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.

Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of ␤-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential ␤-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific ␤-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/␤-catenin but not CBP/␤-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/␤-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/␤-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/␤-catenin interactions in a PKC-dependent manner, likely involving PKC. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting ␤-catenin to modulate adult progenitor cell behavior in disease.

MicroRNA (miRNA) expression is frequently altered in human cancers. To search for epigenetically silenced miRNAs in nonsmall-cell lung cancer (NSCLC), we mapped human miRNAs on autosomal chromosomes and selected 55 miRNAs in silico. We treated six NSCLC cell lines with the DNA methylation inhibitor 5-aza-2 0 -deoxycytidine (5-aza-CdR) and determined the expressions of the 55 miRNAs. Fourteen miRNAs were decreased in the cancer cell lines and were induced after 5-aza-CdR treatment. After a detailed DNA methylation analysis, we found that mir-34b and mir-126 were silenced by DNA methylation. Mir-34b was silenced by the DNA methylation of its own promoter, whereas mir-126 was silenced by the DNA methylation of its host gene, EGFL7. A chromatin immunoprecipitation assay revealed H3K9me2 and H3K9me3 in mir-34b and EGFL7, and H3K27me3 in EGFL7. The overexpression of mir-34b and mir-126 decreased the expression of c-Met and Crk, respectively. The 5-aza-CdR treatment of lung cancer cell line resulted in increased mir-34b expression and decreased c-Met protein. We next analyzed the DNA methylation status of these miRNAs using 99 primary NSCLCs. Mir-34b and mir-126 were methylated in 41 and 7% of all the cases, respectively. The DNA methylation of mir-34b was not associated with c-Met expression determined by immunohistochemistry, but both mir-34b methylation (p 5 0.007) and c-Met expression (p 5 0.005) were significantly associated with lymphatic invasion in a multivariate analysis. The DNA methylation of mir-34b can be used as a biomarker for an invasive phenotype of lung cancer.MicroRNAs (miRNAs) are broadly conserved small noncoding RNA that regulate gene expression by binding to the 3 0 UTR of target mRNAs in a complementary manner. 1Through the posttranscriptional regulation of many target genes, miRNAs are involved in many biological processes, such as development and human carcinogenesis. MicroRNA expression is altered in human cancers, and some miRNAs have oncogenic or tumor suppressive functions in human malignancies, including lung cancer. 2-5Chromosomal deletions or amplifications are important mechanisms of miRNA expression change in cancers. For example, mir-15 and mir-16 are frequently deleted and downregulated in chronic lymphocytic leukemia.2 The mir-17-92 miRNA cluster is amplified and overexpressed in B-cell lymphoma 6 and lung cancer. 4 However, the precise mechanisms responsible for changes in miRNA expression in cancer remain largely unknown.DNA methylation plays an important role in inactivating tumor suppressor genes in many types of human cancers. 7,8 Recently, DNA methylation in cancerous tissue has been shown to cause the silencing of miRNAs located in the vicinity of CpG islands. 9,10 As the epigenetic silencing of tumor suppressor genes is a common event in lung carcinogenesis 11-14 and miRNA expression is altered in lung cancer, 5 we decided to search for epigenetically silenced miRNAs in lung cancer.In our study, we selected 55 candidate miRNAs in silico based on the genome structure and tre...

Background and objectiveThe prevalence and mortality of chronic obstructive pulmonary disease (COPD) in elderly patients are increasing worldwide. Low body mass index (BMI) is a well-known prognostic factor for COPD. However, the obesity paradox in elderly patients with COPD has not been well elucidated. We investigated the association between BMI and in-hospital mortality in elderly COPD patients.MethodsUsing the Diagnosis Procedure Combination database in Japan, we retrospectively collected data for elderly patients (>65 years) with COPD who were hospitalized between July 2010 and March 2013. We performed multivariable logistic regression analysis to compare all-cause in-hospital mortality between patients with BMI of <18.5 kg/m2 (underweight), 18.5–22.9 kg/m2 (low–normal weight), 23.0–24.9 kg/m2 (high–normal weight), 25.0–29.9 kg/m2 (overweight), and ≥30.0 kg/m2 (obesity) with adjustment for patient backgrounds.ResultsIn all, 263,940 eligible patients were identified. In-hospital mortality was 14.3%, 7.3%, 4.9%, 4.3%, and 4.4%, respectively, in underweight, low–normal weight, high–normal weight, overweight, and obese patients. Underweight patients had a significantly higher mortality than low–normal weight patients (odds ratio [OR]: 1.55, 95% confidence interval [CI]: 1.48–1.63), whereas lower mortality was associated with high–normal weight (OR: 0.76, CI: 0.70–0.82), overweight (OR: 0.73, CI: 0.66–0.80), and obesity (OR: 0.67, CI: 0.52–0.86). Higher mortality was significantly associated with older age, male sex, more severe dyspnea, lower level of consciousness, and lower activities of daily living.ConclusionOverweight and obese patients had a lower mortality than low–normal weight patients, which supports the obesity paradox.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.