PMID- 10352093 OWN - NLM STAT- MEDLINE DCOM- 19990728 LR - 20151119 IS - 0884-0431 (Print) IS - 0884-0431 (Linking) VI - 14 IP - 6 DP - 1999 Jun TI - Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3. PG - 856-67 AB - We have recently identified a membrane vitamin D receptor (mVDR) specific for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) 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)2D3-mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24, 25-dihydroxyvitamin D3 (24,25(OH)2D3-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)2D3 with <0.1% affinity for the nVDR (2a, 1alpha-CH2OH-3beta-25D3; 3a, 1alpha-CH2OH-3beta-20-epi-22-oxa-25D3; and 3b, 1beta-CH2OH-3alpha-20-epi-22-oxa-25D3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)2D3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [3H]1,25(OH)2D3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)2D3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)2D3 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 GC MVs. Like 1,25(OH)2D3, 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)2D3 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)2D3 to GC MVs was displaced completely with 1,25(OH)2D3 and analogs 2a, 3a, and 3b, but 24, 25(OH)2D3 only displaced 51% of the bound ligand. 24,25(OH)2D3 displaced 50% of [3H]1,25(OH)2D3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24, 25(OH)2D3 to recognition sites in RC MVs with a Kd of 69.2 fmol/ml and a Bmax of 52.6 fmol/mg of protein. Specific binding for 24, 25(OH)2D3 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)2D3 mVDR mediates the antiproliferative effect of 1,25(OH)2D3 on chondrocytes. It also mediates the 1,25(OH)2D3-dependent stimulation of PKC in GC cells, but not the 2a-dependent increase in RC PKC activity, indicating that 24,25(OH)2D3 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)2D3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)2D3 and 24,25(OH)2D3, their relative distribution is cell maturation-dependent. FAU - Pedrozo, H A AU - Pedrozo HA AD - Department of Orthopaedics, The University of Texas Health Science Center, San Antonio, Texas 78284-7774, USA. FAU - Schwartz, Z AU - Schwartz Z FAU - Rimes, S AU - Rimes S FAU - Sylvia, V L AU - Sylvia VL FAU - Nemere, I AU - Nemere I FAU - Posner, G H AU - Posner GH FAU - Dean, D D AU - Dean DD FAU - Boyan, B D AU - Boyan BD LA - eng GR - CA-44530/CA/NCI NIH HHS/United States GR - DE-05937/DE/NIDCR NIH HHS/United States GR - DE-08603/DE/NIDCR NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. PT - Research Support, U.S. Gov't, P.H.S. PL - England TA - J Bone Miner Res JT - Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research JID - 8610640 RN - 0 (24,25-dihydroxycholecalciferol receptor) RN - 0 (Receptors, Calcitriol) RN - 0 (Sulfur Radioisotopes) RN - 0 (Sulfuric Acid Esters) RN - 10028-17-8 (Tritium) RN - EC 2.7.11.13 (Protein Kinase C) RN - VC2W18DGKR (Thymidine) SB - IM MH - Animals MH - Cells, Cultured MH - Chondrocytes/*metabolism MH - Growth Plate/cytology/*metabolism MH - Male MH - Protein Kinase C/metabolism MH - Rats MH - Rats, Sprague-Dawley MH - Receptors, Calcitriol/*physiology MH - Sulfur Radioisotopes MH - Sulfuric Acid Esters/metabolism MH - Thymidine/metabolism MH - Tritium EDAT- 1999/06/03 00:00 MHDA- 1999/06/03 00:01 CRDT- 1999/06/03 00:00 PHST- 1999/06/03 00:00 [pubmed] PHST- 1999/06/03 00:01 [medline] PHST- 1999/06/03 00:00 [entrez] AID - 10.1359/jbmr.1999.14.6.856 [doi] PST - ppublish SO - J Bone Miner Res. 1999 Jun;14(6):856-67. doi: 10.1359/jbmr.1999.14.6.856.