Ribozyme ablation demonstrates that the cardiac subtype of the voltage- sensitive calcium channel is the molecular transducer of 1,25- dihydroxyvitamin D3-stimulated calcium influx in osteoblastic cells

Abstract

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) stimulates transmembrane influx of Ca2+ through L-type voltage-sensitive Ca2+ channels (VSCCs) in ROS 17/2.8 osteoblastic cells. Ca2+ influx modulates osteoblastic activities including matrix deposition, hormone responsiveness, and Ca2+- dependent signaling. 1,25(OH)2D3 also regulates transcript levels encoding VSCCs. L-type VSCCs are multisubunit complexes composed of a central pore- forming α1 subunit and four additional subunits. The α1 subunit is encoded by one gene in a multimember family, defining tissue-specific subtypes. Osteoblasts synthesize two splice variants of the α(1C) cardiac VSCC subtype; however, the molecular identity of the 1,25(OH)2D3-regulated VSCC remained unknown. We created a ribozyme specifically cleaving α(1C) mRNA. To increase target ablation efficiency, the ribozyme was inserted into U1 small nuclear RNA (snRNA) by engineering the U1 snRNA expression cassette, conferring the ribozyme transcript with stabilizing stem-loops at both sides and the Sm binding site that facilitates localization into nucleoplasm. After transfection of ROS 17/2.8 cells with U1 ribozyme-encoding vector, stable clonal cells were selected in which the expression of α(1C) transcript and protein were strikingly reduced. Ca2+ influx assays in ribozyme transfectants showed selective attenuation of depolarization and 1,25(OH)2D3-regulated Ca2+ responses. We conclude that the cardiac subtype of the L-type VSCC is the transducer of stimulated Ca2+ influx in ROS 17/2.8 osteoblastic cells.