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

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-beta (TGF-beta) in keratinocytes. We report that short-term TGF-beta treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-beta-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-beta. Interestingly, long-term TGF-beta 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 TGF-beta-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-beta and was shown to be directly involved in the TGF-beta-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short-and long-term TGF-beta-mediated regulation of EMT. Oncogene (2012) 31, 2862-2875; doi:10.1038/onc.2011.457; published online 10 October 2011