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Nongenomic Actions of 1,25-Dihydroxyvitamin D₃ in Rat Osteosarcoma Cells: Structure-Function Studies Using Ligand Analogs^*

Department of Biological Chemistry, University of Texas Dental Branch (M.C.F.-C) Houston, Texas 77030
Division of Biomedical Sciences and Department of Biochemistry, University of California (I.S., A. W.N.) Riverside, California 92521

Address all correspondence and requests for reprints to: Dr. Mary C. Farach-Carson, Department of Biological Chemistry, University of Texas Dental Branch, Houston, Texas 77030.

Abstract

Osteoblast-like osteosarcoma cells (ROS 17/2.8) display a rapid transmembrane influx of extracellular calcium after stimulation by 1,25-dihydroxyvitamin D₃ [l,25-(OH)₂D₃] that is mediated largely by the opening of voltage-gated calcium channels. These cells also constitute ely express high numbers (>18,000/cell) of nuclear receptors for this seco-steroid hormone that are involved in the modulation of genomic activity in the osteoblast and in the up-regulation of transcript ion of osteoblast- specific genes such as osteocalcin. The objective of this study was to determine the structural hierarchy of vitamin D₃ analogs with regard to their efficacy as molecular transducers of the genomic and nongenomic pathways that are activated upon treatment of osteoblasts with 1,25-(0H)₂D₃. To test the structural features of the agonist required for initiation of these distinct pathways, a series of ligand analogs and naturally occurring metabolites of 1,25-(OH)₂D₃ were used that contain Aring, D-ring, and side-chain modifications. The abilities of these analogs/metabolites to 1) bind to nuclear receptors and 2) stimulate transmembrane calcium influx were measured. Several analogs (25-hydroxy-16-ene-23-yne-D₃ and 25-hydroxy-23-yne D₃) were found to stimulate Ca²⁺ channel opening, but bind only poorly to the l,25-(OH)₂D₃ nuclear receptor. Conversely, other analogs (l,24-dihydroxy-22-ene-24-cyclopropyl D₃ and 1,25-dihydroxy- 16-ene-23-yne,26,27 F₆-D₃) were found to bind very well to the nuclear receptor, but displayed little or no activity in opening Ca²⁺ channels. Pertusssis toxin, which interferes with coupling of certain ligand-gated receptors to ion channels, failed to block the activation of calcium channels by 1,25-(OH)₂D₃ or active agonist analogs. Our results indicate that there are likely to be distinct nuclear and plasma membrane-associated forms of the l,25-(OH)₂D₃ receptor that are involved in genomic and nongenomic activation of osteoblast activity, respectively. The membrane-associated receptors do not appear to be coupled to pertussis toxin-sensitive G-proteins. (Endocrinology 129: 1876–1884,1991)

Footnotes

^* This work was supported by grants from the Muscular Dystrophy Association, BRSG Grant 2S07-RR-05970 (to M.C.F.-C), and USPHS Grant DK-09012-28 (to A.W.N.). Additional funds for this research were provided by USPHS Grant AR-39273 (to W.T.B.).

Received June 14, 1991.

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