טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentRozenfeld Julia
SubjectThe Human Magnesium Channel (Paracellin-1) Gene: Renal
Epithelial Cell-Specific Expression and
Regulation
DepartmentDepartment of Medicine
Supervisors Professor Israel Zelikovic
Dr. Edna Efraty


Abstract

The kidney is the principal organ responsible for Mg++ balance. Tubular reabsorption of Mg++ is mediated by the Mg++ channel, paracellin-1, encoded by the gene PCLN-1 and exclusively expressed in the kidney. Tubular Mg++ reclamation is modulated by several 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 involvement of the hPCLN-1 promoter in the regulation of Mg++ transport.

Endogenous PCLN-1 mRNA was detected in renal cell lines, but not in non-renal control cells. A 7.5kb hPCLN-1 5’-flanking sequence along with seven 5’-deletion products were cloned into luciferase reporter vectors and transiently transfected into renal and non-renal cells. The highest levels of luciferase activity resulted from transfection of a 5’-flanking 2.5kb fragment (pJ2M). This activity was maximal in OK cells, was orientation-dependent, and was absent in non-renal cells. Deletion analysis and electrophoretic-mobility-shift-assay demonstrated that OK cells contain nuclear proteins, which bind to a 70bp region harboring a single peroxisome-proliferator-response-element (PPRE). Stimulating the 70bp with 1,25(OH)2VitD decreased, and with insulin increased, luciferase activity in a PPRE-dependent fashion. 1,25(OH)2VitD or cyclosporine, but not insulin, decreased luciferase activity in cells transfected with pJ2M. Using mice transgenic for the 2.5kb hPCLN-1 promoter region, the study demonstrated renal medullary-specific expression of the promoter.

We conclude that the PPRE within the 70bp DNA region may play a key role in the cell-specific and regulatory activity of the hPCLN-1 promoter. 1,25(OH)2VitD and cyclosporine may modulate paracellular, paracellin-1-mediated, Mg++ transport at the transcriptional level. The mice transgenic for the hPCLN-1 promoter may serve as an excellent model for investigating the effects of physiological and pathophysiological factors on this promoter.