Role of the mitogen-activated protein kinases in cytokine-mediated inhibition of insulin gene expression

Background: Following islet transplant, inflammatory cells in the vicinity of the transplant graft elaborate cytokines that contribute to islet graft dysfunction. To better understand the mechanism for this effect of cytokines on graft function, we examined the impact of cytokines on intracellular signaling and insulin promoter activity in pancreatic beta cells. Methods: Two pancreatic beta cell lines, RINm5F and MIN6 cells, were transfected with a rat insulin promoter (RIP) luciferase fusion gene and treated with a combination of cytokines, including 5 ng/mL interleukin-1β + 10 ng/mL tumor necrosis factor α + 25 ng/mL interferon-γ. The effect of cytokines on beta cell transcription factors and signaling pathways was analyzed by real-time reverse transcriptase polymerase chain reaction and Western blotting. Results: Treatment for 48 hours with the combination of cytokines decreased insulin 1 messenger ribonucleic acid (mRNA) levels to 51% and 38% and RIP1 activity to 16% and 30% of control levels in RINm5F and MIN6 cells, respectively. The level of mRNAs encoding transcription factors important for insulin gene expression and beta cell function, including MafA, PDX-1, Nkx6.1, and Pax6, was also decreased by cytokine treatment. Cytokines increased phosphorylation of ERK and c-Jun NH₂-terminal kinase (JNK) in RINm5F and MIN6 cells but had no effect on p38 kinase phosphorylation. Neither JNK nor ERK inhibition had a significant effect on cytokine-mediated inhibition of RIP1 activity. Conclusion: Beyond modulating beta cell survival, cytokines inhibit insulin promoter activity, which likely contributes to islet dysfunction following islet transplant. This effect appears to be mediated, in part, via altered expression of transcription factors important for insulin gene expression.