TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes.
p38 has been shown to be involved in TGF-beta-induced gene expression, but the upstream of the signaling pathway leading to the activation of p38 is left undefined. We investigated the pathway in cultured human keratinocytes (HaCat cells). Western blot analysis revealed that TGF-beta induced the activation of p38 within 1 h post TGF-beta treatment. H2O2 also strongly induced p38 activation in a time dependent manner. We also observed that TGF-beta-induced p38 activation was inhibited by PDTC, pyrrolidinedithiocarbamate, a known antioxidant, and DPI, diphenylene iodonium chloride, one of the known NADPH oxidase inhibitors. In contrast, TGF-beta-induced Smad2 phosphorylation was not affected. To test whether reactive oxygen species (ROS) is involved in TGF-beta-induced p38 activation, we examined the generation of ROS and activation of NADPH oxidase. FACS analysis showed that TGF-beta induced generation of ROS in time-dependent manner. DPI, an inhibitor of NADPH oxidase, inhibited TGF-beta-induced ROS production. Lucigenin-based NADPH oxidase assay indicated that TGF-beta-induced NADPH oxidase activity started as early as 5 min following treatment and peaked at about 15 min with induction of about 2-folds. The activity remained elevated up to 1 h. Immunofluorescence microscopy study showed that Rac1, one of the subunits of NADPH oxidase, translocated from cytoplasm to the membrane within 5 min. Pretreatment with DPI dramatically reduced TGF-beta-induced NADPH oxidase activity. Collectively, our data suggest that TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes.
Chiu
C
C
Department of Biology, Providence College, 549 River Ave., Providence, RI 02918, USA.
Maddock
D A
DA
Zhang
Q
Q
Souza
K P
KP
Townsend
A R
AR
Wan
Y
Y
D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D051899:Smad2 Protein; D013859:Thiocarbamates / Q000494:pharmacology; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein / Q000378:metabolism / Q000502:physiology*
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009097:Multienzyme Complexes; D009861:Onium Compounds; D017382:Reactive Oxygen Species; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D013859:Thiocarbamates; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C066229:prolinedithiocarbamate; C007517:diphenyleneiodonium; D011392:Proline; D006861:Hydrogen Peroxide; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D020830:rac1 GTP-Binding Protein