Par-3 modulates intestinal epithelial barrier function through regulating intracellular trafficking of occludin and myosin light chain phosphorylation
Par-3 modulates intestinal epithelial barrier function through regulating intracellular trafficking of occludin and myosin light chain phosphorylation
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DOI：
10.1007/s00535-015-1066-z
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发表时间：
2015-03
影响因子：
6.3
通讯作者：
Min Yu;Songwei Yang;Y. Qiu;Guo-qing Chen;Wensheng Wang;Chao Xu;Wenqian Cai;Lihua Sun;W. Xiao;Hua Yang
中科院分区：
文献类型：
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作者：
Min Yu;Songwei Yang;Y. Qiu;Guo-qing Chen;Wensheng Wang;Chao Xu;Wenqian Cai;Lihua Sun;W. Xiao;Hua Yang
研究背景紧密连接在维持肠屏障功能中起重要作用。分配缺陷蛋白3（Partitioning-deficient protein 3，Par-3）可通过调节紧密连接组装和细胞极性来调节肠屏障功能。然而，其机制仍然没有完全understood.MethodsAdult C57 BL/6小鼠用葡聚糖硫酸钠治疗7天，收获结肠段用于Par-3的免疫荧光染色。用肿瘤坏死因子α（TNF-α）处理Caco-2肠上皮细胞24 h，Western blot和免疫荧光法检测Par-3的表达。此外，用Par-3小干扰RNA处理Caco-2细胞，通过Western印迹分析和免疫荧光研究紧密连接蛋白的表达和亚细胞定位的改变。结果Par-3在葡聚糖硫酸钠诱导的小鼠急性炎症和TNF-α处理的Caco-2细胞中表达下调。通过小干扰RNA消除Par-3表达延迟了Caco-2细胞中肠上皮屏障的发育。这种调节是由于紧密连接蛋白occludin的重新分布，而不是改变了紧密连接蛋白的总水平。Par-3沉默阻断了occludin从高尔基复合体或通过高尔基复合体向细胞表面的运输，并显著诱导occludin在高尔基复合体处积累。重要的是，Par-3可以与MLC相互作用，Par-3的缺失上调MLC激酶的表达和MLC磷酸化，从而导致肠上皮屏障功能障碍.ConclusionsThese结果表明，Par-3通过调节occludin的传递以及抑制MLC磷酸化在肠屏障功能的调节中起着重要作用。
BackgroundTight junctions play a critical role in the maintenance of intestinal barrier function. Partitioning-defective protein 3 (Par-3) can regulate intestinal barrier function through the modulation of tight junction assembly and cell polarity. However, the mechanisms are still not fully understood.MethodsAdult C57BL/6 mice were treated with dextran sulfate sodium for 7 days, and segments of colon were harvested for immunofluorescent staining of Par-3. Caco-2 intestinal epithelial cells were treated with tumor necrosis factor α (TNF-α) for 24 h, and Par-3 expression was detected by Western blot analysis and immunofluorescence. Additionally, Caco-2 cells were treated with Par-3 small interfering RNA, and altered expression and subcellular localization of tight junction proteins were studied by Western blot analysis and immunofluorescence. Furthermore, the interaction between Par-3 and myosin light chain (MLC) was detected by immunoprecipitation.ResultsPar-3 was downregulated in murine dextran sulfate sodium induced acute inflammation and TNF-α-treated Caco-2 cells. Depletion of Par-3 expression by small interfering RNA delayed intestinal epithelial barrier development in Caco-2 cells. This regulation was due to the redistribution of the tight junction protein occludin rather than the altered total levels of tight junction proteins. Par-3 silencing blocked the trafficking of occludin from or through the Golgi complex to the cell surface, and dramatically induced occludin accumulated at the Golgi complex. Importantly, Par-3 can interact with MLC, and loss of Par-3 upregulated MLC kinase expression and MLC phosphorylation, which contributed to intestinal epithelial barrier dysfunction.ConclusionsThese results indicate that Par-3 plays an important role in the modulation of intestinal barrier function by regulating delivery of occludin as well as suppression of MLC phosphorylation.