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*1,25-DIHYDROXYCHOLECALCIFEROL
Endocrinology Vol. 139, No. 2 534-545
Copyright © 1998 by The Endocrine Society

ARTICLES

The Synergistic Effects of Vitamin D Metabolites and Transforming Growth Factor-ß on Costochondral Chondrocytes Are Mediated by Increases in Protein Kinase C Activity Involving Two Separate Pathways1

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

Departments of Orthopaedics (Z.S., V.L.S., D.D.D., B.D.B.), Periodontics (Z.S., B.D.B.), and Biochemistry (B.D.B.), The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7774; and Department of Periodontics (Z.S.), Hebrew University, Hadassah Faculty of Dental Medicine, Jerusalem, Israel 91010

Address all correspondence and requests for reprints to: Barbara D. Boyan, Ph.D., Department of Orthopaedics, The University of TX Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7774 E-Mail: MESSIER@uthscsa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß (TGFß), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFß effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFß on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC.

This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFß showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFß was added to GC cells or when 1,25 plus TGFß were added to GC or RC cells. The addition of 1,25 plus TGFß and 24,25 plus TGFß to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFß on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFß for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFß may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFß only affected PKC activity through G protein.

The results of the present study indicate that vitamin D metabolites and TGFß produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFß was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFß through G protein activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRANSFORMING growth factor ß (TGFß) is a potent stimulator of mesenchymal cell differentiation (1, 2, 3) and was originally purified from demineralized bone based on its ability to stimulate the differentiation of muscle cells into chondrocytes (4). TGFß is also known to regulate committed chondrocytes (5), including alkaline phosphatase-specific activity (6) and proteoglycan production (7, 8). Sporn et al. (9) have suggested that TGFß works in conjunction with other cytokines to modulate their effects, further amplifying its potential biological functions. For example, TGFß enhances endochondral bone formation by bone morphogenetic protein (10).

These latter observations indicate that TGFß promotes expression of the terminally differentiated chondrocyte phenotype. Other data suggest that, although TGFß promotes the early stages of endochondral differentiation, it retards the terminal differentiation of growth plate chondrocytes to a calcifying cartilage phenotype. This is based on the observation that TGFß inhibits phospholipase A2 activity (6), which normally increases as the epiphyseal growth plate calcifies (11). In addition, TGFß has been shown to inhibit collagen (12, 13, 14) and proteoglycan production (15), as well as alkaline phosphatase activity (14), and to increase cell proliferation (6, 16, 17), further suggesting that this factor inhibits the latter stages of chondrocyte maturation.

Three possibilities need to be considered to fully understand how TGFß may elicit seemingly opposing effects on chondrocytes. First, in the studies referenced above, chondrocytes at differing stages of maturation have been used. Differences in cellular response would be consistent with the hypothesis that the stage of cell maturation alters the effect of TGFß (14, 18). Second, TGFß is produced by chondrocytes (6, 8, 14, 17, 19) and, therefore, it may have an autocrine effect, which might vary with the experimental protocol. Third, TGFß has interactive effects with other growth factors known to regulate chondrocyte metabolism, and this may alter the overall response of the cells (14, 20, 21, 22).

The interaction of TGFß with the vitamin D metabolites, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], in modulating the activity of growth plate chondrocytes appears to be cell maturation-specific. When TGFß and 24,25-(OH)2D3 are added together to cultures of resting zone chondrocytes, a synergistic increase in alkaline phosphatase-specific activity is observed. In contrast, no synergistic effect is found when a similar experiment is repeated with growth zone chondrocytes and 1,25-(OH)2D3 and TGFß (6). In general, resting zone chondrocytes respond primarily to 24,25-(OH)2D3 (23, 24, 25, 26, 27, 28, 29, 30), whereas growth zone chondrocytes respond primarily to 1,25-(OH)2D3. Moreover, 24,25-(OH)2D3 causes resting zone cells to acquire responsiveness to 1,25-(OH)2D3, a phenotype characteristic of the growth zone chondrocytes’ phenotype (31). The synergistic increase in alkaline phosphatase seen in resting zone chondrocytes exposed to 24,25-(OH)2D3 and TGFß suggests that these two factors may work in concert to enhance the transition in cell maturation state.

In addition to the synergistic effects of TGFß and 24,25-(OH)2D3 on alkaline phosphatase in resting-zone cell cultures noted above, we have shown that activation of latent TGFß by matrix vesicles produced by growth-zone chondrocytes is stimulated by treatment with 1,25-(OH)2D3 (32) and that TGFß regulates the production of both 1,25-(OH)2D3 and 24,25-(OH)2D3 by resting-zone chondrocytes (30). Further, both vitamin D metabolites (33) and TGFß (34) mediate their effects through activation of protein kinase C (PKC), although vitamin D metabolites elicit a rapid increase in PKC activity, while TGFß elicits a more delayed response.

The present study was designed to examine the interrelationship between these two modulators of growth plate chondrocyte differentiation. We determined whether the synergistic increase in alkaline phosphatase observed in resting-zone cells in response to 24,25-(OH)2D3 and TGFß was cell maturation specific by examining the effect of these factors on growth-zone cells. We also established a time course for the synergistic effect and examined whether matrix vesicles or plasma membranes, or both, were the target of the marked increase in alkaline phosphatase. We examined whether the synergistic increase in enzyme activity correlated with a decrease in cell proliferation or a change in proteoglycan production. The final goal of this study was to elucidate the signal transduction mechanism used for producing this synergistic effect, particularly with respect to PKC as the second messenger.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chondrocyte cultures
The rat costochondral chondrocyte culture system used in this study has been described in detail previously (35). Cells were derived from the resting zone and growth zone of costochondral cartilage from 125-g male Sprague-Dawley rats (Harlan, Indianapolis, IN) and cultured in DMEM containing 10% FBS, 50 µg/ml vitamin C, and antibiotics in an atmosphere of 5% CO2 and 100% humidity at 37 C. Fourth-passage cells were used for all experiments because prior studies have shown that these cells retain their chondrogenic phenotype, including synthesis of type II collagen (26), ability to form cartilage nodules when implanted into nude mouse thigh muscle (36), and differential responsiveness to vitamin D metabolites and a number of other factors (6, 24, 25, 26, 31, 32, 35, 37, 38, 39).

Vitamin D metabolites [10-8 or 10-9 M 1{alpha},25-dihydroxyvitamin D3; 10-7 or 10-8 M 24R,25-dihydroxyvitamin D3] were dissolved in ethanol, since prior in vivo and in vitro studies have shown this to be the solvent of choice for these hormones. Before addition to the culture medium, each hormone stock solution was diluted at least 1:5000 (vol/vol) to minimize the toxic effects of ethanol on the cultures. Each experiment included control cultures that contained ethanol at the highest concentration found in the experimental groups. Both hormones were gifts from Dr. Milan Uskokovic at Hoffman-LaRoche (Nutley, NJ). Recombinant human TGFß1 was obtained from R and D Systems, Inc. (Minneapolis, MN). Growth factor was dissolved in PBS and diluted to the appropriate concentration in DMEM before addition to the cultures. Confluent cultures were incubated in media containing 1,25-(OH)2D3 [10-9 or 10-8 M], 24,25-(OH)2D3 [10-8 or 10-7 M] or TGFß [0.03–0.88 ng/ml], or a combination of both hormone and growth factor. Control cultures contained vehicle for vitamin D alone, TGFß alone, or both vehicles in combination.

[3H]Thymidine incorporation
DNA synthesis was estimated by measuring [3H]thymidine incorporation into trichloroacetic acid-insoluble cell precipitates as described previously (26). 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 and TGFß, vitamin D metabolite, or a combination of the two for 24 h. Two hours before harvest, [3H]thymidine was added. Radioactivity in trichloroacetic acid-precipitable material was measured by liquid scintillation spectroscopy.

[35S]Sulfate incorporation
Proteoglycan synthesis was assessed by measuring [35S]sulfate incorporation by confluent cultures of fourth-passage resting-zone chondrocytes according to the method of O’Keefe et al. (40). In prior studies, we found that the amount of radiolabeled proteoglycan secreted by the chondrocytes into the medium was less than 15% of the total radiolabeled proteoglycan (medium and cell layer) synthesized (41). Because of this, we only examined the effects of hormone and growth factor treatment on [35S]sulfate incorporation in the cell layer. At confluence, fresh medium containing vehicle alone, TGFß, vitamin D, or a combination of the two was added to the cells, and the incubation was 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, the cell layers (cells and matrix) were collected, and the amount of [35S]sulfate incorporated was determined by liquid scintillation spectrometry. The protein content was determined by the method of Lowry et al. (42), and the data were expressed as disintegrations per min/mg protein in the cell layer.

Preparation of the cell layer
Cell layers were prepared by a modification of the method described by Hale et al. (35, 43). At harvest, the medium was decanted, and the cell layer was washed twice with PBS and then removed using a cell scraper. After centrifugation at 500 x g, the cell layer pellet was washed two more times with PBS and resuspended by vortexing in 500 µl deionized water containing 25 µl 1% Triton X-100. Enzyme assays were performed on lysates of the cell layers.

Preparation of matrix vesicle and plasma membrane fractions
Matrix vesicles and plasma membranes were prepared as described previously (24, 35). Plasma membranes were prepared by differential centrifugation of homogenized cells, followed by sucrose-density centrifugation as previously described (24, 35, 44). 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 (42). 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 that is 2- to 10-fold greater than that of the plasma membrane. There is a differential distribution of other plasma membrane marker enzymes in the matrix vesicles as well (24). Contamination of other oganelles in either membrane preparation is minimal.

Assay of alkaline phosphatase activity
Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, alkaline (EC 3.1.3.1)] was measured as a function of release of p-nitrophenol from p-nitrophenylphosphate at pH 10.2 (45). Enzyme activity was assayed in both the cell layer and matrix vesicle and plasma membrane fractions.

Signal transduction
Protein kinase C. Cell layer lysates containing equivalent amounts of protein were mixed for 20 min with a lipid preparation containing phorbol-12-myristate-13-acetate, phosphatidylserine, and Triton X-100-mixed micelles to provide the necessary cofactors and conditions for optimal enzyme activity (46). To this mixture, a high-affinity myelin basic protein peptide 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.

To verify that PKC activity was being measured, cultures were incubated in the presence of 0.01, 0.10, or 1.0 µM chelerythrine (Calbiochem, San Diego, CA) for 90 min. Control cultures contained 10 µl PBS, the chelerythrine vehicle. Chelerythrine has been shown to be a general inhibitor of PKC activity (47, 48, 49).

Role of diacylglycerol (DAG). The role of DAG was examined by adding 0–50 µM R59002 (Calbiochem, San Diego, CA), a specific diacylglycerol kinase inhibitor, to the cultures for 90 min or 24 h (50). Control cultures contained 0.02% ethanol in DMEM, the maximal amount of vehicle added to the cultures treated with the inhibitor.

Role of G proteins. The role of G proteins in mediating the effect of vitamin D metabolites and/or TGFß on PKC-specific activity was assessed by adding 10 ng/ml pertussis toxin (Sigma Chemical Co., St. Louis, MO) to block Gi or 10 ng/ml cholera toxin (Sigma Chemical Co.) to block Gs. Control cultures were treated with the vitamin D metabolite and/or TGFß vehicle alone.

Statistical analysis
Unless otherwise noted, the data presented below are from one of three or more replicate experiments. Each data point represents the mean ± SEM for six cultures. For assays of matrix vesicle or plasma membrane enzyme activity, each data point represents the mean ± SEM for six samples, where each sample is the combined membranes from three cultures. Data were analyzed by ANOVA. Statistical significance was determined using Bonferroni’s t test, with P < 0.05 being considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Alkaline phosphatase activity
When 24,25-(OH)2D3 was added to cultures of growth zone chondrocytes for 24 h, no change in alkaline phosphatase-specific activity was observed (Fig. 1Go, upper panel). In contrast, when TGFß was added to the cultures, a biphasic increase in alkaline phosphatase-specific activity, which was significant at 0.06–0.22 ng/ml, was found (Fig. 1Go, middle panel). In cultures in which both TGFß and 24,25-(OH)2D3 were added to the cells, the effect was similar to that seen with TGFß alone (Fig. 1Go, lower panel). No differences were noted between cultures treated with TGFß plus 10-8 M or 10-9 M 24,25-(OH)2D3. Similarly, when 1,25-(OH)2D3 was added to cultures of resting zone cells, no change in alkaline phosphatase-specific activity was observed (Fig. 2Go, upper panel). TGFß treatment produced the same biphasic response seen in the growth-zone cells (Fig 2Go, middle panel), and combined treatment with both 1,25-(OH)2D3 and TGFß gave the same effect as seen with TGFß alone (Fig. 2Go, lower panel).



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  		Figure 1. The effect of 24,25-(OH)2D3 and TGFß on alkaline phosphatase-specific activity of growth-zone chondrocytes. Confluent, fourth-passage cells were treated with 10-10 to 10-8 M 24,25-(OH)2D3 (upper panel), 0.03 to 0.88 ng/ml TGFß (middle panel), or a combination of the two (lower panel) for 24 h. At harvest, the cell layers were assayed for alkaline phosphatase-specific activity. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *, P < 0.05, treatment vs. control.

 


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  		Figure 2. The effect of 1,25-(OH)2D3 and TGFß on alkaline phosphatase-specific activity of resting-zone chondrocytes. Confluent, fourth-passage cells were treated with 10-10 to 10-8 M 1,25-(OH)2D3 (upper panel), 0.03 to 0.88 ng/ml TGFß (middle panel), or a combination of the two (lower panel) for 24 h. At harvest, the cell layers were assayed for alkaline phosphatase-specific activity. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *, P < 0.05, treatment vs. control.

 
The TGFß-dependent effect on resting-zone cells was observed as early as 3 h after addition to the medium and remained constant through a 48-h exposure, whether or not 24,25-(OH)2D3 was present (Fig. 3Go, upper panel). In contrast, when 24,25-(OH)2D3 was added to resting-zone cultures for 3–48 h, no change in alkaline phosphatase-specific activity was detectable. In cultures receiving both TGFß and 24,25-(OH)2D3, there was a significant increase in alkaline phosphatase-specific activity that was greater than that seen with either growth factor or hormone alone over the entire time course of 3–48 h. When 1,25-(OH)2D3 was added to growth-zone cells, a significant increase in alkaline phosphatase-specific activity was noted at all time points examined (Fig. 3Go, lower panel). The addition of TGFß alone or in combination with 1,25-(OH)2D3 also increased alkaline phosphatase-specific activity over control cultures at all times examined. Moreover, the increase was greater than that seen with 1,25-(OH)2D3 at 3–12 h but, by 24 h, no differences in stimulation between 1,25-(OH)2D3 alone, TGFß alone, or 1,25-(OH)2D3 and TGFß together were noted.



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  		Figure 3. The effect of vitamin D metabolite and TGFß treatment time on alkaline phosphatase-specific activity of resting-zone and growth-zone chondrocytes. Upper panel, Confluent, fourth-passage resting-zone chondrocytes were treated with 10-8 M 24,25-(OH)2D3, 0.22 ng/ml TGFß, or a combination of the two for 3–48 h. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *,P < 0.05, treatment vs. control or 24,25-(OH)2D3 alone; #, P < 0.05, combined treatment vs. TGFß alone. Lower panel, Confluent, fourth-passage growth-zone chondrocytes were treated with 10-8 M 1,25-(OH)2D3, 0.22 ng/ml TGFß, or a combination of the two for 3–48 h. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *, P < 0.05, control vs. all other treatment groups; •, P < 0.05, treatment vs. treatment with 1,25-(OH)2D3 alone.

 
When matrix vesicles were isolated from resting-zone chondrocyte cultures treated with 24,25-(OH)2D3, TGFß, or a combination of the two for 24 h, alkaline phosphatase-specific activity was increased (Fig. 4Go, upper panel). However, plasma membranes isolated from cultures treated with 24,25-(OH)2D3 showed no increase in alkaline phosphatase-specific activity, while those from cultures receiving TGFß alone or the combined treatment contained increased enzyme-specific activity. Moreover, the increase in alkaline phosphatase-specific activity of the combined treatment was significantly greater than that seen with TGFß alone (Fig. 4Go, upper panel).



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  		Figure 4. The effect of TGFß and vitamin D metabolites on matrix vesicle and plasma membrane alkaline phosphatase-specific activity. Confluent, fourth-passage resting-zone chondrocytes were treated with 24,25-(OH)2D3 (upper panel) or 1,25-(OH)2D3 (lower panel) alone or in combination with TGFß. At harvest, matrix vesicles and plasma membranes were isolated and then assayed for alkaline phosphatase-specific activity. Values are the mean ± SEM of six samples, where each sample is the combined membranes from two T75 culture flasks. Data are from one of three identical experiments yielding similar results. *,P < 0.05, treatment vs. untreated control; #, P < 0.05, combined treatment vs. 24,25-(OH)2D3 alone.

 
The addition of 1,25-(OH)2D3 to cultures of resting-zone chondrocytes had no affect on the alkaline phosphatase-specific activity of either membrane fraction (Fig. 4Go, lower panel). The addition of 0.22 ng/ml TGFß to the cultures produced a significant increase in matrix vesicle alkaline phosphatase-specific activity. The effect of combined treatment was the same as that of TGFß alone. Moreover, the addition of 10-8 M 1,25-(OH)2D3 and 0.44 ng/ml TGFß together had no effect on matrix vesicle alkaline phosphatase-specific activity. A trend similar to the one seen with matrix vesicles was also seen with the plasma membranes (Fig. 4Go, lower panel).

[3H]Thymidine incorporation
The addition of 24,25-(OH)2D3 to resting zone cells resulted in a dose-dependent inhibition of [3H]thymidine incorporation that was significant at 10-9-10-8 M (Fig. 5Go, upper panel). In contrast, the addition of TGFß to these cells stimulated a dose-dependent increase in [3H]thymidine incorporation that was significant at 0.22–0.88 ng/ml (Fig. 5Go, middle panel). When both hormone and growth factor were added to the cultures, there was an inhibition of [3H]thymidine incorporation at the lower doses of TGFß (0.03–0.22 ng/ml) and, in general, no effect at the higher doses. However, at 10-9 M 24,25-(OH)2D3 and 0.88 ng/ml TGFß, there was a significant increase in [3H]thymidine incorporation (Fig. 5Go, lower panel). When 1,25-(OH)2D3, TGFß, or a combination of the two was added to growth-zone cells, a similar trend emerged, although no stimulation in [3H]thymidine incorporation was observed at higher doses of TGFß when given simultaneously with 1,25-(OH)2D3 (Fig. 6Go). A time course study was also performed. The addition of TGFß, 24,25-(OH)2D3, or a combination of the two to growth-zone cells showed an effect on [3H]thymidine incorporation after 12, 24, or 48 h (Table 1Go). Similarly, the effect of 24,25-(OH)2D3, TGFß, or a combination of the two on resting-zone cells was also observed after 12, 24, or 48 h (data not shown). Preliminary time course studies examining the effect of 1,25-(OH)2D3 and TGFß on growth-zone cells have displayed results that are similar to those found with 24,25-(OH)2D3 and TGFß (data not shown).



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  		Figure 5. The effect of TGFß and 24,25-(OH)2D3 on [3H]thymidine incorporation by resting-zone chondrocytes. Confluent, fourth-passage cells were treated with 10-10 to 10-8 M 24,25-(OH)2D3 (upper panel), 0.03–0.88 ng/ml TGFß (middle panel), or a combination of the two (lower panel) for 24 h. Two hours before harvest, [3H]thymidine was added to the cultures. At harvest, the cell layers were washed, precipitated with trichloroacetic acid as described in Materials and Methods, and counted in a scintillation counter. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *,P < 0.05, treatment vs. control.

 


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  		Figure 6. The effect of TGFß and 1,25-(OH)2D3 on [3H]thymidine incorporation by growth-zone chondrocytes. Confluent, fourth-passage cells were treated with 10-10 to 10-8 M 1,25-(OH)2D3 (upper panel), 0.03–0.88 ng/ml TGFß (middle panel), or a combination of the two (lower panel) for 24 h. Two hours before harvest, [3H]thymidine was added to the cultures. At harvest, the cell layers were washed, precipitated with trichloroacetic acid as described in Materials and Methods, and counted in a scintillation counter. Values are the mean ± SEM of six cultures. Data are from one of three identical experiments yielding similar results. *, P < 0.05, treatment vs. control.

 

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  		Table 1. The effect of TGFß and 24,25-(OH)2D3 on [3H]thymidine incorporation by growth-zone chondrocytes after varying treatment times

 
[35S]Sulfate incorporation
The addition of 10-7 M 24,25-(OH)2D3 to resting-zone cells produced a significant increase in [35S]sulfate incorporation (Table 2Go). In contrast, TGFß had no effect. When the cultures were treated with both 24,25-(OH)2D3 and TGFß, a significant increase in [35S]sulfate incorporation was observed that was greater than seen with either of the factors alone, with maximal stimulation observed at 0.44 ng/ml TGFß and 10-7 M 24,25-(OH)2D3 (Table 2Go).


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  		Table 2. The effect of TGFß and 24,25-(OH)2D3 on [35S]sulfate incorporation by resting-zone chondrocytes

 
The addition of 10-8 M 1,25-(OH)2D3 to growth-zone cells significantly stimulated [35S]sulfate incorporation (Table 3Go), while TGFß was without effect. Combined treatment with TGFß and 1,25-(OH)2D3 significantly increased [35S]sulfate incorporation to a level that was greater than that attained with either factor by itself. Maximal [35S]sulfate incorporation was observed when 0.44ng/ml TGFß and 10-8 M 1,25-(OH)2D3 were added to the media (Table 3Go).


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  		Table 3. The effect of TGFß and 1,25-(OH)2D3 on [35S]sulfate incorporation by growth-zone chondrocytes

 
PKC activity
To examine the mechanism behind the observed synergistic effect of 24,25-(OH)2D3 and TGFß on resting-zone chondrocytes, the effect of hormone and growth factor on PKC activity was examined. When cultures were treated with TGFß for 12 h, a significant, dose-dependent increase in PKC activity was observed (Fig. 7Go). In contrast, the addition of 24,25-(OH)2D3 for 12 h had no effect on PKC activity. However, when both TGFß and 24,25-(OH)2D3 were added to the cultures for 12 h, a significant increase in PKC activity was observed that was greater than that found with 24,25-(OH)2D3 or TGFß alone. Maximal response was seen in cultures treated with 10-7 M 24,25-(OH)2D3 and 0.88 ng/ml TGFß. Moreover, at the same concentration of vitamin D3, a higher concentration of TGFß showed a significant increase in PKC (Fig. 7Go). The addition of TGFß to the cultures for 11 h caused a significant, dose-dependent increase in PKC activity (Fig. 8Go). When 24,25-(OH)2D3 was added to the cultures during the final 90 min before harvest, a dose-dependent increase in PKC activity was observed, although it was less intense than the effect seen with TGFß. The addition of TGFß for 11 h, followed by treatment with 24,25(OH)2D3 for the final 90 min, resulted in a significant increase in PKC activity that was greater than that seen with any of the factors alone (Fig. 8Go). However, if 24,25-(OH)2D3 was added only for the final 9 min of the culture, no increase in PKC was noted (Fig. 9Go). Moreover, the combination of TGFß for 11 h and 24,25-(OH)2D3 for the final 9 min showed an increase in PKC activity that was similar to that found with TGFß alone (Fig. 9Go). Similarly, the addition of 24,25-(OH)2D3 for the first 90 min had no effect on PKC (Fig. 10Go). The addition of 24,25-(OH)2D3 for only the first 90 min, followed by TGFß for 12 h, showed an increase in PKC activity that was similar to that found with TGFß alone (Fig. 10Go).



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  		Figure 7. PKC-specific activity of resting-zone chondrocyte cell layers after combined treatment with TGFß and 24,25-(OH)2D3 for 12 h. Confluent, fourth- passage resting-zone chondrocytes were treated for 12 h with TGFß (0.22 or 0.88 ng/ml), 24,25-(OH)2D3 (10-7 or 10-8 M), or a combination of the two. At harvest, PKC-specific activity was measured. Values are the mean ± SEM of six cultures. Data are from one of two identical experiments yielding similar results. *, P < 0.05, treatment vs. untreated control. ***, P < 0.05, treatment vs. untreated control or treatment with TGFß or 24,25-(OH)2D3 alone.

 


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  		Figure 8. PKC-specific activity of resting-zone chondrocyte cell layers after pretreatment with TGFß for 11 h, followed by treatment with 24,25-(OH)2D3 for 90 min. Confluent, fourth-passage resting-zone chondrocytes were pretreated for 11 h with TGFß (0.22 or 0.88 ng/ml) or vehicle, followed by treatment for 90 min with 24,25-(OH)2D3 (10-8 or 10-7 M) or vehicle. At harvest, PKC-specific activity was measured. Values are the mean ± SEM of six cultures. Data are from one of two identical experiments yielding similar results. *, P < 0.05, treatment vs. untreated control. ***,P < 0.05, vs. treatment with TGFß for 11 h followed by 24,25-(OH)2D3 vehicle for 90 min or TGFß vehicle for 11 h followed by 24,25-(OH)2D3 for 90 min.

 


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  		Figure 9. PKC-specific activity of resting-zone chondrocyte cell layers after pretreatment with TGFß for 11 h followed by treatment with 24,25-(OH)2D3 for 9 min. Confluent, fourth-passage resting-zone chondrocytes were pretreated for 11 h with TGFß (0.22 or 0.88 ng/ml) or vehicle, followed by treatment for 9 min with 24,25-(OH)2D3 (10-8 or 10-7 M) or vehicle. At harvest, PKC-specific activity was measured. Values are the mean ± SEM of six cultures. Data are from one of two identical experiments yielding similar results. *, P < 0.05, treatment vs. untreated control. ***,P < 0.05, vs. treatment with TGFß for 11 h followed by 24,25-(OH)2D3 vehicle for 9 min or TGFß vehicle for 11 h followed by 24,25-(OH)2D3 for 9 min.

 


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  		Figure 10. PKC-specific activity of resting zone chondrocyte cell layers after pretreatment with 24,25-(OH)2D3 for 90 min followed by treatment with TGFß for 12 h. Confluent, fourth-passage resting-zone chondrocytes were pretreated for 90 min with 24,25-(OH)2D3 (10-8 or 10-7 M) or vehicle, followed by treatment for 12 h with TGFß (0.22 or 0.88 ng/ml) or vehicle. At harvest, PKC-specific activity was measured. Values are the mean ± SEM of six cultures. Data are from one of two identical experiments yielding similar results. *, P < 0.05, treatment vs. untreated control.

 
Effect of pertussis toxin and cholera toxin on PKC activity
The addition of TGFß to cultures of resting-zone chondrocytes produced a dose-dependent stimulation of PKC activity that could be blocked by the addition of pertussis toxin (Fig. 11Go, upper panel). Total inhibition of the TGFß-dependent stimulation in PKC activity was accomplished by 100 ng/ml pertussis toxin. Cholera toxin also inhibited TGFß-induced PKC activation (Fig. 11Go, lower panel).



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  		Figure 11. Effect of pertussis toxin and cholera toxin on PKC-specific activity in TGFß-treated resting-zone chondrocytes. Confluent, fourth-passage resting- zone chondrocytes were treated with medium containing 0.11 or 0.22 ng/ml TGFß in the presence or absence of 1–100 ng/ml pertussis toxin or cholera toxin. At harvest, PKC-specific activity in the cell layer was determined. Data represent the mean ± SEM for six cultures. Data are from one of two identical experiments yielding similar results. *, P < 0.05, treatment vs. control. #, P < 0.05, 0.22 ng/ml TGFß vs. 0.11 ng/ml TGFß.

 
Effect of DAG kinase inhibition (R59022)
The addition of R59022 for 90 min produced a small, but significant, dose-dependent increase in PKC activity (Table 4Go). The addition of TGFß to the cultures for 90 min, in the presence of R59022, produced an effect similar to that seen with the inhibitor alone. The addition of inhibitor for 24 h also resulted in a dose-dependent increase in PKC. When TGFß was added by itself for 24 h, a significant 10-fold increase in PKC activity was found. This increase in PKC activity was not affected by R59022.


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  		Table 4. The effect of DAG kinase inhibition (R59022) on the PKC activity of resting-zone chondrocytes treated with TGFß

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of this study confirm that TGFß and 24,25-(OH)2D3 have a synergistic effect on growth plate chondrocytes that is specific to cells from the reserve zone. We previously showed that TGFß and 24,25-(OH)2D3 elicited a synergistic increase in alkaline phosphatase-specific activity in these cells; in contrast, a synergistic increase in alkaline phosphatase was not seen in growth-zone cultures treated with TGFß and 1,25-(OH)2D3 (6). In the present study, TGFß plus 24,25-(OH)2D3 did not exert an effect different from that of TGFß alone on growth-zone cells, nor did TGFß plus 1,25-(OH)2D3 exert an effect different from that of TGFß alone on resting-zone cells. These results are in agreement with reports demonstrating that the effects of these vitamin D3 metabolites are cell maturation-dependent and metabolite-specific (24, 25).

Other investigators have also shown that TGFß has interactive effects with agents known to regulate chondrocytes (14, 20, 21, 22, 51). The complexity of these interactions may explain, in part, the difference in cellular response among studies. This is particularly the case with respect to TGFß and chondrocytes. In our study, synergistic increases in alkaline phosphatase were observed only in resting-zone cells treated with TGFß plus 24,25-(OH)2D3. Synergistic increases in [35S]sulfate incorporation were also observed in growth-zone cells treated with TGFß plus 1,25-(OH)2D3. Finally, the effects of TGFß and 24,25-(OH)2D3 on [3H]thymidine incorporation were additive, with TGFß being stimulatory (6) and 24,25-(OH)2D3 being inhibitory (26) for both resting-zone and growth-zone chondrocytes. Thus at low TGFß concentrations, the net effect of the combination was inhibitory. This was also the case for growth-zone cells treated with TGFß plus 1,25-(OH)2D3.

The synergistic effects of TGFß and vitamin D metabolites were probably a consequence of the specificity of cellular response to either 1,25-(OH)2D3 or 24,25-(OH)2D3, since both resting-zone cells and growth-zone cells responded to TGFß in a comparable manner. Both TGFß (6) and 24,25-(OH)2D3 (31) cause resting-zone cells to acquire a more differentiated phenotype. The marked increase in alkaline phosphatase observed in cultures exposed to both agents may reflect this phenotypic transition.

Alkaline phosphatase is a plasma membrane ectoenzyme. Mesenchymal cells that produce matrix vesicles, such as osteoblasts (52, 53) and growth plate chondrocytes (38), concentrate this enzyme in the extracellular organelle (54, 55, 56). Packaging of alkaline phosphatase into matrix vesicles is a targeted event and may occur independently of a general increase in plasma membrane alkaline phosphatase (57), although increases in matrix vesicle enzyme would first be detected as an increase in plasma membrane enzyme as the matrix vesicles are released from the cells. TGFß alone causes increases in both plasma membrane and matrix vesicle alkaline phosphatase (24). In contrast, 1,25-(OH)2D3 and 24,25-(OH)2D3 target alkaline phosphatase activity to matrix vesicles produced by growth-zone cells or resting-zone cells, respectively. The synergistic effect of TGFß plus 24,25-(OH)2D3 observed in the present study was targeted to the plasma membranes, not to the matrix vesicles.

These results suggest that the synergistic effect was on cell differentiation, but not on matrix calcification, an event associated with high matrix vesicle alkaline phosphatase (37). This supports previous observations on osteoblasts treated with TGFß plus 1,25-(OH)2D3. These studies found that TGFß inhibited 1,25-(OH)2D3-dependent osteocalcin production whereas collagen production was unaffected (58). The fact that TGFß enhanced cell differentiation and matrix production, but inhibited events associated with calcification, may be why the synergistic effect of TGFß and 24,25-(OH)2D3 on alkaline phosphatase was targeted to the less mature chondrocyte phenotype. In contrast, the synergistic increase in proteoglycan sulfation was observed in both mature [TGFß + 1,25-(OH)2D3] and less mature [TGFß plus 24,25-(OH)2D3] cells, and a proteoglycan-rich extracellular matrix containing sulfated glycosaminoglycans tends to remain uncalcified (59, 60, 61).

The combination of TGFß and 24,25-(OH)2D3 caused a synergistic increase in PKC activity in the resting-zone cells that was dependent on the concentration of both the hormone and the growth factor, suggesting that PKC might be involved in mediating the synergistic effects. Previous studies showed that TGFß increases PKC, but the effect is observed after 9 h of exposure to the growth factor, with peak increases occurring at 12 h (34). 24,25-(OH)2D3 also stimulates PKC, with a peak effect at 90 min (33). The time course of response indicates that PKC is involved in mediating the synergistic effects, but there is a complicated relationship between the two factors, suggesting that indirect mechanisms are responsible. If resting-zone cells are exposed to both TGFß and 24,25-(OH)2D3 for 12 h, the synergistic effect on PKC is observed. If 24,25-(OH)2D3 is given only for the last 90 min, again a synergistic effect is found. However, if 24,25-(OH)2D3 is given for only the first 90 min or the final 9 min, no synergistic response is seen. The vitamin D metabolite alone causes an increase in PKC that occurs after 9 min, peaks at 90 min, and is terminated by 270 min (33); TGFß alone causes an increase in PKC activity that is evident only after 9 h and shows maximal effect at 12 h (34). At 12 h, both TGFß and 24,25-(OH)2D have exerted their effects and a synergistic response occurs.

The last question this study attempted to answer was how 24,25-(OH)2D3 and TGFß can synergistically increase PKC activity. Both factors can increase PKC activity and do so without causing a translocation of cytosolic PKC to the membrane (62, 63). Both factors increase synthesis of plasma membrane PKC, and the major isoform affected is PKC{alpha}. Thus, it is likely that the mechanisms involved in the synergistic response include direct modulation of new PKC{alpha} expression as well as indirect modulation of PKC activity.

It is likely that each agent operates through a different pathway to increase PKC activity. Previously, we found that 24,25-(OH)2D3 activates PKC through production of DAG, not through activation of Gs or Gi proteins (64). In the present study, when the same parameters were examined after the addition of TGFß, it was found that TGFß activates both G proteins, with no effect on DAG production. It is possible that a G protein other than Gs or Gi is affected by 24,25-(OH)2D3. It is clear from this study that TGFß exerts its effects, at least in part, via activation of Gs and Gi. The mechanism by which Gi is operating in this context is intriguing. One possibility is that phospholipase A2 is involved. Pertussis toxin can inhibit phospholipase A2 via the G{alpha}i2 subunit (65, 66, 67). We have shown that changes in phospholipase A2 modulate PKC activity in the chondrocyte cultures (68), and 24,25-(OH)2D3 (25) and TGFß (6) regulate phospholipase A2, suggesting that the effect of pertussis toxin on TGFß-dependent PKC noted in the present study is via phospholipase A2.

It should be noted that the basal level of PKC activity is sensitive to DAG levels because enzyme activity was increased when the DAG kinase inhibitor was added to the cultures. This effect was unrelated to the TGFß-dependent response, however. The inhibitor did not appear to be toxic to the cells, even after long exposures, since cellular response to TGFß was unaffected.

The results of the present study indicate that vitamin D metabolites and TGFß have a synergistic effect that is cell maturation-dependent and metabolite-specific. Moreover, the synergistic effect between TGFß and 24,25-(OH)2D3 was mediated by increased PKC activity through two parallel pathways: 24,25-(OH)2D3 through DAG production and TGFß through Gs and Gi protein activation.


   Acknowledgments
 
The authors appreciate the secretarial assistance of Sandra Messier and Lucinda Flores and the technical assistance of Kimberly Rhame, Roland Campos, and Monica Luna in the preparation of this manuscript.


   Footnotes
 
1 This work was supported by Public Health Service Grants DE-08603 and DE-05937. Back

Received April 22, 1997.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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