Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells
Occludin independently regulates permeability under hydrostatic pressure and cell division in retinal pigment epithelial cells
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DOI：
10.1167/iovs.07-1204
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发表时间：
2008-06-01
影响因子：
4.4
通讯作者：
Antonetti, David A.
中科院分区：
文献类型：
--
作者：
Phillips, Brett E.;Cancel, Limary;Antonetti, David A.
目的。本研究的目的是确定紧密连接蛋白occludin 在扩散压力和静水压力下控制通透性的功能，及其对控制视网膜色素上皮细胞分裂的贡献。方法。 siRNA 抑制人视网膜色素上皮细胞系 ARPE-19 中的 Occludin 表达。通过蛋白质印迹、共聚焦显微镜和 RT-PCR 证实了 occludin 的耗尽。在扩散条件下或施加 10 cm H2O 跨壁压力后，检查单层细胞对荧光标记的 70 kDa 葡聚糖、10 kDa 葡聚糖和 467 Da 四甲基罗丹明 (TAMRA) 的细胞旁通透性。通过氚化胸苷掺入和 Ki67 免疫反应性测定细胞分裂率。使用细胞周期抑制剂来确定细胞分裂的变化是否影响通透性。结果。与对照相比，Occludin 耗竭使弥散性细胞旁通透性增加 15%，达到 467 Da TAMRA，静水压下的通透性增加 50%。相反，用 siRNA 去除 occludin 蛋白不会改变对 70 kDa 和 10 kDa RITC-葡聚糖的扩散渗透性，并且在静水压条件下，occludin 去除的细胞对 70 kDa 葡聚糖的渗透性降低两倍。 Occludin 耗竭还使胸苷掺入增加了 90%，Ki67 阳性细胞增加了 50%。最后，细胞周期抑制剂并没有改变occludin siRNA 对细胞旁通透性的影响。结论。数据表明，occludin 可响应静水压的变化来调节紧密连接的渗透性。此外，这些数据表明 occludin 还有助于细胞分裂的控制，证明了这种紧密连接蛋白的新功能。
PURPOSE. The aim of this study was to determine the function of the tight junction protein occludin in the control of permeability, under diffusive and hydrostatic pressures, and its contribution to the control of cell division in retinal pigment epithelium.METHODS. Occludin expression was inhibited in the human retinal pigment epithelial cell line ARPE-19 by siRNA. Depletion of occludin was confirmed by Western blot, confocal microscopy, and RT-PCR. Paracellular permeability of cell monolayers to fluorescently labeled 70 kDa dextran, 10 kDa dextran, and 467 Da tetramethylrhodamine (TAMRA) was examined under diffusive conditions or after the application of 10 cm H2O transmural pressure. Cell division rates were determined by tritiated thymidine incorporation and Ki67 immunoreactivity. Cell cycle inhibitors were used to determine whether changes in cell division affected permeability.RESULTS. Occludin depletion increased diffusive paracellular permeability to 467 Da TAMRA by 15%, and permeability under hydrostatic pressure was increased 50% compared with control. Conversely, depletion of occludin protein with siRNA did not alter diffusive permeability to 70 kDa and 10 kDa RITC-dextran, and permeability to 70 kDa dextran was twofold lower in occludin-depleted cells under hydrostatic pressure conditions. Occludin depletion also increased thymidine incorporation by 90% and Ki67-positive cells by 50%. Finally, cell cycle inhibitors did not alter the effect of occludin siRNA on paracellular permeability.CONCLUSIONS. The data suggest that occludin regulates tight junction permeability in response to changes in hydrostatic pressure. Furthermore, these data suggest that occludin also contributes to the control of cell division, demonstrating a novel function for this tight junction protein.