HYBRID STRUCTURAL ANALOGS OF 1,25-(OH)(2)D-3 REGULATE CHONDROCYTE PROLIFERATION AND PROTEOGLYCAN PRODUCTION AS WELL AS PROTEIN-KINASE-C THROUGH A NONGENOMIC PATHWAY

1,25-(OH)(2)D-3 and 24,25-(OH)(2)D-3 mediate their effects on chondroc ytes through the classic vitamin D receptor (VDR) as well as through r apid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish betwe en genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid anal ogues of 1,25-(OH)(2)D-3 which have been modified on the A-ring and C, D-ring side chain (1 alpha-(hydroxymethyl)-3 beta-hydroxy-20-epi-22-o xa-26,27-dihomo vitamin D-3 (analogue MCW-YA = 3a) and 1 beta-(hydroxy methyl)-3 alpha-hydroxy-20-epi-22-oxa-26,27-dihomo vita min D-3 (analo gue MCW-YB = 3b) to examine the role of the VDR in response of rat cos tochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 -(OH)(2)D-3 and 24,25-(OH)(2)D-3. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)(2)D- 3. Chondrocyte proliferation ([H-3]-thymidine incorporation), proteogl ycan production ([S-35]-sulfate incorporation), and activity of protei n kinase C (PKC) were measured after treatment with 1,25-(OH)(2)D-3, 2 4,25-(OH)(2)D-3, or the analogues. Both analogues inhibited proliferat ion of both cell types, as did 1,25-(OH)(2)D-3 and 24,25-(OH)(2)D-3. A nalogue 3a had no effect on proteoglycan production by GCs but increas ed that by RCs. Analogue 3b increased proteoglycan production in both CC and RC cultures. Both analogues stimulated PKC in CC cells; however , neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(O H)(2)D-3 and 3a decreased PKC in matrix vesicles from CC cultures, whe reas plasma membrane PKC activity was increased, with 1,25-(OH)(2)D-3 having a greater effect. 24,25-(OH)(2)D-3 caused a significant decreas e in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)(2)D- 3, 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 activ ity. The direct membrane effect is analogue-specific and cell maturati on-dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a memb rane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites. (C) 1997 Wiley-Liss, Inc.