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

Abstract

Transforming growth factor-␤ (TGF␤), as well as the vitamin D 3 metabolites 1,25-dihydroxyvitamin D 3 (1,25) and 24,25-dihydroxyvitamin D 3 (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 D 3 metabolite effects are detected soon after addition to the media, whereas TGF␤ effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGF␤ 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 TGF␤ showed synergistic effects on RC alkaline phosphatasespecific activity (ALPase). No similar effect was found when 24,25 plus TGF␤ was added to GC cells or when 1,25 plus TGF␤ were added to GC or RC cells. The addition of 1,25 plus TGF␤ and 24,25 plus TGF␤ to GC and RC cells, respectively, produced a synergistic increase in [ 35 S]sulfate incorporation and had an additive effect on [ 3 H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGF␤ on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGF␤ 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 TGF␤ 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 TGF␤ only affected PKC activity through G protein.The results of the present study indicate that vitamin D metabolites and TGF␤ produced a synergistic effect that is maturationdependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGF␤ was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGF␤ through G protein activation. (Endocrinology 139: 534 -545, 1998)