Induction of an epithelial to mesenchymal transition in human immortal and malignant keratinocytes by TGF‐β1 involves MAPK, Smad and AP‐1 signalling pathways

Abstract

Recent data indicate that transforming growth factor‐β1 (TGF‐β1) 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‐β1‐induced EMT in human keratinocytes and that epidermal growth factor (EGF) can substitute for mutant Ras. EMT was reversed by the removal of TGF‐β1. Under conditions of TGF‐β1‐induced EMT, cells were growth inhibited by the ligand resulting in G 1 arrest. In cells containing normal Ras, TGF‐β1‐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‐β1. Further, inhibition of the AP‐1 transcriptional complex by [6]‐Gingerol, or by the ectopic expression of JDP2, blocked TGF‐β1‐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‐β1 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‐β1‐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‐β1 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‐β1‐induced EMT. © 2005 Wiley‐Liss, Inc.