We have recently, identified a membrane vitamin D receptor (mVDR) specific for 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) and shown that it mediates the rapid activation of protein kinase C (PKC) in growth zone chondrocytes (GCs). In this study, we examine the role of the 1,25(OH)(2)D-3-mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24,25-dihydroxyvitamin D-3 (24,25(OH)(2)D-3-mVDR). Fourth-passage cultures of growth plate chondrocytes at two distinct stages of endochondral development, resting zone(RC) and growth zone (GC) cells, were used to assess the role of the mVDR in cell proliferation, PKC activation, and proteoglycan sulfation. To preclude the involvement of the nuclear vitamin D receptor (nVDR), we used hybrid analogs of 1,25(OH)(2)D-3 with <0.1% affinity for the nVDR (2a, 1 alpha-CH2OH-3 beta-25D(3); 3a, 1 alpha-CH2OH-3 beta-20-epi-22-oxa-25D(3); and 3b, 1 beta-CH2OH-3 alpha-20-epi-22-oxa-25D(3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)(2)D-3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [H-3]1,25(OH)(2)D-3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)(2)D-3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)(2)D-3 was also investigated in plasma membranes (PMs) from RC and GC cells and GC MVs. In addition, we examined the ability of Ab99 to block the stimulation of PKC by analog 2a in isolated RC PMs as well as the inhibition of PKC by analog 2a in Ge MVs. Like 1,25(OH)(2)D-3, analogs 2a, 3a, and 3b inhibit RC and GC cell proliferation. The effect was dose dependent and could be blocked by Ab99. In GC cells, PKC activity was stimulated maximally by analogs 2a and 3a and very modestly by 3b. The effect of 2a and 3a was similar to that of 1,25(OH)(2)D-3 and was blocked by Ab99, whereas the effect of 3b was unaffected by antibody. In contrast, 2a was the only analog that increased PKC activity in RC cells, and this effect was unaffected by Ab99. Analog 2a had no effect on proteoglycan sulfation in RC cells, whereas analogs 3a and 3b stimulated it and this was not blocked by Ab99. Binding of [3H]1,25(OH),D,to GC MVs was displaced completely with 1,25(OH)(2)D-3 and analogs 2a, 3a, and 3b, but 24,25(OH)(2)D-3 only displaced 51% of the bound ligand. 24,25(OH)(2)D-3 displaced 50% of [H-3]1,25(OH)(2)D-3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24,25(OH)(2)D-3 to recognition sites in RC MVs with a K-d of 69.2 fmol/ml and a B-max of 52.6 fmol/mg of protein. Specific binding for 24,25(OH)(2)D-3 was; also found in RC and GC PMs and GC MVs. GC membranes exhibited lower specific binding than RC membranes; MVs had greater specific binding than PMs in both cell types. 2a caused a dose-dependent increase in PKC activity of RC PMs that was unaffected by Ab99; it inhibited PKC activity in GC MVs, and this effect was blocked by Ab99. The results indicate that the 1,25(OH)(2)D-3 mVDR mediates the antiproliferative effect of 1,25(OH)(2)D-3 on chondrocytes, It also mediates the 1,25(OH)(2)D-3-dependent stimulation of PKC in GC cells, but not the 2a-dependent increase in RC PKC activity, indicating that 24,25(OH)(2)D-3 mediates its effects through a separate receptor. This is supported by the failure of Ab99 to block 2a-dependent stimulation of PKC in isolated PMs. The data demonstrate for the first time the presence of a specific 24,25(OH)(2)D-3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)(2)D-3 and 24,25(OH)(2)D-3, their relative distribution is cell maturation-dependent.
