Key result
Removal or mutation of an NF-κB binding site in the human angiotensinogen promoter region reduced promoter activity in human renal proximal tubular cells.
An NF-κB binding site in the hAGT promoter region is functionally important for hAGT promoter activity in human renal proximal tubular cells, contributing to the regulation of intrarenal angiotensin II.
Should not alter hypertension management; hypothesis-generating for NF-κB regulation of intrarenal angiotensinogen, pending in vivo confirmation.
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)-κB suppressed human (h)AGT expression in human RPTCs. However, the presence and localization of an NF-κB binding site in the hAGT promoter region have not been determined. Therefore, this study was performed to demonstrate that an NF-κB binding 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-κB 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-κB 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-κB binding site. Promoter activity was reduced in the domain mutants M1 (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-κB protein were reduced in M1. These data demonstrate the functional importance of an NF-κB binding site in the hAGT promoter region, which contributes to hAGT promoter activity in human RPTCs.
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Acres et al. (2011) studied this question. Deletion or mutation of NF-κB binding site in hAGT promoter vs. Wild-type hAGT promoter was evaluated on hAGT promoter activity. Removal or mutation of an NF-κB binding site in the human angiotensinogen promoter region reduced promoter activity in human renal proximal tubular cells.
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