title: 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-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.
authors: Department of Biochemistry, University of Crete Medical School, Heraklion, Crete, Greece.
authors: E
authors: E
authors: Papadimitriou
authors: E
authors: E
authors: Vasilaki
authors: C
authors: C
authors: Vorvis
authors: D
authors: D
authors: Iliopoulos
authors: A
authors: A
authors: Moustakas
authors: D
authors: D
authors: Kardassis
authors: C
authors: C
authors: Stournaras
mesh_terms: D015820:Cadherins / Q000378:metabolism; D002478:Cells, Cultured; D058750:Epithelial-Mesenchymal Transition; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics*; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology; D015513:Oncogene Proteins / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D059748:Proteolysis; D034622:RNA Interference; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D015820:Cadherins; D020662:Guanine Nucleotide Exchange Factors; C543845:MIRN24 microRNA, human; D035683:MicroRNAs; C099763:NET1 protein, human; D015513:Oncogene Proteins; D020033:Protein Isoforms; D016212:Transforming Growth Factor beta; D048049:Extracellular Signal-Regulated MAP Kinases; D020742:rhoA GTP-Binding Protein