Osteoblastic voltage-sensitive calcium channels mediate the actions of 1,25-dihydroxyvitamin D3 during bone remodeling

VSCCs open in response to external stimuli, including calcitropic hormones, which alter plasma membrane Ca2+ permeability. Ca2+ that enters cells through these channels serves a second messenger function, eliciting responses including secretion and gene expression changes. In osteoblasts, VSCCs serve as key regulators of Ca2+ permeability and are a major class of calcitropic hormone-sensitive plasma membrane Ca 2+ channels. VSCCs exist as complexes of polypeptide subunits comprised of a pore-forming α₁ subunit, an intracellular β subunit, a dimer of α₂ and δ subunits, and in some tissues, a γ subunit. Previous studies demonstrated that the activity of the α _1C subunit of the L-type VSCC rapidly modulates Ca2+ permeability in proliferating osteoblasts. In this dissertation, I identified which α₁ subunits are expressed in mouse osteoblastic cells and quantified expression changes following 1,25(OH)₂D₃ treatment. Of the nine α₁ subunits expressed in osteoblasts, there are decreases in expression for the α_1C and the T-type α _1G subunits, and an increase in the expression of the T-type α _1H subunit, following 24 hr 1,25(OH)₂D₃ treatment. Ca2+ influx assays corroborate the shift from an L-type dominant to a T-type dominant state. RT-PCR and immunohistochemistry assays measured mRNA and protein expression of auxiliary subunits. Results indicate that osteoblastic cells express multiple β subunits and α₂δ dimers, but no γ subunits. We propose a structure for the functional osteoblast VSCC consisting of α₁, β, α₂δ subunits and devoid of a γ subunit.; The balance of osteoblast and osteoclast activity maintains bone density through the activity of OPG and RANKL. In this dissertation, we measured changes in expression and secretion of OPG in MC3T3-E1 cells and calvarial organ cultures following treatment with 1,25(OH)₂D₃ and inhibitors to VSCCs and Ca2+-regulated signaling pathways. In both systems, OPG secretion significantly decreased following 24 hr 1,25(OH)₂D ₃ treatment, by inhibitors of L-type VSCCs and by CaMK, but not by inhibitors of PKA, MAPK, or other families of VSCCs. OPG secretion is abrogated by transfection with decoy CRE binding sites. My results suggest that OPG secretion is regulated through CaMK signaling maintained by the activity of the L-type VSCC and is mediated through the CRE-binding protein.