Ribozyme ablation demonstrates that the cardiac subtype of the voltage-sensitive calcium channel is the molecular transducer of 1, 25-dihydroxyvitamin D(3)-stimulated calcium influx in osteoblastic cells.
1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) stimulates transmembrane influx of Ca(2+) through L-type voltage-sensitive Ca(2+) channels (VSCCs) in ROS 17/2.8 osteoblastic cells. Ca(2+) influx modulates osteoblastic activities including matrix deposition, hormone responsiveness, and Ca(2+)-dependent signaling. 1, 25(OH)(2)D(3) also regulates transcript levels encoding VSCCs. L-type VSCCs are multisubunit complexes composed of a central pore-forming alpha(1) subunit and four additional subunits. The alpha(1) subunit is encoded by one gene in a multimember family, defining tissue-specific subtypes. Osteoblasts synthesize two splice variants of the alpha(1C) cardiac VSCC subtype; however, the molecular identity of the 1,25(OH)(2)D(3)-regulated VSCC remained unknown. We created a ribozyme specifically cleaving alpha(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 alpha(1C) transcript and protein were strikingly reduced. Ca(2+) influx assays in ribozyme transfectants showed selective attenuation of depolarization and 1, 25(OH)(2)D(3)-regulated Ca(2+) responses. We conclude that the cardiac subtype of the L-type VSCC is the transducer of stimulated Ca(2+) influx in ROS 17/2.8 osteoblastic cells.
Liu
R
R
Department of Basic Sciences, University of Texas-Houston, Dental Branch, Houston, Texas 77030, USA.
Li
W
W
Karin
N J
NJ
Bergh
J J
JJ
Adler-Storthz
K
K
Farach-Carson
M C
MC
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010006:Osteoblasts / Q000378:metabolism*; D016337:RNA, Catalytic / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D020746:Calcium Channels, L-Type; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D002117:Calcitriol; D002118:Calcium