Treatment of mice with EGF and orthovanadate activates cytoplasmic and nuclear MAPK, p70S6k, and p90rsk in the liver.
OBJECTIVE
Although signal transduction pathways activated by EGF have been extensively studied in cultured cells, few such studies have been done in whole animals. In this study, activation of hepatic kinases, phosphatases, and DNA-binding activity of AP-1 was examined after intraperitoneal injections of either EGF or sodium orthovanadate into mice.


METHODS
Cytoplasmic and nuclear proteins, extracted from isolated hepatocytes 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 blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay.


RESULTS
Systemic administration of EGF induced simultaneous increase in the 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 kinases but to a much lesser degree than EGF. In vitro phosphatase assays showed that neither EGF nor orthovanadate induced measurable changes in phosphatase activities. EGF, but not orthovanadate, activated nuclear AP-1 DNA-binding activity in intact liver, indicating that activation of MAPK, p70S6k, and p90rsk by orthovanadate is not sufficient to activate this transcription factor.


CONCLUSIONS
These observations provide groundwork for future studies to examine the role of EGF-induced kinase cascades and transcription factors in liver regeneration and other growth factor-mediated hepatic processes.
Ostrowski
J
J
Department of Gastroenterology, Medical Center for Postgraduate Education in the Maria Sklodowska-Curie Memorial Cancer Center, Walrsaw, Poland. jostrow@warman.com.pl
Woszczyński
M
M
Kowalczyk
P
P
Trzeciak
L
L
Hennig
E
E
Bomsztyk
K
K
D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D007274:Injections, Intraperitoneal; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008808:Mice, Inbred CBA; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D019893:Ribosomal Protein S6 Kinases / Q000235:genetics / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology*
D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D014638:Vanadates; D004815:Epidermal Growth Factor; D004247:DNA; D019893:Ribosomal Protein S6 Kinases; D020928:Mitogen-Activated Protein Kinases; D010744:Phosphoric Monoester Hydrolases