Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2004 Sep-Oct;6(5):603-10.
doi: 10.1593/neo.04241.

Activation of the Erk pathway is required for TGF-beta1-induced EMT in vitro

Lu Xie  1 , Brian K Law, Anna M Chytil, Kimberly A Brown, Mary E Aakre, Harold L Moses
Affiliations

Activation of the Erk pathway is required for TGF-beta1-induced EMT in vitro

Lu Xie et al. Neoplasia. 2004 Sep-Oct.

Abstract

Transforming growth factor-beta1 (TGF-beta1) can be tumor-suppressive through the activation of the Smad-mediated signaling pathway. TGF-beta1 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-beta1-mediated EMT. However, the role of Erk in the induction of TGF-beta1-mediated EMT remains unclear. TGF-beta1 treatment of normal murine mammary gland (NMuMG) epithelial cells 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-beta1, 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-beta1 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-beta1-induced EMT in both cell lines. These data show that TGF-beta1 induces the activation of the Erk signaling pathway, which is required for TGF-beta1-mediated EMT in vitro.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Inhibition of TGF-β1-mediated EMT by the MEK inhibitor, U0126. NMuMG and MCT cells were treated with 4 ng/ml TGF-β1 in the absence or presence of 10 µM U0126 for 24 hours (NMuMG) or 48 hours (MCT). Nontreated cells or cells treated with U0126 alone were used as negative controls. Phase contrast images (x200) were captured to analyze cell morphology. Cells were stained for ZO-1, E-cadherin, and F-actin (red) and were counterstained with Hoechst 33258 (blue) to visualize cell nuclei. Immunofluorescent images were captured at x400 magnification. Upon TGF-β1 treatment, cells elongated, ZO-1 and E-cadherin disappeared from cell junctions, and F-actin stress fibers formed. The MEK inhibitor (U0126) blocked these effects induced by TGF-β1.
Figure 2
Figure 2
U0126 blocks TGF-β1-induced phosphorylation of Erk. NMuMG (A) and MCT (B) cells were incubated with TGF-β1 (4 ng/ml) in the presence or absence of 10 µM U0126 for 24 hours. Cell lysates were analysed by SDS-PAGE and immunoblotted with antibodies to phospho-Raf, total Raf, phospho-MEK, total MEK, phospho-Erk, total Erk, phospho-Smad2, and total Smad2. β1-Actin was used as a control for equal protein loading. TGF-β1 treatment increased levels of phospho-Erk, and U0126 abolished this increase.
Figure 3
Figure 3
U0126 blocks Erk kinase activity induced by TGF-β1. Erk kinase assays were performed on NMuMG and MCT cells treated with TGF-β1 (4 ng/ml) and/or U0126 (10 µM) for 24 hours. Nontreated cells were used as a control. Soluble lysates were immunoprecipitated with Erk1 antibody or rabbit IgG (Rab. IgG) as a control. The immunoprecipitates were subjected to immune complex kinase assays with myelin basic protein as the substrate. The kinase reactions or immunoprecipitates were analyzed by phosphorimaging and immunoblotting with Erk1. NMuMG and MCT cells treated with TGF-β1 had enhanced Erk activation. Treatment with U0126 reduced Erk kinase activity induced by TGF-β1.
Figure 4
Figure 4
Model of TGF-β1-induced Erk activation and EMT being blocked by U0126. TGF-β1 may activate the Erk pathway through upstream factors, such as Ras/Raf, or directly through more downstream factors, such as MEK or Erk. To investigate the role of Erk signaling in TGF-β1-mediated EMT, the MEK1/2 inhibitor, U0126, was used to block Erk phosphorylation in NMuMG and MCT cells. In these cells, TGF-β1 induction of Erk activity and EMT was blocked by U0126. However, TGF-β1 may activate other pathways, such as p38, JNK, PI3-kinase, and RhoA, to induce EMT in other cell types.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Shi Y, Massague J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus. Cell. 2003;113:685–700. - PubMed
    1. Massague J. TGF-beta signal transduction. Annu Rev Biochem. 1998;67:753–791. - PubMed
    1. de Caestecker MP, Piek E, Roberts AB. Role of transforming growth factor-beta signaling in cancer. J Natl Cancer Inst. 2000;92:1388–1402. - PubMed
    1. Atfi A, Djelloul S, Chastre E, Davis R, Gespach C. Evidence for a role of Rho-like GTPases and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in transforming growth factor beta-mediated signaling. J Biol Chem. 1997;272:1429–1432. - PubMed
    1. Engel ME, McDonnell MA, Law BK, Moses HL. Interdependent SMAD and JNK signaling in transforming growth factor-beta-mediated transcription. J Biol Chem. 1999;274:37413–37420. - PubMed

Publication types

MeSH terms

LinkOut - more resources