Glendenning P, Ratajczak T, Prince R L, Garamszegi N, Strehler E E
Department of Medicine, University of Western Australia, Western Australia, Australia.
Biochem Biophys Res Commun. 2000 Nov 2;277(3):722-8. doi: 10.1006/bbrc.2000.3745.
The gene for plasma membrane calcium pump isoform1 (PMCA1) is expressed in calcium-transporting epithelia and bone mesenchymal cells and is upregulated to 1,25-(OH)(2)D(3) in those tissues. A candidate sequence for a vitamin D response element (VDRE) is present within a 1.7-kb promoter region of the human PMCA1 (hPMCA1) gene. We studied hPMCA1 promoter activity in MDBK and ROS 17/2.8 cell lines as PMCA1 mRNA expression is upregulated by 1,25-(OH)(2)D(3) in both. Structural analysis of the putative hPMCA1 VDRE sequence was performed using mobility shift analysis (EMSA) and nuclear extracts from COS-1 cells expressing human VDR (hVDR) and RXRalpha (hRXRalpha). 1,25-(OH)(2)D(3) induced transrepression of the entire 1.7-kb hPMCA1 promoter and of one promoter deletion construct in ROS 17/2.8 cells but not MDBK cells when assayed by luciferase reporter gene assays. Three additional hPMCA1 promoter deletion constructs were unaffected by 1,25-(OH)(2)D(3) in either cell line. While hVDR and hRXRalpha were capable of complexing with a rat osteocalcin DR3 VDRE, EMSA analysis of the potential VDRE from the hPMCA1 gene did not show interaction of either nuclear receptor. Our results indicate tissue-specific sensitivity of the promoter region of the hPMCA1 gene to direct transcriptional downregulation by 1,25-(OH)(2)D(3) and suggest that any positive regulatory VDRE must lie outside of the 1.7-kb core promoter.
质膜钙泵同工型1（PMCA1）基因在钙转运上皮细胞和骨间充质细胞中表达，并且在这些组织中被1,25 -（OH）₂D₃上调。人PMCA1（hPMCA1）基因1.7 kb启动子区域内存在维生素D反应元件（VDRE）的候选序列。我们在MDBK和ROS 17/2.8细胞系中研究了hPMCA1启动子活性，因为在这两种细胞系中PMCA1 mRNA表达均被1,25 -（OH）₂D₃上调。使用迁移率变动分析（EMSA）以及来自表达人维生素D受体（hVDR）和视黄酸X受体α（hRXRα）的COS - 1细胞的核提取物，对假定的hPMCA1 VDRE序列进行了结构分析。当通过荧光素酶报告基因检测进行分析时，1,25 -（OH）₂D₃在ROS 17/2.8细胞中诱导了整个1.7 kb hPMCA1启动子以及一个启动子缺失构建体的反式抑制，但在MDBK细胞中未诱导。另外三个hPMCA1启动子缺失构建体在两种细胞系中均不受1,25 -（OH）₂D₃影响。虽然hVDR和hRXRα能够与大鼠骨钙素DR3 VDRE形成复合物，但对hPMCA1基因潜在VDRE的EMSA分析未显示任何一种核受体的相互作用。我们的结果表明hPMCA1基因启动子区域对1,25 -（OH）₂D₃直接转录下调具有组织特异性敏感性，并表明任何正向调节的VDRE必定位于1.7 kb核心启动子之外。
