the protein. Because matrix vesicles do not contain DNA or RNA, any response to the addition of 1α,25(OH)2D3 by naive membranes would a priori be via nongenomic mechanisms. This would not rule out a role for the 1,25-nVDR, however. Accordingly, we examined matrix vesicles for the presence of the 1,25-nVDR by Western blot. Monensin and PGE2 were purchased from Sigma. The following chemicals were purchased from Calbiochem(San Diego, CA): 1,2-dioctanoyl-sn-glycerol (DOG), arachidonic acid, linolenic acid, pertussis toxin (Giinhibitor), cholera toxin (Gs inhibitor), GDPβS (general G-protein inhibitor), and wortmannin (PLD inhibitor). 1α,25(OH)2D3 and 24R,25(OH)2D3 were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Recombinant nuclear vitamin D receptor (1,25-nVDR) was obtained from Affinity BioReagents, Inc., Golden, CO. Rabbit polyclonal anti-1,25-nVDR and alkaline phosphatase-conjugated anti-rabbit antibodies, as well as polyclonal rabbit antibodies specific for the α, β, δ, ε, and ζ isoforms of PKC, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Nonspecific rabbit IgG1 was obtained from Sigma. Mouse monoclonal antibody to the C terminus of annexin II was obtained from Transduction Laboratories (Lexington, KY), and rabbit polyclonal antibody to the N terminus of annexin II was obtained from Santa Cruz Biotechnology. PKC assay reagents and Dulbecco's modified Eagle's medium were obtained from Life Technologies, Inc. (Gaithersburg, MD). The protein content of each sample was determined using the bicinchoninic acid protein assay reagent (32.Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18578) Google Scholar) obtained from Pierce. 1β,25(OH)2D3 was a generous gift from Dr. Anthony Norman, University of California, Riverside, CA. The rat costochondral chondrocyte culture system used in this study has been described in detail previously (20.Boyan B.D. Schwartz Z. Swain L.D. Carnes Jr., D.L. Zislis T. Bone. 1988; 9: 185-194Crossref PubMed Scopus (176) Google Scholar). Cells from the growth zone (prehypertrophic and upper hypertrophic cell zones) of costochondral cartilage from 125-g male Sprague-Dawley rats (Harlan, Indianapolis, IN) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, vitamin C, and antibiotics. Fourth passage cells were used for all experiments. The characteristics of these cells have been described in a number of publications and have been reviewed (14.Boyan, B. D., Sylvia, V. L., Dean, D. D., Del Toro, F., and Schwartz, Z. (2002) Crit. Rev. Oral Biol. Med., in pressGoogle Scholar, 22.Boyan B.D. Dean D.D. Sylvia V.L. Schwartz Z. Feldman D. Glorieux F.H. Pike J.W. Vitamin D. Academic Press, San Diego, CA1997: 395-421Google Scholar). Matrix vesicles were prepared by differential centrifugation of trypsin digests of the extracellular matrix as previously described (33.Boyan B.D. Schwartz Z. Carnes Jr., D.L. Ramirez V. Endocrinology. 1988; 122: 2851-2860Crossref PubMed Scopus (143) Google Scholar). In addition, plasma membranes were prepared by differential and sucrose gradient centrifugation of cells isolated from the same cultures for comparison. PKC activity was determined using previously described methods (19.Sylvia V.L. Schwartz Z. Ellis E.B. Helm S.H. Gomez R. Dean D.D. Boyan B.D. J. Cell. Physiol. 1996; 167: 380-393Crossref PubMed Scopus (92) Google Scholar, 23.Sylvia V.L. Schwartz Z. Schuman L. Morgan R.T. Mackey S. Gomez R. Boyan B.D. J. Cell. Physiol. 1993; 157: 271-278Crossref PubMed Scopus (99) Google Scholar). To determine total PKC specific activity in each culture, cell layer lysates were used. To determine PKC specific activity of isolated matrix vesicles or plasma membranes, 10 μg of membrane protein were diluted to a final volume of 35 μl and assayed as described for the cell layer lysates. For experiments examining the direct effect of hormones and inhibitors on matrix vesicles and plasma membranes, the protein concentration was adjusted with 0.9% NaCl such that 10 μg of membrane protein were incubated with the vitamin D3 metabolites in a final volume of 50 μl. Following incubation for the times shown below, 35 μl was removed and assayed using the same conditions as for cell layer lysates. To test the hypothesis that 1α,25(OH)2D3modulates matrix vesicle PKC activity through production of new matrix vesicles and incorporation of PKC, confluent fourth passage cultures of growth zone chondrocytes were incubated with 10−9 or 10−8m 1α,25(OH)2D3for 0.2, 1.5, 12, and 24 h. At each time, plasma membranes and matrix vesicles were isolated from the cultures, and PKC specific activity was assayed. To determine whether the organelle-specific effect of 1α,25(OH)2D3 is due to a change in the differential distribution of PKC isoforms during matrix vesicle biogenesis, isoform-specific antibodies were used. Growth zone chondrocytes were treated with 10−8m1α,25(OH)2D3 for 90 min or 24 h, and matrix vesicles and plasma membranes were isolated. Membranes were depleted of individual isoforms by immunoprecipitation, and the supernatant was assayed for remaining PKC activity (19.Sylvia V.L. Schwartz Z. Ellis E.B. Helm S.H. Gomez R. Dean D.D. Boyan B.D. J. Cell. Physiol. 1996; 167: 380-393Crossref PubMed Scopus (92) Google Scholar). Membrane preparations (10 μg of protein/sample) were incubated on ice for 1 h with 6 μl of a 1:10 dilution of nonspecific rabbit IgG1 or isoform-specific anti-PKC rabbit IgG1 in 0.9% saline, resulting in a final antibody dilution of 1:500. Protein G-agarose (10 μl) (Oncogene Science, Inc., Uniondale, NY) was added for 4 h to clear the samples of immunoreactive PKC isoforms and any remaining unbound antibody. Following precipitation of this material, 35 μl of the supernatant was assayed for PKC activity. To determine whether protein transport through the Golgi apparatus is necessary for the PKC activity in matrix vesicles, growth zone chondrocyte cultures were treated for 24 h with 1–100 μm monensin (34.Tartakoff A.M. Vassalli P. J. Exp. Med. 1978; 146: 1332-1345Crossref Scopus (235) Google Scholar). PKC activity was determined in matrix vesicles and plasma membranes as described above. To determine whether the long term effect of 1α,25(OH)2D3 on PKC is regulated, at least in part, through activation of the 1,25-mVDR, growth zone cells were incubated for 24 h with 10−8m 1α,25(OH)2D3 in the presence and absence of Ab99 at a final dilution of 1:500. Ab99 (provided as a generous gift by Dr. Ilka Nemere, Utah State University, Logan, UT) was generated to a synthetic peptide corresponding to the N-terminal 20 amino acids of the 1α,25(OH)2D3-binding protein isolated from the basal lateral membranes of chick intestinal epithelium (35.Nemere I. Ray R. Jia Z. J. Bone Miner. Res. 1996; 11: S312PubMed Google Scholar, 36.Nemere I. Ray R. McManus W. Am. J. Physiol. 2000; 278: E1104-E1114Crossref PubMed Google Scholar). It blocks the rapid effect of 1α,25(OH)2D3 on PKC activity of growth zone chondrocyte cultures as well as the direct effect of 1α,25(OH)2D3 on isolated plasma membranes and matrix vesicles (16.Nemere I. Schwartz Z. Pedrozo H. Sylvia V.L. Dean D.D. Boyan B.D. J. Bone Miner. Res. 1998; 13: 1353-1359Crossref PubMed Scopus (187) Google Scholar). Ab99 also blocks many of the physiological responses of growth zone cells to 1α,25(OH)2D3 (17.Pedrozo H.A. Schwartz Z. Rimes S. Sylvia V.L. Nemere I. Posner G.H. Dean D.D. Boyan B.D. J. Bone Miner. Res. 1999; 14: 856-867Crossref PubMed Scopus (89) Google Scholar). Specificity of the effect was shown using the stereoisomer of 1α,25(OH)2D3, 1β,25(OH)2D3 (provided as a generous gift by Dr. Anthony Norman, University of California, Riverside, CA). Although 1β,25(OH)2D3 blocks 1α,25(OH)2D3-dependent transcaltachia in chick intestine (37.Norman A.W. Nemere I. Muralidharan K.R. Okamura W.H. Biochem. Biophys. Res. Comm. 1992; 189: 1450-1456Crossref PubMed Scopus (54) Google Scholar), it does not affect the rapid increase in PKC due to 1α,25(OH)2D3 in rat growth zone chondrocyte cultures (18.Sylvia V.L. Del Toro Jr., F. Hardin R.R. Dean D.D. Boyan B.D. Schwartz Z. J. Steroid Biochem. Mol. Biol. 2001; 78: 261-274Crossref PubMed Scopus (45) Google Scholar). Matrix vesicles were isolated from growth zone chondrocyte cultures that had been treated for 24 h with 10−8m1β,25(OH)2D3 ± Ab99. PKC activity was measured as described above. Specificity was also examined using 24R,25(OH)2D3, a metabolite of vitamin D that does not elicit a rapid increase in PKC in growth zone chondrocyte cultures (23.Sylvia V.L. Schwartz Z. Schuman L. Morgan R.T. Mackey S. Gomez R. Boyan B.D. J. Cell. Physiol. 1993; 157: 271-278Crossref PubMed Scopus (99) Google Scholar), nor does it affect PKC when incubated with matrix vesicles or plasma membranes isolated from growth zone chondrocyte cultures (19.Sylvia V.L. Schwartz Z. Ellis E.B. Helm S.H. Gomez R. Dean D.D. Boyan B.D. J. Cell. Physiol. 1996; 167: 380-393Crossref PubMed Scopus (92) Google Scholar). For these studies, growth zone chondrocytes were treated with 10−7m24R,25(OH)2D3 ± Ab99 for 24 h. Matrix vesicles were isolated, and PKC activity was determined. To determine whether signaling pathways that mediat
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