An octamer motif is required for activation of the inducible nitric oxide synthase promoter in pancreatic beta-cells

Abstract

Nitric oxide, generated by the inducible form of nitric oxide synthase ( iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction in type 1 diabetes mellitus. We have previously shown that cytokine-induced iNOS expression is cycloheximide (CHX) sensitive and requires nuclear factor-kappaB (NF-kappaB) activation. In the present study, we show that an octamer motif located 20 bp downstream of the proximal NF-kappaB binding site in the rat iNOS promoter is critical for IL-1beta and interferon-gamma induction of promoter activity in rat primary beta-cells and insulin-producing RINm5F cells. In gel shift assays, the octamer motif bound constitutively the transcription factor Oct1. Neither Oct1 nor NF-kappaB binding activities were blocked by CHX, suggesting that other factor(s) synthesized in response to IL-1beta contribute to iNOS promoter induction. The high mobility group (HMG)-I(Y) protein also bound the proximal iNOS promoter region. HMG-I(Y) binding was decreased in cells treated with CHX and HMG-I( Y) silencing by RNA interference reduced IL-1beta-induced iNOS promoter activity. These results suggest that Oct1, NF-kappaB, and HMG-I( Y) cooperate for transactivation of the iNOS promoter in pancreatic beta-cells.