The airway epithelium of smokers acquires pathological phenotypes, including basal cell (BC) and/or goblet cell hyperplasia, squamous metaplasia, structural and functional abnormalities of ciliated cells, decreased number of secretoglobin (SCGB1A1)-expressing secretory cells, and a disordered junctional barrier. In this study, we hypothesized that smoking alters airway epithelial structure through modification of BC function via an EGF receptor (EGFR)-mediated mechanism. Analysis of the airway epithelium revealed that EGFR is enriched in airway BCs, whereas its ligand EGF is induced by smoking in ciliated cells. Exposure of BCs to EGF shifted the BC differentiation program toward the squamous and epithelial-mesenchymal transition-like phenotypes with down-regulation of genes related to ciliogenesis, secretory differentiation, and markedly reduced junctional barrier integrity, mimicking the abnormalities present in the airways of smokers in vivo. These data suggest that activation of EGFR in airway BCs by smoking-induced EGF represents a unique mechanism whereby smoking can alter airway epithelial differentiation and barrier function.airway epithelial barrier | progenitor cell | cigarette smoking

Smoking-induced global distal-to-proximal reprogramming of the SAE represents a novel pathologic feature of COPD and is mediated by exaggerated epidermal growth factor/epidermal growth factor receptor signaling in SAE BCs.

These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

The airway epithelium of cigarette smokers undergoes dramatic remodeling with hyperplasia of basal cells (BC) and mucus-producing cells, squamous metaplasia, altered ciliated cell differentiation and decreased junctional barrier integrity, relevant to chronic obstructive pulmonary disease and lung cancer. In this study, we show that epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) is induced by smoking in human airway epithelium as a result of epidermal growth factor (EGF)-driven squamous differentiation of airway BC stem/progenitor cells. In turn, AREG induced a unique EGFR activation pattern in human airway BC, distinct from that evoked by EGF, leading to BC- and mucous hyperplasia, altered ciliated cell differentiation and impaired barrier integrity. Further, AREG promoted its own expression and suppressed expression of EGF, establishing an autonomous self-amplifying signaling loop in airway BC relevant for promotion of EGF-independent hyperplastic phenotypes. Thus, EGF-AREG interplay in airway BC stem/progenitor cells is one of the mechanisms that mediates the interconnected pathogenesis of all major smoking-induced lesions in the human airway epithelium.

Recent studies suggest that the epithelium might modulate the contractility of smooth muscle. However, the mechanisms underlying this regulation are unknown. The present study investigated the regulation of smooth muscle contraction by the epithelium in rat vas deferens and the possible factor(s) involved. Exogenously applied ATP inhibited electrical field stimulation (EFS)-evoked smooth muscle contraction in an epithelium-dependent manner. As the effects of ATP on smooth muscle contractility were abrogated by inhibitors of prostaglandin synthesis, but not by those of nitric oxide synthesis, prostaglandins might mediate the effects of ATP. Consistent with this idea, PGE 2 inhibited EFS-evoked smooth muscle contraction independent of the epithelium, while ATP and UTP induced the release of PGE 2 from cultured rat vas deferens epithelial cells, but not smooth muscle cells. The ATP-induced PGE 2 release from vas deferens epithelial cells was abolished by U73122, an inhibitor of phospholipase C (PLC) and BAPTA AM, a Ca 2+ chelator. ATP also transiently increased [Ca 2+ ] i in vas deferens epithelial cells. This effect of ATP on [Ca 2+ ] i was independent of extracellular Ca 2+ , but abolished by the P2 receptor antagonist RB2 and U73122. In membrane potential measurements using a voltage-sensitive dye, PGE 2 , but not ATP, hyperpolarized vas deferens smooth muscle cells and this effect of PGE 2 was blocked by MDL12330A, an adenylate cyclase inhibitor, and the chromanol 293B, a blocker of cAMP-dependent K + channels. Taken together, our results suggest that ATP inhibition of vas deferens smooth muscle contraction is epithelium dependent. The data also suggest that ATP activates P2Y receptor-coupled Ca 2+ mobilization leading to the release of PGE 2 from epithelial cells, which in turn activates cAMP-dependent K + channels in smooth muscle cells leading to the hyperpolarization of membrane voltage and the inhibition of vas deferens contraction. Thus, the present findings suggest a novel regulatory mechanism by which the epithelium regulates the contractility of smooth muscle.

In the process of seeking novel lung host defense regulators by analyzing genome-wide RNA sequence data from normal human airway epithelium, we detected expression of POU2AF1, a known transcription co-factor previously thought to be expressed only in lymphocytes. Lymphocyte contamination of human airway epithelial samples obtained by bronchoscopy and brushing was excluded by immunohistochemistry staining, the observation of up-regulation of POU2AF1 in purified airway basal stem/progenitor cells undergoing differentiation and analysis of differentiating single basal cell clones. Lentivirus-mediated up-regulation of POU2AF1 in airway basal cells induced up-regulation of host defense genes, including MX1, IFIT3, IFITM and known POU2AF1 downstream genes HLA-DRA, ID2, ID3, IL6, BCL6. Interestingly, expression of these genes paralleled changes of POU2AF1 expression during airway epithelium differentiation in vitro, suggesting POU2AF1 helps to maintain a “host defense tone” even in pathogen-free condition. Cigarette smoke, a known risk factor for airway infection, suppressed POU2AF1 expression both in vivo in humans and in vitro in human airway epithelial cultures, accompanied by deregulation of POU2AF1 downstream genes. Finally, enhancing POU2AF1 expression in human airway epithelium attenuated the suppression of host defense genes by smoking. Together, these findings suggest a novel function of POU2AF1 as a potential regulator of host defense genes in the human airway epithelium.

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CXCL14, a recently described epithelial cytokine, plays putative multiple roles in inflammation and carcinogenesis. In the context that chronic obstructive pulmonary disease (COPD) and lung cancer are both smoking-related disorders associated with airway epithelial disorder and inflammation, we hypothesized that the airway epithelium responds to cigarette smoking with altered CXCL14 gene expression, contributing to the disease-relevant phenotype. Using genome-wide microarrays with subsequent immunohistochemical analysis, the data demonstrate that the expression of CXCL14 is up-regulated in the airway epithelium of healthy smokers and further increased in COPD smokers, especially within hyperplastic/metaplastic lesions, in association with multiple genes relevant to epithelial structural integrity and cancer.In vitro experiments revealed that the expression of CXCL14 is induced in the differentiated airway epithelium by cigarette smoke extract, and that epidermal growth factor mediates CXCL14 upregulation in the airway epithelium through its effects on the basal stem/progenitor cell population. Analyses of two independent lung cancer cohorts revealed a dramatic up-regulation of CXCL14 expression in adenocarcinoma and squamous-cell carcinoma. High expression of the COPD-associated CXCL14-correlating cluster of genes was linked in lung adenocarcinoma with poor survival. These data suggest that the smoking-induced expression of CXCL14 in the airway epithelium represents a novel potential molecular link between smoking-associated airway epithelial injury, COPD, and lung cancer.Keywords: CXCL14; airway epithelium; smoking; COPD; lung cancerThe airway epithelium, a pseudostratified layer of epithelial cells lining the tracheobronchial tree, is the initial cell population that interacts with inhaled environmental contaminants (1, 2). Among these, cigarette smoke, with its 4,000 compounds and greater than 10 14 free radicals per puff, is a major stressor for airway epithelial cells, and is causally associated with the development of chronic obstructive pulmonary disease (COPD) and lung cancer (1,(3)(4)(5)(6)(7)(8).COPD is a smoking-related disorder defined by irreversible airflow obstruction, associated with structural alterations in the small airways (9, 10), including the small airway epithelium (SAE), which becomes hyperplastic, secretes excessive amounts of mucus, and undergoes metaplastic changes (10-12). In addition to the epidemiological data establishing COPD as a risk factor for lung cancer, several lines of evidence indicate that the molecular mechanisms of COPD pathogenesis are relevant to smoking-induced lung cancer (5,7,8). Importantly, the airway epithelial pathology associated with COPD exhibits remarkable similarities to the pre-neoplastic phenotypes of smoking-induced lung cancer, with a continuum of hyperplastic, metaplastic, and dysplastic changes gradually evolving into an invasive phenotype (13).Studies using mouse models suggest that COPD and lung cancer are both driven, in part, by chronic inflamma...