A-ring analogues of 1,25-(OH)2D3 with low affinity for the vitamin D receptor modulate chondrocytes via membrane effects that are dependent on cell maturation
✍ Scribed by Daniel M. Greising; Zvi Schwartz; Gary H. Posner; Victor L. Sylvia; David D. Dean; Barbara D. Boyan
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 283 KB
- Volume
- 171
- Category
- Article
- ISSN
- 0021-9541
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✦ Synopsis
1,25-(OH) 2 D 3 (1,25) and 24,25-(OH) 2 D 3 (24,25) 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 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 examine the role of the VDR in the response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocyte proliferation ([ 3 H]-thymidine incorporation), proteoglycan production ([ 35 S]-sulfate incorporation), and second messenger activation (activity of protein kinase C) were measured after treatment with 10 08 M 1,25, 10 07 M 24,25, or the analogues at 10 09 -10 06 M. Both analogues inhibited proliferation of both cell types, as did 1,25 and 24,25. Neither 2a nor 2b had an effect on proteoglycan production by GCs or RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC) that was not inhibited by cycloheximide or actinomycin D in either GC 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 matrix vesicles, extracellular organelles associated with the initial stages of calcification, in culture that are regulated by vitamin D metabolites. Since these organelles contain no DNA or RNA, they provide an excellent model for studying the mechanisms used by vitamin D metabolites to mediate their nongenomic effects. When matrix vesicles were isolated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was similar to that found with 1,25-(OH) 2 D 3 . Plasma membranes contained increased 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 no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures