Claudins, the integral tight junction (TJ) proteins that regulate paracellular permeability and cell polarity, are frequently dysregulated in cancer; however, their role in neoplastic progression is unclear. Here, we demonstrated that knockout of Cldn18, a claudin family member highly expressed in lung alveolar epithelium, leads to lung enlargement, parenchymal expansion, increased abundance and proliferation of known distal lung progenitors, the alveolar epithelial type II (AT2) cells, activation of Yes-associated protein (YAP), increased organ size, and tumorigenesis in mice. Inhibition of YAP decreased proliferation and colony-forming efficiency (CFE) of Cldn18-/- AT2 cells and prevented increased lung size, while CLDN18 overexpression decreased YAP nuclear localization, cell proliferation, CFE, and YAP transcriptional activity. CLDN18 and YAP interacted and colocalized at cell-cell contacts, while loss of CLDN18 decreased YAP interaction with Hippo kinases p-LATS1/2. Additionally, Cldn18-/- mice had increased propensity to develop lung adenocarcinomas (LuAd) with age, and human LuAd showed stage-dependent reduction of CLDN18.1. These results establish CLDN18 as a regulator of YAP activity that serves to restrict organ size, progenitor cell proliferation, and tumorigenesis, and suggest a mechanism whereby TJ disruption may promote progenitor proliferation to enhance repair following injury.

Claudins are integral tight junction (TJ) proteins that contribute to cell polarity and regulate paracellular permeability to ions and solutes. Claudin 18 (CLDN18) is one of the most highly expressed claudin family members in lung alveolar epithelium. To investigate the role of CLDN18 in alveolar homeostasis, we recently generated Cldn18−/− mice that demonstrate increased lung solute permeability and alveolar fluid clearance (AFC) compared to wild type (WT) controls. Lungs of Cldn18−/− mice are markedly enlarged due to increased abundance and proliferation of alveolar epithelial type II (AT2) cells, known progenitors of distal lung epithelium, with resultant parenchymal expansion. Cldn18−/− AT2 cells grown with MLg fibroblasts in 3‐dimensional (3D) culture show increased colony forming efficiency (CFE), suggesting increased progenitor capacity. Given its known role in regulating stem/progenitor cell proliferation and organ size, we investigated a potential role for Yes‐associated protein (YAP) signaling in mediating the proliferative phenotype of Cldn18−/− lung progenitors. Progenitor cell activation was accompanied by activation of YAP, as evidenced by increased nuclear YAP, increased expression of YAP target genes in lungs of Cldn18−/− mice, and increased YAP and decreased phospho‐YAP (p‐YAP) by western analysis in isolated Cldn18−/− AT2 cells and by immunofluorescence in Cldn18−/− AT2 cells in 3D culture. Treatment with the YAP inhibitor verteporfin (VP, 100 mg/kg) reduced AT2 cell proliferation in vivo (EdU+NKX2.1+/total cells: 1.36 ± 0.10% vehicle vs 0.93 ± 0.06% VP, p<0.05) and decreased lung size (lung dry weight/body weight ratios (mg/g): 3.56 ± 0.08 vehicle vs 2.93 ± 0.13 VP, p<0.05) in Cldn18−/− mice. Inhibition of YAP with VP (0.75 μM) or shRNA decreased colony size (~50% vs control) and number (30–50% vs control) and reduced proliferation (Ki67+ cells at 10.7 ± 0.4% VP vs 29.2 ± 3.7% vehicle) of Cldn18−/− AT2 cells in 3D culture, while overexpression of CLDN18 decreased YAP nuclear localization, cell proliferation, CFE and YAP activity. These results reveal a novel role for YAP signaling in regulation of distal lung epithelial progenitor cell homeostasis and identify a role for TJ proteins, in particular CLDN18, in regulating YAP activity and organ size. Overall, they suggest a mechanism whereby growth‐promoting signals are transduced from TJ to the nucleus that has important implications for modulating stem/progenitor cell function and regeneration following injury.Support or Funding InformationNational Institutes of Health, Hastings and Whittier Foundations

Claudin proteins are major constituents of epithelial and endothelial tight junctions (TJs) that regulate paracellular permeability to ions and solutes. Claudin 18, a member of the large claudin family, is highly expressed in lung alveolar epithelium. To elucidate the role of claudin 18 in alveolar epithelial barrier function, we generated claudin 18 knockout (C18 KO) mice. C18 KO mice exhibited increased solute permeability and alveolar fluid clearance (AFC) compared with wild-type control mice. Increased AFC in C18 KO mice was associated with increased β-adrenergic receptor signaling together with activation of cystic fibrosis transmembrane conductance regulator, higher epithelial sodium channel, and Na-K-ATPase (Na pump) activity and increased Na-K-ATPase β1 subunit expression. Consistent with in vivo findings, C18 KO alveolar epithelial cell (AEC) monolayers exhibited lower transepithelial electrical resistance and increased solute and ion permeability with unchanged ion selectivity. Claudin 3 and claudin 4 expression was markedly increased in C18 KO mice, whereas claudin 5 expression was unchanged and occludin significantly decreased. Microarray analysis revealed changes in cytoskeleton-associated gene expression in C18 KO mice, consistent with observed F-actin cytoskeletal rearrangement in AEC monolayers. These findings demonstrate a crucial nonredundant role for claudin 18 in the regulation of alveolar epithelial TJ composition and permeability properties. Increased AFC in C18 KO mice identifies a role for claudin 18 in alveolar fluid homeostasis beyond its direct contributions to barrier properties that may, at least in part, compensate for increased permeability.