Contribution of a Nuclear Factor-KAPPA?Binding Site to Human Angiotensinogen Promoter Activity in Renal Proximal Tubular Cells

摘要

Intrarenal angiotensinogen (AGT) is expressed highly in renal proximal tubular cells (RPTCs) and contributes to the regulation of intrarenal angiotensin II levels. Inhibition of nuclear factor (NF)-KAPPAB suppressed human (h)AGT expression in human RPTCs. However, the presence and localization of an NF-KAPPABbinding site in the hAGT promoter region have not been determined. Therefore, this study was performed to demonstrate that an NF-KAPPABbinding site in the hAGT promoter region contributes to hAGT promoter activity in human RPTCs. The hAGT promoter region was cloned from -4358 to +122 and deletion analysis was performed. A possible NF-kappaB binding site was removed from the hAGT promoter region (M1) and mutated (M2). Human RPTCs were transfected, and hAGT promoter activity was determined by luciferase assay. The identity of DNA binding proteins from binding assays were determined by Western blot. Progressive 5'-end deletions demonstrated removal of a distal promoter element in hAGT_-2414/+122 reduced promoter activity (0.61 +-0.12, ratio to hAGT_-4358/+122). Inhibition of NF-kappa suppressed promoter activity in hAGT_-4358/+122 (0.51 +-0.14, ratio to control) and hAGT_-3681/+122 (0.48+-0.06, ratio to control) but not in the construct without the NF-kappaB binding site. Promoter activity was reduced in the domain mutants Ml (0.57+-0.08, ratio to hAGT_-4358/+122) and M2 (0.61+-0.16, ratio to hAGT_-4358/+122). DNA binding levels of NF-KAPPA?protein were reduced in Ml. These data demonstrate the functional importance of an NF-KAPPA?binding site in the hAGT promoter region, which contributes to hAGT promoter activity in human RPTCs.