Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia.
Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia.
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DOI：
10.1371/journal.ppat.1005382
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发表时间：
2016-01
期刊：
影响因子：
6.7
通讯作者：
Crowe SE
中科院分区：
文献类型：
--
作者：
den Hartog G;Chattopadhyay R;Ablack A;Hall EH;Butcher LD;Bhattacharyya A;Eckmann L;Harris PR;Das S;Ernst PB;Crowe SE
在感染期间产生活性氧（ROS）是导致微生物杀灭的直接宿主防御。APE 1是由ROS诱导的多功能蛋白质，并且在诱导后，保护免受ROS介导的DNA损伤。Rac 1和NAPDH氧化酶（Nox 1）是感染后ROS产生的重要贡献者，与胃肠道上皮损伤相关。本研究的目的是确定在氧化应激过程中，APE 1是否调节Rac 1和Nox 1的功能。用幽门螺杆菌或沙门氏菌感染胃或结肠上皮细胞（野生型或抑制APE 1），并评估Rac 1和NADPH氧化酶依赖性超氧化物的产生。Rac 1和APE 1的相互作用进行了测量，通过免疫共沉淀，共聚焦显微镜和邻近连接试验（PLA）在细胞系或活检标本。显着更高水平的活性氧产生的APE 1缺陷的人胃和结肠细胞系和原代胃上皮细胞相比，控制细胞感染后，无论是胃或肠道病原体。H. pylori在所有细胞类型中激活Rac 1和Nox 1，但在APE 1抑制的细胞中激活更高。APE 1过表达降低H. pylori诱导的ROS生成、Rac 1活化和Nox 1表达。我们确定APE 1的作用是通过其N-末端赖氨酸残基与Rac 1相互作用介导的，从而抑制Nox 1的表达和ROS的产生。APE 1通过调节Rac 1和Nox 1，是细菌感染期间胃肠上皮中氧化应激的负调节剂。我们的研究结果暗示APE 1在新的分子相互作用，调节微生物感染引起的早期应激反应。 胃粘膜的幽门螺杆菌感染在很大程度上是终身的，导致免疫细胞的持续刺激。这导致产生活性氧物质（ROS），其产生以杀死细菌，但同时ROS调节宿主中的细胞事件。然而，ROS的长时间产生与DNA损伤有关，这最终可能导致癌症的发展。我们研究了胃和肠细胞中一种称为APE-1的分子，它在遇到ROS时被激活。我们的研究结果表明，APE 1限制了形成胃肠道衬里的细胞中ROS的产生。APE 1通过抑制分子Rac 1的活化来调节ROS的产生。APE 1抑制ROS产生发生在幽门螺杆菌感染胃细胞和沙门氏菌感染肠细胞后。这些数据表明，APE 1抑制消化道内部细胞中活性氧的产生。
Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections. Helicobacter pylori infection of the gastric mucosa is largely lifelong leading to continued stimulation of immune cells. This results in the generation of reactive oxygen species (ROS) which are produced to kill bacteria, but at the same time ROS regulate cellular events in the host. However, prolonged generation of ROS has been implicated in damage of DNA, which ultimately could lead to the development of cancer. We studied a molecule known as APE-1 in gastric and intestinal cells, which is activated upon encounter of ROS. Our results show that APE1 limits the production of ROS in cells that form the lining of the gastrointestinal tract. APE1 regulates ROS production by inhibiting activation of the molecule Rac1. Inhibition of ROS production by APE1 occurred after infection of gastric cells with Helicobacter pylori and after Salmonella infection of intestinal cells. These data demonstrate that APE1 inhibits production of ROS in cells that line the inside of the digestive tract.