Key Points: Chloride channels are important for proper hydration of the airway surface. TMEM16A protein is an important component of calcium‐activated chloride channels. Interleukin‐4, a cytokine that induces mucous cell metaplasia, also upregulates calcium‐dependent chloride secretion in human bronchial epithelial cells. In bronchial epithelial cells treated with interleukin‐4, we found that TMEM16A protein becomes highly expressed in goblet but not in ciliated cells. Upregulation of TMEM16A by interleukin‐4 may be important for secretion and proper expansion of mucins. Abstract  The TMEM16A protein has a potential role as a Ca²⁺‐activated Cl⁻ channel (CaCC) in airway epithelia where it may be important in the homeostasis of the airway surface fluid. We investigated the function and expression of TMEM16A in primary human bronchial epithelial cells and in a bronchial cell line (CFBE41o–). Under resting conditions, TMEM16A protein expression was relatively low. However, TMEM16A silencing with short‐interfering RNAs caused a marked inhibition of CaCC activity, thus demonstrating that a low TMEM16A expression is sufficient to support Ca²⁺‐dependent Cl⁻ transport. Following treatment for 24–72 h with interleukin‐4 (IL‐4), a cytokine that induces mucous cell metaplasia, TMEM16A protein expression was strongly increased in approximately 50% of primary bronchial epithelial cells, with a specific localization in the apical membrane. IL‐4 treatment also increased the percentage of cells expressing MUC5AC, a marker of goblet cells. Interestingly, MUC5AC was detected specifically in cells expressing TMEM16A. In particular, MUC5AC was found in 15 and 60% of TMEM16A‐positive cells when epithelia were treated with IL‐4 for 24 or 72 h, respectively. In contrast, ciliated cells showed expression of the cystic fibrosis transmembrane conductance regulator Cl⁻ channel but not of TMEM16A. Our results indicate that TMEM16A protein is responsible for CaCC activity in airway epithelial cells, particularly in cells treated with IL‐4, and that TMEM16A upregulation by IL‐4 appears as an early event of goblet cell differentiation. These findings suggest that TMEM16A expression is particularly required under conditions of mucus hypersecretion to ensure adequate secretion of electrolytes and water.

Goblet cell hyperplasia, a feature of asthma and other respiratory diseases, is driven by the Th-2 cytokines IL-4 and IL-13. In human bronchial epithelial cells, we find that IL-4 induces the expression of many genes coding for ion channels and transporters, including TMEM16A, SLC26A4, SLC12A2, and ATP12A. At the functional level, we find that IL-4 enhances calcium- and cAMP-activated chloride/bicarbonate secretion, resulting in high bicarbonate concentration and alkaline pH in the fluid covering the apical surface of epithelia. Importantly, mucin release, elicited by purinergic stimulation, requires the presence of bicarbonate in the basolateral solution and is defective in cells derived from cystic fibrosis patients. In conclusion, our results suggest that Th-2 cytokines induce a profound change in expression and function in multiple ion channels and transporters that results in enhanced bicarbonate transport ability. This change is required as an important mechanism to favor release and clearance of mucus.

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins.

Cystic fibrosis (CF) is a chronic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes for a channel expressed at the apical surface of epithelial tissues. Defective chloride and bicarbonate secretion, arising from CFTR mutations, cause a multi-organ disease. In the airways, impaired ion transport results in a thick mucus, dehydration of the periciliar region and bacterial infections. Over the last years, basic research has sustained a great effort to identify therapies that are able to correct defective CFTR. For this purpose, in vitro cell models have played a key role in the study of mechanisms of the disease and to assess CFTR modulator therapies. Cultures of human primary bronchial epithelia are considered a physiologically relevant disease model due to their ability to maintain most of the morphological and functional characteristics of the airway epithelium in vivo. Despite their value, these cells are limited by the availability of human lung tissue and by the complexity of the culture procedure. However, primary human nasal cells can be considered as an alternative model for the study of CF pathophysiology since they are easier to obtain and recapitulate the properties of bronchial cultures. Over the years, several groups have optimized a protocol with key steps to culture and fully amplify differentiated primary airway epithelia. Our approach provides epithelia monolayers grown on porous filters, characterized by high transepithelial electrical resistance and an electrical potential difference. These parameters are required to perform electrophysiological experiments devoted to the study of ion transport mechanisms in airway epithelia. The aim of this study was to describe different methods to expand and differentiate isolated cells into fully polarized monolayers of airway epithelium, in order to provide an optimized protocol to support physiopathology analysis and to evaluate therapeutic strategies.