STAT5 activation by human GH protects insulin-producing cells against interleukin-1beta, interferon-gamma and tumour necrosis factor-alpha-induced apoptosis independent of nitric oxide production

Abstract

The proinflammatory cytokines interleukin-1beta (IL-1beta), interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) are toxic to pancreatic beta-cells and are implicated in the pathogenesis of type 1 diabetes. We have previously found that GH and prolactin (PRL) stimulate both proliferation and insulin production in pancreatic beta-cells and rat insulin-producing INS-1 cells. Here we report that human (h) GH can prevent the apoptotic effects of IL-1beta, IFN-gamma and TNF-alpha in INS-1 and INS-1E cells. Using adenovirus-mediated gene transfer, we found that the anti-apoptotic effect of hGH is abrogated by expression of a dominant negative signal transducer and activator of transcription (STAT5) mutant in INS-1E cells. hGH and the cytotoxic cytokines was found to additively increase suppressor of cytokine signalling-3 mRNA expression after 4 h of exposure. In order to identify possible targets for the STAT5-mediated protection of INS-1E cells, we studied the effect of hGH on activation of the transcription factors STAT1 and nuclear factor-kappaB (NF-kappaB) by IFN-gamma and IL-1beta+TNF-alpha respectively. Gel retardation experiments showed that hGH affects neither IFN-gamma+TNF-alpha-induced STAT1 DNA binding nor IL-1beta and IFN-gamma+TNF-alpha-induced NFkappaB DNA binding. The lack of influence of hGH on cytokine-mediated activation of STAT1 and NFkappaB is in accordance with the finding that hGH had only a minor effect on cytokine-induced inducible nitric oxide synthase (iNOS) gene expression and in fact augmented the IL-1beta-stimulated nitric oxide production. As the anti-apoptotic Bcl-xL gene has been shown to harbour a STAT5-binding element we measured the expression of Bcl-xL as well as the pro-apoptotic Bax. We found that hGH increased the Bcl-xL/Bax ratio both in the absence and in the presence of cytotoxic cytokines. In conclusion, these results suggested that GH and PRL protect beta-cells against cytotoxic cytokines via STAT5-dependent mechanisms distal to iNOS activation possibly at the level of Bcl-xL.