Zyxin Mediates Actin Fiber Reorganization in Epithelial–Mesenchymal Transition and Contributes to Endocardial Morphogenesis

Abstract

Epithelial-mesenchymal transition (EMT) confers destabilization of cell-cell adhesion and cell motility required for morphogenesis or cancer metastasis. Here we report that zyxin, a focal adhesion-associated LIM protein, is essential for actin reorganization for cell migration in TGF-␤1-induced EMT in normal murine mammary gland (NMuMG) cells. TGF-␤1 induced the relocation of zyxin from focal adhesions to actin fibers. In addition, TGF-␤1 up-regulated zyxin via a transcription factor, Twist1. Depletion of either zyxin or Twist1 abrogated the TGF-␤1-dependent EMT, including enhanced cell motility and actin reorganization, indicating the TGF-␤1-Twist1-zyxin signal for EMT. Both zyxin and Twist1 were predominantly expressed in the cardiac atrioventricular canal (AVC) that undergoes EMT during heart development. We further performed ex vivo AVC explant assay and revealed that zyxin was required for the reorganization of actin fibers and migration of the endocardial cells. Thus, zyxin reorganizes actin fibers and enhances cell motility in response to TGF-␤1, thereby regulating EMT. INTRODUCTIONEpithelial-mesenchymal transition (EMT) is an essential event during embryogenesis for the formation of many tissues including the mesoderm, for migration of neural crest cells, and for development of the heart valves and septa. The picture emerging from diverse EMT-related studies suggests that precise molecular and cellular control of EMT is complex and context-dependent (Nakaya et al., 2008). An example of developmentally regulated EMT occurs during the initial stages of cardiac morphogenesis. At embryonic day 9.5 (E9.5), endocardial cells undergo EMT ("endocardial EMT"); delaminating from the endothelial sheet, invading the matrix tissue called cardiac jelly, and engaging in endocardial cushion cellularization required for valve and septum formation (Chang et al., 2004). Because a number of congenital heart diseases are caused by abnormal atrioventricular canal (AVC) development (Bruneau, 2008), understanding of the molecular basis of AVC morphogenesis has been long sought, but still not fully achieved. Several EMTinducing transcription factors such as Snail (Cano et al., 2000) and Twist1 (Ma et al., 2005) are expressed in endocardium during development. Although among them, Twist1 is well analyzed as a potent EMT inducer during cancer metastasis (Yang et al., 2004); however, the role for Twist1 in AVC development is not fully understood.An important hallmark of EMT is an increase in cell motility with actin cytoskeletal rearrangement. On EMT induction, the structure of actin cytoskeleton changes dynamically from cortical actin network to stress fiber (Zavadil and Bottinger, 2005). Cell motility also depends on the adhesion of the cell to the substratum where the integrin family molecules bind to the extracellular matrix to form focal adhesion complexes. Stress fibers link the focal adhesion to retract the cells. Furthermore, the nascent focal complexes at the leading edge of the migrating cells are needed for cell crawlin...

Tead2 expression levels control Yap/Taz subcellular distribution, zyxin expression, and epithelial-mesenchymal transition

“…Immunofluorescence microscopy analysis confirmed increased levels of zyxin localized along stress fibers that are formed during TGFb-induced EMT in Py2T cells and at focal adhesion sites (Fig. 5D), as previously reported (Mori et al, 2009).…”

“…Zyxin is an actin regulatory protein that localizes to sites of focal adhesions and stress fibers in response to mechanical cues to facilitate actin polymerization and generation of traction force (Beckerle, 1997;Hirata et al, 2008;Smith et al, 2010). In line with this functional role, zyxin upregulation during EMT in NMuMG cells has been reported to be essential for cell migration (Mori et al, 2009), and given that forced expression of Tead2 promotes cancer cell migration or invasion, and metastatic outgrowth of Py2T cells in the lung (Fig. 4), we decided to focus on zyxin as a Tead2 target gene.…”

“…Zyxin associates with the actin cytoskeleton and therefore can be localized at the apical membrane, in focal adhesions and on stress fibers (Beckerle, 1997). Notably, zyxin is required for the rearrangement of the actin cytoskeleton during TGFb-induced EMT of NMuMG cells, thereby enabling cell migration (Mori et al, 2009). Zyxin upregulation could therefore be at least one of the mechanisms by which Tead transcriptional activity controls the induction of a migratory and invasive cellular phenotype during EMT.…”

Epithelial-mesenchymal transitions: insights from development

“…TGF signaling is linked to actin remodeling, possibly in part through zyxin, a focal adhesionassociated LIM protein implicated in EndMT under TGF-Twist1-zyxin regulation (Mori et al, 2009). In mouse embryos, BMP2 signaling drives EndMT, as shown by the absence of a cardiac cushion in endothelial Smad4 knockout mice (Moskowitz et al, 2011).…”

Epithelial-Mesenchymal Transition Induced by Senescent Fibroblasts

“…A significant characteristic of the EMT is an increase in cellular motility. This increase is due in part to rearrangements in the actin cytoskeleton [7], and the replacement of cytokeratin intermediate filaments by vimentin intermediate filaments, which transforms the cell from a cuboidal to a spindle shape [2]. Several inducers of EMTs have been found, and there is evidence that EMTs are important during the progression of tumor cells to metastatic stages.…”