Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway

Abstract

1,25-(OH)2D3 and 24,25-(OH)2D3 mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)2D3 which have been modified on the A-ring and C,D-ring side chain (1 alpha-(hydroxymethyl)-3 beta-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YA = 3a) and 1 beta-(hydroxymethyl)-3 alpha-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)2D3 and 24,25-(OH)2D3. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)2D3. Chondrocyte proliferation ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)2D3, 24,25-(OH)2D3, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)2D3 and 24,25-(OH)2D3. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)2D3 and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)2D3 having a greater effect. 24,25-(OH)2D3 caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)2D3, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation-dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites.