Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor κB-mediated gene expression in insulin-producing INS-1E cells

Aims/hypothesis: The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1 beta, and by nitric oxide (NO)-dependent and - independent effector mechanisms. IL-1 beta activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NF kappa B) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase ( iNOS) and for IL-1 beta-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NF kappa B activation in beta cells. Materials and methods: The MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1 beta-induced iNOS production and kappa B inhibitor protein (I kappa B) degradation were examined by western blotting. NF kappa B DNA binding was investigated by electrophoretic mobility shift assay, while NF kappa B-induced gene transcription was evaluated by gene reporter assays. Results: Inhibition of the MAPKs did not affect I kappa B degradation or NF kappa B DNA binding. However, inhibition of ERK reduced NF kappa B-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NF kappa B complex seemed critical, as evaluated by amino acid mutation analysis. Conclusions/interpretation: ERK activity is required for NF kappa B-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NF kappa B. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.