Inhibition of MAP kinase activity moderates changes in expression and function of Cx32 but not claudin-1 during DNA synthesis in primary cultures of rat hepatocytes.
The signal transduction pathways and activation of the MAP kinase or PI3 kinase signaling cascade regulate a variety of cellular processes, including proliferation and differentiation in hepatocytes. To elucidate the mechanisms of signal transmission required for the regulation of gap and tight junctions during DNA synthesis in rat hepatocytes, we determined changes of expression and function of gap and tight junctions of cells grown in primary culture, using inhibitors of signaling pathways for MAP kinase (PD98059) and PI3 kinase (LY294002). During the stimulation of DNA synthesis induced by epidermal growth factor (EGF), immunoreactivity and mRNAs of gap junction protein Cx32 and of tight junction protein claudin-1 markedly decreased with reduction of gap junctional intercellular communication (GJIC) and the fence function of tight junctions. In Western blots, whole-cell lysate of claudin-1 protein decreased and phosphorylated Cx32 protein in the insoluble fraction of Triton X-100 increased during the stimulation of DNA synthesis. During reinhibition of DNA synthesis, the changes of Cx32 and claudin-1 returned to control levels, as did both functions. In treatment with the inhibitors before DNA synthesis, PD98059 inhibited the changes of expression and function of Cx32, but not claudin-1, without inhibition of cell growth, whereas LY294002 completely inhibited cell growth. These findings indicate that the PI3 kinase pathway rather than the MAP kinase pathway plays an important role for EGF-induced proliferation of rat hepatocytes, and that changes of Cx32 in hepatocytes during the stimulation of DNA synthesis may be in part controlled through MAP kinase. Furthermore, Cx32, but not claudin-1, protein may be a target of activated MAP kinase in hepatocytes.
Kojima
Takashi
T
Department of Pathology, Sapporo Medical University School of Medicine, S1, W17, Sapporo 060-8556, Japan. ktakashi@sapmed.ac.jp
Yamamoto
Toshinobu
T
Lan
Mengdong
M
Murata
Masaki
M
Takano
Ken-Ichi
K
Go
Mitsuru
M
Ichimiya
Shingo
S
Chiba
Hideki
H
Sawada
Norimasa
N
D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D062445:Claudin-1; D017630:Connexins / Q000378:metabolism*; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D019108:Tight Junctions / Q000187:drug effects / Q000378:metabolism; D000097002:Gap Junction beta-1 Protein
D002867:Chromones; D062445:Claudin-1; D017630:Connexins; D004791:Enzyme Inhibitors; D004815:Epidermal Growth Factor; D005419:Flavonoids; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000097002:Gap Junction beta-1 Protein; C568114:Cldn1 protein, rat; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one