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

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 NFkappaB family. TNF-alpha and IL-1beta 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 NFkappaB in cytokine-elicited iNOS transcription, we cloned a 661 bp genomic rat DNA fragment, which contains 497 bp of the proximal iNOS promoter. An NFkappaB-binding site identical to that described for the murine sequence was identified and used for electrophoretic mobility shift experiments. We found that binding of NFkappaB is strongly induced in mesangial cells by both IL-1beta and TNF-alpha. While endothelin-1 blocks cytokine-induced iNOS expression, it has no influence on the binding pattern of NFkappaB. We conclude from these data that transcription of iNOS in mesangial cells requires additional signals besides activation of NFkappaB.