Transcriptional regulation of the small GTPase RhoB gene by TGF{beta}-induced signaling pathways.
The purpose of the present study was to investigate the mechanism of transcriptional induction of the small GTPase RhoB gene by the transforming growth factor beta (TGFbeta) signaling pathway and the role of this regulation in TGFbeta-induced cell migration. To achieve our goals, we utilized a combination of siRNA-mediated gene silencing, adenovirus-mediated gene transfer receptor and MAPK inhibition, transactivation assays, and DNA-protein interaction assays in human HaCaT keratinocytes. We found that the RhoB gene is a direct transcriptional target of TGFbeta. We show that TGFbeta activates an early MEK/ERK pathway and that this activation is required for the recruitment of Smad3 to a novel, nonclassical, Smad binding element in the proximal RhoB promoter, in a p53-dependent manner. This element is overlapping with a CCAAT box that constitutively binds nuclear factor Y. Mutagenesis of this site abolished the Smad-mediated transactivation of the RhoB promoter. Finally, silencing of RhoB gene expression via siRNA or utilization of a dominant negative form of RhoB significantly inhibited TGFbeta-induced migration of HaCaT keratinocytes and DU145 prostate cancer cells. Our findings establish RhoB as a direct transcriptional target of TGFbeta in human keratinocytes and identify an important role of RhoB in TGFbeta-induced cell migration.-Vasilaki, E., Papadimitriou, E., Tajadura, V., Ridley, A. J., Stournaras, C., Kardassis, D. Transcriptional regulation of the small GTPase RhoB gene by TGFbeta-induced signaling pathways.
Vasilaki
Eleftheria
E
Department of Biochemistry, School of Medicine, University of Crete, GR-71003, Heraklion, Greece.
Papadimitriou
Elsa
E
Tajadura
Virginia
V
Ridley
Anne J
AJ
Stournaras
Christos
C
Kardassis
Dimitris
D
D000199:Actins / Q000235:genetics / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology; D002070:Butadienes / Q000494:pharmacology; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D047369:Chromatin Immunoprecipitation; D004149:Dioxoles / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051785:Smad Proteins / Q000378:metabolism; D051900:Smad3 Protein / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D000199:Actins; D001549:Benzamides; D002070:Butadienes; D004149:Dioxoles; D009570:Nitriles; D034741:RNA, Small Interfering; D018125:Receptors, Transforming Growth Factor beta; D051785:Smad Proteins; D051900:Smad3 Protein; D016212:Transforming Growth Factor beta; D016159:Tumor Suppressor Protein p53; C113580:U 0126; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein