Membrane-mediated increases in protein kinase C (PKC) activity and PKC-dependent physiological responses of growth plate chondrocytes to vitamin D metabolites depend on the state of endochondral maturation; 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3] regulates growth zone (GC) cells, whereas 24R,25-(OH)2D3 regulates resting zone (RC) cells. Different mechanisms, including protein kinase A signaling, mediate the effects of 1α,25-(OH)2D3 and 24R,25-(OH)2D3 on PKC, suggesting that different mechanisms may also regulate any MAPK involvement in the physiological responses. This study used confluent cultures of rat costochondral chondrocytes as a model. 1α,25-(OH)2D3 stimulated MAPK specific activity in GC in a time- and dose-dependent manner, evident within 9 min. 24R,25-(OH)2D3 stimulated MAPK in RC; increases were dose dependent, occurred after 9 min, and were greatest at 90 min. In both cells the effect was due to ERK1/2 activation (p42 > p44 in GC; p42 = p44 in RC). MAPK activation was dependent on PKC, but not protein kinase A. The effect of 1α,25-(OH)2D3 required phospholipase C, and the effect of 24R,25-(OH)2D3 required phospholipase D. Inhibition of cyclooxygenase activity reduced the effect of 1α,25-(OH)2D3 on MAPK in GC and enhanced the effect of 24R,25-(OH)2D3 in RC. Based on MAPK inhibition with PD98059, ERK1/2 MAPK mediated the effect of 24R,25-(OH)2D3 on [3H]thymidine incorporation and [35S]sulfate incorporation by RC, but only partially mediated the effect of 1α,25-(OH)2D3 on GC. ERK1/2 was not involved in the regulation of alkaline phosphatase specific activity by either metabolite. This paper supports the hypothesis that 1α,25-(OH)2D3 regulates the physiology of GC via rapid membrane-mediated signaling pathways, and some, but not all, of the response to 1α,25-(OH)2D3 is via the ERK family of MAPKs. In contrast, 24R,25-(OH)2D3 exerts its effects on RC via PKC-dependent MAPK. Whereas 1α,25-(OH)2D3 increases MAPK activity via phospholipase C and increased prostaglandin production, 24R,25-(OH)2D3 increases MAPK via phospholipase D and decreased prostaglandin production. The cell specificity, metabolite stereospecificity, and the dependence on PKC argue for the participation of membrane receptors for 1α,25-(OH)2D3 and 24R,25-(OH)2D3 in the regulation of ERK1/2 in the growth plate.
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