Activation of the Erk Pathway Is Required for TGF-β1-Induced EMT In Vitro

TLDR

Transforming growth factor‑β1 (TGF‑β1) can act tumor‑suppressively via Smad signaling but also promotes tumor progression by inducing epithelial‑to‑mesenchymal transition (EMT) through additional pathways, including MAPK signaling, though the specific role of Erk in TGF‑β1‑mediated EMT remains unclear. In normal murine mammary gland epithelial cells, TGF‑β1 upregulates Ras, Raf, MEK1/2, and Erk1/2 expression, and after 24–48 h of treatment, both NMuMG and mouse cortical tubule cells undergo EMT, evidenced by morphological changes, loss of tight junction proteins, and actin stress fiber formation. TGF‑β1 increases phosphorylated Erk and Erk kinase activity, and inhibition of MEK with U0126 blocks Erk activation and prevents TGF‑β1‑induced EMT in both cell lines, demonstrating that Erk signaling is required for TGF‑β1‑mediated EMT in vitro.

Abstract

Transforming growth factor-β1 (TGF-β1) can be tumorsuppressive through the activation of the Smadmediated signaling pathway. TGF-β1 can also enhance tumor progression by stimulating epithelial-tomesenchymal 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-α1-mediated EMT. However, the role of Erk in the induction of TGF-β1-mediated EMT remains unclear. TGF-β1 treatment of normal murine mammary gland (NMuMG) epithelial cells resulted in increased gene expression of Ras, Raf, MEK1/2, and Erki/2, as shown by microarray analysis and real-time polymerase chain reaction. Upon 24 and 48 hours of treatment with TGIF-α1, NMuMG and mouse cortical tubule (MCT) epithelial cells underwent EMT as shown by changes in cell morphology, delocalization of zonula occludens-1 and E-cadherin from cell-cell junctions, and formation of actin stress fibers. TGF-β1 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-β1 -induced EMT in both cell lines. These data show that TGF-β1 induces the activation of the Erk signaling pathway, which is required for TGF-β1 -mediated EMT in vitro.