THE SYNERGISTIC EFFECTS OF VITAMIN-D METABOLITES AND TRANSFORMING-GROWTH-FACTOR-BETA ON COSTOCHONDRAL CHONDROCYTES ARE MEDIATED BY INCREASES IN PROTEIN-KINASE-C ACTIVITY INVOLVING 2 SEPARATE PATHWAYS

Transforming growth factor-beta (TGF beta), as well as the vitamin D-3 metabolites 1,25-dihydroxyvitamin D-3 (1,25) and 24,25-dihydroxyvitam in D-3 (24,25), regulate chondrocyte differentiation and maturation du ring endochondral bone formation. Both the growth factor and secostero ids also affect protein kinase C (PXC) activity, although each has its own unique time course of enzyme activation. Vitamin D-3 metabolite e ffects are detected soon after addition to the media, whereas TGF beta effects occur over a longer term. The present study examines the inte rrelation between the effects of 1,25, 24,25, and TGF beta on chondroc yte differentiation, matrix production, and proliferation. We also exa mined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is media ted by PKC. This study used a chondrocyte culture model developed in o ur laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less m ature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGF beta showed synergistic effects on RC alkaline phosphatase-sp ecific activity (ALPase). No similar effect was found when 24,25 plus TGF beta was added to GC cells or when 1,25 plus TGF beta were added t o GC or RC cells. The addition of 1,25 plus TGF beta and 24,25 plus TG F beta to GC and RC cells, respectively, produced a synergistic increa se in [S-35] sulfate incorporation and had an additive effect on [H-3] thymidine incorporation. To examine the signal transduction pathway in volved in producing the synergistic effect of 24,25 and TGF beta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGF beta for 12 h produced a synergistic increase in PKC activity. Mor eover, a similar effect was found when 24,25 was added for only the la st 90 min of a 12-h incubation. However, a synergistic effect could no t 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 TGF beta may media te the observed synergistic increase in PKC activity, the pathways pot entially 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 TGF beta only affected PKC ac tivity through G protein. The results of the present study indicate th at vitamin D metabolites and TGF beta produced a synergistic effect th at is maturation-dependent and hormone-specific. Moreover, the synergi stic effect between 24,25 and TGF beta was mediated by activation of P KC through two parallel pathways: 24,25 through diacylglycerol product ion and TGF beta through G protein activation.