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

Oncogene volume 31, pages 2862–2875 (2012)Cite this article

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.

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Acknowledgements

This work was supported by the PENED program of the Greek Secretariat for Research and Technology (Grant no. PENED03-688). We thank Dr EA Papakonstanti for valuable comments.

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Authors and Affiliations

  1. Department of Biochemistry, University of Crete Medical School, Heraklion, Crete, Greece

    E Papadimitriou, E Vasilaki, D Kardassis & C Stournaras

  2. Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute and Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA

    C Vorvis & D Iliopoulos

  3. Ludwig Institute for Cancer Research,

    E Vasilaki & A Moustakas

  4. Uppsala University, Uppsala, Sweden

    E Vasilaki & A Moustakas

  5. Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden

    A Moustakas

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Correspondence to C Stournaras.

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Papadimitriou, E., Vasilaki, E., Vorvis, C. et al. Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-β and miR-24: role in epithelial-to-mesenchymal transition. Oncogene 31, 2862–2875 (2012). https://doi.org/10.1038/onc.2011.457

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