Cigarette Smoke Extract Induces a Phenotypic Shift in Epithelial Cells; Involvement of HIF1α 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-β and HIF1α 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-β signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α 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, HIF1α signaling appears to play an important role in this process.

Involvement of c-Jun N-Terminal Kinase in TNF-α–Driven Remodeling

“…We have shown that hypoxia signaling is involved in the induction of a phenotypic shift of epithelial cells by cigarette smoke extract, especially with respect to the mesenchymal transition (8). In the current study, in the context of TNF-a-induced EMT, no role for hypoxia signaling could be demonstrated.…”

“…We have shown a role for c-Jun N-terminal kinase (JNK) 1 in TGF-b1-induced EMT in lung epithelial cells, as the induction of this phenotypic shift was blunted in JNK1 knockout cells (7). Furthermore, our group recently demonstrated that cigarette smoke extract also has the ability to induce such a phenotypic shift, in which hypoxia inducible factor (HIF1a) signaling was implicated (8).…”

The Association between Smoking and Epiretinal Membrane

“…Although the pathogenesis of ERM has not yet been fully elucidated, the formation and progression of ERM can be regarded as a fibrotic process because the pathologic findings include increased ECM protein deposition and membrane contraction in which myofibroblasts play a crucial role1. Several studies have demonstrated that exposure to cigarette smoke extracts can provoke activation of the TGF β pathway and the epithelial-mesenchymal transition (EMT)4445, and thereby up-regulate numerous genes involved in fibrosis and the accumulate of ECM28 and these processes are also known to plays a crucial roles in the pathogenesis of ERM. However, the results of epidemiologic studies of the relationship between smoking and risk of ERM have remained inconclusive.…”

MAP3K19 Is Overexpressed in COPD and Is a Central Mediator of Cigarette Smoke-Induced Pulmonary Inflammation and Lower Airway Destruction

“…Beas-2B cells were cultured in BEBM media (Lonza Inc., Allandale, NJ, USA) supplemented with a BEGM kit (Lonza Inc.) without the gentamycin-amphotericin B mix. Cigarette smoke extract (CSE) was made essentially by the method detailed in Eurlings et al [30]. Filters were removed from 3R4F Research cigarettes (University of Kentucky, Lexington, KY) and CSE was made by bubbling the smoke of one cigarette into 10 mls of full media using a linear pump (2 mL/sec).…”