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

Barbara D. Boyan, Lynda F. Bonewald, Victor L. Sylvia, Ilka Nemere, Dennis Larsson, Anthony W. Norman, Jennifer Rosser, David D. Dean, Zvi Schwartz

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64 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)235-246
Number of pages12
JournalSteroids
Volume67
Issue number3-4
DOIs
Publication statusPublished - Feb 26 2002

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Keywords

  • 1α,25-(OH)D
  • 24R,25-(OH)D
  • Membrane receptors
  • Osteoblasts
  • Protein kinase C

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Endocrinology
  • Pharmacology
  • Clinical Biochemistry
  • Organic Chemistry

Cite this

Boyan, B. D., Bonewald, L. F., Sylvia, V. L., Nemere, I., Larsson, D., Norman, A. W., ... Schwartz, Z. (2002). Evidence for distinct membrane receptors for 1α,25-(OH)2D3 and 24R,25-(OH)2D3 in osteoblasts. Steroids, 67(3-4), 235-246. https://doi.org/10.1016/S0039-128X(01)00160-X