Treatment of mice with EGF and orthovanadate activates cytoplasmic and nuclear MAPK, p70S6k, and p90rsk in the liver

Citation
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
Citations number
41
Categorie Soggetti
Gastroenerology and Hepatology","da verificare
Journal title
JOURNAL OF HEPATOLOGY
ISSN journal
01688278 → ACNP
Volume
32
Issue
6
Year of publication
2000
Pages
965 - 974
Database
ISI
SICI code
0168-8278(200006)32:6<965:TOMWEA>2.0.ZU;2-F
Abstract
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.