The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding.
The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding.
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DOI：
10.1053/j.gastro.2011.01.004
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发表时间：
2011-04
期刊：
影响因子：
29.4
通讯作者：
Watson AJ
中科院分区：
文献类型：
--
作者：
Marchiando AM;Shen L;Graham WV;Edelblum KL;Duckworth CA;Guan Y;Montrose MH;Turner JR;Watson AJ
肿瘤坏死因子（TNF）增加肠上皮细胞脱落和凋亡，潜在地挑战胃肠腔和内部组织之间的屏障。我们研究了紧密连接重建和屏障维持的机制，以及细胞骨架调节分子在TNF诱导的脱落过程中的作用。我们研究了野生型和转基因小鼠，表达荧光标记的蛋白增强绿色荧光蛋白-闭合蛋白或单体红色荧光蛋白1-ZO-1。注射高剂量TNF（7.5µg，i. p.）后，进行剖腹手术，打开小肠段，通过视频共聚焦显微镜观察粘膜。药理学抑制剂和基因敲除小鼠被用来确定caspase激活，肌动球蛋白，和微管重塑和上皮脱落中的膜运输的作用。检测到的变化包括紧密连接蛋白ZO-1的重新分布和闭塞脱落细胞的侧膜。这些蛋白质最终在脱落细胞周围形成一个漏斗，定义了屏障保存的位点。紧密连接蛋白、E-钙粘蛋白、F-肌动蛋白、肌球蛋白II、Rho相关激酶（ROCK）和肌球蛋白轻链激酶（MLCK）也被募集到侧膜。启动脱落需要半胱天冬酶活性、肌球蛋白运动活性和微管，而完成该过程需要微丝重塑和ROCK、MLCK和发动蛋白II活性。在TNF诱导的细胞脱落过程中维持上皮屏障是一个复杂的过程，涉及微管、微丝和膜运输的整合以去除凋亡细胞。这一过程伴随着顶端连接复合物蛋白的重新分布，以在侧膜之间形成细胞间屏障并维持粘膜功能。
Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance, as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein–occludin or monomeric red fluorescent protein1–ZO-1. After injection of high doses of TNF (7.5µg, i.p.), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. Changes detected included redistribution of the tight junction proteins ZO-1 and occluding to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.
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