Activation of the Erk Pathway Is Required for TGF-b1–Induced EMT In Vitro1

Transforming growth factor-B1 (TGF-B1) can be tumor-suppressive through the activation of the Smad-mediated signaling pathway. TGF-B1 can also enhance tumor progression by stimulating epithelial-to-mesenchymal transition (EMT) through additional pathways. EMT is characterized by the acquisition of a fibroblast-like cell morphology, dissolution of tight junctions, disruption of adherence junctions, and formation of actin stress fibers. There is evidence linking the activation of mitogen-activated protein kinase pathways to the induction of TGF-B1–mediated EMT. However, the role of Erk in the induction of TGF-B1–mediated EMT remains unclear. TGF-B1 treat-ment of mouse mammary gland epithelial cells (NMuMG) resulted in increased gene expression of Ras, Raf, MEK1/2, and Erk1/2, as shown by microarray analysis and real-time polymerase chain reaction. Upon 24 and 48 hours of treatment with TGF-B1, NMuMG and mouse proximal tubule epithelial cells underwent EMT as shown by changes in cell mor-phology, delocalization of zonula occludens-1 and E-cadherin from cell–cell junctions, and formation of actin stress fibers. TGF-B1 treatment also resulted in increased levels of phosphorylated Erk and Erk kinase activity. Treatment with an MEK inhibitor, U0126, inhibited increased Erk phosphorylation and kinase activity, and blocked TGF-B1–induced EMT in both cell lines. These data show that TGF-B1 induces the activation of the Erk signaling pathway, which is required for TGF-B1–mediated EMT in vitro