REGULATION BY CYTOKINES OF THE INDUCIBLE NITRIC-OXIDE SYNTHASE PROMOTER IN INSULIN-PRODUCING CELLS

Cytokines could contribute to beta-cell damage in Type I diabetes mell itus. The radical nitric oxide, generated by the inducible form of nit ric oxide synthase (iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction. In rat pancreatic islets and insulin-producing cell lines, interleukin-1 beta (IL-1 beta) induces expression of iNOS mRNA and increases NO production, an effect potentiated by interferon -gamma (IFN-gamma). In human islet cells both IL-1 beta and IFN-gamma are required for iNOS expression. We have shown previously that both t he transcription factors nuclear factor-kappa B (NF-kappa B) and inter feron regulatory factor-1 (IRF-1) are activated by cytokines in rodent and human islets but there is no direct information on the regulation of the iNOS promoter in insulin-producing cells. We presently investi gated the effects of cytokines on iNOS transcriptional regulation in b oth rat insulin-producing RINm5F cells and in primary FACS-purified ra t beta cells. Transient transfection experiments with the 1.5-kb rat p romoter region and 5 ' deletants of it showed that a distal region ext ending up to -1002 bp, and containing a distal and a proximal nuclear factor-kappa B (NF-kappa B) binding site, a gamma-interferon activated site (GAS) and two adjacent IFN-stimulated response elements (ISRE), is required for IL-1 beta induction and IFN-gamma potentiation of iNOS activation. Site-mutation analysis showed that both the distal and pr oximal NF-kappa B and GAS are necessary for IL-1 beta-induced iNOS exp ression in RINm5F cells. In these cells IFN-gamma potentiation is most ly mediated by GAS and ISRE, suggesting a role for the IFN-gamma-induc ed transcription factors Stat1 alpha (which binds GAS) and IRF-1 (whic h binds ISRE), which may cooperate with NF-kappa B induced by IL-1 bet a for INOS activation. In primary beta cells both NF-kappa B binding s ites are required for IL-1 beta-induced iNOS promoter activation. In t hese cells IFN-gamma neither increased IL-1 beta-induced iNOS promoter activity nor INOS mRNA expression but it induced a twofold increase i n NO production. The present results unveiled the nature of the promot er binding sites necessary for iNOS expression in rodent beta cells. T his information could be relevant for the development of new strategie s aimed at preventing cytokine-induced iNOS expression and consequent beta-cell damage.