IGFs stimulate zebrafish cell proliferation by activating MAP kinase and PI3-kinase-signaling pathways.
Insulin-like growth factor (IGF)-I and -II have been cloned from a number of teleost species, but their cellular actions in fish are poorly defined. In this study, we show that both IGF-I and -II stimulated zebrafish embryonic cell proliferation and DNA synthesis in a concentration-dependent manner, whereas insulin had little mitogenic activity. Affinity cross-linking and immunoblotting studies revealed the presence of IGF receptors with the characteristics of the mammalian type I IGF receptor. Competitive binding assay results indicated that the binding affinities of the zebrafish IGF-I receptors to IGF-I, IGF-II, and insulin are 1.9, 2.6, and >190 nM, indicating that IGF-I and -II bind to the IGF-I receptor(s) with approximately equal high affinity. To further investigate the cellular mechanism of IGF actions, we have studied the effects of IGFs on two major signal transduction pathways: mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3 kinase). IGFs activated MAPK in zebrafish embryonic cells in a dose-dependent manner. This activation occurred within 5 min of IGF-I stimulation and disappeared after 1 h. IGF-I also caused a concentration-dependent activation of protein kinase B, a downstream target of PI3 kinase, this activation being sustained for several hours. Inhibition of MAPK activation by the MAPK kinase inhibitor PD-98059 inhibited the IGF-I-stimulated DNA synthesis. Similarly, use of the PI3 kinase inhibitor LY-294002 also inhibited IGF-I-stimulated DNA synthesis. When both the MAPK and PI3 kinase pathways were inhibited using a combination of these compounds, the IGF-I-stimulated DNA synthesis was completely negated. These results indicate that both IGF-I and -II are potent mitogens for zebrafish embryonic cells and that activation of both the MAPK and PI3 kinase-signaling pathways is required for the mitogenic action of IGFs in zebrafish embryonic cells.
University of Michigan, Department of Biology, Natural Science Building, Ann Arbor, MI 48109, USA.
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Pozios
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Ding
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Degger
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Upton
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Duan
D000818:Animals; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004625:Embryo, Nonmammalian; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007335:Insulin-Like Growth Factor II / Q000494:pharmacology*; D007700:Kinetics; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017526:Receptor, IGF Type 1 / Q000187:drug effects / Q000502:physiology*; D017527:Receptor, IGF Type 2 / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D012474:Salmon; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D015027:Zebrafish
D002867:Chromones; D005419:Flavonoids; D007328:Insulin; D009025:Morpholines; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D007334:Insulin-Like Growth Factor I; D007335:Insulin-Like Growth Factor II; D019869:Phosphatidylinositol 3-Kinases; D017526:Receptor, IGF Type 1; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one