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Non‐NF‐κB elements are required for full induction of the rat type II nitric oxide synthase in vascular smooth muscle cells
Author(s) -
Zhang Hanfang,
Teng Xingwu,
Snead Connie,
Catravas John D
Publication year - 2000
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0703284
Subject(s) - microbiology and biotechnology , nitric oxide synthase , nf κb , biology , vascular smooth muscle , promoter , transfection , binding site , nitric oxide , chemistry , gene expression , signal transduction , biochemistry , gene , endocrinology , smooth muscle
We have investigated the role of the NF‐κB binding sites and other promoter elements beyond NF‐κB in iNOS induction in rat vascular smooth muscle cells (SMC). Rat aortic SMC transfected with iNOS promoter constructs with either mutation or deletion of the downstream NF‐κB site exhibited about 50% reduction in promoter activity in response to a cytokine mixture, whereas either mutation or deletion of the upstream NF‐κB site reduced promoter activity by 90%, suggesting that the latter site is the most important, and that co‐existence of two NF‐κB sites is necessary for iNOS induction. Nuclear NF‐κB activity was robustly induced by TNF‐α. However, TNF‐α alone did not induce iNOS promoter activity, protein expression, or nitrite production, indicating that NF‐κB activation alone is not sufficient for iNOS induction. The construct up to −890 bp, containing the downstream NF‐κB site, exhibited little response to cytokines. The construct up to −1.0 kb, containing the two NF‐κB sites exhibited only 22% of full promoter activity. The regions −1001 to −1368 bp and −2 to −2.5 kb contributed an additional 43 and 22% promoter activity, respectively. Internal deletion or reversal of the orientation of −1001 to −1368 bp in the full promoter resulted in 40% reduction in promoter activity. These data suggest that the co‐existence of two NF‐κB sites is essential for core promoter activity, but that full induction of the rat SMC iNOS gene requires other elements located between −1.0 to −1.37 and −2.0 to −2.5 kb of the promoter.British Journal of Pharmacology (2000) 130 , 270–278; doi: 10.1038/sj.bjp.0703284

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