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Endocrinology Vol. 139, No. 4 1825-1834
Copyright © 1998 by The Endocrine Society

ARTICLES

The Effect of Prostaglandin E2 on Costochondral Chondrocyte Differentiation Is Mediated by Cyclic Adenosine 3',5'-Monophosphate and Protein Kinase C1

Z. Schwartz, R. M. Gilley2, V. L. Sylvia, D. D. Dean and B. D. Boyan

Departments of Periodontics (Z.S., R.M.G., B.D.B.), Orthopedics (Z.S., V.L.S., D.D.D., B.D.B.), and Biochemistry (B.D.B.), University of Texas Health Science Center, San Antonio, Texas 78284; Hebrew University (Z.S.), 91010 Jerusalem, Israel; and Lackland Air Force Base (R.M.G.), San Antonio, Texas 78246

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: messier{at}uthscsa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recent studies indicate that vitamin D metabolites exert rapid effects on growth plate chondrocytes via changes in PG production and protein kinase C (PKC) activity. This suggests that these two products of vitamin D action may be interrelated. To test this hypothesis, we examined the effect of PGE2 on rat costochondral resting zone and growth zone cartilage cells and determined whether the effects of PGE2 are mediated by changes in the level of cAMP and/or PKC activity, whether there is a relationship between cAMP production and PKC activity, and whether cell maturation-specific effects are involved. Confluent, fourth passage resting zone and growth zone cartilage cell cultures were incubated in DMEM containing 10% FBS, 50 µg/ml vitamin C, and 1% antibiotics. The PGE2 concentration was varied from 0.007–15 ng/ml. Low concentrations of PGE2 caused a dose-dependent increase in cell number and [3H]thymidine incorporation and stimulated alkaline phosphatase specific activity. These effects were comparable in resting zone and growth zone cartilage cells at the same PGE2 concentrations. At higher concentrations, PGE2 caused a general increase in the synthesis of collagenase-digestible protein and noncollagenase-digestible protein in resting zone cartilage cells and of collagenase-digestible protein in growth zone cartilage cells, resulting in a net increase in the percent collagen synthesis for both cell types. cAMP production was increased over the entire range of chondrocyte response. Prevention of cAMP metabolism with the protein kinase A inhibitors H-8 and H-89 blocked the PGE2-dependent inhibition of PKC in resting zone cartilage cells in a dose-dependent manner. H-8 alone had no effect on PKC in resting zone cartilage cells, but stimulated PKC activity in growth zone cartilage cells; H-89 alone stimulated PKC activity in resting zone cartilage cells. These results suggest that low levels of PGE2 promote differentiation, whereas high doses promote an anabolic response; PGE2 increases cAMP production and PKC activity in a cell maturation-dependent manner; PGE2 exerts its effects via cAMP production and PKC activity; and regulation of PGE2-dependent PKC is via cAMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PROSTAGLANDINS (PGs) are important regulators of cellular events in a number of tissues, including cartilage (1). In articular cartilage, PGs have been associated with tissue destruction because they inhibit the production of proteoglycans (2) and stimulate the degradation of extracellular matrix (3). PGs also play a role in cartilage formation. In rat condylar cartilage, PGE2 stimulates the differentiation of prechondroblasts to chondrocytes (4). Similarly, PGE2 stimulates chondrogenesis of undifferentiated limb bud mesenchymal cells (5), and PGs have been shown to increase the cartilaginous tissue mass in a fracture-healing model (6).

It is likely that PGs function in cartilage as autocrine factors. PGs of the E series have been identified in normal growth plate cartilage (7). Homogenates of growth plate cartilage contain PG synthetase activity (8), and growth plate chondrocyte cultures produce PGE2 constitutively as well as in response to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (9). Moreover, growth plate chondrocytes respond to exogenous PGE2 in culture. In some instances, the data have been contradictory. Chick growth plate chondrocytes have been shown to respond to PGE2 with increased thymidine incorporation and alkaline phosphatase activity (7) and proteoglycan production (10), as well as with decreased alkaline phosphatase activity and collagen production (10). Some of these differences may be due to variations in PGE2 concentration or to differences in the growth plate maturation state of the chondrocytes used.

The mechanism of PG action in cartilage appears to involve cAMP. PGs have been shown to stimulate the accumulation of cAMP in chondrocytes isolated from rabbit ribs (11) and rat epiphysis (12). The amount of cAMP produced in response to PGE2 varies with different stages of chondrocyte maturation in the growth plate (13, 14). During chondrogenesis, limb bud mesenchymal cells show a dramatic increase in PGE2 and cAMP levels before their expression of a cartilage cell phenotype, suggesting a role for PGE2 and cAMP in these events (13, 15).

PGE2 may function as a second messenger for a number of regulatory factors that modulate growth plate development. This is supported by the fact that several factors, including interleukin-1, tumor necrosis factor, fibroblast growth factor, phorbol myristate acetate, and bradykinin stimulate PG production in chondrocytes (16, 17, 18, 19, 20). Recently, we showed that inhibition of PG production with indomethacin blocks the stimulation of protein kinase C (PKC) by 1,25-(OH)2D3 in growth zone chondrocytes, whereas inhibition of prostaglandin production causes an increase in the stimulation of PKC activity by 24,25-(OH)2D3 in resting zone chondrocyte cultures (21, 22). Thus, it appears that changes in PG modulate cellular response to 1,25-(OH)2D3 and 24,25-(OH)2D3, suggesting that PGE2 may act as an autocrine and paracrine regulator of chondrocyte metabolism in the growth plate.

In the present study, we evaluated the direct effects of PGE2 on chondrocyte proliferation, differentiation, and matrix production to determine whether these effects were dependent on the state of maturation of the cell. We also examined whether PGE2 regulates the production of cAMP and/or PKC activity to assess whether the effects of the cytokine are mediated through either or both of these mechanisms. Moreover, we determined whether the effects of PGE2 on cAMP and PKC are chondrocyte maturation dependent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chondrocyte cultures
The culture system used in this study has been described in detail previously (23, 24, 25). Chondrocytes were derived from the resting zone (reserve zone) and growth zone (prehypertrophic and upper hypertrophic cell zones) of the costochondral cartilage of 125-g male Sprague-Dawley rats. Cells were cultured in DMEM containing 10% FBS, 1% penicillin-streptomycin, and 50 µg/ml vitamin C in an atmosphere of 5% CO2 and 100% humidity at 37 C. Fourth passage cells were used for all experiments because previous studies have shown that these cells retain their chondrogenic phenotype, including the ability to form cartilage nodules when implanted into nude mouse thigh muscle and differential responsiveness to vitamin D metabolites and a number of other factors, through this number of passages in culture (26, 27).

For each experiment, confluent cultures of fourth passage resting zone or growth zone chondrocytes were treated with 0.007–15 ng/ml PGE2 (1 ng/ml PGE2 = 2.84 nM PGE2; Sigma Chemical Co., St. Louis, MO). The factor was dissolved in absolute ethanol and then diluted by at least 1000-fold in culture medium immediately before adding to the cultures. To determine a time course for the effect of PGE2 on the cells, chondrocytes were incubated with PGE2 for 10, 60, 360 (6 h), 720 (12 h), 1440 (24 h), and 2880 (48 h) min.

Cell proliferation
The effect of PGE2 on proliferation was determined by measuring cell number at harvest and by assessing [3H]thymidine incorporation as described previously (28). For these latter experiments, quiescence was induced by incubating confluent cultures for 48 h in DMEM containing 1% FBS. The medium was then replaced with DMEM containing 1% FBS PGE2 for 24 h. Four hours before harvest, [3H]thymidine was added. Radioactivity in trichloroacetic acid (TCA)-precipitable material was measured by liquid scintillation spectroscopy.

Alkaline phosphatase
Cell layer. Resting zone and growth zone chondrocytes were cultured in 24-well culture dishes (Corning, Corning, NY). At harvest, the media were decanted, and the cell layers were washed twice with PBS before removal with a cell scraper according to the method of Hale et al. (29), as described previously (23, 30). The cell layer pellets were collected by centrifugation, washed twice with PBS, and resuspended by vortexing in 500 µl deionized water containing 25 µl 1% Triton X-100. Enzyme assays were then performed using these cell layer lysates.

Preparation of membranes. Matrix vesicle and plasma membrane fractions were prepared as described previously (24). At harvest, the culture media were removed and replaced by 1% trypsin in Hanks’ Balanced Salt Solution. Cells were separated from the trypsin digest by centrifugation and resuspended in Hanks’ Balanced Salt Solution. Chondrocytes were then homogenized, and plasma membranes were prepared (31). Matrix vesicles were isolated from the supernatant of the 500 x g centrifugation prepared above by differential centrifugation (32, 33). After being assayed for protein content (34), plasma membranes and matrix vesicles were suspended in 0.9% NaCl and frozen at -70 C. Matrix vesicles isolated in this manner typically exhibit greater than 2-fold enrichment of alkaline phosphatase specific activity compared with the plasma membranes (23, 24, 30, 35) and have a transmis-sion electron microscopic appearance consistent with matrix vesicles in vivo (24).

Alkaline phosphatase activity. Alkaline phosphatase specific activity (orthophosphoric monoester phosphohydrolase alkaline; EC 3.1.3.1) in both the cell layer and membrane fractions was measured as a function of release of para-nitrophenol from para-nitrophenylphosphate at pH 10.2 (36).

RNA synthesis
RNA synthesis was estimated by measuring [3H]uridine incorporation into TCA-insoluble cell precipitates as described previously (28). The media of confluent, fourth passage cells were replaced with DMEM containing 1% FBS and varying quantities of PGE2 for 5 h. Two hours before harvest, [3H]uridine (1 µCi/ml) was added to the cultures, and radioactivity in TCA-precipitable material was measured by liquid scintillation spectroscopy.

Collagen production
Incorporation of labeled proline into collagenase-digestible protein (CDP) and noncollagenase-digestible protein (NCP) was used as a measure of collagen synthesis (37), as described previously (28). At confluence, cells were labeled for 24 h with 5 µCi L-[3H]proline (New England Nuclear, Boston, MA) in 0.5 ml medium. Proteins present in the media or cell layer were precipitated with 0.1 ml 100% TCA containing 10% tannic acid, washed three times with 10% TCA-1% tannic acid, and then washed twice with ice-cold acetone. The final pellet was dissolved in 500 µl 0.05 N NaOH. The amount of radiolabeled proline incorporated into CDP and NCP was determined by digesting the protein with purified clostridial collagenase (28, 38). Data were calculated with respect to both cell number and protein content of the fraction (cell layer or medium) being examined.

cAMP production
To determine whether the effect of PGE2 is mediated by cAMP, the amount of cAMP produced by the cells in response to PGE2 was measured. At confluence, the cells were washed twice with 500 µl DMEM and then preincubated for 30 min in 500 µl DMEM containing 0.2 mM isobutylmethylxanthine (Sigma Chemical Co., St. Louis, MO), which is a cAMP phosphodiesterase inhibitor (39, 40). The preincubation media were removed, and the cells were incubated with experimental media. At harvest, cellular cAMP was extracted with 500 µl 90% n-propan-1-ol at 4 C for 24 h. The extract was evaporated and reconstituted in 100 µl acetate buffer. The cAMP content of the chondrocytes was measured by RIA using an assay kit (BT-300) purchased from Biomedical Technologies (Stoughton, MA).

PKC assay
To examine whether the effect of PGE2 is mediated by PKC, PKC specific activity was measured in the cultures as described previously (41). Cell layer lysates were prepared by washing the cultures once with cold PBS, followed by lysis in 0.3 ml RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA; 1 mM phenylmethylsulfonylfluoride; and 1% Nonidet P-40 detergent). Aliquots of the cell layer lysates (35 µl) were then incubated for 20 min with a lipid preparation (5 µl) containing 0.3 mg/ml phosphatidylserine, 10 µM phorbol 12-myristate 13-acetate, and Triton X-100 mixed micelles, which provides the necessary cofactors and conditions for optimal activity (41, 42, 43). To this mixture a high affinity myelin basic protein peptide (8 µM) and [32P]ATP (25 µCi/ml) were added to a final assay volume of 50 µl. After a 10-min incubation in a 30 C water bath, samples were spotted onto phosphocellulose discs, which were then washed twice with 1% phosphoric acid and once with distilled water to remove unincorporated label before placement in a scintillation counter. The phosphocellulose disc strongly bound the myelin basic protein peptide substrate, which became 32P labeled during the PKC reaction, and unincorporated [32P]ATP was washed free of the discs during the phosphoric acid wash step (42).

Role of cAMP in PKC activation
To determine whether cAMP production mediates the effect of PGE2 on PKC activity, resting zone and growth zone chondrocytes were incubated with 0–15 ng/ml PGE2 in the presence and absence of 1 µM H-8 (Calbiochem, La Jolla, CA) (44). PKC activity was measured as described above. Because H-8 can affect PKC at high concentrations (44), we also investigated the effects of N-[2-(p-bromocinnamylamine)ethyl]-5-isoquinolinesulfonamide (H-89) (Calbiochem), which is a selective inhibitor of cAMP production (45). For these experiments, confluent cultures of fourth passage, resting zone cells were incubated in the presence and absence of 0.75 ng/ml PGE2 and 0.05, 0.5, or 5.0 µM H-8 or 0.01, 0.1, or 1.0 µM H-89.

Statistical analysis
The data presented are from one of three or more replicate experiments. Each data point represents the mean ± SEM for six individual cultures (cell layers) or six membrane samples (where each sample represents the membranes from one T-150 flask). The data were analyzed by ANOVA, and statistical significance was determined by comparing each data point to the control using Bonferroni’s modification of Student’s t test, using P < 0.05 confidence limits. Treatment/control ratios were determined from the results of five separate experiments, and statistical significance was determined using the Wilcoxon assigned rank test (P < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cell proliferation
PGE2 caused a biphasic increase in cell number, which was significant at 0.23–3.75 ng/ml for both resting zone and growth zone chondrocyte cultures (Table 1Go). These data were confirmed by measurement of [3H]thymidine incorporation. PGE2 caused an increase in [3H]thymidine incorporation at concentrations of 0.12–3.75 ng/ml (Fig. 1Go). Similar effects were observed when resting zone chondrocytes were treated with PGE2, although the increase in [3H]thymidine incorporation was found at concentrations of 0.12–1.87 ng/ml (Fig. 1Go). A time-course study showed that the stimulatory effect of PGE2 on [3H]thymidine incorporation by both cell types was found only after addition of the factor for 24 or 48 h (Fig. 2Go).


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  		Table 1. Cell number of growth zone (GC) and resting zone (RC) chondrocyte cultures incubated with PGE2 for 24 h

 


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  		Figure 1. Effect of PGE2 on [3H]thymidine incorporation by growth zone and resting zone chondrocytes. Quiescence was induced in confluent, fourth passage growth zone (GC) and resting zone (RC) chondrocytes by incubation for 48 h in medium containing 1% FBS. The cultures were then treated with varying concentrations of PGE2 (0.007–15 ng/ml) for 24 h; 4 h before harvest, [3H]thymidine was added, and incorporation into TCA-precipitable material determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 


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  		Figure 2. Effect of varying PGE2 treatment times on [3H]thymidine incorporation by growth zone and resting zone chondrocytes. Quiescence was induced in confluent, fourth passage resting zone (A) and growth zone (B) chondrocytes by incubation for 48 h in medium containing 1% FBS. The cultures were then treated with varying concentrations of PGE2 (0.015–15 ng/ml) for 5, 12, 24, and 48 h; 4 h before harvest, [3H]thymidine was added, and incorporation into TCA-precipitable material was determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 
Alkaline phosphatase specific activity
When PGE2 was added to either resting zone or growth zone chondrocytes, only 0.24–0.48 ng/ml produced a significant increase in alkaline phosphatase specific activity (Fig. 3AGo). The results of five different experiments, expressed as treatment/control ratios, showed similar results (Fig. 3BGo). Further, the effects were found only after exposing the cells for 12 or 24 h (Fig. 4Go). PGE2 also caused a dose-dependent increase in alkaline phosphatase specific activity in matrix vesicles and plasma membranes isolated from growth zone and resting zone chondrocyte cultures treated with 0.24–0.94 ng/ml PGE2 for 24 h (Fig. 5Go).



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  		Figure 3. Effect of PGE2 on cell layer alkaline phosphatase specific activity of growth zone and resting zone chondrocytes. Confluent, fourth passage growth zone (GC) and resting zone (RC) chondrocytes were treated with varying concentrations of PGE2 (0.007–15 ng/ml) for 24 h, and alkaline phosphatase specific activity in the cell layer was determined. Data from a representative experiment are shown in A, and the treatment/control ratios for five experiments are shown in B. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control in A and treatment vs. 1 in B.

 


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  		Figure 4. Effect of PGE2 on cell layer alkaline phosphatase specific activity of growth zone and resting zone chondrocytes. Confluent, fourth passage resting zone (A) and growth zone (B) chondrocytes were treated with varying concentrations of PGE2 (0.03–0.94 ng/ml) for 10, 60, 360 (6 h), 720 (12 h), and 1440 (24 h) min, and alkaline phosphatase specific activity in the cell layer was determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 


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  		Figure 5. Effect of PGE2 treatment on alkaline phosphatase specific activity of matrix vesicles and plasma membranes isolated from growth zone and resting zone chondrocyte cultures. Confluent, fourth passage resting zone (A) and growth zone (B) chondrocytes were treated with varying concentrations of PGE2 (0.06–15 ng/ml) for 24 h. Matrix vesicles (MV) and plasma membranes (PM) were isolated from the cell layers and then assayed for alkaline phosphatase specific activity. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six membrane preparations, each preparation being the membranes from two cultures. *, P < 0.05, treatment vs. control.

 
RNA synthesis
PGE2 caused a dose-dependent increase in [3H]uridine incorporation for both growth zone and resting zone chondrocytes (Fig. 6Go). A significant increase was found at concentrations of 0.94–15 ng/ml PGE2 in growth zone chondrocytes and 1.85–15 ng/ml in resting zone chondrocytes. The effect on [3H]uridine incorporation was time dependent (data not shown). Increased RNA synthesis was observed at 5, 12, and 24 h for both cell types and at the highest PGE2 concentration at 3 h in growth zone cells.



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  		Figure 6. Effect of PGE2 on [3H]uridine incorporation by growth zone and resting zone chondrocytes. Confluent, fourth passage growth zone (GC) and resting zone (RC) chondrocytes were treated with varying concentrations of PGE2 (0.007–15 ng/ml) for 5 h; 2 h before harvest, [3H]uridine was added, and incorporation into TCA-precipitable material was determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 
Collagen production
PGE2 increased the percent collagen production at concentrations of 0.94–15 ng/ml, with maximal collagen production at 7.5 ng/ml in both cell types (Fig. 7AGo). In growth zone cell cultures, both CDP and NCP production increased (Fig. 7Go, B and C). However, in resting zone chondrocyte cultures, the significant increase in the percent collagen production was the result of a significant increase in CDP only (Fig. 7Go, B and C).



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  		Figure 7. Effect of PGE2 on matrix production by growth zone and resting zone chondrocytes. Confluent, fourth passage growth zone (GC) and resting zone (RC) chondrocytes were treated with varying concentrations of PGE2 (0.007–15 ng/ml) for 24 h, and the percent collagen production (A), CDP production (B), and NCP production (C) were determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 
cAMP production
PGE2 induced both growth zone and resting zone chondrocytes to produce increased levels of cAMP over a wide range of concentrations; this was significant from 0.12–15 ng/ml (Fig. 8Go). In growth zone cells, the increase in cAMP production was bimodal and greater than that in resting zone cells at concentrations of 0.12–1.85 ng/ml. The effect was time dependent in both cell types, occurring within the first 10 min of exposure of the chondrocytes to PGE2 (Fig. 9Go).



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  		Figure 8. Effect of PGE2 on cAMP production of growth zone (GC) and resting zone (RC) chondrocytes. Confluent, fourth passage growth zone and resting zone chondrocytes were treated with varying concentrations of PGE2 (0.007–15 ng/ml) for 10 min, and cAMP production was determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 


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  		Figure 9. Effect of varying PGE2 treatment time on cAMP production by resting zone and growth zone chondrocytes. Confluent, fourth passage resting zone (RC; A) and growth zone (GC; B) chondrocytes were treated with varying concentrations of PGE2 (0.06–15 ng/ml) for 10, 60, 180, 360 (6 h), and 720 (12 h) min, and cAMP production were determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 
PKC activity
The addition of PGE2 to growth zone chondrocytes for 9 min significantly increased PKC specific activity at all concentrations examined. Maximal stimulation was seen at 0.24 and 15 ng/ml PGE2 (Fig. 10AGo). In contrast, PGE2 significantly inhibited PKC specific activity in a dose-dependent fashion when added to resting zone chondrocytes. At 90 min, PGE2 had a similar inhibitory effect on resting zone chondrocytes; however, in growth zone chondrocytes, the increase in PKC specific activity was found only at concentrations of 0.15–0.94 ng/ml PGE2, and the magnitude of the stimulation was reduced (Fig. 10BGo).



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  		Figure 10. Effect of PGE2 on PKC activity of resting zone and growth zone chondrocytes. Confluent, fourth passage growth zone (GC) and resting zone (RC) chondrocytes were treated with varying concentrations of PGE2 (0.015–15 ng/ml) for 9 (A) or 90 (B) min, and PKC specific activity in the cell layer was determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control.

 
Treatment of resting zone cells with 1 µM H-8 prevented the inhibitory effect of PGE2 on PKC specific activity in resting zone chondrocyte cultures (Fig. 11AGo). By itself, H-8 did not exert a dose-dependent effect on PKC activity in resting zone cells, even at 5 µM (Fig. 12AGo). However, in cultures treated with H-8 and 0.75 ng/ml PGE2, H-8 had a concentration-dependent effect on PKC activity, blocking the PGE2-dependent inhibition and, at the highest concentration of H-8, stimulating PKC over control levels. In contrast, H-89 had a slight stimulatory effect on PKC at 1 µM and only partially inhibited the dose-dependent inhibition of PKC by PGE2 (Fig. 12BGo). Addition of 1 µM H-8 to growth zone chondrocytes significantly increased PKC activity in the absence of PGE2 (Fig. 11BGo). In growth zone cell cultures treated with H-8 and PGE2, PKC levels were comparable to those in cultures exposed to H-8 alone, and the effect of the combination of H-8 and PGE2 was either equal to or greater than the stimulatory effects of PGE2 alone.



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  		Figure 11. Effect of H-8 on PGE2-induced PKC activity of resting zone and growth zone chondrocytes. Confluent, fourth passage resting zone (A) and growth zone (B) chondrocytes were treated with varying concentrations of PGE2 (0.015–15 ng/ml) in the presence or absence of 1 µM H-8 for 90 min (resting zone cells) or 9 min (growth zone cells), and PKC specific activity in the cell layer was determined. The data are from one of three representative experiments, each yielding similar results. Values are the mean ± SEM for six cultures. *, P < 0.05, treatment vs. control; #, P < 0.05, control vs. 1 µM H-8.

 


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  		Figure 12. Comparison of the concentration-dependent effects of H-8 and H-89 on PKC activity of resting zone chondrocytes in the presence and absence of PGE2. Confluent, fourth passage resting zone (RC) chondrocytes were treated with 0.75 ng/ml PGE2 or control medium in the presence or absence of H-8 (A) or H-89 (B) for 90 min, and PKC specific activity in the cell layer was determined. The values are the mean ± SEM for six cultures. *, P < 0.05, treatment with PGE2vs. control medium; #, P < 0.05, treatment with PKA inhibitor vs. no PKA inhibitor.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results show that PGE2 regulates a number of aspects of costochondral cartilage cell behavior, confirming previous studies showing that PGE2 has a direct effect on chondrocytes from the growth plate (7, 10). In the present study, PGE2 stimulated chondrocyte growth, including an increase in cell number and [3H]thymidine incorporation as well as an increase in RNA synthesis and protein production. Both CDP and NCP production were stimulated. In addition, PGE2 appeared to regulate cell differentiation, as evidenced by increased alkaline phosphatase specific activity.

This is an unusual finding because differentiation is most commonly associated with decreased proliferation. Although it is possible that some of the increase in enzyme activity may have been due to a general increase in extracellular matrix vesicles attendant with the overall increase in matrix, there appeared to be a specific effect on this enzyme activity due to enhanced cell differentiation. Increased alkaline phosphatase specific activity was observed at 0.24 and 0.48 ng/ml PGE2, whereas changes in CDP or NCP required PGE2 concentrations of 0.94 ng/ml or greater. Moreover, peak stimulation of alkaline phosphatase specific activity of matrix vesicles and plasma membranes was observed in cultures treated with 0.24–0.94 ng/ml PGE2, but not at higher doses of the mediator. Thus, the lower concentrations of PGE2 appeared to favor differentiation, whereas the higher concentrations seemed to have a general anabolic effect on the cells.

The effects of PGE2 were dose dependent, with the optimal concentration varying with each parameter. For example, both RNA production and matrix synthesis (NCP, CDP, and percent collagen) were stimulated by high PGE2 concentrations (0.94–15.0 ng/ml), whereas the effects on [3H]thymidine incorporation (0.12–1.8 ng/ml) and alkaline phosphatase (0.24–0.48 ng/ml) were observed at much lower concentrations. These results suggest that PGE2 exerts a continuum of response rather than an on/off effect. The sensitivity of the chondrocytes to time and concentration, as well as differences in the methods and animals used may explain the differences in some of the results reported in the literature (7, 10).

This hypothesis is supported by the observation that PGE2 increases cAMP production over a wide range of concentrations, including those that modulate the parameters examined in this study. Furthermore, the effect of PGE2 on cAMP production is dose dependent. This suggests that PGE2 regulates cAMP production, and at different levels, cAMP will stimulate different aspects of cell behavior. Increased cAMP leads to increased protein kinase A (PKA) activity; thus, inhibition of PKA should decrease the effects of PGE2 on the cells. This was indeed the case. Inhibition of PKA with H-8 and H-89 blocked the PGE2 inhibition of PKC activity normally seen in resting zone chondrocytes. These results are in agreement with previous studies that found that PGE2 stimulated differentiation of limb bud mesodermal cells via production of cAMP (5, 13, 14, 15, 46).

The effects of PGE2 on cell proliferation and differentiation as well as on the induction of cAMP were not dependent on the maturation state of the chondrocytes. This supports our observation that exogenous PGE2 stimulates alkaline phosphatase specific activity in both cell types. However, basal production of PGE2 by resting zone and growth zone chondrocytes as well as production of PGE2 in response to 1,25-(OH)2D3 and 24,25-(OH)2D3 are maturation dependent (47). These latter observations led us to hypothesize that at least part of the effects of these vitamin D metabolites is mediated through PGE2 (21, 22). Moreover, exogenous PGE2 stimulates PKC activity in growth zone cells, but inhibits PKC activity in resting zone cells (21). Taken together, these data suggest that cell maturation-specific effects of PGE2, such as those noted for NCP and CDP synthesis, cAMP production, and regulation of PKC, may be due in part to differences in the endogenous production of this PG. As discussed below, they may also be due in part to the interrelationship between cAMP and PKC.

PGE2 caused a comparable fold increase in alkaline phosphatase in the plasma membranes and matrix vesicles. This suggests that PGE2 not only modulates cellular enzyme activity, but also activity in organelles released to the matrix that participate in matrix maturation and calcification (33, 48, 49, 50). The amount of activity in the matrix vesicles was markedly higher than that in plasma membranes, suggesting that the greatest effect of PGE2 was on enzyme in the matrix. As 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate matrix vesicle alkaline phosphatase and phospholipase A2 activities (24, 35), the present data support the hypothesis that the vitamin D metabolites exert their effects through the production of PGE2.

The results of the present study also indicate that PGE2 operates via a PKC-dependent mechanism. The effect of PGE2 on PKC is cell maturation specific. In growth zone cells, PGE2 exerts a bimodal regulatory effect, with the overall result of increased PKC activity. In contrast, PGE2 had an inhibitory effect on PKC activity in the resting zone cells. Activation and inhibition of phospholipase A2, the enzyme that catalyzes the release of arachidonic acid, which is the rate-limiting step in PG production (51), had a similar cell maturation-specific effect on PKC activity in these cells (21).

There appears to be an interrelationship between the effects of PGE2 on cAMP production and those on PKC activity. Treatment of resting zone cells with either H-8 or H-89, both of which inhibit PKA, the protein kinase regulated by cAMP, blocks the inhibition of PKC activity by PGE2 in resting zone cells. By itself, H-8 had no effect on PKC activity, even at relatively high concentrations, whereas H-89 was stimulatory. Moreover, H-8 completely blocked the PGE2-dependent effect, whereas H-89 was only partially effective. Because H-89 is a potent and specific inhibitor of PKA and H-8 may also affect PKC at high concentrations, we used a 5-fold lower concentration of H-89 for our experiments. This may have accounted in part for the difference in cell response to these inhibitors. It is possible that some of the effects at high concentrations of H-8 may have been due to inhibition of PKC, not only to inhibition of PKA (44), but for cultures treated with H-89, only the contribution of PKA played a role.

In growth zone cells, the interrelationship of PKA and PKC appears to be complex. Treatment of the cells with 1 µM H-8 caused an increase in PKC in control cultures, suggesting that cAMP may inhibit the activity of this enzyme. H-8 is not reported to inhibit PKC at this concentration (44), and the fact that PKC activity was stimulated supports this. In cells treated with H-8 and PGE2 together, the effect was comparable to that seen with H-8 alone. This suggests that the effect of PGE2 is mediated by cAMP, and the rate of production and the amount produced regulate PKC in a cell maturation-dependent manner. These results are in agreement with previous studies in other systems showing that cAMP activates various protein kinases, affecting a variety of cell characteristics (52, 53, 54, 55, 56, 57, 58).


   Acknowledgments
 
The authors thank Ms. Sandra Messier for her help in preparing the manuscript, and Ms. Kimberly Rhame for her 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, Texas). This research was done in partial fulfillment of the requirements for the M.S. degree of R.M.G. and does not necessarily reflect the opinions of the U.S. Air Force. Back

2 Fellow at the Air Force Institute of Technology. Back

Received September 12, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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