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

Abstract

1,25-(OH)2D3 (1,25) and 24,25-(OH)2D3(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 ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and second messenger activation (activity 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 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)2D3. 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)2D3. Analogue 2b had no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures were treated with 24,25-(OH)2D3, a significant decrease in PKC activity was observed. No change in enzyme activity was found for either 1,25-(OH)2D3 or the analogues. PKC activity in the plasma membrane fraction, however, was increased by 24,25-(OH)2D3 as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D receptor, can affect cell proliferation and PKC activity, but not proteoglycan production. The direct membrane effect is analogue specific and cell maturation dependent. Further, by eliminating the VDR-mediated component of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites.

Physiological Importance of the 1,25(OH)2D3 Membrane Receptor and Evidence for a Membrane Receptor Specific for 24,25(OH)2D3

“…(12,24) The results presented here indicate that this nVDR-independent mechanism is a membrane-mediated pathway that involves the novel 1,25(OH) 2 D 3 mVDR described previously. (23) By preventing ligand binding to the mVDR with Ab99, the antiproliferative effects of the 1,25(OH) 2 D 3 analogs were prevented.…”

“…(16) In culture, 24,25(OH) 2 D 3 and analog 2a elicit peak increases in PKC in RCs at 90 minutes. (11,24) Thus, while cell culture experiments discussed above indicate that resting zone PKC is regulated through a receptor other than the 1,25(OH) 2 …”

“…Previous studies (24) showed that analog 2a acts like 1,25(OH) 2 D 3 when incubated directly with GC MVs, causing a decrease in PKC activity, but has no effect on PKC in MVs isolated from RC cultures. In contrast, 2a stimulates PKC activity in PMs isolated from RC cells in a manner comparable to 24,25(OH) 2 D 3 , whereas 1,25(OH) 2 D 3 has no effect.…”

“…1␣-(hydroxymethyl)-3␤,25-dihydroxyvitamin D 3 , referred to as 2a, is an A-ring-modified analog of 1,25(OH) 2 D 3 synthesized as described previously. (24)(25)(26) The other two hybrid analogs, 1␣-(hydroxymethyl)-3␤-hydroxy-20-epi-22-oxa-26,27-dihomovitamin D 3 (MCW-Ya) and 1␤-(hydroxymethyl)-3␣-hydroxy-20-epi-22-oxa-26,27-dihomovitamin D 3 , referred to as here 3a and 3b, respectively, also have C-D side chain modifications and are diastereoisomers of each other. (25)(26)(27)(28) Previous studies showed that 2a exhibits 1,25(OH) 2 D 3 -like effects in cultures of GC cells and 24,25(OH) 2 D 3 -like effects in cultures of RC cells.…”

“…These events result in a rapid increase in protein kinase C (PKC) activity, (10) which is independent of new protein synthesis (11) and can be elicited by hybrid analogs of 1,25(OH) 2 D 3 , which exhibit <0.1% binding affinity for the VDR. (12) Another important, but less studied, metabolite of 25-hydroxyvitamin D 3 (24,25(OH) 2 D 3 ) has been considered a metabolic end product of 25(OH)D 3 without physiological importance. This is due to the fact that 25(OH)D 3 is preferentially metabolized to 24,25(OH) 2 D 3 when plasma levels of 1,25(OH) 2 D 3 are high and calcium levels are normal.…”