Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells
Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells
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DOI：
10.1152/ajpcell.00581.2004
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发表时间：
2005-06-01
影响因子：
5.5
通讯作者：
Schneeberger, EE
中科院分区：
文献类型：
--
作者：
Yu, ASL;McCarthy, KM;Schneeberger, EE
occludin （Occ）在紧密连接中的作用尚不明确。为了深入了解其在上皮细胞生物学中的作用，我们通过稳定表达短干扰RNA来抑制Madin-Darby犬肾II细胞中的occludin水平。occludin的抑制与claudins-1和-7的减少以及claudins-3和-4的增加有关。Claudin-2水平未受影响。在受抑制的Occ （Occ-）克隆中，紧密连接“栅栏”功能没有受损，这是通过膜内bodipy -鞘磷脂扩散来确定的。最显著的变化与细胞骨架的控制和紧密连接的“门”功能有关。Occ-克隆从单层中挤出凋亡细胞的能力降低，这表明凋亡细胞的邻居要么无法感知它们的存在，要么无法协调挤出细胞所需的细胞骨架活动。为了进一步测试肌动蛋白细胞骨架活性依赖于occludin存在的程度，Occ-和Occ+单层被耗尽了胆固醇。先前的研究表明，胆固醇消耗与肌动蛋白细胞骨架的重组和上皮电阻的下降有关。与对照Occ （Occ+）细胞相比，在胆固醇缺失的Occ-单层细胞中，经上皮电阻没有显著下降，它们不能产生Rho-GTP，这是一种参与调节肌动蛋白细胞骨架的信号分子。虽然所有无性系的稳态跨上皮电阻相似，但Occ-单层膜对一价和二价无机阳离子的紧密结通透性增加。此外，对一价有机阳离子的渗透率也有不成比例的大幅增加，达到6.96直径。氯化物渗透率不受影响，甘露醇通量变化不大。数据表明，occludin通过Rho信号通路转导外部（凋亡细胞）和膜内（快速胆固醇消耗）信号，进而引发肌动蛋白细胞骨架的重组。在缺乏occludin的情况下，信号传导受损也可能改变紧密连接链的动态行为，这反映在对大型有机阳离子的渗透性增加上；然而，由claudin形成的离子孔的渗透性受到的影响较小。
The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ-) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ- clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ- and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ- monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ- monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected.