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Open Access Research article

c-Jun N terminal kinase modulates NOX-4 derived ROS production and myofibroblasts differentiation in human breast stromal cells

Nicolás Tobar1, Marcela Toyos1, Carla Urra1, Nicolás Méndez1, Rodrigo Arancibia2, Patricio C Smith2 and Jorge Martínez1*

Author Affiliations

1 Laboratorio de Biología Celular y Molecular, INTA, Universidad de Chile, El Líbano 5524, Macul, Santiago, Chile

2 Laboratorio de Fisiología Periodontal, Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile

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BMC Cancer 2014, 14:640  doi:10.1186/1471-2407-14-640

Published: 30 August 2014

Abstract

Background

Hard consistency, developed under the influence of tumor cell factors, is a characteristic feature of a breast tumor. Activation of resident fibroblasts leading to a myofibroblast phenotype is the principal feature that orchestrates this fibrotic process. The aim of this study was to assess the effects induced by TGF-β1, a growth factor abundantly present in tumor microenvironment, on the molecular mechanisms that mediate myofibroblastic differentiation of normal human mammary fibroblasts.

Methods

We used an immortalized fibroblastic cell line derived from normal mammary tissue (RMF-EG cells) to study the effect of TGF-β1 in the expression of α-SMA and CTGF as markers of myofibroblastic differentiation. The influence of redox status and JNK activity on TGF-β1-induced transcriptional activity was measured by a luciferase reporter assay. We also used a shRNA approach to evaluate the influence of NOX4 in myofibroblastic differentiation.

Results

TGF-β1 stimulates the expression of myofibroblast markers α-SMA and CTGF. Using a NOX inhibitor (DPI) and cells expressing a shRNA for NOX4, we demonstrated that TGF-β1 promotes an oxidative environment that favors myofibroblastic differentiation. We also found that activation of c-Jun N-terminal kinase is required for TGF-β1-dependent expression of CTGF, NOX4 and α-SMA.

Conclusions

Human mammary stromal fibrosis, evaluated by the expression of early and late markers as CTGF and α-SMA, depends on the activation of JNK signaling pathway. Our results show that JNK activation is an early event that precedes the increase in ROS levels leading to myofibroblastic differentiation and tumor fibrosis, suggesting that inhibition of JNK may be used a method to interrupt the development of tumor desmoplasia.

Keywords:
Fibrosis; NOX-4; JNK; ROS