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Endocrinology Vol. 140, No. 7 2991-3002
Copyright © 1999 by The Endocrine Society

ARTICLES

Arachidonic Acid Directly Mediates the Rapid Effects of 24,25-Dihydroxyvitamin D3 Via Protein Kinase C and Indirectly through Prostaglandin Production in Resting Zone Chondrocytes1

Z. Schwartz, V. L. Sylvia, D. Curry, M. H. Luna, D. D. Dean and B. D. Boyan

Departments of Orthopedics (Z.S., V.L.S., D.C., M.H.L., D.D.D., B.D.B.), Periodontics (Z.S., B.D.B.), and Biochemistry (B.D.B.), University of Texas Health Science Center, San Antonio, Texas 78284; Department of Periodontics, Lackland Air Force Base (D.C.), San Antonio, Texas 78236; and Department of Periodontics, Hebrew University Hadassah Faculty of Dental Medicine (Z.S.), Jerusalem, Israel 91010

Address all correspondence and requests for reprints to: Barbara D. Boyan, Ph.D., Department of Orthopedics, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7774. E-mail: boyanb{at}uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKC{alpha}; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKC{alpha} was inhibited, and matrix vesicle PKC{zeta} was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. {gamma}-Linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A NUMBER of studies have now shown that the vitamin D metabolite 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] has a direct effect on chondrocytes derived from the resting zone of growth plate cartilage both in vivo and in vitro (1, 2, 3, 4, 5, 6). The mechanisms involved in this regulation include traditional receptor-mediated genomic pathways (7) as well as rapid membrane-mediated events, some of which do not require new gene expression or protein synthesis. Within minutes of exposure of resting zone cells to 24,25-(OH)2D3, release of arachidonic acid is decreased (4), calcium ion flux is altered (5), plasma membrane fluidity is decreased (6), and production of diacylglycerol via phospholipase D is increased (8).

After 24 h of treatment with 24,25-(OH)2D3, there is a decrease in PGE2 (9), and matrix vesicle phospholipase A2 (PLA2) is also decreased (3). Because arachidonic acid production is the rate-limiting step in prostanoid production (10), this decrease in PGE2 may be related to the initial decrease in arachidonic acid release. Our experiments have used two time frames for analysis; therefore, it is not known whether the decrease in arachidonic acid release seen within the first 15 min of exposure is related directly to the inhibition of PLA2 and decreased production of PGE2 noted at 24 h.

24,25-(OH)2D3 also stimulates protein kinase C (PKC) specific activity in resting zone chondrocytes. Maximal increased activity is observed at 90 min, and both new gene expression and protein synthesis are required (7). The activity of PKC{alpha} in the plasma membrane is increased, in part due to new PKC synthesis, but also in part to translocation of cytosolic PKC{alpha}. When isolated plasma membranes are incubated directly with 24,25-(OH)2D3, PKC{alpha} activity is also increased (11), indicating that already existing enzyme can be regulated by nongenomic mechanisms. Similarly, in cultures treated with the seco-steroid, PKC{zeta}, the activity of matrix vesicles increases due to new protein synthesis and matrix vesicle production. In contrast, when isolated matrix vesicles are incubated directly with 24,25-(OH)2D3, PKC{zeta} is inhibited through nongenomic mechanisms.

These experiments demonstrate that regulation of PKC may involve more than one signal transduction pathway. They also indicate that changes in PKC activation play a role in the membrane-mediated signal transduction involved in 24,25-(OH)2D3 action and suggest that there may be a relationship between changes in PG production and PKC. Indeed, when PG production is inhibited by the cyclooxygenase inhibitor indomethacin, PKC activity is increased. There is a synergistic increase when 24,25-(OH)2D3 is included in the medium (8), supporting the hypothesis of a causal relationship. In addition, resting zone chondrocytes are regulated by exogenous PGE2 (12), indicating that modulation of PGE2 concentration by 24,25-(OH)2D3 may be part of the regulatory mechanism.

It is also possible that arachidonic acid itself acts as a mediator of 24,25-(OH)2D3 action, as not all of the effects of 24,25-(OH)2D3 are blocked by indomethacin (8). We recently demonstrated that arachidonic acid has a direct and rapid effect on PKC activity in costochondral growth plate chondrocytes (13, 14). Exogenous arachidonic acid inhibits PKC activity in resting zone cell cultures. Agents that stimulate arachidonic acid production cause a decrease in PKC, and agents that inhibit arachidonic acid production stimulate PKC activity. Although some of this effect is due to subsequent metabolism of arachidonic acid to PG, time-course studies suggest that at least some of the effect is due to the arachidonic acid itself. Arachidonic acid has also been shown to have direct effects on cells in other model systems (15, 16, 17, 18, 19, 20, 21, 22), potentially through new gene expression, as specific retinoic acid X (RXR) receptors have been found (23, 24, 25, 26). These observations led us to examine whether arachidonic acid plays a direct role in mediating the action of 24,25-(OH)2D3 in resting zone chondrocytes and to determine the mechanisms by which arachidonic acid exerts its effects on these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
The vitamin D3 metabolite 24R,25-(OH)2D3 was a gift from Dr. Milan Uskokovic of Hoffmann-La Roche, Inc. (Nutley, NJ). 24R,25-(OH)2D3 stock solutions were dissolved in ethanol and diluted at least 1:5000 (vol/vol) with culture medium before addition to the cultures. Arachidonic acid, {gamma}-linolenic acid, 1,2-dioctanoyl-sn-glycerol (DOG), the PKC inhibitor chelerythrine (27), and the protein kinase A (PKA) inhibitor H8 (28) were obtained from Calbiochem (San Diego, CA). Palmitic acid, nonspecific rabbit IgG1, and indomethacin, an inhibitor of cyclooxygenase, were purchased from Sigma Chemical Co. (St. Louis, MO). The lipoxygenase inhibitors, nordihydroguaiaretic acid (NDGA), which selectively inhibits 5-lipoxygenase (29), and esculetin, which preferentially inhibits 12- and 15-lipoxygenases (30), were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA). [3H]Thymidine, [35S]sulfate and [32P]ATP were obtained from NEN-DuPont (Boston, MA). PKC assay reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). PKC isoform-specific antibodies to PKC{alpha}, -ß, -{delta}, -{epsilon}, and -{zeta} were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Protein G-agarose was obtained from Oncogene Sciences, Inc. (Uniondale, NY). The protein content of each sample was determined using the bicinchoninic acid protein assay reagent (31) obtained from Pierce Chemical Co. (Rockford, IL).

Experimental design
To characterize cellular response to arachidonic acid, confluent cultures of fourth passage resting zone chondrocytes were treated with 1, 10, or 100 µM arachidonic acid in the absence or presence of 10-7 M 24,25-(OH)2D3. After a 24-h incubation, [3H]thymidine incorporation, alkaline phosphatase specific activity, and proteoglycan sulfation were assessed. To examine the mechanisms by which arachidonic acid modulates PKC activity, confluent cultures of resting zone cells were incubated with 1, 10, or 100 µM arachidonic acid in the presence and absence of 10-8 M 24,25-(OH)2D3 for 9, 90, or 270 min. The direct effect of arachidonic acid with or without 24,25-(OH)2D3 on specific isoforms of PKC in isolated plasma membranes and matrix vesicles was determined using isoform-specific PKC antibodies. The effects of arachidonic acid were compared with those of the diacylglycerol, DOG, a known activator of PKC (32), to examine the roles of phospholipase C and/or phospholipase D in the mechanism. To verify that the effects of arachidonic acid were due to the fatty acid and not to its metabolites, indomethacin (10-7 M) was used to block cyclooxygenase activity and PG production. In addition, we determined whether PKA was involved, as PGE2 is known to mediate many of its effects through a PKA-dependent mechanism. For these experiments, the inhibitor H8 (28) was added to the cultures in the presence of 24,25-(OH)2D3 with or without arachidonic acid, and [3H]thymidine incorporation, alkaline phosphatase specific activity, [35S]sulfate incorporation, and PKC specific activity were measured. Alternatively, to establish whether leukotriene production was involved, the cells were treated with the lipoxygenase inhibitors, NDGA (2, 20, and 40 µM) and esculetin (0.1, 1, and 10 µM). Cells were also treated with indomethacin and esculetin in combination with arachidonic acid. The specificity of arachidonic acid in mediating the 24,25-(OH)2D3 response was assessed by treating the cells or isolated membrane preparations with 1, 10, or 100 µM {gamma}-linolenic acid or palmitic acid in the presence and absence of 24,25-(OH)2D3. To demonstrate that 24,25-(OH)2D3-dependent increases in PKC mediate the physiological response of resting zone cells to the vitamin D metabolite, we incubated the cultures for 24 h with DOG (1, 10, and 100 µM) with or without the PKC inhibitor chelerythrine (10 µM) in the presence and absence of 24,25-(OH)2D3 and measured the effect on alkaline phosphatase specific activity.

Chondrocyte cultures
The culture system has been previously described in detail (33). Chondrocytes were obtained from resting zone (reserve zone) costochondral cartilage of 125-g male Sprague Dawley rats. Resting zone chondrocyte cultures were seeded at an initial density of 10,000 cells/cm2. Cells were cultured at 37 C in DMEM containing 10% FBS, 1% penicillin-streptomycin-fungizone, and 50 µg/ml sodium ascorbate in an atmosphere of 5% CO2 and 100% humidity for 7–10 days. At confluence, cells were subcultured using the same plating density and allowed to return to confluence. For all experiments, confluent, third passage cultures were subpassaged into 24-well microtiter plates and grown to confluence. Previous studies have demonstrated a retention of differential phenotypic markers through fourth passage (34, 35).

Preparation of matrix vesicle and plasma membrane fractions
Matrix vesicles and plasma membranes were prepared as described previously (33, 36). Plasma membranes were prepared by differential centrifugation of homogenized cells, followed by sucrose density centrifugation (33, 36, 37). Matrix vesicles were isolated by differential centrifugation of the supernatant of the trypsin-digested matrix obtained at the time of cell harvest. Both fractions were suspended in 0.9% NaCl, and protein content was determined (31). All assays were conducted on membranes pooled from two separate cultures (i.e. two T75 flasks). These techniques result in matrix vesicle preparations that are enriched in alkaline phosphatase specific activity, which is 2–10 times greater than that of the plasma membrane. There is a differential distribution of other plasma membrane marker enzymes in the matrix vesicles as well (36). Contamination of other organelles in either membrane preparation is minimal.

Cell function assays
[3H]Thymidine incorporation. DNA synthesis was estimated by measuring [3H]thymidine incorporation into trichloroacetic acid (TCA)-insoluble cell precipitates as described previously (2). Quiescence was induced by incubating confluent cultures for 48 h in DMEM containing 1% FBS. The medium was then replaced with experimental medium, and the incubation was continued for another 24 h. Four hours before harvest, [3H]thymidine was added. Radioactivity in TCA-precipitable material was measured by liquid scintillation spectroscopy.

Alkaline phosphatase specific activity. Alkaline phosphatase (orthophosphoric monoester phosphohydrolase, alkaline; EC 3.1.3.1) specific activity was measured in cell layer lysates (38) as a function of release of para-nitrophenol from para-nitrophenylphosphate at pH 10.2 (39).

[35S]Sulfate incorporation. Proteoglycan synthesis was assessed by measuring [35S]sulfate incorporation by fourth passage resting zone chondrocytes according to the method of O’Keefe et al. (40), as modified by us (41). At confluence, experimental media were added to the cells, and the incubation continued for an additional 24 h. Four hours before harvest, 50 µl DMEM containing 18 µCi/ml [35S]sulfate and 0.814 mM carrier sulfate were added to each culture. At harvest, the conditioned media were removed and the amount of [35S]sulfate incorporated into the cell layers was determined by liquid scintillation spectrometry. The protein content of each sample was determined using the bicinchoninic acid protein assay reagent (31), and the data are expressed as disintegrations per min/mg protein in the cell layer.

PKC enzyme assay
Cell layer lysates: PKC specific activity was measured in cell layer lysates as previously described (7).

Isolated membranes: The direct effects of arachidonic acid on PKC activity were tested using matrix vesicles and plasma membranes that had been isolated from confluent, fourth passage resting zone cultures as described previously (1, 11, 36). Matrix vesicles or plasma membranes (1 mg/ml in 0.9% NaCl containing 10% FBS) were incubated in the absence (vehicle only) or presence of 1–100 µM arachidonic acid for 9–270 min at 37 C and then assayed for PKC specific activity.

PKC translocation
Confluent fourth passage resting zone cells were treated with control medium or medium containing 10 or 100 µM DOG or 10 or 100 µM arachidonic acid for 90 min. Membrane and cytosol fractions were isolated using a modification (42) of the procedure used to investigate PKC translocation in osteosarcoma cells (43).

Determination of PKC Isoforms
Isoform-specific antibodies were used to examine the distribution of PKC isoforms in culture lysates as well as in isolated plasma membranes and matrix vesicles as previously described (11, 44). Resting zone cells were treated with either control medium or with 100 µM arachidonic acid. The following antibodies were used: polyclonal rabbit antibodies specific for the {alpha}-, ß-, {delta}-, {epsilon}-, and {zeta}-isoforms and nonspecific rabbit IgG1.

Statistical analysis
The data presented are from one representative experiment that was repeated three or more times giving similar results. Each data point represents the mean ± SEM for six individual cultures (cell layers) or six membrane samples (where each sample represents the combined membranes from two T75 flasks). Significance between groups was determined by Bonferroni’s t test using P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of arachidonic acid on 24,25-(OH)2D3- dependent responses
Arachidonic acid exerted dose-dependent effects on the resting zone cells (Fig. 1Go). [3H]Thymidine incorporation was increased (10–100 µM), and alkaline phosphatase activity was decreased (10–100 µM), but [35S]sulfate incorporation was unaffected. When arachidonic acid was added to cultures together with 24,25-(OH)2D3, the 24,25-(OH)2D3-dependent inhibition of [3H]thymidine incorporation was reversed, as was the stimulation of alkaline phosphatase and [35S]sulfate incorporation. For [3H]thymidine incorporation and alkaline phosphatase, this effect was seen at 1 µM arachidonic acid, whereas for [35S]sulfate incorporation, inhibition of the 24,25-(OH)2D3-dependent effect required 10–100 µM arachidonic acid.



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  		Figure 1. Effect of arachidonic acid and 24,25-(OH)2D3 on [3H]thymidine incorporation, alkaline phosphatase specific activity, and [35S]sulfate incorporation in resting zone chondrocytes. Fourth passage cells were treated with control medium or medium containing 1, 10, or 100 µM arachidonic acid in the absence or presence of 10-7 M 24,25-(OH)2D3 or 24,25-(OH)2D3 plus the PKA inhibitor H8 (1 µM) for 24 h and then assayed for changes in [3H]thymidine incorporation (top panel), alkaline phosphatase specific activity (middle panel), and [35S]sulfate incorporation (bottom panel) as described in Materials and Methods. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *, P < 0.05, cultures treated with arachidonic acid vs. untreated control; #, P < 0.05, cultures treated with 24,25-(OH)2D3 or 24,25-(OH)2D3 plus H8 vs. untreated control; •, P < 0.05, cultures treated with 24,25-(OH)2D3 plus H8 vs. 24,25-(OH)2D3 alone.

 
The effects of arachidonic acid on the resting zone cells were not mediated through PKA, although 24,25-(OH)2D3 does mediate some of its effects through a PKA-dependent mechanism (Fig. 1Go). The PKA inhibitor, H8, had no effect on the 24,25-(OH)2D3-dependent decrease in [3H]thymidine incorporation or on the stimulatory action of arachidonic acid on this parameter. Whereas H8 enhanced the stimulatory effects of 24,25-(OH)2D3 on alkaline phosphatase specific activity at low concentrations of arachidonic acid, as the concentration of arachidonic acid increased, the effect of H8 on 24,25-(OH)2D3-stimulated alkaline phosphatase was lost. Although H8 had no effect on 24,25-(OH)2D3-stimulated [35S]sulfate incorporation at low concentrations of arachidonic acid, in cultures treated with 10 µM arachidonic acid, the presence of H8 preserved the effect of 24,25-(OH)2D3 on the cells; this was lost at higher concentrations of arachidonic acid.

PKC
Arachidonic acid inhibited both basal and 24,25-(OH)2D3-induced PKC activity at 9, 90, and 270 min of treatment (Fig. 2Go). As noted previously (7), 24,25-(OH)2D3 exerted its maximal effects on PKC at 90 min; addition of 100 µM arachidonic acid decreased the 24,25-(OH)2D3-stimulated enzyme activity by approximately 50%. At 9 and 270 min, PKC activity in the arachidonic acid-treated cultures was approximately 25–30% below basal levels regardless of whether 24,25-(OH)2D3 was present. When cells were treated with 24,25-(OH)2D3 plus H8, PKC activity at 9 and 90 min was increased compared with that after 24,25-(OH)2D3 treatment alone. Addition of arachidonic acid inhibited the H8-dependent increase. At 270 min, H8 had no additional effect over 24,25-(OH)2D3 alone, but it prevented the inhibition of PKC due to arachidonic acid.



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  		Figure 2. Time course of the effect of arachidonic acid on basal and 24,25-(OH)2D3-stimulated PKC activity in resting zone chondrocytes. Confluent, fourth passage, resting zone chondrocyte cultures were treated with 100 µM arachidonic acid (AA), 10-8 M 24,25-(OH)2D3 (24 25 ), or a combination of the two in the presence or absence of 1 µM H8 for 9, 90, or 270 min. At harvest, the cell layers were assayed for PKC specific activity. Data represent the mean ± SEM of six cultures. *, P < 0.05, treatment vs. untreated control for a particular time point; #, P < 0.05, media containing 24,25-(OH)2D3 plus H8 vs. 24,25-(OH)2D3 alone.

 
The effect of arachidonic acid on PKC activity did not require new gene expression, as it could be observed in isolated membranes incubated directly with the fatty acid (Fig. 3Go, top). As noted previously (11), 24,25-(OH)2D3 inhibited matrix vesicle PKC. In contrast, arachidonic acid caused a dose-dependent increase in PKC and reversed the effects of 24,25-(OH)2D3 in a dose-dependent manner. When 24,25-(OH)2D3 was added to isolated plasma membranes, the expected increase in PKC activity was observed. Arachidonic acid alone caused a slight decrease in basal levels of PKC and inhibited the 24,25-(OH)2D3-dependent increase in a dose-dependent manner.



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  		Figure 3. Effect of arachidonic acid on basal and 24,25-(OH)2D3-stimulated PKC activity in isolated matrix vesicles (MV) and plasma membranes (PM) from resting zone chondrocytes. Matrix vesicles (left panels) and plasma membranes (right panels) were isolated, incubated with control medium, 10-8 M 24,25-(OH)2D3, or 10 or 100 µM arachidonic acid (AA) in the presence or absence of 10-8 M 24,25-(OH)2D3 for 90 min (top panels) or with control medium, 10-8 M 24,25-(OH)2D3, or 100 µM arachidonic acid (AA) in the presence or absence of 10-8 M 24,25-(OH)2D3 for 9, 90, or 270 min (bottom panels) and then assayed for PKC specific activity. Data represent the mean ± SEM of PKC activity in membranes from a representative experiment (n = 6). *, P < 0.05, treatment vs. control for a particular concentration of arachidonic acid (top panels) or time point (bottom panels); •, P < 0.05, membranes treated with arachidonic acid vs. those not treated with arachidonic acid (top panels) or membranes treated with 24,25-(OH)2D3 and arachidonic acid vs. those treated with arachidonic acid alone (bottom panels).

 
The direct effects of arachidonic acid on membrane PKC were time dependent as well (Fig. 3Go, bottom). In matrix vesicles, 24,25-(OH)2D3 caused a 50% reduction in PKC below baseline at all times examined. Maximal stimulation of PKC by arachidonic acid was seen at 9 min, and elevated levels were still evident at 90 min, but by 270 min, activity had almost returned to basal levels. When matrix vesicles were treated with arachidonic acid and 24,25-(OH)2D3 together, enzyme activity at 9 and 90 min was reduced to baseline, but not below. However, at 270 min, PKC activity was reduced to levels below baseline that were only slightly greater than the levels seen in membranes treated with 24,25-(OH)2D3 alone.

Maximal stimulation of plasma membrane PKC by 24,25-(OH)2D3 occurred by 9 min; there was a time-dependent decline, but even at 270 min, enzyme activity was still elevated more than 2-fold over the baseline value. When plasma membranes were treated with arachidonic acid, no effect on PKC was observed at any time examined. Arachidonic acid caused a time-dependent inhibition of the 24,25-(OH)2D3-stimulated PKC at 9 and 90 min, but this effect was gone by 270 min, and even at the early time points, arachidonic acid reduced PKC by less than 10%.

Arachidonic acid specifically affected the {alpha}-isoform of PKC in cell layer lysates (data not shown). Only the anti-PKC{alpha} antibody inhibited PKC activity in lysates of resting zone cell cultures. Normal rabbit IgG had no effect on the arachidonic acid-dependent inhibition of PKC, nor did antibodies specific for the ß, {delta}, {epsilon}, or {zeta} PKC isoforms. However, anti-PKC{alpha} reduced PKC activity by 79% in control cultures and by 72% in arachidonic acid-treated cultures.

The isoform responsible for the direct effect of arachidonic acid on membrane PKC was dependent on the membrane fraction examined (Fig. 4Go). In matrix vesicles, only anti-PKC{zeta} antibodies blocked PKC activity. This antibody also blocked the arachidonic acid-dependent increase as well as the 24,25-(OH)2D3-dependent decrease in PKC noted above. In contrast, anti-PKC{alpha} prevented the 24,25-(OH)2D3-stimulated increases in PKC noted in isolated plasma membranes when the vitamin D metabolite was used alone or in combination with arachidonic acid; if anything, the anti-PKC{alpha} antibody had a further inhibitory effect on the arachidonic acid-dependent decrease in PKC. Plasma membrane PKC was unaffected by any other PKC isoform-specific antibody. The effects of the isoform-specific antibodies on matrix vesicle PKC{zeta} and plasma membrane PKC{alpha} correlated with the expected time course for the arachidonic acid- and 24,25-(OH)2D3-dependent effects (Table 1Go).



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  		Figure 4. Effect of anti-PKC isoform-specific antibodies on PKC activity in isolated membrane fractions treated with arachidonic acid or 24,25-(OH)2D3 (24 25 ). Matrix vesicles (MV; top panel) or plasma membranes (PM; bottom panel) derived from resting zone chondrocyte cultures were treated with either control medium or medium containing 100 µM arachidonic acid (AA), 10-8 M 24,25-(OH)2D3, or the two in combination for 90 min in the presence of control (no added IgG), IgG1 (nonspecific rabbit IgG1), or anti-PKC isoform-specific antibodies and then assayed. Data represent the mean ± SEM of PKC activity remaining in the supernatant. *, P < 0.05 vs. membranes treated with control medium (i.e. 24,25 vehicle); #, P < 0.05 vs. membranes treated with 24,25-(OH)2D3 alone.

 

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  		Table 1. Effects of AA and 24,25-(OH)2D3 on PKC isoenzymes in matrix vesicles and plasma membranes isolated from resting zone chondrocyte cultures

 
Translocation of PKC from the cytosol to the membrane did not appear to be involved in the arachidonic acid-dependent changes in enzyme activity (Table 2Go). Arachidonic acid reduced cytosolic PKC by 50%, but caused only a slight decrease in membrane PKC activity that was not statistically significant. When the resting zone cells were treated with DOG, a known PKC activator, enzyme activity in the cytosol increased by approximately 30%, whereas the membrane enzyme activity increased by more than 6-fold, indicating translocation of cytosolic PKC to the membrane.


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  		Table 2. Effects of AA and DOG for 90 min on PKC specific activity in cytosol and solubilized membranes from fourth passage rat costochondral chondrocytes

 
Arachidonic acid metabolites
Some of the effects of arachidonic acid on PKC may have involved PG production. Treatment of the resting zone cells with indomethacin caused the expected increase in PKC activity noted previously (8), and when used with 24,25-(OH)2D3, the effects were additive (Fig. 5Go). Arachidonic acid caused a dose-dependent inhibition of the indomethacin-stimulated PKC, but even at the highest concentrations of arachidonic acid, the stimulatory effect of indomethacin was not completely blocked.



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  		Figure 5. Effect of arachidonic acid on PKC activity of resting zone chondrocyte cultures treated with 24,25-(OH)2D3 in the presence and absence of the cyclooxygenase inhibitor indomethacin (Indo). Confluent, fourth passage, resting zone chondrocyte cultures were treated with control medium or medium containing 1, 10, or 100 µM arachidonic acid (AA) in the presence or absence of 10-8 M 24,25-(OH)2D3 (24 25 ), 10-7 M indomethacin, or a combination of the two for 90 min. At harvest, the cell layers were assayed for PKC specific activity. Data represent the mean ± SEM of six cultures. *, P < 0.05, cultures treated with 24,25, Indo, or 24,25 plus Indo vs. untreated control; #, P < 0.05, cultures treated with arachidonic acid vs. cultures not treated with arachidonic acid.

 
Leukotrienes did not appear to be involved in the arachidonic acid-dependent response (data not shown). Neither of the lipoxygenase inhibitors had any effect on basal levels of PKC or on the arachidonic acid-dependent inhibition of PKC. Furthermore, there was no effect on the indomethacin-stimulated PKC regardless of whether arachidonic acid was present.

Fatty acid specificity
{gamma}-Linolenic acid caused a dose-dependent inhibition of PKC activity regardless of whether 24,25-(OH)2D3 was present (Fig. 6Go). At low levels, {gamma}-linolenic acid had no effect on the 24,25-(OH)2D3-stimulated enzyme activity, but at higher concentrations, the reduction in 24,25-(OH)2D3-stimulated PKC was similar to that observed in the absence of the vitamin D metabolite. At 100 µM {gamma}-linolenic, PKC activity was reduced to below baseline with and without 24,25-(OH)2D3. In contrast, palmitic acid had no effect on baseline PKC or on 24,25-(OH)2D3-stimulated activity (data not shown).



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  		Figure 6. The effect of {gamma}-linolenic acid on PKC activity in control and 24,25-(OH)2D3-treated resting zone chondrocytes. Confluent, fourth passage cells were treated with control medium or medium containing 1, 10, or 100 µM {gamma}-linolenic acid in the absence or presence of 10-8 M 24,25-(OH)2D3. At harvest, the cell layers were assayed for PKC specific activity. Data represent the mean ± SEM of six cultures. *, P < 0.05, treatment with fatty acid vs. control; #, P < 0.05, treatment with 24,25-(OH)2D3 vs. control.

 
The dose-dependent effects of {gamma}-linolenic acid were also noted in isolated plasma membranes incubated directly with the fatty acid (Table 3Go). There was a dose-dependent decrease in plasma membrane PKC that was significant at 100 µM {gamma}-linolenic acid. The fatty acid had no effect on the 24,25-(OH)2D3-dependent stimulation of plasma membrane PKC, however. In matrix vesicles, the opposite effects were seen (Fig. 7Go, top). {gamma}-Linolenic acid caused a dose-dependent increase in PKC and partially blocked the inhibition of the matrix vesicle enzyme caused by 24,25-(OH)2D3. These effects were time dependent (Fig. 7Go, bottom). Maximal stimulation of PKC by {gamma}-linolenic acid was seen at 9 min. Even though the effect of {gamma}-linolenic decreased at 90 min, it was still significantly higher than baseline and remained at this level over 270 min. In combination with 24,25-(OH)2D3, {gamma}-linolenic acid blocked the inhibition of PKC at all time points, and at 270 min, PKC activity was comparable to that seen with {gamma}-linolenic acid alone. Palmitic acid had no effect on either plasma membrane or matrix vesicle PKC regardless of whether 24,25-(OH)2D3 was present (data not shown).


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  		Table 3. Effects of {gamma}-linolenic acid and 24,25-(OH)2D3 on PKC specific activity of resting zone (RC) plasma membranes

 


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  		Figure 7. The effect of {gamma}-linolenic acid on PKC activity in control and 24,25-(OH)2D3-treated matrix vesicles isolated from resting zone chondrocytes. Matrix vesicles were isolated and then treated with control medium or medium containing 10 or 100 µM {gamma}-linolenic acid for 90 min (top panel) or with 10-8 M 24,25-(OH)2D3, 100 µM {gamma}-linolenic acid, or a combination of the two for 9, 90, or 270 min (bottom panel). At harvest, the cell layers were assayed for PKC specific activity. Data represent the mean ± SEM of six membrane preparations. *, P < 0.05, treatment vs. control; •, P < 0.05, medium containing {gamma}-linolenic acid vs. medium without {gamma}-linolenic acid (top panel).

 
Role of diacylglycerol
DOG caused a dose-dependent increase in PKC that was unaffected by the PKA inhibitor H8 (Fig. 8Go). The effect was blocked by the PKC inhibitor chelerythrine in a dose-dependent manner. As the concentration of DOG increased, the inhibitory effect of chelerythrine was reduced. Indomethacin caused a slight increase in the DOG-dependent stimulation of PKC in the resting zone cells. Whereas DOG had a stimulatory effect on the plasma membrane PKC activity (Table 2Go), it had a dose-dependent inhibitory effect on matrix vesicle PKC (Fig. 9Go, top). When isolated matrix vesicles were treated with DOG and 24,25-(OH)2D3 together, the inhibition noted in the presence of the vitamin D metabolite alone dominated the response. These effects were time dependent (Fig. 9Go, bottom). At 9 min, DOG had no effect on PKC activity, whereas complete inhibition was caused by 24,25-(OH)2D3. At 90 min, DOG reduced basal PKC by 29%, but by 270 min, activity in the presence of DOG was comparable to that seen in the untreated membranes. In contrast, when DOG and 24,25-(OH)2D3 were used in combination, PKC activity in the matrix vesicles was completely inhibited at all times examined.



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  		Figure 8. Effect of DOG on PKC activity in resting zone chondrocyte cultures in the presence of various inhibitors. Confluent, fourth passage cells were treated with control medium or medium containing 1 µM H8 (PKA inhibitor), 10 µM chelerythrine (Chel; PKC inhibitor), or 10-7 M indomethacin (Indo; cyclooxygenase inhibitor) for 90 min in the absence or presence of 1, 10, or 100 µM DOG. At harvest, the cell layers were assayed for PKC specific activity. Data represent the mean ± SEM of six cultures. *, P < 0.05, treatment with DOG vs. treatment without DOG; #, P < 0.05, treatment vs. control for each concentration of DOG.

 


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  		Figure 9. The effect of DOG on PKC activity in control and 24,25-(OH)2D3-treated matrix vesicles isolated from resting zone chondrocytes. Matrix vesicles were isolated and then treated with control medium or medium containing 10-8 M 24,25-(OH)2D3 in the presence or absence of 10 or 100 µM DOG for 90 min (top panel) or with 10-8 M 24,25-(OH)2D3, 100 µM DOG, or a combination of the two for 9, 90, or 270 min (bottom panel). At harvest, the membranes were assayed for PKC specific activity. Data represent the mean ± SEM of six membrane preparations. *, P < 0.05, 24,25-(OH)2D3 treatment vs. untreated control (top panel) or treatment vs. control for a particular time (bottom panel); •, P < 0.05, membranes treated with DOG vs. untreated control.

 
Requirement for PKC in mediating the effects of 24,25-(OH)2D3
The effect of 24,25-(OH)2D3 on alkaline phosphatase specific activity is mediated by PKC (Fig. 10Go). DOG caused a dose-dependent increase in alkaline phosphatase activity that was completely blocked at all concentrations by addition of chelerythrine to the cells. The maximal 6-fold increase in enzyme activity observed in cultures treated with 100 µM DOG was not different from the 6-fold stimulation caused by 10-7 M 24,25-(OH)2D3. The addition of chelerythrine to the 24,25-(OH)2D3-stimulated cultures reduced enzyme activity approximately 50% regardless of whether DOG was present.



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  		Figure 10. The requirement for PKC in mediating the 24,25-(OH)2D3-dependent increase in alkaline phosphatase specific activity. Confluent, fourth passage cultures of resting zone chondrocytes were incubated for 24 h with 0, 1, 10, or 100 µM DOG with or without 10-7 M 24,25-(OH)2D3 (24 25 ). Chelerythrine (Chel; 10 µM) was added to half of the cultures. Alkaline phosphatase specific activity of the cell layer lysates was determined. Data are the mean ± SEM of six cultures. •, P < 0.05, DOG vs. 0 µM DOG; *, P < 0.05, cultures with chel vs. cultures without chel.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study shows that arachidonic acid has a direct and specific regulatory effect on resting zone chondrocytes. It caused an increase in [3H]thymidine incorporation, as has been noted in other models (45). Moreover, in Swiss 3T3 fibroblasts, arachidonic acid induces expression of early growth response genes c-fos and Egr-1 (46). In contrast to its stimulatory effect on [3H]thymidine incorporation in resting zone cells, arachidonic acid decreased alkaline phosphatase specific activity and had no effect on proteoglycan production. This is commensurate with the observed increase in proliferation, as proliferation is frequently inversely correlated with differentiation.

The effects of arachidonic acid on the cells were opposite those of 24,25-(OH)2D3. This vitamin D metabolite inhibits proliferation and stimulates alkaline phosphatase specific activity and proteoglycan sulfation. When used in combination, the effect of arachidonic acid predominated, causing dose-dependent decreases in the response of resting zone cells to 24,25-(OH)2D3. This would suggest that these two mediators operate through separate mechanisms. We have shown previously that at least part of the response to 24,25-(OH)2D3 is mediated through the production of PG (9), with exogenous PGE2 exerting some of its effects through PKA (12). In the present study, the PKA inhibitor H8 had no effect on proliferation, but it preserved the stimulatory effect of 24,25-(OH)2D3 on alkaline phosphatase and proteoglycan sulfation at lower concentrations of arachidonic acid, but not at higher concentrations of the fatty acid. These observations suggest that some of the effects of arachidonic acid are via PG, but there is a direct mode of action that is independent of the subsequent metabolism of the fatty acid as well.

Our results confirm our previous observation that arachidonic acid exerts direct effects on PKC in resting zone cells (13). Moreover, these effects modulate PKC response to 24,25-(OH)2D3, suggesting a role for arachidonic acid in mediating the effects of the vitamin D metabolite on the PKC signal transduction pathway. Part of the effect of arachidonic acid in mediating the response of chondrocytes to 24,25-(OH)2D3 appears to be direct and partly involves a PKA-dependent mechanism, potentially via the production of PG. This hypothesis is based on the observation that arachidonic acid inhibited PKC below baseline levels by 9 min and maintained this inhibition through 270 min. However, arachidonic acid could only partially reduce the stimulatory effects of 24,25-(OH)2D3; when resting zone cells were treated with 24,25-(OH)2D3 plus H8, there was a further increase in PKC activity, and inclusion of arachidonic acid only reduced the effect to levels seen with 24,25-(OH)2D3 alone. Thus, the direct effect of arachidonic acid does not appear to involve PKA, whereas the role of arachidonic acid in the cell response to 24,25-(OH)2D3 does. Lopez-Ruiz et al. (19) showed that the effect of arachidonic acid is mediated by PKC in rat Leydig cells, demonstrating that its rapid effects proceed through this pathway.

As PGs mediate some of their effects on the resting zone cells via a PKA-dependent pathway (12), our results suggested that PGs might be involved in the cellular response to 24,25-(OH)2D3, but not to arachidonic acid directly. This hypothesis was substantiated by the fact that there was no change in the dose-dependent effect of arachidonic acid on PKC activity even when arachidonic metabolism to PG was blocked by indomethacin (9, 12). Moreover, it is unlikely that the effect of arachidonic acid was due to metabolism of the fatty acid via the lipoxygenase pathway to leukotrienes. Neither the lipoxygenase inhibitor esculetin, which preferentially inhibits 12- and 15-lipoxygenases (30), nor NDGA, which selectively inhibits 5-lipoxygenase (29), had any effect on the arachidonic acid inhibition of PKC activity in the resting zone cell cultures. When both cyclooxygenase and lipoxygenase pathways were inhibited with a combination of indomethacin and esculetin, the arachidonic acid effect was still seen. These data demonstrate strongly that part of the effect of arachidonic acid on chondrocytes involves a mechanism that is independent of its metabolism to PGs or leukotrienes.

The mechanisms by which arachidonic acid regulates PKC activity in the resting zone cells are complex. The arachidonic acid-sensitive isoform in resting zone cells is PKC{alpha}, the same isoform that is sensitive to 24,25-(OH)2D3 (11). Whereas 24,25-(OH)2D3 stimulates PKC{alpha} activity, arachidonic acid causes an inhibition. Arachidonic acid causes a decrease in the cytosolic stores of PKC without a corresponding translocation of this enzyme to the cell membrane, suggesting that it down-regulates PKC gene expression and/or protein synthesis. That arachidonic acid has a genomic effect has been shown in rat liver cells (47) as well as in other systems (48, 49, 50, 51). Arachidonic acid appears to exert its nuclear effect via RXR receptors (26), and RXR receptor mechanisms also participate in cellular response to vitamin D (52, 53). In contrast to arachidonic acid, diacylglycerol, which is involved in the 24,25-(OH)2D3-dependent stimulation of PKC in resting zone cells, caused an increase in cytosolic PKC together with a marked increase in membrane PKC, indicative of translocation.

This inhibitory effect of arachidonic acid on PKC is similar to our previous finding that exogenous PGE2 causes a decrease in PKC activity (8). However, the present study suggests that only a minor component of the response to arachidonic acid may be mediated through PKA. This suggests that there may be time-dependent differences in the mechanisms by which arachidonic acid and its metabolites exert their effects on PKC, particularly as they relate to 24,25-(OH)2D3 action on the cells. We have shown previously that 24,25-(OH)2D3-dependent stimulation of PKC involves phospholipase D-dependent production of diacylglycerol (8). Here, we show that the effect of DOG on PKC in the resting zone cells is unaffected by H8, indicating that PKA is not involved and, by inference, that PGE2 is not required. When indomethacin is included, DOG-stimulated PKC activity is enhanced, suggesting that PGE2 is inhibitory. Thus, the rapid decrease in arachidonic acid release noted in response to 24,25-(OH)2D3 (4) may permit an initial stimulation of PKC via translocation of existing cytosolic PKC to the membrane. As phospholipids are metabolized, resulting in arachidonic acid release and metabolism, the arachidonic acid will exert its effects through direct action on the cells as well as through downstream action of its metabolites. The outcome will be a down-regulation of the rapid PKC stimulation, but not of the genomic response through the nuclear receptors. This latter effect would account for the long term effects of arachidonic acid as cytosolic stores are replenished. Figure 11Go summarizes key elements of this hypothesis.



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  		Figure 11. Key elements of the proposed membrane-mediated mechanism of 24,25-(OH)2D3 action in resting zone chondrocytes. 24,25-(OH)2D3 interacts with a specific receptor in the plasma membrane activating phospholipase D (PLD), resulting in the release of diacylglycerol (DAG) and choline-phosphate (Chol-P). DAG then stimulates PKC and promotes its translocation to the plasma membrane. There is a rapid influx of Ca2+ ions and inhibition of PLA2; arachidonic (AA) release is initially decreased, resulting in increased PKC activity and reduction of PGE2 production, which also increases PKC activity. The initial decrease in AA release is followed by an increase. AA is metabolized to PGE2 through the constitutive action of cyclooxygenase 1. PGE2 acts on its receptor(s), resulting in cAMP production and activation of PKA, ultimately leading to new gene expression. AA also acts directly by inhibiting PKC activity and ultimately, through its own nuclear receptors, modulates phenotypic expression of the cell. This provides a mechanism for downregulating PKC-dependent phosphorylation events (Protein P Ser/Thr) and new gene expression. Alkaline phosphatase activity is increased by PKC, but whether this is due to direct regulation of existing enzyme or through new alkaline phosphatase specific activity appears to be mediated through PKA.

 
If this hypothesis is valid, then there must be a rapid effect of arachidonic acid on existing membrane PKC. This is, in fact, the case. Arachidonic acid elicits a rapid, membrane-specific change in PKC activity that is the opposite of that elicited by 24,25-(OH)2D3. Arachidonic acid caused a reduction in the 24,25-(OH)2D3-dependent stimulation of plasma membrane PKC by 9 min. Moreover, 24,25-(OH)2D3 could only partially block the arachidonic acid-dependent stimulation of PKC noted in matrix vesicles at 9 min. These effects of arachidonic acid and 24,25-(OH)2D3 on the membrane enzyme are nongenomic, as there is no possibility of new gene expression or protein synthesis in this assay. The effects of the two modulators appear to be on the enzyme directly, as both arachidonic acid and 24,25-(OH)2D3 affect PKC{alpha} in the plasma membranes, and both affect PKC{zeta} in the matrix vesicles.

The specific characteristics of arachidonic acid that are responsible for its effects are not yet known. {gamma}-Linolenic acid also inhibited PKC activity in resting zone chondrocyte cultures. These data suggest a similar mechanism of action for the structurally similar {gamma}-linolenic acid. As {gamma}-linolenic acid is a precursor in the biosynthesis of arachidonic acid (10), and no inhibitor is available to block this conversion, we cannot rule out metabolism of {gamma}-linolenic acid to arachidonic acid. The effects of {gamma}-linolenic acid on plasma membrane and matrix vesicle PKC were less robust than those noted in response to arachidonic acid, particularly with respect to 24,25-(OH)2D3. This may have been due to the requirement for metabolism to arachidonic acid, to the alternative metabolism of {gamma}-linolenic acid to PGE1, or to subtle differences in the interaction of {gamma}-linolenic acid with the membranes and PKC, all of which could blunt the membrane effect. In contrast to {gamma}-linolenic acid, palmitic acid had no effect on PKC activity, suggesting that the specificity of the fatty acid structure is important in PKC activation. This concept is supported by the work of other investigators indicating that the molecular structure of fatty acids (e.g. chain length and degree of saturation) may influence the specificity of their interactions with PKC (54, 55).

The effect of 24,25-(OH)2D3 on PKC is central to its modulation of the physiological response of resting zone chondrocytes to this vitamin D metabolite. In this study, both diacylglycerol and 24,25-(OH)2D3 caused a 6-fold increase in alkaline phosphatase activity, indicating that this enzyme is regulated at least in part through PKC-mediated pathways. Whereas chelerythrine reduced the diacylglycerol-dependent increase to baseline values, it only reduced the 24,25-(OH)2D3-dependent increase by 50%. Thus, part of the effect of 24,25-(OH)2D3 may be through other mechanisms, including inhibition of arachidonic acid release and PGE2 production as well as via traditional nuclear receptor pathways.

Our studies and those of other laboratories (56, 57) have indicated an important role for 24,25-(OH)2D3 in modulating phenotypic expression of rat resting zone chondrocytes in culture and in vivo. Moreover, the chondrocytes contain 24-hydroxylase (58) and 14C-labeled 24,25-(OH)2D3D3 is localized to the growth plate in rats injected with [14C]25-hydroxyvitamin D3 (59), suggesting that local production of this metabolite may also be important. Recent studies describing the 24-hydroxylase knockout mouse did not show a major defect in the growth plate of the long bones, but the mice failed to develop a mandible, suggesting a genetic lesion in the formation of Meckle’s cartilage (60). Our model examines costochondral cartilages from adult rats, and the knockout mice are an embryonic model; thus, the correlation between our two sets of findings is not yet clear.


   Acknowledgments
 
The authors thank Ms. Sandra Messier for her help in preparing the manuscript, and Ms. Kimberly Rhame and Dr. Zhi Chang for their technical assistance.


   Footnotes
 
1 This work was supported by USPHS Grants DE-05937 and DE-08603 and the Center for the Enhancement of the Biology/Biomaterials Interface at the University of Texas Health Science Center (San Antonio, TX). D.C. is a fellow in the Air Force Institute of Technology. This research was performed in partial fulfillment of the requirements for his M.S. degree and does not necessarily reflect the opinions of the U.S. Air Force. Back

Received August 31, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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