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

Oncogene. 2012 Jun 7;31(23):2862-75. doi: 10.1038/onc.2011.457. Epub 2011 Oct 10.


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-β (TGF-β) in keratinocytes. We report that short-term TGF-β treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-β-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-β. Interestingly, long-term TGF-β 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-β-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-β and was shown to be directly involved in the TGF-β-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short- and long-term TGF-β-mediated regulation of EMT.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cadherins / metabolism
  • Cells, Cultured
  • Epithelial-Mesenchymal Transition*
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Gene Expression Regulation*
  • Guanine Nucleotide Exchange Factors / metabolism
  • Humans
  • Keratinocytes / cytology
  • Keratinocytes / metabolism
  • Kidney / cytology
  • Kidney / metabolism
  • MicroRNAs / genetics*
  • Neoplasms / metabolism
  • Neoplasms / pathology
  • Oncogene Proteins / genetics*
  • Oncogene Proteins / metabolism
  • Phosphorylation
  • Protein Isoforms
  • Proteolysis
  • RNA Interference*
  • Signal Transduction
  • Transforming Growth Factor beta / pharmacology*
  • rhoA GTP-Binding Protein / metabolism*


  • Cadherins
  • Guanine Nucleotide Exchange Factors
  • MIRN24 microRNA, human
  • MicroRNAs
  • NET1 protein, human
  • Oncogene Proteins
  • Protein Isoforms
  • Transforming Growth Factor beta
  • Extracellular Signal-Regulated MAP Kinases
  • rhoA GTP-Binding Protein