Evidence for distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in osteoblasts.
1 alpha,25-(OH)(2)D(3) 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)(2)D(3) also stimulates PKC in chondrocytes, but through other membrane mechanisms. This study examined the hypothesis that osteoblasts possess distinct membrane receptors for 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) that are involved in the activation of PKC and that receptor expression varies as a function of cell maturation state. 1 alpha,25-(OH)(2)D(3) 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)(2)D(3) 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 1 alpha,25-(OH)(2)D(3). 1 alpha,25-(OH)(2)D(3) 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)(2)D(3) on PKC was stereospecific; 24S,25-(OH)(2)D(3) had no effect. These results demonstrate that response to 1 alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) depends on osteoblast maturation state and suggest that specific and distinct membrane receptors are involved.
Boyan
Barbara D
BD
Department of Orthopaedics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA. BoyanB@UTHSCSA.edu
Bonewald
Lynda F
LF
Sylvia
Victor L
VL
Nemere
Ilka
I
Larsson
Dennis
D
Norman
Anthony W
AW
Rosser
Jennifer
J
Dean
David D
DD
Schwartz
Zvi
Z
D015650:24,25-Dihydroxyvitamin D 3 / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000378:metabolism / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism; D014407:Tumor Cells, Cultured
C031424:24,25-dihydroxycholecalciferol receptor; D018167:Receptors, Calcitriol; D015650:24,25-Dihydroxyvitamin D 3; D011493:Protein Kinase C; D002117:Calcitriol