EFFECTS OF THE VITAMIN-D-3 ANALOG -ALPHA,25-DIHYDROXYVITAMIN-D-3-3-BETA-BROMOACETATE ON RAT OSTEOSARCOMA CELLS - COMPARISON WITH 1-ALPHA,25-DIHYDROXYVITAMIN D-3

The actions of the hormonal form of vitamin D, 1 alpha,25-dihydroxyvit amin D-3 [1 alpha,25-(OH)(2)D-3], are mediated by both genomic and non genomic mechanisms. Several vitamin D synthetic analogs have been deve loped in order to identify and characterize the site(s) of action of 1 alpha,25-(OH)(2)D-3 in many cell types including osteoblastic cells. We have compared the effects of 1 alpha,25-(OH)(2)D-3 and a novel 1 al pha,25-(OH)(2)D-3 bromoester analog (1,25-(OH)(2)-BE) that covalently binds to vitamin D receptors. Rat osteosarcoma cells that possess (ROS 17/2.8) or lack (ROS 24/1) the classic intracellular vitamin D recept or were studied to investigate genomic and nongenomic actions. in ROS 17/2.8 cells plated at low density, the two vitamin D compounds (1 x 1 0(-8) M) caused increased cell proliferation, as assessed by DNA synth esis and total cell counts. Northern blot analysis revealed that the m itogenic effect of both agents was accompanied by an increase in stead y-state osteocalcin mRNA levels, but neither agent altered alkaline ph osphatase mRNA levels in ROS 17/2.8 cells. ROS 17/2.8 cells responded to 1,25-(OH)(2)-BE but not the natural ligand with a significant incre ase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatmen t. Treatment of ROS 17/2.8 cells with the bromoester analog also resul ted in a significant decrease in alkaline phosphatase-specific activit y. To compare the nongenomic effects of 1 alpha,25-(OH)(2)D-3 and 1,25 -(OH)2-BE, intracellular calcium was measured in ROS 24/1 cells loaded with the fluorescent calcium indicator Quin 2. At 2 x 10(-8) M, both 1 alpha,25-(OH)(2)D-3 and 1,25-(OH)(2)-BE increased intracellular calc ium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH) (2)-BE are similar to those of 1 alpha,25-(OH)(2)D-3, and since 1,25-( OH)(2)-BE has more potent effects on osteoblast function than the natu rally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of ce llular vitamin D receptors. (C) 1996 Wiley-Liss, Inc.