Oxidative Modification of Nuclear Mitogen-activated Protein Kinase Phosphatase 1 Is Involved in Transforming Growth Factor β1-induced Expression of Plasminogen Activator Inhibitor 1 in Fibroblasts

Rui-Ming Liu,Jinah Choi,Jian-He Wu,Kimberly A Gaston Pravia,Karen M Lewis,Jeffrey D Brand,N.S Reyes Mochel,David M Krzywanski,J David Lambeth,James S Hagood,Henry Jay Forman,Victor J Thannickal,Edward M Postlethwait

doi:10.1074/jbc.m110.111732

Abstract

Transforming growth factor beta (TGF-beta) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-beta. The molecular mechanisms whereby TGF-beta increases ROS production and ROS modulate the signaling processes of TGF-beta, however, remain poorly defined. In this study, we show that TGF-beta1 stimulates NADPH oxidase 4 (Nox4) expression and ROS generation in the nucleus of murine embryo fibroblasts (NIH3T3 cells). This is associated with an increase in protein thiol modification and inactivation of MAPK phosphatase 1 (MKP-1), a nuclear phosphatase. Furthermore, knockdown of MKP-1 using small interfering RNA enhances TGF-beta1-induced phosphorylation of JNK and p38 as well as the expression of plasminogen activator inhibitor 1 (PAI-1), a TGF-beta-responsive gene involved in the pathogenesis of many diseases. Knockdown of Nox4 with Nox4 small interfering RNA, on the other hand, reduces TGF-beta1-stimulated ROS production, p38 phosphorylation, and PAI-1 expression. TGF-beta also increased the nuclear level of Nox4 protein as well as PAI-1 expression in human lung fibroblasts (CCL-210 cells), suggesting that TGF-beta may induce PAI-1 expression by a similar mechanism in human lung fibroblasts. In summary, in this study we have identified nuclear MAPK phosphatase MKP-1 as a novel molecular target of ROS in TGF-beta signaling pathways. Our data suggest that increased generation of ROS by Nox4 mediates TGF-beta1-induced PAI-1 gene expression at least in part through oxidative modification and inhibition of MKP-1 leading to a sustained activation of JNK and p38 MAPKs.

Highlights

Transforming growth factor ␤ (TGF-␤) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-␤
We show that TGF-␤1 increases the expression and activity of NADPH oxidase 4 (Nox4) in the nucleus of NIH3T3 cells, which is associated with increased nuclear ROS production, thiol modifications, and inhibition of a nuclear phosphatase MAPK phosphatase 1 (MKP-1)
TGF-␤1 Increases Nuclear Nox4 Expression and Activity in NIH3T3 Cells— it has been well documented that TGF-␤ increases ROS production in various cell types, the mechanism and precise location(s) of ROS production remain unclear

Summary

Introduction

Transforming growth factor ␤ (TGF-␤) stimulates reactive oxygen species (ROS) production in various cell types, which mediates many of the effects of TGF-␤. We show that TGF-␤1 stimulates NADPH oxidase 4 (Nox4) expression and ROS generation in the nucleus of murine embryo fibroblasts (NIH3T3 cells) This is associated with an increase in protein thiol modification and inactivation of MAPK phosphatase 1 (MKP-1), a nuclear phosphatase. Our data suggest that increased generation of ROS by Nox mediates TGF-␤1-induced PAI-1 gene expression at least in part through oxidative modification and inhibition of MKP-1 leading to a sustained activation of JNK and p38 MAPKs. Transforming growth factor ␤ (TGF-␤), a multi-functional protein, plays an essential role in the development of fibrosis under various pathological conditions involved in almost all organ systems. NADPH oxidases (Noxs) are a family of heme-containing proteins that transfer electrons from NAD(P)H to molecular O2 to form ROS Their biological functions are still largely unknown, emerging evidence indicates that Nox family enzymes are key contributors to ROS generation in both phagocytic and nonphagocytic cells. Whether inactivation of MKPs by ROS/RNS contributes to the sustained activation of MAPKs and increased expression of TGF-␤-responsive genes remains to be determined

Results

Discussion

Conclusion