The human paracellin-1 gene (hPCLN-1): renal epithelial cell-specific expression and regulation

Abstract

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)2vitamin D decreased luciferase activity by 52%. This effect of 1,25(OH)2vitamin 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)2vitamin D may modulate paracellular, paracellin-1-mediated, Mg2+transport at the transcriptional level. 1,25(OH)2vitamin D exerts its activity on the hPCLN-1 promoter likely via the PPRE site.