A-RING ANALOGS OF 1,25-(OH)(2)D-3 WITH LOW-AFFINITY FOR THE VITAMIN-D-RECEPTOR MODULATE CHONDROCYTES VIA MEMBRANE EFFECTS THAT ARE DEPENDENT ON CELL MATURATION

1,25-(OH)(2)D-3 (1,25) and 24,25-(OH)(2)D-3 (24,25) mediate their effe cts on chondrocytes through the classic vitamin D receptor (VDR) as we ll as through rapid membrane-mediated mechanisms, which result in both nongenomic and genomic effects. In intact cells, it is difficult to d istinguish between genomic responses via the VDR and genomic and nonge nomic responses via membrane-mediated pathways. In this study, we used two analogues of 1,25 that have been modified on the A-ring (2a, 2b) and are only 0.1% as effective in binding to the VDR as 1,25, to exami ne the role of the VDR in the response of rat costochondral resting zo ne (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocy te proliferation ([H-3]-thymidine incorporation), proteoglycan product ion ([S-35]-sulfate incorporation), and second messenger activation (a ctivity of protein kinase C) were measured after treatment with 10(-8) M 1,25, 10(-7) M 24,25, or the analogues at 10(-9)-10(-6) M Both anal ogues inhibited proliferation of both cell types, as did 1,25 and 24,2 5. Neither 2a nor 2b had an effect on proteoglycan production by GCs o r RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC ) that was not inhibited by cycloheximide or actinomycin D in either G C or RC cells. 2b, on the other hand, had no effect on PKC activity in RCs and only a slight stimulatory effect in GCs. Both cells produce m atrix vesicles, extracellular organelles associated with the initial s tages of calcification, in culture that are regulated by vitamin D met abolites. Since these organelles contain no DNA or RNA, they provide a n excellent model for studying the mechanisms used by vitamin D metabo lites to mediate their nongenomic effects. When matrix vesicles were i solated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was simi lar to that found with 1,25-(OH)(2)D-3. Plasma membranes contained inc reased PKC activity after treatment with 2a, but the magnitude of the effect was less than that seen with 1,25-(OH)(2)D-3. Analogue 2b had n o affect on PKC activity in either membrane fraction. When matrix vesi cles from resting zone chondrocyte cultures were treated with 24,25-(O H)(2)D-3, a significant decrease in PKC activity was observed. No chan ge in enzyme activity was found for either 1,25-(OH)(2)D-3 or the anal ogues. PKC activity in the plasma membrane fraction, however, was incr eased by 24,25-(OH)(2)D-3 as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D recep tor, can affect cell proliferation and PKC activity, but not proteogly can production. The direct membrane effect is analogue specific and ce ll maturation dependent. Further, by eliminating the VDR-mediated comp onent of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenom ic effects of vitamin D metabolites. (C) 1997 Wiley-Liss, Inc.