Cloning and sequencing of the proximal promoter of the rat iNOS gene: activation of NFκB is not sufficient for transcription of the iNOS gene in rat mesangial cells

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Abstract
It has previously been shown that expression of the inducible form of NO synthase (EC 1.14.23) is controlled at the transcriptional level and that induction of iNOS transcription is dependent on activation of transcription factors of the NFκB family. TNF-α and IL-1β synergistically stimulate iNOS transcription in rat glomerular mesangial cells. We have recently reported that endothelin-1 completely blocks cytokine-induced iNOS expression at the transcriptional level. To further investigate the molecular mechanisms and the role of NFκB in cytokine-elicited iNOS transcription, we cloned a 661 bp genomic rat DNA fragment, which contains 497 bp of the proximal iNOS promoter. An NFκB-binding site identical to that described for the murine sequence was identified and used for electrophoretic mobility shift experiments. We found that binding of NFκB is strongly induced in mesangial cells by both IL-1β and TNF-α. While endothelin-1 blocks cytokine-induced iNOS expression, it has no influence on the binding pattern of NFκB. We conclude from these data that transcription of iNOS in mesangial cells requires additional signals besides activation of NFκB.

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