C. Chiu et al., TGF-beta-induced p38 activation is mediated by Rac1-regulated generation of reactive oxygen species in cultured human keratinocytes, INT J MOL M, 8(3), 2001, pp. 251-255
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.