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Regulatory Role of PI 3-Kinase on Expression of Cdk4 and p27, Nuclear Localization of Cdk4, and Phosphorylation of p27 in Corneal Endothelial Cells

¹From the Doheny Eye Institute and the ²Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, California.

PURPOSE. FGF-2 is a potent mitogen of rabbit corneal endothelial cells (CECs). This study was undertaken to investigate whether PI 3-kinase participates in cell cycle regulation in response to stimulation with FGF-2 in CECs.

METHODS. Cell proliferation was assayed by counting the cells. Subcellular localization of proteins was determined by immunofluorescent staining and expression of cyclin-dependent kinase 4 (Cdk4), p27^Kip1 (p27), phosphatidylinositol 3 (PI 3)-kinase, protein kinase B/Akt (Akt), and ß-actin was analyzed by immunoblot. PI 3-kinase activity was determined by measuring production of phosphatidylinositol-3-phosphate. LY294002 was used to inhibit PI 3-kinase.

RESULTS. CEC required prolonged and continuous exposure to FGF-2. FGF-2 at 10 ng/mL markedly stimulated PI 3-kinase enzyme activity, and stimulation with FGF-2 also caused activation of Akt. LY294002 inhibited both cell proliferation and PI 3-kinase activity in a concentration-dependent manner. The role of PI 3-kinase in cell cycle stimulation was determined: FGF-2 markedly upregulated expression of Cdk4 and stimulated translocation of Cdk4 into nuclei, whereas LY294002 markedly blocked upregulation of Cdk4 expression, and the inhibitor facilitated nuclear export of Cdk4. In contrast, FGF-2 significantly downregulated expression of p27 and facilitated phosphorylation of p27. LY294002 completely blocked the action of FGF-2 on the expression and phosphorylation of p27.

CONCLUSIONS. These data indicate that PI 3-kinase ultimately leads to activation of the cell cycle machinery in response to FGF-2. It does so by upregulating expression of Cdk4, facilitating the nuclear import of Cdk4, and sequestering Cdk4 in the nuclei as it simultaneously downregulates expression of p27 and facilitates the proteolysis of the molecule by phosphorylation.

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