Role of Reactive Oxygen Species in TGF-β1-Induced Mitogen-Activated Protein Kinase Activation and Epithelial-Mesenchymal Transition in Renal Tubular Epithelial Cells

TLDR

Epithelial‑mesenchymal transition drives renal tubulointerstitial fibrosis, and TGF‑β1 is the principal inducer that requires Smad and MAPK phosphorylation. The study aimed to determine how reactive oxygen species influence TGF‑β1‑induced MAPK activation and EMT in rat proximal tubular epithelial cells. NRK‑52E cells were stimulated with TGF‑β1 or H₂O₂, with or without antioxidants, NADPH oxidase inhibitors, mitochondrial inhibitors, and MAPK inhibitors, and assayed over 96 h. TGF‑β1 triggered ROS production, Smad2, p38 MAPK, and ERK1/2 phosphorylation, α‑SMA and fibronectin up‑regulation, and E‑cadherin down‑regulation, all of which were suppressed by antioxidants, MAPK inhibitors, and NADPH oxidase inhibitors, showing that ROS mediate TGF‑β1‑induced EMT via MAPK‑dependent Smad activation.

Abstract

Epithelial-mesenchymal transition (EMT) plays an important role in renal tubulointerstitial fibrosis and TGF-beta1 is the key inducer of EMT. Phosphorylation of Smad proteins and/or mitogen-activated protein kinases (MAPK) is required for TGF-beta1-induced EMT. Because reactive oxygen species (ROS) are involved in TGF-beta1 signaling and are upstream signaling molecules to MAPK, this study examined the role of ROS in TGF-beta1-induced MAPK activation and EMT in rat proximal tubular epithelial cells. Growth-arrested and synchronized NRK-52E cells were stimulated with TGF-beta1 (0.2 to 20 ng/ml) or H(2)O(2) (1 to 500 microM) 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-beta1 increased dichlorofluorescein-sensitive cellular ROS, phosphorylated Smad 2, p38 MAPK, extracellular signal-regulated kinases (ERK)1/2, alpha-smooth muscle actin (alpha-SMA) expression, and fibronectin secretion and decreased E-cadherin expression. Antioxidants effectively inhibited TGF-beta1-induced cellular ROS, phosphorylation of Smad 2, p38 MAPK, and ERK, and EMT. H(2)O(2) reproduced all of the effects of TGF-beta1 with the exception of Smad 2 phosphorylation. Chemical inhibition of ERK but not p38 MAPK inhibited TGF-beta1-induced Smad 2 phosphorylation, and both MAPK inhibitors inhibited TGF-beta1- and H(2)O(2)-induced EMT. Diphenyleneiodonium, apocynin, and rotenone also significantly inhibited TGF-beta1-induced ROS. Thus, this data suggest that ROS play an important role in TGF-beta1-induced EMT primarily through activation of MAPK and subsequently through ERK-directed activation of Smad pathway in proximal tubular epithelial cells.