Regulation of the 1b isoform of the plasma membrane calcium pump by 1,25-dihydroxyvitamin D3 in rat osteoblast-like cells.
The first isogene of the plasma membrane calcium pump (PMCA1) is expressed on the apical plasma membrane of osteoblasts, but its regulation by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has not been studied in this cell type. We studied 1,25(OH)2D3 effects on PMCA1 function, protein, messenger RNA (mRNA), and isoform expression in osteoblasts. Of seven rat and human immortalized osteoblast-like cell lines studied, PMCA1 mRNA expression was confirmed in all. Only ROS 17/2.8 cells expressed measurable PMCA1 protein by Western analysis. Immunocytochemistry indicated that PMCA1 was expressed predominantly on the plasma membrane of ROS 17/2.8 cells. The 1,25(OH)2D3 but not 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] treatment of confluent ROS 17/2.8 cells resulted in an approximate 3- to 5-fold dose-dependent increase in PMCA1 expression of message and protein as assessed by Western and Northern analysis and vesicular 45Ca uptake of membrane vesicles. 1,25(OH)2D3 had no effect on PMCA1 posttranscriptional splicing. The 1b isoform of PMCA was expressed under all experimental conditions. 1,25(OH)2D3 favored increased expression of the 5.5 kilobases (kb) over the 7.5-kb PMCA1b transcript, with a 2-fold proportional increase in the smaller transcript relative to the larger transcript evident at the highest dose of 1,25(OH)2D3 studied. The resultant proportional increase in the smaller 5.5-kb transcript may increase mRNA stability and account for the increase in PMCA1b protein and function with 1,25(OH)2D3. These data provide evidence for the role of 1,25(OH)2D3 and PMCA1b in the regulation of calcium transport in bone cells.
Department of Medicine, University of Western Australia, Perth, Australia.
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Glendenning
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Ratajczak
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IM
Dick
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RL
Prince
D015650:24,25-Dihydroxyvitamin D 3 / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002117:Calcitriol / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins; D002454:Cell Differentiation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects / Q000378:metabolism; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D053499:Plasma Membrane Calcium-Transporting ATPases; D012326:RNA Splicing; D012333:RNA, Messenger / Q000187:drug effects; D051381:Rats; D014158:Transcription, Genetic; D015854:Up-Regulation
C506952:ATP2B1 protein, human; D027682:Cation Transport Proteins; D016895:Culture Media, Serum-Free; D007527:Isoenzymes; D012333:RNA, Messenger; D015650:24,25-Dihydroxyvitamin D 3; D053499:Plasma Membrane Calcium-Transporting ATPases; C506955:Atp2b1 protein, rat; D000252:Calcium-Transporting ATPases; D002117:Calcitriol