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J Biol Chem, Vol. 275, Issue 12, 8711-8718, March 24, 2000

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*

Riting LiuDagger §, Wei Li, Norman J. KarinDagger , Joel J. Bergh, Karen Adler-StorthzDagger , and Mary C. Farach-Carson||

From the Dagger  Department of Basic Sciences, University of Texas-Houston, Dental Branch, Houston, Texas 77030 and the  Department of Biological Sciences, University of Delaware, Newark, Delaware 19716

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 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)2D3-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. 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.

* This work was supported in part by National Institutes of Health Grant AR39273 (to William T. Butler and M. C. F.-C.) and a grant from NASA/Texas Medical Center (to M. C. F.-C. and N. J. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Biological Sciences, University of Delaware, Newark, DE 19716.

|| To whom correspondence should be addressed: Dept. of Biological Sciences, University of Delaware, 304 Wolf Hall, Newark, DE 19716. Tel.: 302-831-2277; Fax: 302-831-2281; E-mail: farachca@udel.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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