Effects of the vitamin D3 analog 1 alpha, 25-dihydroxyvitamin D3-3 beta-bromoacetate on rat osteosarcoma cells: comparison with 1 alpha, 25-dihydroxyvitamin D3.
The actions of the hormonal form of vitamin D, 1 alpha, 25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2 D3], are mediated by both genomic and nongenomic mechanisms. Several vitamin D synthetic analogs have been developed in order to identify and characterize the site(s) of action of 1 alpha, 25-(OH)2D3 in many cell types including osteoblastic cells. We have compared the effects of 1 alpha, 25-(OH)2D3 and a novel 1 alpha, 25-(OH)2D3 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 receptor 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 10(-8) M) caused increased cell proliferation, as assessed by DNA synthesis and total cell counts. Northern blot analysis revealed that the mitogenic effect of both agents was accompanied by an increase in steady-state osteocalcin mRNA levels, but neither agent altered alkaline phosphatase 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 increase in osteocalcin secretion after 72, 96, 120, and 144 hr of treatment. Treatment of ROS 17/2.8 cells with the bromoester analog also resulted in a significant decrease in alkaline phosphatase-specific activity. To compare the nongenomic effects of 1 alpha, 25-(OH)2D3 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)2D3 and 1, 25-(OH)2-BE increased intracellular calcium within 5 min. Both the genomic and nongenomic actions of 1,25-(OH)2-BE are similar to those of 1 alpha,25-(OH)2D3, and since 1,25-(OH)2-BE has more potent effects on osteoblast function than the naturally occurring ligand due to more stable binding, this novel vitamin D analog may be useful in elucidating the structure and function of cellular vitamin D receptors.
Department of Orthopedics and Physical Rehabilitation, University of Massachusetts Medical Center, Worcester 01655, USA.
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Van Auken
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Buckley
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Ray
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Holick
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Baran
D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D009374:Neoplasms, Experimental / Q000378:metabolism / Q000473:pathology*; D015675:Osteocalcin / Q000378:metabolism; D012516:Osteosarcoma / Q000378:metabolism / Q000473:pathology*; D051381:Rats; D018167:Receptors, Calcitriol / Q000378:metabolism; D014407:Tumor Cells, Cultured
C104313:1,25-dihydroxyvitamin D3-3-bromoacetate; D018167:Receptors, Calcitriol; D015675:Osteocalcin; D002117:Calcitriol; D002118:Calcium