Regulation of human angiotensinogen expression by nuclear factor-KB in human renal proximal tubular cells
Description
Intrarenal angiotensinogen (AGT), which is expressed highly in renal proximal tubular cells (RPTCs), plays an important role in the regulation of intrarenal angiotensin II levels. A nuclear factor-kappa B (NF-kappaB) binding site has been identified in the rat AGT promoter region and in vivo treatment with an NF-kappaB inhibitor attenuates the augmentation of AGT in kidneys of angiotensin II-infused rats. Furthermore, inhibition of NF-kappaB activity suppresses human AGT (hAGT) expression in human RPTCs. However, the presence of an NF-kappaB binding site in the hAGT promoter region has not been determined. Therefore, these studies were performed to identify an NF-kappaB binding site in the hAGT promoter region and demonstrate that activated NF-kappaB augments hAGT expression in human RPTCs The hAGT promoter region was cloned from -4,358 to +122 and deletion analysis was performed. Bioinformatics analysis of the clone indicated a 10-bp NF-kappaB binding site at -2,515 to -2,506. The 10-bp NF-kappaB binding site was removed from the hAGT promoter region in the hAGT promoter construct mutant-1 (M1) and mutated in mutant-2 (M2). The promoter activity in transfected human RPTCs was confirmed by dual luciferase assay. The identity of DNA binding proteins from binding assays was confirmed by western blot analysis. hAGT mRNA expression was determined by real-time quantitative RT-PCR. Progressive 5'-end deletions demonstrated that removal of a distal promoter element in hAGT_-2,414/+122 reduced hAGT promoter activity (0.61 +/- 0.12, ratio to hAGT_-4,358/+122). Treatment with an NF-kappaB inhibitor suppressed promoter activity in hAGT_-4,358/+122 (0.51 +/- 0.14, ratio to untreated) and hAGT_-3,681/+122 (0.48 +/- 0.06, ratio to untreated) but not in hAGT_-2,414/+122 which did not possess the NF-kappaB binding site. Promoter activity was reduced in mutants M1 (0.57 +/- 0.08, ratio to hAGT_-4,358/+122) and M2 (0.61 +/- 0.16, ratio to hAGT_-4,358/+122). DNA binding levels of NF-kappaB protein were reduced in M1. Furthermore, activation of NF-kappaB by IkappaB siRNA augmented hAGT mRNA expression (1.4 +/- 0.24, ratio to control) In conclusion, these studies demonstrate that the hAGT promoter region possesses a functional NF-kappaB binding site, which contributes to hAGT regulation, and that the activation of NF-kappaB augments hAGT expression in human RPTCs