Jensen Janne, Galsgaard Elisabeth D, Karlsen Allan E, Lee Ying C, Nielsen Jens H
Department of Medical Biochemistry and Genetics, University of Copenhagen, Panum Institute, Blegdamsvej 3, DK 2200 Copenhagen N, Denmark.
J Endocrinol. 2005 Oct;187(1):25-36. doi: 10.1677/joe.1.06086.
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.
促炎细胞因子白细胞介素-1β（IL-1β）、干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）对胰腺β细胞具有毒性，并与1型糖尿病的发病机制有关。我们之前发现，生长激素（GH）和催乳素（PRL）可刺激胰腺β细胞和大鼠胰岛素分泌细胞INS-1细胞的增殖及胰岛素生成。在此我们报告，人（h）GH可预防IL-1β、IFN-γ和TNF-α对INS-1和INS-1E细胞的凋亡作用。利用腺病毒介导的基因转移，我们发现INS-1E细胞中显性负性信号转导子和转录激活子（STAT5）突变体的表达可消除hGH的抗凋亡作用。发现hGH与细胞毒性细胞因子在作用4小时后可相加性增加细胞因子信号抑制因子3 mRNA的表达。为了确定STAT5介导的INS-1E细胞保护作用的可能靶点，我们分别研究了hGH对IFN-γ和IL-1β+TNF-α激活转录因子STAT1和核因子-κB（NF-κB）的影响。凝胶阻滞实验表明，hGH既不影响IFN-γ+TNF-α诱导的STAT1 DNA结合，也不影响IL-1β和IFN-γ+TNF-α诱导的NF-κB DNA结合。hGH对细胞因子介导的STAT1和NF-κB激活缺乏影响，这与hGH对细胞因子诱导的诱导型一氧化氮合酶（iNOS）基因表达只有轻微影响且实际上增强了IL-1β刺激的一氧化氮生成这一发现一致。由于抗凋亡的Bcl-xL基因已被证明含有一个STAT5结合元件，我们检测了Bcl-xL以及促凋亡的Bax的表达。我们发现，无论有无细胞毒性细胞因子存在，hGH均可增加Bcl-xL/Bax比值。总之，这些结果表明，GH和PRL通过可能在Bcl-xL水平上iNOS激活远端的STAT5依赖性机制保护β细胞免受细胞毒性细胞因子的损伤。
