Increased intracellular Cl^- concentration improves airway epithelial migration by activating the RhoA/ROCK Pathway

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 CFTR_inh-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 CFTR_inh-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.