1alpha,25-Dihydroxyvitamin D/3 (1alpha,25-(OH)2/D3) exerts its effects by both genomic and rapid (seconds to minutes) mechanisms. The genomic effects are mediated by an intracellular receptor, the vitamin D receptor (VDR). Binding of the lalpha,25-(OH)2/D3-VDR complex to vitamin D response elements in promoter regions of vitamin D regulated genes modifies the activity of the genes, resulting in altered protein synthesis. The rapid effects appear to be mediated by interaction with a membrane signaling system that does not involve the VDR. 1alpha,25- (OH)2/D3 exerts rapid effects in osteoblast-like cells that lack the VDR and binds specifically to their plasma membrane. Excess non-radioactive ligand inhibits binding to a membrane protein, molecular weight 33 kDa, pI 7.0. The physiologic role of the rapid effects is demonstrated by their modulation of hormone-induced increments in osteocalcin (OC) gene transcription in osteoblast-like cells. Inhibition of the rapid effects of 1alpha,25-(OH)2/D3 blocks the hormone-induced increases in OC mRNA transcription, and OC and osteopontin (OPN) mRNA steady-state levels. These observations form the basis of our hypothesis that the rapid effects of the steroid hormone, 1alpha,25-(OH)2/D3, are mediated by a signaling system which does not involve the classical VDR and play an important role in the hormone-induced regulation of osteoblasts. We will focus on 2 Specific Aims. (1) What is the identity of the plasma membrane receptor for 1alpha,25-(OH)2/D3? We will use preparative 2-D gel electrophoresis to obtain sufficient quantities of the protein to allow its sequencing and identification. Polyclonal antibodies will be prepared to identify representation of the peptide in tissues and for use as a ligand to block the rapid actions of 1alpha,25-(OH)2/D3. The receptor will be cloned and function addressed by overexpression and antisense strategies in intact cells. (2) How do the rapid effects modulate gene transcription? We will functionally identify and characterize the promoter region(s) of the OC gene that are responsive to the rapid effects of 1alpha,25-(OH)2/D3. OC promoter-CAT constructs in stably transfected ROS 17/2.8 cells will be used to define the region responsive to the rapid actions. Gel mobility shift assays, deoxyribonuclease-1 protection mapping (quantitative and qualitative), and methylation interference assays will further characterize the rapid effect response region. We will confirm that the promoter region is operative in another vitamin D responsive gene, OPN. The proposed studies are of significance because they will define the signaling system that mediates the rapid actions of 1alpha,25-(OH)2/3D and the promoter region(s) of vitamin D responsive genes that are functionally altered by the rapid actions. Understanding the mechanisms by which the rapid effects of 1alpha25-(OH)2/D3 modify osteoblast function will provide new avenues for targeted therapeutic approaches to disorders of bone formation.
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