The steroid 1alpha,25(OH)2 vitamin D3 (1,25D) is a systemic hormone with bone anabolic effects. 1,25D promotes the synthesis of bone matrix and its mineralization by acting on osteoblasts. This occurs via interaction with the vitamin D receptor (VDR) and modulation of gene transcription. In addition, 1,25D acts rapidly (sec-min) at the plasma membrane level, where it activates cytoplasmic signaling pathways and ion channel functions. I recently demonstrated that 1,25D-potentiation of chloride currents is coupled to a stimulation of secretion of matrix proteins in osteoblasts expressing a functional VDR. However, the precise molecular mechanisms of these 1,25D effects remain only partially understood. The long-term goal of this proposal is to elucidate 1,25D non-genomic mechanisms of bone formation in osteoblasts. My working hypothesis, which I propose as a new investigator, is that signal transduction triggered by 1,25D acting at a membrane-associated VDR leads to a non-genomic rapid exocytotic response, which is coupled to chloride channel activation in osteoblasts. This explains in part the anabolic effects of the hormone in bone. The first specific aim of this proposal investigates two parallel signal transduction pathways recruited by a membrane-associated VDR in osteoblasts: a) Galpha q/cAMP/PKA/CI- channel phosphorylation/exocytosis, and b) Galpha q/PLC/IP3/calcium/exocytosis. The second specific aim studies the osteoblastic CIC-3 gene, its protein product, and coupling to exocytosis. This will be studied in osteoblasts obtained from VDR WT and KO mice, the latter expressing a rachitic phenotype. Although the primary focus of this proposal is on basic research, the objective is to identify molecular targets in the treatment of bone pathologies characterized by decreased bone mass and mineralization. This typifies skeletal diseases such as osteoporosis and osteomalacia, respectively. Osteoporosis in particular affects a large sector of the aging American population and constitutes a significant economic burden.
