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Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1? in mesenchymal transition.

ABSTRACT: In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-? and HIF1? signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-? signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1? knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1? signaling appears to play an important role in this process.

SUBMITTER: Eurlings IM

PROVIDER: S-EPMC4199572 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1α in mesenchymal transition.

Eurlings Irene M J IM Reynaert Niki L NL van den Beucken Twan T van den Beucken Twan T Gosker Harry R HR de Theije C C CC Verhamme Fien M FM Bracke Ken R KR Wouters Emiel F M EF Dentener Mieke A MA

PloS one 20141016 10

In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as wer ...[more]

PMID: 25329389

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Project description:BackgroundCigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and lung cancer. Epithelial-mesenchymal transition (EMT) is an essential pathophysiological process in COPD and plays an important role in airway remodeling, fibrosis, and malignant transformation of COPD. Previous studies have indicated FERMT3 is downregulated and plays a tumor-suppressive role in lung cancer. However, the role of FERMT3 in COPD, including EMT, has not yet been investigated.MethodsThe present study aimed to explore the potential role of FERMT3 in COPD and its underlying molecular mechanisms. Three GEO datasets were utilized to analyse FERMT3 gene expression profiles in COPD. We then established EMT animal models and cell models through cigarette smoke (CS) or cigarette smoke extract (CSE) exposure to detect the expression of FERMT3 and EMT markers. RT-PCR, western blot, immunohistochemical, cell migration, and cell cycle were employed to investigate the potential regulatory effect of FERMT3 in CSE-induced EMT.ResultsBased on Gene Expression Omnibus (GEO) data set analysis, FERMT3 expression in bronchoalveolar lavage fluid was lower in COPD smokers than in non-smokers or smokers. Moreover, FERMT3 expression was significantly down-regulated in lung tissues of COPD GOLD 4 patients compared with the control group. Cigarette smoke exposure reduced the FERMT3 expression and induces EMT both in vivo and in vitro. The results showed that overexpression of FERMT3 could inhibit EMT induced by CSE in A549 cells. Furthermore, the CSE-induced cell migration and cell cycle progression were reversed by FERMT3 overexpression. Mechanistically, our study showed that overexpression of FERMT3 inhibited CSE-induced EMT through the Wnt/β-catenin signaling.ConclusionsIn summary, these data suggest FERMT3 regulates cigarette smoke-induced epithelial-mesenchymal transition through Wnt/β-catenin signaling. These findings indicated that FERMT3 was correlated with the development of COPD and may serve as a potential target for both COPD and lung cancer.

Project description:Recent epidemiological studies demonstrate that both active and involuntary exposure to tobacco smoke increase the risk of breast cancer. Little is known, however, about the molecular mechanisms by which continuous, long term exposure to tobacco smoke contributes to breast carcinogenesis because most previous studies have focused on short term treatment models. In this work we have set out to investigate the progressive transforming effects of tobacco smoke on non-tumorigenic mammary epithelial cells and breast cancer cells using in vitro and in vivo models of chronic cigarette smoke exposure.We show that both non-tumorigenic (MCF 10A, MCF-12A) and tumorigenic (MCF7) breast epithelial cells exposed to cigarette smoke acquire mesenchymal properties such as fibroblastoid morphology, increased anchorage-independent growth, and increased motility and invasiveness. Moreover, transplantation experiments in mice demonstrate that treatment with cigarette smoke extract renders MCF 10A cells more capable to survive and colonize the mammary ducts and MCF7 cells more prone to metastasize from a subcutaneous injection site, independent of cigarette smoke effects on the host and stromal environment. The extent of transformation and the resulting phenotype thus appear to be associated with the differentiation state of the cells at the time of exposure. Analysis by flow cytometry showed that treatment with CSE leads to the emergence of a CD44(hi)/CD24(low) population in MCF 10A cells and of CD44+ and CD49f + MCF7 cells, indicating that cigarette smoke causes the emergence of cell populations bearing markers of self-renewing stem-like cells. The phenotypical alterations induced by cigarette smoke are accompanied by numerous changes in gene expression that are associated with epithelial to mesenchymal transition and tumorigenesis.Our results indicate that exposure to cigarette smoke leads to a more aggressive and transformed phenotype in human mammary epithelial cells and that the differentiation state of the cell at the time of exposure may be an important determinant in the phenotype of the final transformed state.

Project description:Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-?1 (TGF-?1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-?1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen ?, ?-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-?1 release. TGF-?1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-?1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-?1 induced morphological changes, decreased E-cadherin, but increased collagen ? and cell migration, a process that was reversed by the inhibitor of ?/epsilon casein kinase I, PF-670462. TGF-?1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-?1-induced collagen ? upregulation. Cigarette smoke (CS) increased TGF-?1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-?1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-?1-induced collagen ? expression, as well as TGF-?1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-?1-mediated EMT, linked to a TGF-?1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD.

Dataset Information

Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1? in mesenchymal transition.

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Cigarette smoke extract induces a phenotypic shift in epithelial cells; involvement of HIF1α in mesenchymal transition.

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