Role of reactive oxygen species in TGF-beta1-induced mitogen-activated protein kinase activation and epithelial-mesenchymal transition in renal tubular epithelial cells.
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.
Rhyu
Dong Young
DY
Ewha Womans University College of Pharmacy, 11-1 Daehyun-dong, Sedaimun-gu, Seoul 120-750, Korea.
Yang
Yanqiang
Y
Ha
Hunjoo
H
Lee
Geun Taek
GT
Song
Jae Sook
JS
Uh
Soo-taek
ST
Lee
Hi Bahl
HB
D000199:Actins / Q000378:metabolism; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects* / Q000502:physiology; D008648:Mesoderm / Q000166:cytology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009395:Nephritis, Interstitial / Q000378:metabolism / Q000473:pathology*; D016877:Oxidants / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051899:Smad2 Protein; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000199:Actins; D000975:Antioxidants; D015820:Cadherins; D004268:DNA-Binding Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D005353:Fibronectins; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D016877:Oxidants; D017382:Reactive Oxygen Species; D051899:Smad2 Protein; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D048051:p38 Mitogen-Activated Protein Kinases; C497784:Smad2 protein, rat; C508962:Tgfb1 protein, rat; C498862:Acta2 protein, rat