Samarin Stanislav N, Ivanov Andrei I, Flatau Gilles, Parkos Charles A, Nusrat Asma
Epithelial Pathobiology Research Unit, Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA.
Mol Biol Cell. 2007 Sep;18(9):3429-39. doi: 10.1091/mbc.e07-04-0315. Epub 2007 Jun 27.
Apical junctional complex (AJC) plays a vital role in regulation of epithelial barrier function. Disassembly of the AJC is observed in diverse physiological and pathological states; however, mechanisms governing this process are not well understood. We previously reported that the AJC disassembly is driven by the formation of apical contractile acto-myosin rings. In the present study, we analyzed the signaling pathways regulating acto-myosin-dependent disruption of AJC by using a model of extracellular calcium depletion. Pharmacological inhibition analysis revealed a critical role of Rho-associated kinase (ROCK) in AJC disassembly in calcium-depleted epithelial cells. Furthermore, small interfering RNA (siRNA)-mediated knockdown of ROCK-II, but not ROCK-I, attenuated the disruption of the AJC. Interestingly, AJC disassembly was not dependent on myosin light chain kinase and myosin phosphatase. Calcium depletion resulted in activation of Rho GTPase and transient colocalization of Rho with internalized AJC proteins. Pharmacological inhibition of Rho prevented AJC disassembly. Additionally, Rho guanine nucleotide exchange factor (GEF)-H1 translocated to contractile F-actin rings after calcium depletion, and siRNA-mediated depletion of GEF-H1 inhibited AJC disassembly. Thus, our findings demonstrate a central role of the GEF-H1/Rho/ROCK-II signaling pathway in the disassembly of AJC in epithelial cells.
顶端连接复合体（AJC）在调节上皮屏障功能中起着至关重要的作用。在多种生理和病理状态下均观察到AJC的解体；然而，控制这一过程的机制尚未完全清楚。我们之前报道过，AJC的解体是由顶端收缩性肌动蛋白-肌球蛋白环的形成所驱动的。在本研究中，我们利用细胞外钙耗竭模型分析了调节肌动蛋白-肌球蛋白依赖性AJC破坏的信号通路。药理学抑制分析显示，Rho相关激酶（ROCK）在钙耗竭的上皮细胞中AJC解体过程中起关键作用。此外，小干扰RNA（siRNA）介导的ROCK-II而非ROCK-I的敲低减弱了AJC的破坏。有趣的是，AJC的解体并不依赖于肌球蛋白轻链激酶和肌球蛋白磷酸酶。钙耗竭导致Rho GTP酶激活以及Rho与内化的AJC蛋白短暂共定位。对Rho的药理学抑制可防止AJC解体。此外，钙耗竭后Rho鸟嘌呤核苷酸交换因子（GEF）-H1转位至收缩性F-肌动蛋白环，并且siRNA介导的GEF-H1缺失抑制了AJC解体。因此，我们的研究结果表明GEF-H1/Rho/ROCK-II信号通路在上皮细胞AJC解体中起核心作用。
