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EP Kay, H Lee; Role of PI3-kinase on the Expression of Cdk4 and p27 in FGF-2-mediated Mitogenic Signaling Pathway in Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):856.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine whether FGF-2-mediated PI 3-kinase activity accounts for cell cycle progression in corneal endothelial cells. Methods: Cell proliferation was assayed using a colorimetric method for determining the number of viable cells. Expression of proteins was analyzed by immunoblotting and subcellular localization was determined by immunofluorescent staining. PI 3-kinase enzyme activity was determined by measuring the level of phosphoinositol 3-phosphate. Results: FGF-2 stimulated PI 3-kinase activity in a biphasic fashion; the initial stimulatory response was observed during the first hour of cell treatment with FGF-2; the late response was observed after 16 hours of treatment, peaking at 24 hours. FGF-2 stimulation caused a biphasic activation of protein kinase B/Akt, similar to that observed in PI 3-kinase activity. LY294002 inhibited both cell proliferation and PI 3-kinase activity in a concentration-dependent manner. The role of PI 3-kinase linking to the cell cycle in response to FGF-2 stimulation was determined: FGF-2 markedly upregulated synthesis of Cdk4 and stimulated translocation of Cdk4 into nuclei, whereas LY294002 markedly blocked both actions of FGF-2. On the other hand, FGF-2 significantly downregulated the expression of p27 and facilitated phosphorylation of p27. LY294002 blocked the downregulation of FGF-2 on p27 expression and completely abolished the action of FGF-2 on phosphorylation of p27 in a time-dependent manner. Conclusions: These data indicate that PI 3-kinase leads to activation of the cell cycle machinery in response to FGF-2. It does so by upregulating Cdk4 expression and facilitating the nuclear import of Cdk4 as it simultaneously downregulates p27 expression and facilitates the proteolysis of the molecule via phosphorylation.
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