Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF-beta1 involves MAPK, Smad and AP-1 signalling pathways.
Recent data indicate that transforming growth factor-beta1 (TGF-beta1) can act to promote tumour progression in the late stages of carcinogenesis. The mechanism by which this occurs is unknown although a ligand-induced epithelial-mesenchymal transition (EMT) is thought to be important. In this study, we demonstrate that active Ras is required for TGF-beta1-induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF-beta1. Under conditions of TGF-beta1-induced EMT, cells were growth inhibited by the ligand resulting in G1 arrest. In cells containing normal Ras, TGF-beta1-activated ERK and p38 mitogen-activated protein kinases (MAPKs), and levels of activation were further increased by co-treatment with EGF. Inhibition of MAPK pathways and Smad2/3 signalling blocked the induction of EMT by TGF-beta1. Further, inhibition of the AP-1 transcriptional complex by [6]-Gingerol, or by the ectopic expression of JDP2, blocked TGF-beta1-induced EMT and conversely, stimulation of AP-1 by 12-O-tetradecanoylphorbol 13-acetate (TPA) substituted for EGF in the induction of EMT by TGF-beta1 in cells containing normal Ras. The presence of oncogenic Ras, the treatment of cells with EGF, or the treatment of cells with TPA to activate AP-1, potentiated TGF-beta1-induced Smad-dependent transcription, an effect that was attenuated by the inhibition of MAPKs and AP-1. The results demonstrate that active Ras and TGF-beta1 co-operate to reversibly induce EMT in human keratinocytes by mechanisms that involve MAPKs, Smad2/3 and AP-1. Further we demonstrate that MAPK/AP-1 signalling enhances Smad transcriptional activity under conditions associated with TGF-beta1-induced EMT.
Davies
Maria
M
Department of Oral and Dental Science, University of Bristol, Bristol BS1 2LY, United Kingdom. maria.davies@bristol.ac.uk
Robinson
Max
M
Smith
Emily
E
Huntley
Suzy
S
Prime
Stephen
S
Paterson
Ian
I
D015153:Blotting, Western; D002396:Catechols; D002453:Cell Cycle; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005233:Fatty Alcohols / Q000494:pharmacology; D011905:Genes, ras / Q000502:physiology; D020939:Zingiber officinale; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D008156:Luciferases / Q000378:metabolism; D008648:Mesoderm / Q000166:cytology* / Q000378:metabolism; D009153:Mutagens / Q000494:pharmacology; D010766:Phosphorylation; D010957:Plasmids; D015398:Signal Transduction; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology; D051900:Smad3 Protein; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D002396:Catechols; D004268:DNA-Binding Proteins; D005233:Fatty Alcohols; D009153:Mutagens; C497790:SMAD3 protein, human; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C007845:gingerol; D008156:Luciferases; D048051:p38 Mitogen-Activated Protein Kinases