Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-β and miR-24: role in epithelial-to-mesenchymal transition

Abstract

In the present study we analyzed the regulation of the two isoforms of the RhoA-specific guanine nucleotide exchange factor Net1 by transforming growth factor-b (TGF-b) in keratinocytes. We report that short-term TGF-b treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-b-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-b. Interestingly, long-term TGF-b treatment resulted in Net1 mRNA downregulation and Net1A protein degradation by the proteasome. Furthermore, we identified the microRNA miR-24 as a novel post-transcriptional regulator of Net1A expression. Silencing of Net1A resulted in disruption of E-cadherin-and zonula occludens-1 (ZO-1)-mediated junctions, as well as expression of the transcriptional repressor of E-cadherin, Slug and the mesenchymal markers N-cadherin, plasminogen activator inhibitor-1 (PAI-1) and fibronectin, indicating that late TGFb-induced downregulation of Net1A is involved in epithelial-to-mesenchymal transition (EMT). Finally, miR-24 was found to be implicated in the regulation of the EMT program in response to TGF-b and was shown to be directly involved in the TGF-b-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short-and long-term TGF-b-mediated regulation of EMT.

Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

“…6A,B). This is consistent with the established role of Net1 isoforms in controlling RhoA activation in other cell types (Carr et al, 2013a;Carr et al, 2013b;García-Mata et al, 2007;Lee et al, 2010;Murray et al, 2008;Papadimitriou et al, 2012). By contrast, expression of wildtype Net1A restored F-actin content close to that observed in wild-type MEFs.…”

“…By contrast, expression of wildtype Net1A restored F-actin content close to that observed in wild-type MEFs. This is consistent with a predominant role for Net1A over the Net1 isoform in controlling cellular RhoA activity (Carr et al, 2013a;Carr et al, 2013b;Papadimitriou et al, 2012). Importantly, expression of Net1A K83/95R did not efficiently rescue F-actin accumulation, whereas Net1A K83/95Q restored F-actin staining (Fig.…”

“…For example, in addition to nuclear sequestration, cells can downregulate Net1 enzymatic activity by Pak1-dependent phosphorylation (Alberts et al, 2005). Net1A is also subject to ubiquitylation and proteasome-mediated degradation (Carr et al, 2009;Papadimitriou et al, 2012), and inhibition of proteasome function extends the duration of Net1A localization outside the nucleus following integrin ligation (Carr et al, 2013a). In this regard, it is not clear whether proteasomal degradation and acetylation-dependent regulation of Net1A nuclear re-import are competing mechanisms to control Net1A activity, or if they are functionally related.…”

“…Two isoforms of Net1 exist in most cells, Net1 and Net1A, which are identical except for their N-terminal regulatory domains. Previous studies have shown that Net1 expression is required for cell motility and extracellular matrix (ECM) invasion (Hayashi et al, 2013;Leyden et al, 2006;Murray et al, 2008), and this phenotype is mainly due to the actions of the Net1A isoform (Carr et al, 2013;Dutertre et al, 2010;Papadimitriou et al, 2012). Net1A accomplishes this by controlling RhoAdependent phosphorylation of the myosin light chain regulatory subunit and focal adhesion kinase activation (Carr et al, 2013).…”

Neuroepithelial transforming gene 1 functions as a potential prognostic marker for patients with non-small cell lung cancer

“…Silencing of Net-1 by small interfering RNAs (siRNAs) was shown to inhibit cancer cell motility, proliferation and extracellular matrix invasion (8)(9)(10). A growing number of studies have suggested that there may be cross-talk between the Net-1 and transforming growth factor β signaling pathways in actin cytoskeletal re-organization (15)(16)(17). However, to the best of our knowledge, a comprehensive profiling of Net-1 expression and function in advanced NSCLC has not been performed.…”