J. Ostrowski et al., Treatment of mice with EGF and orthovanadate activates cytoplasmic and nuclear MAPK, p70S6k, and p90rsk in the liver, J HEPATOL, 32(6), 2000, pp. 965-974
Background/Aims: Although signal transduction pathways activated by EGF hav
e been extensively studied in cultured cells, few such studies have been do
ne in whole animals. In this study, activation of hepatic kinases, phosphat
ases, and DNA-binding activity of AP-1 was examined after intraperitoneal i
njections of either EGF or sodium orthovanadate into mice.
Methods: Cytoplasmic and nuclear proteins, extracted from isolated hepatocy
tes or whole liver tissue, were immunoprecipitated with either anti-ERK1/2,
anti-70S6k, or anti-p90rsk antibodies and kinase activities were measured
using specific substrates, Kinase protein levels was evaluated by Western b
lot analysis, AP-1 DNA binding activity was measured by electrophoretic mob
ility shift assay.
Results: Systemic administration of EGF induced simultaneous increase in th
e activities of cytoplasmic and nuclear MAPK, p70S6k, and p90rsk. MAPK and
p70S6k were more potently activated in the cytosol while p90rsk activation
was more pronounced in the nucleus. Orthovanadate also activated these kina
ses but to a much lesser degree than EGE In vitro phosphatase assays showed
that neither EGF nor orthovanadate induced measurable changes in phosphata
se activities. EGF, but not orthovanadate, activated nuclear AP-1 DNA-bindi
ng activity in intact liver, indicating that activation of MAPK, p70S6k, an
d p90rsk by orthovanadate is not sufficient to activate this transcription
factor,
Conclusion: These observations provide groundwork for future studies to exa
mine the role of EGF-induced kinase cascades and transcription factors in l
iver regeneration and other growth factor-mediated hepatic processes.