Osteoprotegerin expression and secretion are regulated by calcium influx through the L-type voltage-sensitive calcium channel

Our previous studies showed that 1,25-dihydroxyvitamin D-3 [1,25(OH)(2)D-3] modulates the activity of the Ca-V1.2 alpha-subunit of the L-type voltage-sensitive calcium channel (VSCC) by two temporally distinct mechanisms. First, 1,25(OH)(2)D-3 rapidly modulates local Ca2+ permeability in the plasma membrane of the proliferating osteoblast. Second, treatment with 1,25(OH)(2)D-3 reduces biosynthesis of Ca-V1.2 such that transcript levels are half of original levels after 24 h. Osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) provide important regulatory mechanisms for controlling osteoclastogenesis and Ca2+ homeostasis. Because they often control Ca2+-activated secretion, we investigated the possibility that L-type VSCCs might regulate basal OPG and RANKL secretion in osteoblasts. We also studied 1,25(OH)(2)D-3 effects on OPG and RANKL expression. To address this, we measured changes in expression and secretion of OPG and RANKL in MC3T3-E1 cells and calvarial organ cultures after treatment with 1,25(OH)(2)D-3, VSCC inhibitors, and inhibitors of Ca2+-regulated signaling. RANKL production was increased in calvarial cultures by 1,25(OH)(2)D-3 but was essentially undetectable in the medium of MC3T3-E1 cells. In contrast, OPG secretion in both systems was significantly reduced after 24 h treatment with 1,25(OH)(2)D-3, by inhibitors of L-type VSCCs and calmodulin-sensitive protein kinases but not by inhibitors of protein kinase A, MAPKs, or other families of VSCCs. OPG secretion was abrogated by transfection with decoy cAMP response element binding sites. Our results suggest that OPG secretion is regulated through calmodulin-sensitive protein kinase signaling that depends on the activity of the L-type VSCC and is mediated through the cAMP response element-binding protein.