Regulation of Cytoskeletal Actin During Airway Epithelial Cell Differentiation

Epithelial cell differentiation is regulated by growth and transcription factors that direct programs required for specialized epithelial cell function. Central to cell function is cell shape, which is determined by cytoskeletal actin. We hypothesized that during airway epithelial cell differentiation specific factors are required for directing unique patterns in the actin cytoskeleton of specialized cell types. We utilized a primary culture model of mouse airway epithelial cells to generate ciliated and non-ciliated cell phenotypes. Cytoskeletal actin was visualized by fluorescent microscopy using fluorescent-labeled phalloidin and epithelial cell differentiation was identified using cell-specific fluorescent labeled antibodies. We found a distinct pattern of actin restricted to the apical aspect of ciliated cells. Evaluation of airway epithelial cells during differentiation revealed an apical network of actin present prior to the appearance of cilia that progressively organized during cell maturation. To determine if cilia localization plays a role in the formation of this apical actin network we utilized Foxj1 deficient airway epithelial cells that fail to properly produce apical cilia. In these mutant cells we found disorganized apical actin, suggesting apical actin is linked to basal bodies and rootlets of cilia. In addition to cilia, specialized salt and water channels and growth factor receptors on the apical membrane provide critical functions for mucus and host defense. These apical proteins are linked to actin through specialized scaffolding proteins, including ezrin, which is localized in the apical aspect of ciliated airway epithelial cells. To determine if apical ezrin can be tethered by apical membrane proteins, or alternatively is dependent on binding to cytoskeletal actin, we disrupted actin in mature airway epithelial cells using cytochalasin B. This treatment resulted in loss of the apical actin network and subsequent loss of apically localized ezrin, suggesting that apical proteins cannot independently fix ezrin to the apical membrane. Apical localization of ezrin has also been shown to be dependent on Rho kinase. To determine if Rho kinase also plays a role in maintenance of the apical actin network, we treated differentiating airway epithelial cells with Rho kinase inhibitor Y-27623 to interrupt apical localization of ezrin. This resulted in disruption of apical ezrin, but minimal alteration of the apical actin network, indicating that apical actin localization is independent of apical ezrin. Taken together, these data indicate that an apical actin network develops early during airway epithelial cell differentiation in a subpopulation of cells that are programmed for specialized functions including ciliogenesis, and is dependent on transcription factor Foxj1 and Rho kinase. Interruption of either regulatory factor by injury, genetic mutation, or airway infection disrupts apical ezrin and normal airway epithelial cell differentiation required for post-defense and lung health.