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 keratinocyte s (HaCat cells). Western blot analysis revealed that TGF-beta induced the a ctivation of p38 within I h post TGF-beta treatment. H2O2 also strongly ind uced p38 activation in a time dependent manner. We also observed that TGF-b eta -induced p38 activation was inhibited by PDTC, pyrrolidinedithiocarbama te, a known antioxidant, and DPI, diphenylene iodonium chloride, one of the known NADPH oxidase inhibitors. In contrast, TGF-beta -induced Smad2 phosp horylation 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 N ADPH oxidase, inhibited TGF-beta -induced ROS production. Lucigenin-based N ADPH oxidase assay indicated that TGF-beta -induced NADPH oxidase activity started as early as 5 min following treatment and peaked at about 15 min wi th induction of about 2-folds. The activity remained elevated up to I h. Im munofluorescence microscopy study showed that Rac1, one of the subunits of NADPH oxidase, translocated from cytoplasm to the membrane within 5 min. Pr etreatment with DPI dramatically reduced TGF-beta -induced NADPH oxidase ac tivity. Collectively, our data suggest that TGF-beta -induced p38 activatio n is mediated by Rac1-regulated generation of reactive oxygen species in cu ltured human keratinocytes.