Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells

Abstract

Cytokines could contribute to beta-cell damage in Type I diabetes mellitus. The radical nitric oxide, generated by the inducible form of nitric oxide synthase (iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction. In rat pancreatic islets and insulin-producing cell lines, interleukin-1β (IL-1β) induces expression of iNOS mRNA and increases NO production, an effect potentiated by interferon-β (IFN-γ). In human islet cells both IL-1β and IFN-γ are required for iNOS expression. We have shown previously that both the transcription factors nuclear factor-κB (NF-κB) and interferon 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 investigated the effects of cytokines on iNOS transcriptional regulation in both rat insulin-producing RINm5F cells and in primary FACS-purified rat beta cells. Transient transfection experiments with the 1.5-kb rat promoter region and 5' deletants of it showed that a distal region extending up to -1002 bp, and containing a distal and a proximal nuclear factor-κB (NF-κB) binding site, a γ-interferon activated site (GAS) and two adjacent IFN-stimulated response elements (ISRE), is required for IL-1β induction and IFN-γ potentiation of iNOS activation. Site-mutation analysis showed that both the distal and proximal NF-κB and GAS are necessary for IL-1β-induced iNOS expression in RINmSF cells. In these cells IFN-γ potentiation is mostly mediated by GAS and ISRE, suggesting a role for the IFN-γ-induced transcription factors Stat1α (which binds GAS) and IRF-1 (which binds ISRE), which may cooperate with NF-κB induced by IL-1β for iNOS activation. In primary beta cells both NF-κB binding sites are required for IL-1β-induced iNOS promoter activation. In these cells IFN-γ neither increased IL-1β-induced iNOS promoter activity nor iNOS mRNA expression but it induced a twofold increase in NO production. The present results unveiled the nature of the promoter binding sites necessary for iNOS expression in rodent beta cells. This information could be relevant for the development of new strategies aimed at preventing cytokine-induced iNOS expression and consequent beta-cell damage.