Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: Role of the microtubule cytoskeleton
Oxidant-induced intestinal barrier disruption and its prevention by growth factors in a human colonic cell line: Role of the microtubule cytoskeleton
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DOI：
10.1016/s0891-5849(00)00160-x
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发表时间：
2000-03-01
影响因子：
7.4
通讯作者：
Keshavarzian, A
中科院分区：
文献类型：
--
作者：
Banan, A;Choudhary, S;Keshavarzian, A
活性氧代谢产物（ROM）在炎症性肠病（IBD）的发炎粘膜中增加，并且可能导致这种疾病中肠屏障功能的丧失。生长因子（GF）具有保护作用。但破坏和保护机制仍然难以捉摸。在本研究中，我们假设微管（一个关键的细胞骨架元素）在ROM诱导的肠屏障功能障碍的分子机制和GF介导的保护中发挥关键作用。利用人结肠细胞系（Caco-2）的单层，我们评估了ROM（H2 O2或HOCl）在存在或不存在GF（表皮生长因子[EGF];转化生长因子-α [TGF-α]）的情况下对肠屏障功能、微管蛋白（微管结构蛋白）和微管稳定性的影响。还处理了两种高灵敏度的蛋白质免疫印迹单层：分级聚合的微管蛋白（S2;稳定性指数）;单体微管蛋白（SI;破坏指数），以检测微管蛋白的氧化和分解/组装。ROM暴露导致微管蛋白氧化显著增加，稳定的S2聚合微管蛋白减少，不稳定的S1单体微管蛋白增加。一致地，每种ROM以剂量依赖性方式损害微管细胞骨架并破坏屏障功能。GF预处理不仅增加了S2稳定的微管蛋白和减少微管蛋白的氧化，但也同时，防止破坏微管和屏障功能的损失暴露于ROM的单层。抗GF-受体的抗体和GF-受体酪氨酸激酶抑制剂废除GF保护，表明表皮生长因子受体（EGFR）信号通路的参与。正如预测的那样，秋水仙碱，微管组装的抑制剂，引起屏障功能障碍，并防止GF保护，而紫杉醇，微管稳定剂，模仿GF的保护作用。因此，组织和稳定的微管细胞骨架似乎是至关重要的氧化剂诱导的粘膜屏障功能障碍和保护肠屏障介导的GF。因此，微管可能是用于开发治疗IBD的药物的有用靶点。(C)2000 Elsevier Science Inc.
Reactive oxygen metabolites (ROM) are increased in the inflamed mucosa of inflammatory bowel disease (IBD) and may contribute to loss of intestinal barrier function iir this disorder. Growth factors (GF) are protective. But the mechanisms of disruption and protection remain elusive. In the present investigation, we hypothesized that the microtubules (a critical cytoskeletal element) play a key role in the molecular mechanism of intestinal barrier dysfunction induced by ROM and in GF-mediated protection. Utilizing monolayers of a human colonic cell line (Caco-2), we evaluated the effects of ROM (H2O2 or HOCl), in the presence or absence of GF (epidermal growth factor [EGF]; transforming growth factor-alpha [TGF-alpha]), on intestinal barrier function, tubulin (microtubule structural protein), and microtubule stability. Monolayers were also processed for two highly sensitive western immunoblots: fractionated polymerized tubulin (S2; an index of stability); monomeric tubulin (SI; an index of disruption) to detect the oxidation and disassembly/assembly of tubulin. ROM exposure led to a significant increase in the oxidation of tubulin, decrease in the stable S2 polymerized tubulin, and increase in the unstable S1 monomeric tubulin. In concert, each ROM in a dose dependent manner damaged the microtubule cytoskeleton and disrupted barrier function. GF pretreatment not only increased the S2 stable tubulin and decreased tubulin oxidation but also, concomitantly, prevented the disruption of microtubules and loss of barrier function in monolayers exposed to ROM. Antibody against the GF-receptor and inhibitors of GF-receptor tyrosine kinase abolished GF protection, indicating the involvement of epidermal growth factor receptor (EGFR) signaling pathway. As predicted, colchicine, an inhibitor of microtubule assembly, caused barrier dysfunction and prevented GF protection whereas taxol, a microtubule-stabilizing agent, mimicked the protective effects of GF. Thus, organization and stability of the microtubule cytoskeleton appears to be critical to both oxidant-induced mucosal barrier dysfunction and protection of intestinal barrier mediated by GF. Therefore, microtubules may be useful targets for development of drugs for the treatment of IBD. (C) 2000 Elsevier Science Inc.