MAR interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide
MAR interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide
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DOI：
10.1042/bj20050959
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发表时间：
2006-01-01
影响因子：
4.1
通讯作者：
Rao, R
中科院分区：
文献类型：
--
作者：
Basuroy, S;Seth, A;Rao, R
丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）途径是参与表皮生长因子（epithelial growth factor，EGF）受体介导的细胞生长和分化的主要细胞内信号传导途径。在Caco-2细胞单层中，研究了MAPK活性在EGF介导的保护TJ（紧密连接）免受H2 O2损伤的机制中的新功能。EGF介导的H2 O2诱导的细胞旁通透性增加的预防与H2 O2诱导的Tyr-磷酸化，Thr-去磷酸化和occludin和ZO-1（zonula occludin-1）的细胞再分布的预防有关。EGF也防止过氧化氢诱导的破坏肌动蛋白细胞骨架和解离occludin和ZO-1从肌动蛋白丰富的洗涤剂不溶性馏分。MEK（MAPK/ERK激酶，其中ERK代表细胞外信号相关激酶）抑制剂PD 98059和U 0126完全阻断EGF对TJ的这些保护作用。EGF迅速增加洗涤剂可溶性组分中磷酸化MEK（p-MEK）和洗涤剂不溶性组分中磷酸化ERK（p-ERK）的水平。将p-ERK与occludin共定位并共免疫沉淀。GST（glutathione S-transferase）pull-down检测显示闭合蛋白的C-末端尾与Caco-2细胞提取物中的p-ERK结合。使用重组蛋白的成对结合研究表明，ERK 1直接与闭合蛋白的C-末端尾部相互作用。因此，本研究表明，ERK与C-末端区域的occludin和介导的预防H2 O2诱导的破坏TJs的EGF相互作用。
The MAPK (mitogen-activated protein kinase) pathway is a major intracellular signalling pathway involved in EGF (epithelial growth factor) receptor-mediated cell growth and differentiation. A novel function of MAPK activity in the mechanism of EGF-mediated protection of TJs (tight junctions) from H2O2 was examined in Caco-2 cell monolayers. EGF-mediated prevention of H2O2-induced increase in paracellular permeability was associated with the prevention of H2O2-induced Tyr-phosphorylation, Thr-dephosphorylation and cellular redistribution of occludin and ZO-1 (zonula occludin-1). EGF also prevented H2O2-induced disruption of the actin cytoskeleton and the dissociation of occludin and ZO-1 from the actin-rich detergent-insoluble fractions. MEK (MAPK/ERK kinase, where ERK stands for extracellular signal related kinase) inhibitors, PD98059 and U0126, completely blocked these protective effects of EGF on TJs. EGF rapidly increased the levels of phosphorylated MEK (p-MEK) in detergent-soluble fractions and phosphorylated ERK (p-ERK) in detergent-insoluble fractions. p-ERK was colocalized and co-immunoprecipitated with occludin. GST (glutathione S-transferase) pull-down assay showed that the C-terminal tail of occludin binds to p-ERK in Caco-2 cell extracts. Pairwise binding studies using recombinant proteins demonstrated that ERK1 directly interacts with the C-terminal tail of occludin. Therefore the present study shows that ERK interacts with the C-terminal region of occludin and mediates the prevention of H2O2-induced disruption of TJs by EGF.