Evidence for distinct membrane receptors for 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ in osteoblasts

1α,25-(OH)₂D₃ exerts its effects on chondrocytes and enterocytes via nuclear receptors (1,25-nVDR) and a separate membrane receptor (1,25-mVDR) that activates protein kinase C (PKC). 24R,25-(OH)₂D₃ also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1α,25-(OH)₂D₃ stimulated PKC in well differentiated (UMR-106, MC-3T3-E1) and moderately differentiated (ROS 17/2.8) osteoblast-like cells, and in cultures of fetal rat calvarial (FRC) cells and 2T3 cells treated with rhBMP-2 to promote differentiation. 24R,25-(OH)₂D₃ stimulated PKC in FRC and 2T3 cultures that had not been treated to induce differentiation, and in ROS 17/2.8 cells. MG63 cells, a relatively undifferentiated osteoblast-like cell line, had no response to either metabolite. Ab99, a polyclonal antibody generated to the chick enterocyte 1,25-mVDR, but not a specific antibody to the 1,25-nVDR, inhibited response to 1α,25-(OH)₂D₃. 1α,25-(OH)₂D₃ exhibited specific binding to plasma membrane preparations from cells demonstrating a PKC response to this metabolite that is typical of positive cooperativity. Western blots of these membrane proteins reacted with Ab99, and the Ab99-positive protein had an Mr of 64 kDa. There was no cross-reaction with antibodies to the C- or N-terminus of annexin II. The effect of 24,25-(OH)₂D₃ on PKC was stereospecific; 24S,25-(OH)₂D₃ had no effect. These results demonstrate that response to 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.