The LIM protein zyxin modulates TGF-β1-induced cell migration

Introduction: Epithelial-to-mesenchymal transition (EMT), the reversible phenotypic switching of epithelial to fibroblast-like cells, is an important process implicated in the development of fibrosis in several organs, including the lung. Enhanced TGF-β1 signalling contributes to lung fibrosis, a disease characterized by accumulation of activated (myo)fibroblasts and excessive extracellular matrix deposition. TGF-β1 is an important regulator of these processes. Recently, we have performed a Smad3 chromatin immunoprecipitation (ChIP) screen in the alveolar epithelial type II (ATII) A549 cells and identified the LIM protein zyxin gene as a novel target of TGF-β1/Smad3-dependent gene expression. Zyxin is a zinc-binding protein that localizes to focal adhesions and the actin cytoskeleton.

Methods/Results: Induction of EMT by TGF-β1 in A549 cells was assessed by reduction of the epithelial cell marker E-cadherin and marked induction of the mesenchymal markers fibronectin and vimentin. This effect was accompanied by a phospho-Smad3-dependent expression of zyxin mRNA. Knockdown of zyxin (using siRNA) revealed decreased formation of actin stress fibers, alterations in focal adhesion formation, and mislocalization of the actin-regulatory protein VASP, as analyzed by confocal laser scanning microscopy. Further, TGF-β1 treatment of zyxin-depleted A549 cells displayed significantly increased integrin α5β1 expression levels accompanied by enhanced adhesion to the extracellular matrix and increased cell motility, as observed by time-lapse live cell imaging.

Conclusions: We have identified zyxin as a novel target of TGF-β/Smad3-dependent gene expression in alveolar epithelial A549 cells. Zyxin expression is important for regulation of epithelial cell adhesion, cell motility and EMT.