Increased intracellular Cl- concentration improves airway epithelial migration by activating the RhoA/ROCK Pathway
Increased intracellular Cl- concentration improves airway epithelial migration by activating the RhoA/ROCK Pathway
复制标题
细胞内 Cl- 浓度增加通过激活 RhoA/ROCK 通路改善气道上皮迁移
DOI：
10.7150/thno.46002
复制
发表时间：
2020-01-01
期刊：
影响因子：
12.4
通讯作者：
Qin, Xiaoqun
中科院分区：
文献类型：
--
作者：
Huang, Wenjie;Tan, Meiling;Qin, Xiaoqun
在气道中，Cl-是最丰富的阴离子，并且在跨上皮转运中起关键作用。氯离子通道（CLC），如囊性纤维化跨膜传导调节因子（CFTRs），anoctamin-1和CLC-2的异常表达和激活与细胞迁移能力的相关性表明，有缺陷的Cl-转运和上皮伤口修复之间的关系。然而，细胞内Cl-和气道创伤修复能力之间是否存在相关性迄今尚未探讨，这种关系中涉及的潜在机制尚未完全确定。研究方法：本工作采用氯离子敏感的荧光探针（N-[乙氧羰基甲基]-6-甲氧基溴化喹啉）测定细胞内氯离子浓度（[Cl-]i）的变化。结果如下：我们发现，高[Cl-]i钳夹和用CLC抑制剂CFTR阻断剂CFTRinh-172和氯细胞内通道抑制剂IAA 94处理1小时增加了气道上皮细胞中的细胞内Cl-浓度（[Cl-]i）。这种作用改善了上皮细胞迁移。此外，细胞中[Cl-]i的增加促进了F-肌动蛋白重组，降低了细胞硬度，并改善了RhoA激活和LIMK 1/2磷酸化。用ROCK抑制剂Y-27632和ROCK 1 siRNA处理显著减弱了[Cl-]i增加对LIMK 1/2活化和细胞迁移的影响。此外，细胞内Ca 2+浓度不受[Cl-]i钳位缓冲液和CFTRinh-172和IAA 94的影响。结论：综上所述，这些结果表明，气道上皮细胞中的Cl-积累可以激活RhoA/ROCK/LIMK级联反应，以诱导F-肌动蛋白重组，下调细胞刚度，并改善上皮迁移。
In the airway, Cl- is the most abundant anion and is critically involved in transepithelial transport. The correlation of the abnormal expression and activation of chloride channels (CLCs), such as cystic fibrosis transmembrane conductance regulators (CFTRs), anoctamin-1, and CLC-2, with cell migration capability suggests a relationship between defective Cl- transport and epithelial wound repair. However, whether a correlation exists between intracellular Cl- and airway wound repair capability has not been explored thus far, and the underlying mechanisms involved in this relationship are not fully defined. Methods: In this work, the alteration of intracellular chloride concentration ([Cl-]i) was measured by using a chloride-sensitive fluorescent probe (N-[ethoxycarbonylmethyl]-6-methoxyquinolium bromide). Results: We found that clamping with high [Cl-]i and 1 h of treatment with the CLC inhibitor CFTR blocker CFTRinh-172 and chloride intracellular channel inhibitor IAA94 increased intracellular Cl- concentration ([Cl-]i) in airway epithelial cells. This effect improved epithelial cell migration. In addition, increased [Cl-]i in cells promoted F-actin reorganization, decreased cell stiffness, and improved RhoA activation and LIMK1/2 phosphorylation. Treatment with the ROCK inhibitor of Y-27632 and ROCK1 siRNA significantly attenuated the effects of increased [Cl-]i on LIMK1/2 activation and cell migration. In addition, intracellular Ca2+ concentration was unaffected by [Cl-]i clamping buffers and CFTRinh-172 and IAA94. Conclusion: Taken together, these results suggested that Cl- accumulation in airway epithelial cells could activate the RhoA/ROCK/LIMK cascade to induce F-actin reorganization, down-regulate cell stiffness, and improve epithelial migration.