Kitiphongspattana Kajorn, Khan Tarannum A, Ishii-Schrade Katrin, Roe Michael W, Philipson Louis H, Gaskins H Rex
Department of Medicine, The University of Chicago, Chicago, USA.
Am J Physiol Endocrinol Metab. 2007 Jun;292(6):E1543-54. doi: 10.1152/ajpendo.00620.2006. Epub 2007 Jan 30.
Higher requirements for disulfide bond formation in professional secretory cells may affect intracellular redox homeostasis, particularly during an endoplasmic reticulum (ER) stress response. To assess this hypothesis, we investigated the effects of the ER stress response on the major redox couple (GSH/GSSG), endogenous ROS production, expression of genes involved in ER oxidative protein folding, general antioxidant defense, and thiol metabolism by use of the well-validated MIN6 beta-cell as a model and mouse islets. The data revealed that glucose concentration-dependent decreases in the GSH/GSSG ratio were further decreased significantly by ER-derived oxidative stress induced by inhibiting ER-associated degradation with the specific proteasome inhibitor lactacystin (10 microM) in mouse islets. Notably, minimal cell death was observed during 12-h treatments. This was likely attributed to the upregulation of genes encoding the rate limiting enzyme for glutathione synthesis (gamma-glutamylcysteine ligase), as well as genes involved in antioxidant defense (glutathione peroxidase, peroxiredoxin-1) and ER protein folding (Grp78/BiP, PDI, Ero1). Gene expression and reporter assays with a NO synthase inhibitor (Nomega-nitro-L-arginine methyl ester, 1-10 mM) indicated that endogenous NO production was essential for the upregulation of several ER stress-responsive genes. Specifically, gel shift analyses demonstrate NO-independent binding of the transcription factor NF-E2-related factor to the antioxidant response element Gclc-ARE4 in MIN6 cells. However, endogenous NO production was necessary for activation of Gclc-ARE4-driven reporter gene expression. Together, these data reveal a distinct protective role for NO during the ER stress response, which helps to dissipate ROS and promote beta-cell survival.
专业分泌细胞中二硫键形成的更高要求可能会影响细胞内的氧化还原稳态，尤其是在内质网（ER）应激反应期间。为了评估这一假设，我们使用经过充分验证的MIN6β细胞作为模型和小鼠胰岛，研究了ER应激反应对主要氧化还原对（GSH/GSSG）、内源性ROS产生、参与ER氧化蛋白折叠的基因表达、一般抗氧化防御和硫醇代谢的影响。数据显示，在小鼠胰岛中，通过用特异性蛋白酶体抑制剂乳胞素（10μM）抑制ER相关降解诱导的ER衍生氧化应激，进一步显著降低了葡萄糖浓度依赖性的GSH/GSSG比值下降。值得注意的是，在12小时的处理过程中观察到最小程度的细胞死亡。这可能归因于编码谷胱甘肽合成限速酶（γ-谷氨酰半胱氨酸连接酶）的基因以及参与抗氧化防御（谷胱甘肽过氧化物酶、过氧化物酶1）和ER蛋白折叠（Grp78/BiP、PDI、Ero1）的基因的上调。使用一氧化氮合酶抑制剂（Nω-硝基-L-精氨酸甲酯，1-10 mM）进行的基因表达和报告基因分析表明，内源性一氧化氮的产生对于几种ER应激反应基因的上调至关重要。具体而言，凝胶迁移分析表明转录因子NF-E2相关因子与MIN6细胞中的抗氧化反应元件Gclc-ARE4的结合不依赖于一氧化氮。然而，内源性一氧化氮的产生对于激活Gclc-ARE4驱动的报告基因表达是必要的。总之，这些数据揭示了一氧化氮在ER应激反应期间的独特保护作用，这有助于消除ROS并促进β细胞存活。
