Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M110398200 on January 22, 2002

J. Biol. Chem., Vol. 277, Issue 14, 11828-11837, April 5, 2002
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/14/11828    most recent
M110398200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwartz, Z.
Right arrow Articles by Boyan, B. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwartz, Z.
Right arrow Articles by Boyan, B. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

1alpha ,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*

Zvi SchwartzDagger §, Victor L. Sylvia§, Dennis Larsson§||, Ilka Nemere||, David Casasola§, David D. Dean§, and Barbara D. Boyan§

From the Dagger  Hebrew University, Jerusalem 91120, Israel, § The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229-3900, and || Utah State University, Logan, Utah 84322

Received for publication, October 29, 2001, and in revised form, January 9, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Matrix vesicles are extracellular organelles involved in mineral formation that are regulated by 1alpha ,25(OH)2D3. Prior studies have shown that protein kinase C (PKC) activity is involved in mediating the effects of 1alpha ,25(OH)2D3 in both matrix vesicles and plasma membranes. Here, we examined the regulation of matrix vesicle PKC by 1alpha ,25(OH)2D3 during biogenesis and after deposition in the matrix. When growth zone costochondral chondrocytes were treated for 9 min with 1alpha ,25(OH)2D3, PKCzeta in matrix vesicles was inhibited, while PKCalpha in plasma membranes was increased. In contrast, after treatment for 12 or 24 h, PKCzeta in matrix vesicles was increased, while PKCalpha in plasma membranes was unchanged. The effect of 1alpha ,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 1alpha ,25(OH)2D3 membrane vitamin D receptor (1,25-mVDR) was involved, since a specific antibody blocked the 1alpha ,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 1alpha ,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 1alpha ,25(OH)2D3. The rapid effect of 1alpha ,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 1alpha ,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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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, 2). The physiological importance of this is not yet well understood. 1alpha ,25(OH)2D3 exerts direct effects on matrix vesicles isolated from the extracellular matrix of growth plate chondrocytes (3), 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 1alpha ,25(OH)2D3 causes increased alkaline phosphatase specific activity, which is associated with the onset of calcification (4). In addition, phospholipase A2 (PLA2)1 specific activity is increased (5), which may lead to a loss of membrane integrity and the release of proteinases capable of remodeling the matrix (6, 7). One of the matrix metalloproteinases that are present in matrix vesicles, stromelysin-1 (MMP-3), has been shown to activate latent transforming growth factor beta -1 (TGF-beta 1) in a 1alpha ,25(OH)2D3-dependent manner (8).

The mechanisms involved in the regulation of matrix vesicles by 1alpha ,25(OH)2D3 are not known. 1alpha ,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 1alpha ,25(OH)2D3 on the organelles.

Recent studies show that 1alpha ,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 (9-13). Many of the physiological responses of the chondrocytes to 1alpha ,25(OH)2D3 are blocked by inhibiting PKC (14). 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) (15-17). The rapid effects of 1,25(OH)2D3 are stereospecific; only the 1alpha ,25(OH)2D3 isomer elicits an increase in PKC or regulates the signaling pathways that lead to the increase in PKC (18), indicating a 1,25(OH)2D3 membrane receptor (1,25-mVDR)-mediated mechanism is involved.

The hypothesis that 1alpha ,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). Moreover, the effect of 1alpha ,25(OH)2D3 on PKC is blocked by Ab99, just as it is in the cell. However, 1alpha ,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), even though the same receptor is involved.

The purpose of the present study was to examine the mechanisms that regulate 1alpha ,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. PKCzeta predominates in matrix vesicles, whereas PKCalpha predominates in plasma membranes (19). The two membrane fractions differ in other ways as well, including phospholipid composition (20) and basal membrane fluidity (3).

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 PKCalpha , whereas the responsive isoform in matrix vesicles is PKCzeta . 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 1alpha ,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, 21). Moreover, matrix vesicle composition, including phospholipids and enzyme activities, and regulation of matrix vesicle function differs between the two cell types (see Refs. 14, 22 for reviews).

It is likely that 1alpha ,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 1alpha ,25(OH)2D3 for 24 h, long enough for new gene expression and matrix vesicle synthesis, matrix vesicle PKC activity is increased (23). While 1alpha ,25(OH)2D3 has been shown to regulate the distribution of matrix proteinases in matrix vesicles (24), it is not known if the increase in matrix vesicle PKC is due to preferential incorporation of specific isoforms of the enzyme. PKCalpha is sensitive to Ca2+ ions and to phospholipid, whereas PKCzeta is insensitive to both co-factors (25), yet both Ca2+ ions and phospholipid are present at relatively high levels in the growth plate extracellular matrix (26), particularly in the growth zone. Thus, it is possible that other isoforms may be involved in the matrix vesicle response to 1alpha ,25(OH)2D3. For example, in renal epithelial cells, 1alpha ,25(OH)2D3 has been shown to increase PKCbeta activity (27). Other aspects of the signaling pathway by which 1alpha ,25(OH)2D3 modulates matrix vesicle PKC may differ as well. G-protein, specifically Gq but not Gi or Gs, mediates the effect of 1alpha ,25(OH)2D3 on cellular PKC (21); whether this is the case for matrix vesicle PKC is unknown.

Phospholipid metabolism plays a major role in the mechanism of 1alpha ,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). 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). Their phospholipid composition is distinct from that of the plasma membrane as well (29-31). 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). Thus, it is likely that phospholipid metabolism may play a different role in the mechanism by which 1alpha ,25(OH)2D3 modulates PKC activity in the extracellular organelle.

This study tested the hypothesis that 1alpha ,25(OH)2D3 regulates matrix vesicle PKC activity in multiple ways. The vitamin D metabolite first increases the amount of PKCzeta incorporated during matrix vesicle biogenesis though genomic mechanisms. Once the matrix vesicles are released into the matrix, 1alpha ,25(OH)2D3 acts directly on the matrix vesicle via the 1,25-mVDR, reducing PKCzeta 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 1alpha ,25(OH)2D3 on the plasma membrane also modulate PKC activity in the extracellular organelle.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Experimental Design-- We used two experimental models to examine the regulation of matrix vesicle PKC by 1alpha ,25(OH)2D3. In the first set of experiments, we tested the hypothesis that 1alpha ,25(OH)2D3 regulates the differential distribution of PKC isoforms during matrix vesicle biogenesis. Rat costochondral growth zone cartilage cells were treated with 1alpha ,25(OH)2D3 for up to 24 h. Matrix vesicles and plasma membranes were then isolated from the cultures. The 1alpha ,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 1alpha ,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), 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 1alpha ,25(OH)2D3 on 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 1alpha ,25(OH)2D3 in the presence of the phosphatidylinositol-specific (PI-PLC) inhibitor U73122.

The second model used matrix vesicles isolated from cultures not previously treated with 1alpha ,25(OH)2D3 to examine the mechanism of the direct effect of the secosteroid. Matrix vesicles were incubated with 1alpha ,25(OH)2D3 ± inhibitors of signal transduction pathways shown previously to mediate the activation of PKCalpha 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 GDPbeta S 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 PKCalpha 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 1alpha ,25(OH)2D3 was assessed using Ab99. Specificity of the response was established using 1beta ,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 1alpha ,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.

Reagents-- 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 (Gi inhibitor), cholera toxin (Gs inhibitor), GDPbeta S (general G-protein inhibitor), and wortmannin (PLD inhibitor). 1alpha ,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 alpha , beta , delta , epsilon , and zeta  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) obtained from Pierce. 1beta ,25(OH)2D3 was a generous gift from Dr. Anthony Norman, University of California, Riverside, CA.

Chondrocyte Cultures-- The rat costochondral chondrocyte culture system used in this study has been described in detail previously (20). 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, 22).

Membrane Isolation-- Matrix vesicles were prepared by differential centrifugation of trypsin digests of the extracellular matrix as previously described (33). In addition, plasma membranes were prepared by differential and sucrose gradient centrifugation of cells isolated from the same cultures for comparison.

Protein Kinase C-- PKC activity was determined using previously described methods (19, 23). 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 1alpha ,25(OH)2D3 modulates 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 10-8 M 1alpha ,25(OH)2D3 for 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 1alpha ,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 10-8 M 1alpha ,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). 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). PKC activity was determined in matrix vesicles and plasma membranes as described above. To determine whether the long term effect of 1alpha ,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 10-8 M 1alpha ,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 1alpha ,25(OH)2D3-binding protein isolated from the basal lateral membranes of chick intestinal epithelium (35, 36). It blocks the rapid effect of 1alpha ,25(OH)2D3 on PKC activity of growth zone chondrocyte cultures as well as the direct effect of 1alpha ,25(OH)2D3 on isolated plasma membranes and matrix vesicles (16). Ab99 also blocks many of the physiological responses of growth zone cells to 1alpha ,25(OH)2D3 (17).

Specificity of the effect was shown using the stereoisomer of 1alpha ,25(OH)2D3, 1beta ,25(OH)2D3 (provided as a generous gift by Dr. Anthony Norman, University of California, Riverside, CA). Although 1beta ,25(OH)2D3 blocks 1alpha ,25(OH)2D3-dependent transcaltachia in chick intestine (37), it does not affect the rapid increase in PKC due to 1alpha ,25(OH)2D3 in rat growth zone chondrocyte cultures (18). Matrix vesicles were isolated from growth zone chondrocyte cultures that had been treated for 24 h with 10-8 M 1beta ,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), nor does it affect PKC when incubated with matrix vesicles or plasma membranes isolated from growth zone chondrocyte cultures (19). For these studies, growth zone chondrocytes were treated with 10-7 M 24R,25(OH)2D3 ± Ab99 for 24 h. Matrix vesicles were isolated, and PKC activity was determined.

To determine whether signaling pathways that mediate the rapid action of 1alpha ,25(OH)2D3 on PKC in growth zone chondrocytes also mediate the downstream increase in matrix vesicle PKC, we examined the role of PI-PLC. Cultures were treated with 10-8 M 1alpha ,25(OH)2D3 ± the inhibitor U73122 (0, 0.1, 1, or 10 µM). Matrix vesicles were isolated, and PKC activity was determined.

Direct Action of 1alpha ,25(OH)2D3 on Matrix Vesicle PKC-- To determine whether the direct effect of 1alpha ,25(OH)2D3 on PKC activity in matrix vesicles is also mediated by PI-PLC, matrix vesicles were incubated with 10-8 M 1alpha ,25(OH)2D3 alone, 10 µM U73122 alone, or the two in combination for 3, 9, 30, or 90 min, and PKC activity was determined. In addition, we examined the direct effect of diacylglycerol (DAG) on matrix vesicle PKC. DAG is the product of PLC action; it is an activator of PKC in growth zone cells and mediates the rapid increase in PKC activity in response to 1alpha ,25(OH)2D3 (38). Matrix vesicles were incubated with 1, 10, or 100 µM DOG for 9 min in the presence and absence of 10-8 M 1alpha ,25(OH)2D3, and PKC activity was measured.

DAG can also be produced through the action of PLD on phosphatidylcholine (39). To determine whether PLD is involved in regulation of matrix vesicle PKC, matrix vesicles were incubated for 9 min with 1alpha ,25(OH)2D3 ± 0.1, 1, or 10 µM wortmannin, a specific inhibitor of PLD activity (40, 41). Wortmannin at higher concentrations can also inhibit PI 3-kinase (42); however, PI 3-kinase is not affected at the concentrations used in the present study (43).

To determine whether 1alpha ,25(OH)2D3 exerts its direct effects on matrix vesicle PKC through a G-protein-dependent mechanism, matrix vesicles were incubated for 9 min with 10-8 M 1alpha ,25(OH)2D3 in the presence of the general G-protein inhibitor GDPbeta S (1, 10, or 100 µM), cholera toxin to inhibit Gs (1,10, or 100 µM), or pertussis toxin to inhibit Gi (1, 10, or 100 µm).

The rapid effect of 1alpha ,25(OH)2D3 on PKC in growth zone chondrocytes is mediated by the action of arachidonic acid (44). In addition, linolenic acid, which is the precursor of arachidonic acid, also exerts a stimulatory effect on PKC activity in the cells, as does the arachidonic acid metabolite PGE2 (45). To determine whether one or more of these lipid mediators exerts an effect on matrix vesicle PKC, matrix vesicles were incubated with 1, 10, or 100 µM arachidonic acid, 1, 10, or 100 µM linolenic acid, or 0.015, 0.06, or 0.24 ng/ml PGE2 in the presence and absence of 10-8 M 1alpha ,25(OH)2D3 for 9, 90, or 270 min before assaying PKC activity.

To determine whether the 1,25-mVDR mediates the direct effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC, matrix vesicles from growth zone chondrocyte cultures were treated with 10-10 to 10-8 M 1alpha ,25(OH)2D3 for 9 min with a 1:500 final dilution of 1,25-mVDR-specific antibody Ab99 or with rabbit IgG1. Matrix vesicles isolated from resting zone cell cultures were incubated with 10-9 to 10-7 M 24R,25(OH)2D3 for 90 min in the presence and absence of Ab99 as a control.

We also determined if the direct effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC involves annexin II, which has been purported to be a receptor for this hormone in osteoblasts (46). For these experiments, matrix vesicles isolated from growth zone chondrocyte cultures were treated with various dilutions of antibodies to the C terminus or N terminus of annexin II in the presence and absence of 10-8 M 1alpha ,25(OH)2D3 for 9 min. PKC specific activity was then assayed as described above.

1,25-nVDR-- To determine whether the traditional 1,25-nVDR is present in matrix vesicles or plasma membranes, we examined matrix vesicles and plasma membranes from confluent fourth passage growth zone cell cultures by Western blot analysis after SDS-PAGE. In addition, we examined the cytosol/nuclear fraction obtained during plasma membrane purification. Isolated membranes and the cytosol/nuclear fraction were solubilized in sample buffer, and 20 µg of protein was separated on 7.5% gradient acrylamide gels (47). Blots of the gels were probed with anti-1,25-nVDR antibody (1:5000 dilution, Santa Cruz Biotechnology) or nonspecific IgG. Immunoreactive bands were visualized by an alkaline phosphatase-conjugated polyclonal anti-rabbit secondary antibody.

Statistical Management of Data-- For each experiment, each data point represents the means ± S.E. for six individual cultures. Each experiment was repeated two or more times to ensure the validity of the data. The data presented are from a single representative experiment. Significance between groups was determined by analysis of variance and post hoc testing performed using Bonferroni's modification of Student's t test for multiple comparisons. p values less than 0.05 were considered significant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1alpha ,25(OH)2D3 regulated matrix vesicle PKC in growth zone chondrocyte cultures in a dose- and time-dependent manner. At 0.2 and 1.5 h, 1alpha ,25(OH)2D3 caused a dose-dependent decrease in matrix vesicle PKC specific activity (Fig. 1A). However, at 12 and 24 h, 1alpha ,25(OH)2D3 dose dependently increased PKC activity in these organelles by 100% over control levels. In con-trast, 1alpha ,25(OH)2D3 affected plasma membrane PKC only at the early time points, causing a concentration-dependent increase in enzyme activity within 0.2 h (Fig. 1B). Activity remained elevated at 1.5 h in cultures treated with 10-8 M 1alpha ,25(OH)2D3, but by 12 h, specific activity had returned to control levels.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 1.   PKC specific activity of matrix vesicles (A) and plasma membranes (B) isolated from growth zone chondrocyte cultures that were treated with 1alpha ,25(OH)2D3 for varying periods of time. All values are the means ± S.E. for membranes from six independent cultures. Data are from one of two separate experiments, both with comparable results. *, p < 0.05, versus control; #, p < 0.05, 10-9 M versus 10-8 M 1alpha ,25(OH)2D3.

The predominant PKC isoform present in matrix vesicles was PKCzeta . In control cultures at 90 min, anti-PKCzeta antibody reduced activity by ~80%, whereas none of the other isoform-specific antibodies reduced activity (Fig. 2A). In cultures treated with 1alpha ,25(OH)2D3 for 90 min, the anti-PKCzeta antibody further reduced PKC specific activity to less than 10% of levels observed in cultures treated with IgG1, indicating that PKCzeta was the 1alpha ,25(OH)2D3-sensitive isoform. At 24 h, the 1alpha ,25(OH)2D3-dependent increase in PKC activity was also due to PKCzeta (Fig. 2B). The anti-PKCzeta antibody reduced 1alpha ,25(OH)2D3-stimulated PKC activity to below baseline levels, although levels still remained higher than in control cultures following precipitation of PKCzeta with the isoform-specific antibody.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of PKC isoform-specific antibodies on PKC specific activity in matrix vesicles isolated from growth zone chondrocyte cultures that were treated with 1alpha ,25(OH)2D3 for 90 min (A) or 24 h (B). Values are the means ± S.E. for matrix vesicle membranes isolated from six independent cultures. Data are from one of two separate experiments, both with comparable results. *, p < 0.05, versus control or preimmune IgG; #, p < 0.05, control versus 1alpha ,25(OH)2D3.

Regardless of the time point examined, the predominant PKC isoform present in plasma membranes was PKCalpha based on the reduction in enzyme activity following incubation with the anti-PKCalpha antibody (Fig. 3). None of the other isoform-specific antibodies used caused a decrease in control cells. PKCalpha was also the isoform sensitive to 1alpha ,25(OH)2D3, based on the 70% reduction in total PKC specific activity in plasma membranes isolated from growth zone cells treated with the secosteroid for 90 min. However, the anti-PKCalpha antibodies did not completely reduce PKC activity to levels seen in control cells.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of PKC isoform-specific antibodies on PKC specific activity in plasma membranes isolated from growth zone chondrocyte cultures that were treated with 1alpha ,25(OH)2D3 for 90 min (A) or 24 h (B). Values are the means ± S.E. for plasma membranes isolated from six independent cultures. Data are from one of two separate experiments, both with comparable results. *, p < 0.05, versus control or preimmune IgG; #, p < 0.05, control versus 1alpha ,25(OH)2D3.

Incorporation of PKC activity in matrix vesicles was dependent upon protein transport through the Golgi. Treatment of the cultures for 24 h with monensin resulted in a dose-dependent decrease in matrix vesicle PKC specific activity (Fig. 4). In contrast, PKC activity of the plasma membranes was unaffected by treatment of the cultures with monensin.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of monensin on incorporation of PKC activity into newly synthesized membranes. Growth zone chondrocyte cultures were treated with monensin for 24 h, the matrix vesicles (MV) and plasma membranes (PM) were isolated, and PKC activity was determined. Values are the means ± S.E. for membranes from six independent cultures. Data are from one of two separate experiments, both with comparable results. *, p < 0.05, versus control; #, p < 0.05, PM versus MV.

The stimulatory effect of 1,25(OH)2D3 on matrix vesicle PKC was receptor-mediated. Not only was it stereospecific, but it was metabolite-specific. Neither 1beta ,25(OH)2D3 nor 24R,25(OH)2D3 elicited the response (Fig. 5A). Ab99 blocked the increase in matrix vesicle PKC due to 1alpha ,25(OH)2D3, but had no effect on PKC in control cultures or in cultures treated with 1beta ,25(OH)2D3 or 24R,25(OH)2D3, indicating that the 1,25-mVDR was involved.


View larger version (19K):
[in this window]
[in a new window]
  		Fig. 5.   Stereospecific effect of vitamin D3 metabolites and effect of PLC inhibitor on PKC activity in matrix vesicles. A, growth zone chondrocyte cultures were treated for 24 h with 10-8 M 1alpha ,25(OH)2D3 or 1beta ,25(OH)2D3 or 10-7 M 24R,25(OH)2D3 in the presence/absence of Ab99. B, growth zone chondrocyte cultures were treated for 24 h with 10-8 M 1alpha ,25(OH)2D3 in the presence/absence of the PLC inhibitor U73122. At harvest, the matrix vesicles were isolated and then assayed for PKC specific activity. Values are the means ± S.E. for six independent cultures. Data are from one of two separate experiments, both with comparable results. *, p < 0.05, versus control; #, p < 0.05, +Ab99 versus -Ab99 (A) or 1alpha ,25(OH)2D3 versus control (B).

Part of the long-term effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC involves a PI-PLC-dependent mechanism. U73122 caused a dose-dependent decrease in matrix vesicle PKC activity (Fig. 5B). At the highest concentration of the PI-PLC inhibitor, there was a 30% reduction in the 1alpha ,25(OH)2D3-stimulated PKC activity.

The direct effect of 1alpha ,25(OH)2D3 on PKC in isolated matrix vesicles was also mediated by the 1,25-mVDR. When matrix vesicles were incubated with 1alpha ,25(OH)2D3, Ab99 prevented the dose-dependent decrease in PKC activity, even at the highest concentration of the secosteroid (Fig. 6A). Ab99 did not alter the 24R,25(OH)2D3-dependent decrease in PKC observed in matrix vesicles isolated from resting zone cell cultures (data not shown), indicating that the effect of the antibody was specific to the 1,25-mVDR and not due to a nonspecific interaction with the matrix vesicle or PKCzeta . Moreover, Ab99 caused a dose-dependent reversal of the direct effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC, but increasing concentrations of Ab99 did not affect PKC activity of control matrix vesicles (Table I). Furthermore, the direct effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC was not via annexin II. Antibodies to either the C or N terminus of the protein had no effect on 1alpha ,25(OH)2D3-dependent PKC inhibition, nor did they affect PKC activity of control matrix vesicles (Table I).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 6.   Role of membrane-mediated signaling mechanisms in the 1alpha ,25(OH)2D3-dependent decrease in matrix vesicle PKC. A, role of 1,25-mVDR. Matrix vesicles were treated for 9 min with 1alpha ,25(OH)2D3 in the presence/absence of Ab99. B, role of PLC. Matrix vesicles were treated with PI-PLC inhibitor (U73122; 10 µM), 1alpha ,25(OH)2D3 (10-8 M), or the two together for 3, 9, 30, or 90 min. C, role of G-protein. Matrix vesicles were treated for 9 min with GDPbeta S in the presence or absence of 10-8 M 1alpha ,25(OH)2D3. Values are the means ± S.E. for six separate membrane preparations, each from an individual culture. Data are from one of two independent experiments, both with comparable results. *, p < 0.05, versus untreated control (A); versus 3 min (B); or versus no G-protein inhibitor (C); #, p < 0.05, Ab99 versus control (A); versus control or U73122 for a particular time (B); or versus 1alpha ,25(OH)2D3 (C).

                              
View this table:
[in this window]
[in a new window]
 
Table I
Effect of antibodies to the C and N termini of annexin II and antibody Ab99 to the 1,25-mVDR on 1alpha ,25(OH)2D3-dependent PKC activity of matrix vesicles isolated from cultures of growth zone chondrocytes
Data represent the mean ± S.E. of six samples, where each sample represents the matrix vesicle membranes derived from one T-150 flask of confluent, fourth passage growth zone cells. Data are from one of two independent experiments, both of which had comparable results. Matrix vesicles were incubated directly with nonspecific IgG or antiserum, 1alpha ,25(OH)2D3 (1,25), or 1alpha ,25(OH)2D3 + antibody. After nine mins, PKC specific activity was determined.

PI-PLC was not involved in the 1alpha ,25(OH)2D3-dependent decrease in matrix vesicle PKC since U73122 did not alter the inhibitory effect of the secosteroid (Fig. 6B). In addition, PKC in control matrix vesicles was not sensitive to PLC inhibition by U73122. Moreover, matrix vesicle PKC activity was insensitive to DOG, whether the organelles were treated with 1alpha ,25(OH)2D3 or not (data not shown), indicating that diacylglycerol was not involved. The direct effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC was also not due to the action of PLD. Wortmannin did not alter enzyme activity in control matrix vesicles, nor did it reverse the inhibition of PKC due to 1alpha ,25(OH)2D3 (data not shown).

1alpha ,25(OH)2D3 mediates its direct effect on matrix vesicle PKC via a G-protein-dependent mechanism. Treatment of matrix vesicles with the general G-protein inhibitor GDPbeta S resulted in an increase in PKC specific activity of control matrix vesicles and a reversal of the 1alpha ,25(OH)2D3-dependent decrease in PKC (Fig. 6C). The G-protein responsible is neither Gi nor Gs, since cholera toxin and pertussis toxin had no effect on PKC activity of control or 1alpha ,25(OH)2D3-treated matrix vesicles (data not shown).

Matrix vesicle PKC was directly regulated by linolenic acid. Linolenic acid caused a rapid, dose-dependent increase in matrix vesicle PKC, whereas this fatty acid did not alter enzyme activity of isolated plasma membranes (Fig. 7A). The increase in matrix vesicle PKC was time-dependent (Fig. 7B). By 90 min, the direct effect on matrix vesicles was reduced by 30%. Even at 270 min, however, linolenic still exerted a stimulatory effect on matrix vesicle PKC compared with control matrix vesicles.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 7.   Direct effect of linolenic acid on PKC activity in isolated matrix vesicles and plasma membranes. Matrix vesicles (MV) and plasma membranes (PM) from growth zone chondrocyte cultures were treated with varying doses of linolenic acid for 9 min (A). Alternatively, matrix vesicles were treated with 10 µM linolenic acid for 9, 90, or 270 min (B). Values are the means ± S.E. for membranes isolated from six independent cultures. Data are from one of two independent experiments, both with comparable results. *, p < 0.05, versus no linolenic acid (A) or 9 min (B); #, p < 0.05, MV versus PM (A) or control versus linolenic acid (B); , p < 0.05, versus 1 µM linolenic acid (A).

Arachidonic acid also directly regulated matrix vesicle PKC activity. In both control matrix vesicles and in matrix vesicles treated directly with 1alpha ,25(OH)2D3, arachidonic acid caused a rapid increase in PKC (Fig. 8A). The effect of arachidonic acid was time-dependent (Fig. 8B). Maximal increases in PKC were found in matrix vesicles incubated with arachidonic acid for 90 min. Moreover, only at this time point was the 1alpha ,25(OH)2D3-dependent decrease in PKC restored to control levels by arachidonic acid. In contrast to the stimulatory effects of linolenic acid and arachidonic acid on PKC specific activity, the arachidonic acid metabolite PGE2 had no effect on enzyme activity in control matrix vesicles (data not shown). Similarly, PGE2 did not alter the inhibitory effect of 1alpha ,25(OH)2D3.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 8.   Direct effect of arachidonic acid on PKC activity in matrix vesicles. Matrix vesicles were treated with arachidonic acid for 9 min in the presence/absence of 10-8 M 1alpha ,25(OH)2D3 (1,25) (A). Alternatively, matrix vesicles were treated with arachidonic acid (100 µM), 1alpha ,25(OH)2D3 (10-8 M), or the two together for 9, 90, or 270 min (B). Values are the means ± S.E. for membranes isolated from six independent cultures. Data are from one of two independent experiments, both with comparable results. *, p < 0.05, versus untreated control (A) or versus 9 min (B); #, p < 0.05, +1alpha ,25(OH)2D3 versus -1alpha ,25(OH)2D3 (A) or versus untreated control for a particular treatment time (B).

The direct effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC was not due to the nuclear VDR. Matrix vesicles and plasma membranes isolated from growth zone chondrocyte cultures contained no detectable immunoreactive 1,25-nVDR (Fig. 9). The cytosol/nuclear fraction remaining after matrix vesicle and plasma membrane isolation did contain 1,25-nVDR immunoreactivity. Immunoreactive bands were seen at 63 and 297 kDa in the growth zone chondrocyte cytosol/nuclear fraction as compared with recombinant 1,25-nVDR, which was detected as a single band with a molecular mass of 63 kDa.


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 9.   Western blot analysis of nuclear vitamin D receptor (1,25-nVDR) in growth zone chondrocyte (GC) membrane fractions. Matrix vesicles (MV), plasma membranes (PM), and cytosol/nuclear fractions (Cyto+Nuc) were analyzed for the presence of the 1,25-nVDR. The 63-kDa band present in the cytosol/nuclear fraction was verified to be nVDR by comparison to recombinant human 1,25-nVDR (nVDR lanes).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results of this study indicate that 1alpha ,25(OH)2D3 exerts its effects on matrix vesicle PKC activity through both direct and indirect mechanisms. The initial effect of the secosteroid is to cause the rapid decrease in existing matrix vesicle PKC activity by down-regulating PKCzeta . It is likely that this is due to direct action of 1alpha ,25(OH)2D3 on the matrix vesicle membrane. At later time points, however, 1alpha ,25(OH)2D3 causes an increase in matrix vesicle PKCzeta activity, suggesting that PKC is regulated at the time of matrix vesicle formation through genomic mechanisms.

The fact that monensin-treated cultures exhibit reduced PKC activity in their matrix vesicles, but not in their plasma membranes, indicates that less PKC is transported and packaged within matrix vesicles. This observation suggests that matrix vesicles are formed within the Golgi, independently of the plasma membrane. Studies using somatic cell hybrids to examine the role of alkaline phosphatase in osteoblasts demonstrated that alkaline phosphatase is packaged in matrix vesicles even when expression of the plasma membrane enzyme is extinguished (48), supporting this hypothesis.

At least part of the downstream effect of 1alpha ,25(OH)2D3 on matrix vesicle PKC is mediated by the 1,25-mVDR. Treatment of the cultures for 24 h with Ab99 blocked the 1alpha ,25(OH)2D3-dependent increase in matrix vesicle PKC activity. The failure of either 1beta ,25(OH)2D3 or 24R,25(OH)2D3 to stimulate matrix vesicle PKC or for Ab99 to inhibit matrix vesicle PKC in cells treated with 1beta ,25(OH)2D3 or 24R,25(OH)2D3 demonstrates the specificity of the 1,25-mVDR-activated response. It is possible that the effect of Ab99 was directly on matrix vesicles already present in the extracellular matrix. However, it is more likely that it blocked the plasma membrane 1,25-mVDR since it inhibited the 1alpha ,25(OH)2D3-dependent increase in enzyme activity, which is cell-mediated.

Further evidence that the 1,25-mVDR is involved is the decrease in 1alpha ,25(OH)2D3-dependent matrix vesicle PKC noted in growth zone cells in the presence of U73122. This PI-PLC inhibitor also blocks the rapid effects of 1alpha ,25(OH)2D3 on PKC activity that are mediated by the 1,25-mVDR in growth zone cells (23). However, U73122 did not affect existing PKC activity in matrix vesicles incubated directly with the secosteroid, indicating that the changes in matrix vesicle PKC observed in cultures treated with 1alpha ,25(OH)2D3 for 24 h were not due to direct effects of the secosteroid on existing matrix vesicles. Instead, they were due to indirect effects involving 1,25-mVDR-dependent PKC synthesis and packaging in matrix vesicles through PLC-dependent signaling pathways, resulting in new PKC expression. Because the primary isoform active in matrix vesicles at 90 min and 24 h is PKCzeta , the data suggest that PI-PLC activity is necessary for the production of PKCzeta -enriched matrix vesicles rather than having a direct effect on existing matrix vesicle PKCzeta . This is consistent with the matrix vesicle PKC being PKCzeta because it is diacylglycerol-independent and would not require the production of diacylglycerol by PLC (25). Similarly, DOG, a potent PKC-activating form of diacylglycerol, was shown in this study to be ineffective at activating matrix vesicle PKC activity when matrix vesicles were treated directly with the compound.

It is likely that MAP kinase was involved in the 1alpha ,25(OH)2D3-dependent increase in matrix vesicle PKC activity. 1alpha ,25(OH)2D3 has been shown to mediate its effects on enterocytes (49) and osteoblasts (50) via MAP kinase signaling pathways, and the rapid increase in cellular PKC in response to 1alpha ,25(OH)2D3 can result in increased MAP kinase activity (51). Although 1alpha ,25(OH)2D3 mediates many of its physiological effects on growth zone chondrocytes through mechanisms that include the 1,25-mVDR-dependent signaling pathways (17), it is important to note that traditional 1,25-nVDR mechanisms are also likely to be involved in the packaging of matrix vesicle PKC in the present study. 1,25-nVDRs are present in the cells, as demonstrated by the presence of immunoreactive receptors in Western blots of the cytosol/nuclear fraction of the growth zone chondrocytes. The dependence of the downstream increase in matrix vesicle PKC activity on the 1,25-mVDR suggests that the action of 1alpha ,25(OH)2D3 on these two receptors is interrelated.

1alpha ,25(OH)2D3 also exerts its direct effects on matrix vesicle PKC through receptor-mediated mechanisms. The 1,25-mVDR is involved since Ab99 reversed the 1alpha ,25(OH)2D3-dependent decrease in PKC activity. The 1,25-nVDR does not appear to play a role in the direct action of 1alpha ,25(OH)2D3 on matrix vesicles, however. We failed to identify the 1,25-nVDR on Western blots of either the matrix vesicles or plasma membranes isolated from growth zone chondrocyte cultures, although it was present in the cytosol/nuclear fraction of these cells.

Annexin II has also been reported to be a membrane receptor for 1alpha ,25(OH)2D3 (46), and annexin II is present in matrix vesicles produced by chick growth plate chondrocytes (52) and osteoblast-like cells (53). It is unlikely that annexin II is responsible for the 1alpha ,25(OH)2D3-dependent effects on matrix vesicles, however. Antibodies to annexin II failed to block the direct inhibition of matrix vesicle PKC by 1alpha ,25(OH)2D3. This confirms our earlier observation using osteoblast cell lines (53). Ab99 did not cross react with annexin II in Western blots of matrix vesicles produced by osteoblast cultures, nor did antibodies to annexin II block the 1alpha ,25(OH)2D3-dependent increase in PKC in these cells.

The mechanism by which 1alpha ,25(OH)2D3 regulates matrix vesicle PKC are different from those that mediate the regulation of cellular PKC. PI-PLC is not involved, nor is diacylglycerol. PLD is also not involved, based on the lack of an effect of wortmannin either on PKC activity of control matrix vesicles or on PKC activity of 1alpha ,25(OH)2D3-treated matrix vesicles. In contrast, both cellular PKC and matrix vesicle PKC are regulated by G-protein-dependent mechanisms. As noted for intact cells (38), neither Gs nor Gi is involved, either in maintenance of PKC activity in control matrix vesicles or in the decrease noted in matrix vesicles treated with 1alpha ,25(OH)2D3. However, inhibition of G-protein activity with GDPbeta S blocks the effects of 1alpha ,25(OH)2D3. Thus, Gq is the likely candidate because it is not sensitive to pertussis toxin or cholera toxin (54). This is the case for intact cells as well (21).

1alpha ,25(OH)2D3 causes an increase in matrix vesicle phospholipase A2 activity (28), suggesting that release of arachidonic acid might be involved in the mechanism of its action on matrix vesicle PKC. However, the direct effect of 1alpha ,25(OH)2D3 is a decrease in PKC activity, and exogenous arachidonic acid elicits a rapid increase. Others have noted that arachidonic acid can stimulate PKC directly, particularly the PKCalpha isoform (55), explaining in part how this fatty acid can increase PKC activity in growth zone chondrocyte cultures (44). It is likely that arachidonic acid did not cause an increase in PKCzeta activity of the isolated matrix vesicles because PKCzeta is relatively insensitive to the fatty acid (56). It is more likely that arachidonic acid is exerting its stimulatory effect on the low levels of PKCalpha present in the organelles (19).

One way the chondrocytes might be able to activate matrix vesicle PKC activity after the matrix vesicles are formed and released into the extracellular matrix would be to secrete a regulatory factor that could act remotely on the organelles. We have shown that 1,25(OH)2D3 is produced by the cells, suggesting that it could serve this purpose. 1alpha ,25(OH)2D3 can also act back on the cell or matrix vesicle to stimulate the rapid release of arachidonic acid. We have previously reported that 1alpha ,25(OH)2D3 stimulates arachidonic acid turnover in growth zone chondrocytes (57) and increases PLA2 activity in the cells (5). Thus, a regulatory feedback loop can be established. 1alpha ,25(OH)2D3 decreases matrix vesicle PKCzeta , and arachidonic acid released by the action of 1alpha ,25(OH)2D3 increases PKCalpha activity.

If anything, the stimulatory effect of linolenic acid on matrix vesicle PKC activity is even more robust than the effect of its metabolite, arachidonic acid. Moreover, the effect of linolenic acid is specific to matrix vesicles; when plasma membranes were incubated directly with this fatty acid, no change in PKC activity was observed. Linolenic acid has been shown to be a potent activator of PKCzeta (56). The significance of this is unclear at this time.

In contrast to the stimulatory effects of arachidonic acid and its biosynthetic precursor linolenic acid, the arachidonic acid metabolite PGE2 had no effect on matrix vesicle PKC. In intact cultures, PGE2 mediates the stimulatory effects of 1alpha ,25(OH)2D3 on PKCalpha through its action on EP1 receptors (18). Whether EP1 is also present in matrix vesicles is not yet known, nor is it known if matrix vesicles contain cyclooxygenase activity.

Fig. 10 illustrates our current model of 1alpha ,25(OH)2D3 regulation of matrix vesicle PKC activity. 1alpha ,25(OH)2D3 binds to the 1,25-mVDR on the plasma membrane, triggering the production of matrix vesicles containing increased PKCzeta activity through genomic mechanisms mediated by PKC and MAP kinase as well as by the 1,25-nVDR. PLA2 activity is also increased, generating arachidonic acid, which acts on matrix vesicles in the extracellular matrix, increasing PKC activity. In addition, 1alpha ,25(OH)2D3 acts directly on matrix vesicles, activating the 1,25-mVDR and causing a rapid decrease in PKCzeta activity through a mechanism involving Gq. The combination of an increase in PKCalpha and a decrease in PKCzeta modulates activity of matrix metalloproteinases present in the matrix vesicles (58), leading to growth factor activation, matrix remodeling, maturation, calcification, and endochondral ossification (8, 24, 59).


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 10.   Proposed model of matrix vesicle regulation by 1alpha ,25(OH)2D3. 1alpha ,25(OH)2D3 activates the 1,25-mVDR, initiating a rapid increase in PKCalpha activity via PLC-dependent production of DAG and translocation of PKCalpha to the membrane. The ensuing phosphorylation cascade and subsequent binding of 1alpha ,25(OH)2D3 to the 1,25-nVDR results in new PKC expression and packaging into matrix vesicles. Matrix vesicles continue to mature in the matrix, eventually supporting the formation of calcium phosphate crystals on the inner leaflet of the membrane, eventual loss of membrane integrity, and exposure of matrix vesicle contents to the extravesicular environment. Arachidonic acid released by cellular phospholipase A2 stimulates matrix vesicle PKC, while 1alpha ,25(OH)2D3 secreted by the cell inhibits matrix vesicle PKCzeta . MV = matrix vesicles; AA = arachidonic acid.

In summary, these results show that the 1,25-mVDR is present in matrix vesicles from growth zone chondrocytes, whereas the 1,25-nVDR is not. 1alpha ,25(OH)2D3 can regulate matrix vesicle PKC activity both directly and indirectly. Both processes involve the 1,25-mVDR, but use different pathways to elicit their effects. 1alpha ,25(OH)2D3-dependent activation of the 1,25-mVDR in growth zone cells results in an increase in production of new matrix vesicles that are enriched in PKCzeta through a mechanism involving PI-PLC. In matrix vesicles, 1alpha ,25(OH)2D3 mediates its 1,25-mVDR-dependent effects on PKCzeta through a mechanism that involves Gq but not PI-PLC or PLD. Matrix vesicle PKC is also stimulated by arachidonic acid and linolenic acid, suggesting additional pathways for modulating the activities of the extracellular organelles. These findings demonstrate that matrix vesicles are unique organelles that can be differentially regulated at the time of their production and at sites distant from the cell surface, either through direct action of 1alpha ,25(OH)2D3 or by regulatory factors such as arachidonic acid. This demonstrates the complexity of the role of 1alpha ,25(OH)2D3 in chondrocyte biology and indicates that 1alpha ,25(OH)2D3 acts both through nuclear and membrane receptors to regulate the biomineralization processes.

    ACKNOWLEDGEMENTS

We acknowledge the contributions of Sandra Messier to the preparation of the manuscript and Ming-Jun Gu and Daaron Spears for technical assistance. We thank Dr. Ilka Nemere (Utah State University, Logan, UT) for the gift of Ab99 and Dr. Anthony Norman (University of California, Riverside, CA) for the gift of 1beta ,25(OH)2D3.

    FOOTNOTES

* This research was supported by United States Public Health Service Grants DE-08603 and DE-05937 and the Center for the Enhancement of the Biology/Biomaterials Interface at University of Texas Health Science Center at San Antonio.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Orthopaedics, MSC 7774, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229-3900. Tel.: 210-567-6326; Fax: 210-567-6295; E-mail: BoyanB@uthscsa.edu.

Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M110398200

    ABBREVIATIONS

The abbreviations used are: PLA2, phospholipase A2; PKC, protein kinase C; PLC, phospholipase C; PGE2, prostaglandin E2; PI, phosphatidylinositol; PLD, phospholipase D; mVDR, membrane vitamin D receptor; nVDR, nuclear vitamin D receptor; DOG, 1,2-dioctanoyl-sn-glycerol; DAG, diacylglycerol; MAP, mitogen-activated protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Schwartz, Z., Brooks, B. P., Swain, L. D., Del Toro, F., Norman, A. W., and Boyan, B. D. (1992) Endocrinology 130, 2495-2504[Abstract/Free Full Text]
2. Pedrozo, H. A., Boyan, B. D., Mazock, J., Dean, D. D., Gomez, R., and Schwartz, Z. (1999) Calcif. Tissue Int. 64, 50-56[CrossRef][Medline] [Order article via Infotrieve]
3. Swain, L. D., Schwartz, Z., Caulfield, K., Brooks, B. P., and Boyan, B. D. (1993) Bone 14, 609-617[Medline] [Order article via Infotrieve]
4. Schwartz, Z., Knight, G., Swain, L. D., and Boyan, B. D. (1988) J. Biol. Chem. 263, 6023-6026[Abstract/Free Full Text]
5. Schwartz, Z., and Boyan, B. D. (1988) Endocrinology 122, 2191-2198[Abstract/Free Full Text]
6. Dean, D. D., Schwartz, Z., Muniz, O. E., Gomez, R., Swain, L. D., Howell, D. S., and Boyan, B. D. (1992) Calcif. Tissue Int. 50, 342-349[CrossRef][Medline] [Order article via Infotrieve]
7. Einhorn, T. A., Hirschman, A., Kaplan, C., Nashed, R., Devlin, V. J., and Warman, J. (1989) J. Orthop. Res. 7, 792-805[CrossRef][Medline] [Order article via Infotrieve]
8. Maeda, S., Dean, D. D., Schwartz, Z., and Boyan, B. D. (2001) J. Bone Miner. Res. 16, 1281-1290[CrossRef][Medline] [Order article via Infotrieve]
9. Boyan, B. D., Sylvia, V. L., Dean, D. D., Pedrozo, H., Del Toro, F., Nemere, I., Posner, G. H., and Schwartz, Z. (1999) Steroids 64, 129-136[CrossRef][Medline] [Order article via Infotrieve]
10. de Boland, A. R., and Boland, R. L. (1994) Cell Signal. 6, 717-724[CrossRef][Medline] [Order article via Infotrieve]
11. Morelli, S., de Boland, A. R., and Boland, R. (1993) Biochem. J. 289, 675-679
12. Facchinetti, M. M., and de Boland, A. R. (1999) Cell Signal. 11, 39-44[CrossRef][Medline] [Order article via Infotrieve]
13. Facchinetti, M. M., Boland, R., and de Boland, A. R. (1998) J. Lipid Res. 39, 197-204[Abstract/Free Full Text]
14. Boyan, B. D., Sylvia, V. L., Dean, D. D., Del Toro, F., and Schwartz, Z. (2002) Crit. Rev. Oral Biol. Med., in press
15. Nemere, I., Dormanen, M. C., Hammond, M. W., Okamura, W. H., and Norman, A. W. (1994) J. Biol. Chem. 269, 23750-23756[Abstract/Free Full Text]
16. Nemere, I., Schwartz, Z., Pedrozo, H., Sylvia, V. L., Dean, D. D., and Boyan, B. D. (1998) J. Bone Miner. Res. 13, 1353-1359[CrossRef][Medline] [Order article via Infotrieve]
17. Pedrozo, H. A., Schwartz, Z., Rimes, S., Sylvia, V. L., Nemere, I., Posner, G. H., Dean, D. D., and Boyan, B. D. (1999) J. Bone Miner. Res. 14, 856-867[CrossRef][Medline] [Order article via Infotrieve]
18. Sylvia, V. L., Del Toro, F., Jr., Hardin, R. R., Dean, D. D., Boyan, B. D., and Schwartz, Z. (2001) J. Steroid Biochem. Mol. Biol. 78, 261-274[CrossRef][Medline] [Order article via Infotrieve]
19. Sylvia, V. L., Schwartz, Z., Ellis, E. B., Helm, S. H., Gomez, R., Dean, D. D., and Boyan, B. D. (1996) J. Cell. Physiol. 167, 380-393[CrossRef][Medline] [Order article via Infotrieve]
20. Boyan, B. D., Schwartz, Z., Swain, L. D., Carnes, D. L., Jr., and Zislis, T. (1988) Bone 9, 185-194[Medline] [Order article via Infotrieve]
21. Schwartz, Z., Sylvia, V. L., Luna, M. H., DeVeau, P., Whetstone, R., Dean, D. D., and Boyan, B. D. (2001) Steroids 66, 683-694[CrossRef][Medline] [Order article via Infotrieve]
22. Boyan, B. D., Dean, D. D., Sylvia, V. L., and Schwartz, Z. (1997) in Vitamin D (Feldman, D. , Glorieux, F. H. , and Pike, J. W., eds) , pp. 395-421, Academic Press, San Diego, CA
23. Sylvia, V. L., Schwartz, Z., Schuman, L., Morgan, R. T., Mackey, S., Gomez, R., and Boyan, B. D. (1993) J. Cell. Physiol. 157, 271-278[CrossRef][Medline] [Order article via Infotrieve]
24. Dean, D. D., Boyan, B. D., Muniz, O. E., Howell, D. S., and Schwartz, Z. (1996) Calcif. Tissue Int. 59, 109-116[CrossRef][Medline] [Order article via Infotrieve]
25. Newton, A. C. (1995) J. Biol. Chem. 270, 28495-28498[Free Full Text]
26. Boyan, B. D., Schwartz, Z., Howell, D. S., Naski, M., Ranly, D. M., Sylvia, V. L., and Dean, D. D. (2002) in Disorders of Bone and Mineral Metabolism (Coe, F. L. , and Favus, M. J., eds), 2nd Ed. , Lippincott, Williams, and Wilkins, Inc., Philadelphia, PA, in press
27. Simboli-Campbell, M., Gagnon, A., Franks, D. J., and Welsh, J. (1994) J. Biol. Chem. 269, 3257-3264[Abstract/Free Full Text]
28. Schwartz, Z., Schlader, D. L., Swain, L. D., and Boyan, B. D. (1988) Endocrinology 123, 2878-2884[Abstract/Free Full Text]
29. Boyan, B. D., and Ritter, N. M. (1984) Calcif. Tissue Int. 36, 332-337[CrossRef][Medline] [Order article via Infotrieve]
30. Peress, N. S., Anderson, H. C., and Sajdera, S. W. (1974) Calcif. Tissue Res. 14, 275-281[CrossRef][Medline] [Order article via Infotrieve]
31. Swain, L. D., Schwartz, Z., and Boyan, B. D. (1992) Bone Miner. 17, 192-196[CrossRef][Medline] [Order article via Infotrieve]
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., and Klenk, D. C. (1985) Anal. Biochem. 150, 76-85[CrossRef][Medline] [Order article via Infotrieve]
33. Boyan, B. D., Schwartz, Z., Carnes, D. L., Jr., and Ramirez, V. (1988) Endocrinology 122, 2851-2860[Abstract/Free Full Text]
34. Tartakoff, A. M., and Vassalli, P. (1978) J. Exp. Med. 146, 1332-1345[Abstract/Free Full Text]
35. Nemere, I., Ray, R., and Jia, Z. (1996) J. Bone Miner. Res. 11, S312.
36. Nemere, I., Ray, R., and McManus, W. (2000) Am. J. Physiol. 278, E1104-E1114[Abstract/Free Full Text]
37. Norman, A. W., Nemere, I., Muralidharan, K. R., and Okamura, W. H. (1992) Biochem. Biophys. Res. Comm. 189, 1450-1456[CrossRef][Medline] [Order article via Infotrieve]
38. Sylvia, V. L., Schwartz, Z., Curry, D. B., Chang, Z., Dean, D. D., and Boyan, B. D. (1998) J. Bone Miner. Res. 13, 559-569[CrossRef][Medline] [Order article via Infotrieve]
39. Park, S. K., Provost, J. J., Dae Bae, C., Ho, W. T., and Exton, J. H. (1997) J. Biol. Chem. 272, 29263-29271[Abstract/Free Full Text]
40. Carrasco-Marin, E., Alvarez-Dominguez, C., and Leyva-Cobian, F. (1994) Eur. J. Immunol. 24, 2031-2039[Medline] [Order article via Infotrieve]
41. Mollinedo, F., Gajate, C., and Flores, I. (1994) J. Immunol. 153, 2457-2469[Abstract]
42. Arcaro, A., and Wymann, M. P. (1993) Biochem. J. 296, 297-301
43. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994) J. Biol. Chem. 269, 5241-5248[Abstract/Free Full Text]
44. Boyan, B. D., Sylvia, V. L., Curry, D., Chang, Z., Dean, D. D., and Schwartz, Z. (1998) J. Cell. Physiol. 176, 516-524[CrossRef][Medline] [Order article via Infotrieve]
45. Schwartz, Z., Gilley, R. M., Sylvia, V. L., Dean, D. D., and Boyan, B. D. (1998) Endocrinology 139, 1825-1834[Abstract/Free Full Text]
46. Baran, D. T., Quail, J. M., Ray, R., Leszyk, J., and Honeyman, T. (2000) J. Cell. Biochem. 78, 34-46[CrossRef][Medline] [Order article via Infotrieve]
47. Racz, A., and Barsony, J. (1999) J. Biol. Chem. 274, 19352-19360[Abstract/Free Full Text]
48. Leach, R. J., Schwartz, Z., Johnson-Pais, T. L., Dean, D. D., Luna, M. H., and Boyan, B. D. (1995) J. Bone Miner. Res. 10, 1614-1624[Medline] [Order article via Infotrieve]
49. de Boland, A. R., and Norman, A. W. (1998) J. Cell. Biochem. 69, 470-482[CrossRef][Medline] [Order article via Infotrieve]
50. Schwartz, Z., Lohmann, C. H., Vocke, A. K., Sylvia, V. L., Cochran, D. L., Dean, D. D., and Boyan, B. D. (2001) J. Biomed. Mater. Res. 56, 417-426[CrossRef][Medline] [Order article via Infotrieve]
51. Duggan, J., Sylvia, V. L., Dean, D. D., Boyan, B. D., and Schwartz, Z. (2001) J. Dent. Res. 80, 44
52. Kirsch, T., Harrison, G., Golub, E. E., and Nah, H. D. (2000) J. Biol. Chem. 275, 35577-35583[Abstract/Free Full Text]
53. Boyan, B. D., Bonewald, L. F., Sylvia, V. L., Nemere, I., Larsson, D., Norman, A. W., Rosser, J., Dean, D. D., and Schwartz, Z. (2002) Steroids, in press
54. Shah, B. H., MacEwan, D. J., and Milligan, G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1886-1890[Abstract/Free Full Text]
55. Lopez-Ruiz, M. P., Choi, M. S., Rose, M. P., West, A. P., and Cooke, B. A. (1992) Endocrinology 130, 1122-1130[Abstract/Free Full Text]
56. Kochs, G., Hummel, R., Meyer, D., Hug, H., Marme, D., and Sarre, T. (1993) Euro. J. Biochem. 216, 597-606[Medline] [Order article via Infotrieve]
57. Schwartz, Z., Swain, L. D., Ramirez, V., and Boyan, B. D. (1990) Biochim. Biophys. Acta 1027, 278-286[Medline] [Order article via Infotrieve]
58. Schmitz, J. P., Schwartz, Z., Sylvia, V. L., Dean, D. D., Calderon, F., and Boyan, B. D. (1996) J. Cell. Physiol. 168, 570-579[CrossRef][Medline] [Order article via Infotrieve]
59. Boyan, B. D., Schwartz, Z., Park-Snyder, S., Dean, D. D., Yang, F., Twardzik, D., and Bonewald, L. F. (1994) J. Biol. Chem. 269, 28374-28381[Abstract/Free Full Text]

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Sci SignalHome page
M. T. Mizwicki and A. W. Norman
The Vitamin D Sterol-Vitamin D Receptor Ensemble Model Offers Unique Insights into Both Genomic and Rapid-Response Signaling
Sci. Signal., June 16, 2009; 2(75): re4 - re4.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
P. Kumar, Q. Wu, K. L. Chambliss, I. S. Yuhanna, S. M. Mumby, C. Mineo, G. G. Tall, and P. W. Shaul
Direct Interactions with G{alpha}i and G{beta}{gamma} Mediate Nongenomic Signaling by Estrogen Receptor {alpha}
Mol. Endocrinol., June 1, 2007; 21(6): 1370 - 1380.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
J. A. Huhtakangas, C. J. Olivera, J. E. Bishop, L. P. Zanello, and A. W. Norman
The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1{alpha},25(OH)2-Vitamin D3 in Vivo and in Vitro
Mol. Endocrinol., November 1, 2004; 18(11): 2660 - 2671.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
S. M. Vaingankar, T. A. Fitzpatrick, K. Johnson, J. W. Goding, M. Maurice, and R. Terkeltaub
Subcellular targeting and function of osteoblast nucleotide pyrophosphatase phosphodiesterase 1
Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1177 - C1187.
[Abstract] [Full Text] [PDF]

Home page
 J. Nutr.Home page
D. M. Peehl, A. V. Krishnan, and D. Feldman
Pathways Mediating the Growth-Inhibitory Actions of Vitamin D in Prostate Cancer
J. Nutr., July 1, 2003; 133(7): 2461S - 2469.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
277/14/11828    most recent
M110398200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Schwartz, Z.
Right arrow Articles by Boyan, B. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Schwartz, Z.
Right arrow Articles by Boyan, B. D.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement