MMP-2 and MMP-9 mediate cigarette smoke extract-induced epithelial-mesenchymal transition in airway epithelial cells via EGFR/Akt/GSK3β/β-catenin pathway: Amelioration by fisetin

Abstract

Matrix metalloproteinases (MMPs) have been implicated in EMT but their role in the regulation of cigarette smoke-induced EMT in airway epithelium is not clear. We have therefore investigated the potential role of MMP-2 and -9 in cigarette smoke extract (CSE) induced EMT using A549 lung epithelial cells and human small airway epithelial cells (SAEC). The cells were treated with different concentration of CSE, and MTT and trypan blue assays, acridine orange-ethidium bromide assay, gelatin zymography, Western blotting, immunofluorescence studies, Boyden-chamber assay, wound healing assay and air-liquid interface (ALI) culture were used to assess different cellular and molecular changes associated with EMT. The results depict that CSE increased the cytotoxicity along with a concurrent increase in the expression and activity of MMP-2 and -9. CSE further altered EMT markers like E-cadherin, N-cadherin, vimentin, and the molecular modulators of EMT such as β-catenin and pGSK-3β. Further, CSE also upregulated EGFR, AKT, and ERK1/2 in airway epithelial cells. SB-3CT, a known inhibitor of MMP-2 and -9, altered and reversed the expression of markers of EMT and kinases, validating the role of MMP-2 and -9 in CSE-induced EMT. Fisetin, a plant-derived bioflavonoid, also reversed the expression of EMT markers and molecular regulators in a similar fashion as SB-3CT. In summary, this study highlights the role of MMP-2 and -9 in CSE-induced EMT and curate its molecular cascade through EGFR/AKT/ERK/β-catenin axis, which could be restored by MMP-2 and -9 inhibitor and fisetin. Fisetin is hitherto unknown to modulate CSE-induced MMPs activity in airway epithelial cells, and our study suggests its potential role as a therapeutic approach in CSE-induced EMT in lung epithelial cells.