The human paracellin-1 gene (hPCLN-1): renal epithelial cell-specific expression and regulation.
Tubular reabsorption of Mg2+ is mediated by the tight junction protein paracellin-1, which is encoded by the gene PCLN-1 (CLDN16) and exclusively expressed in the kidney. Tubular Mg2+ reclamation is modulated by many hormones and factors. The aim of this study was to define regulatory elements essential for renal tubular cell-specific expression of human PCLN-1 (hPCLN-1) and to explore the effect of Mg2+ transport modulators on the paracellin-1 gene promoter. Endogenous paracellin-1 mRNA and protein were detected in renal cell lines opossom kidney (OK), HEK293, and MDCT, but not in the fibroblast cell line NIH3T3. A 7.5-kb hPCLN-1 5'-flanking DNA sequence along with seven 5'-deletion products were cloned into luciferase reporter vectors and transiently transfected into the renal and nonrenal cells. The highest levels of luciferase activity resulted from transfection of a 5'-flanking 2.5-kb fragment (pJ2M). This activity was maximal in OK cells, was orientation dependent, and was absent in NIH3T3 cells. Mg2+ deprivation significantly increased pJ2M-driven activity in transfected OK cells, whereas Mg2+ load decreased it compared with conditions of normal Mg2+. Deletion analysis along with electrophoretic mobility-shift assay demonstrated that OK cells contain nuclear proteins, which bind a 70-bp region between -1633 and -1703 of major functional significance. Deleting this 70-bp segment, which contains a single peroxisome proliferator-response element (PPRE), or mutating the PPRE, caused a 60% reduction in luciferase activity. Stimulating the 70-bp sequence with 1,25(OH)2 vitamin D decreased luciferase activity by 52%. This effect of 1,25(OH)2 vitamin D was abolished in the absence of PPRE or in the presence of mutated PPRE. We conclude that the PPRE within this 70-bp DNA region may play a key role in the cell-specific and regulatory activity of the hPCLN-1 promoter. Ambient Mg2+ concentration and 1,25(OH)2 vitamin D may modulate paracellular, paracellin-1-mediated, Mg2+ transport at the transcriptional level. 1,25(OH)2 vitamin D exerts its activity on the hPCLN-1 promoter likely via the PPRE site.
Efrati
Edna
E
Laboratory of Developmental Nephrology, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Arsentiev-Rozenfeld
Julia
J
Zelikovic
Israel
I
D024506:5' Flanking Region; D000818:Animals; D018929:Cell Culture Techniques; D057167:Claudins; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D007684:Kidney Tubules / Q000502:physiology*; D008274:Magnesium / Q000493:pharmacokinetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D009893:Opossums; D011401:Promoter Regions, Genetic / Q000235:genetics; D019108:Tight Junctions; D014807:Vitamin D / Q000494:pharmacology
D057167:Claudins; D008565:Membrane Proteins; C502010:claudin 16; D014807:Vitamin D; D008274:Magnesium