Role of reactive oxygen species in TGF-β1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells

Abstract

Epithelial-mesenchymal transition (EMT) plays an important role in renal tubulointerstitial fibrosis and TGF-β1 is the key inducer of EMT. Phosphorylation of Smad proteins and/or mitogen-activated protein kinases (MAPK) is required for TGF-β1-induced EMT. Because reactive oxygen species (ROS) are involved in TGF-β1 signaling and are upstream signaling molecules to MAPK, this study examined the role of ROS in TGF-β1-induced MAPK activation and EMT in rat proximal tubular epithelial cells. Growth-arrested and synchronized NRK-52E cells were stimulated with TGF-β1 (0.2 to 20 ng/ml) or H2O2 (1 to 500 μM) in the presence or absence of antioxidants (N-acetylcysteine or catalase), inhibitors of NADPH oxidase (diphenyleneiodonium and apocynin), mitochondrial electron transfer chain subunit I (rotenone), and MAPK (PD 98059, an MEK [MAP kinase/ERK kinase] inhibitor, or p38 MAPK inhibitor) for up to 96 h. TGF-β1 increased dichlorofluorescein-sensitive cellular ROS, phosphorylated Smad 2, p38 MAPK, extracellular signal-regulated kinases (ERK)1/2, α-smooth muscle actin (α-SMA) expression, and fibronectin secretion and decreased E-cadherin expression. Antioxidants effectively inhibited TGF-β1-induced cellular ROS, phosphorylation of Smad 2, p38 MAPK, and ERK, and EMT. H2O 2 reproduced all of the effects of TGF-β1 with the exception of Smad 2 phosphorylation. Chemical inhibition of ERK but not p38 MAPK inhibited TGF-β1-induced Smad 2 phosphorylation, and both MAPK inhibitors inhibited TGF-β1- and H2O2-induced EMT. Diphenyleneiodonium, apocynin, and rotenone also significantly inhibited TGF-β1-induced ROS. Thus, this data suggest that ROS play an important role in TGF-β1-induced EMT primarily through activation of MAPK and subsequently through ERK-directed activation of Smad pathway in proximal tubular epithelial cells. Copyright © 2005 by the American Society of Nephrology.