Regulatory role of PI 3-kinase on expression of Cdk4 and p27, nuclear localization of Cdk4, and phosphorylation of p27 in corneal endothelial cells.
OBJECTIVE
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 beta-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.
Lee
Hyung Taek
HT
Doheny Eye Institute, Los Angeles, California 90033, USA.
Kay
EunDuck P
EP
D000199:Actins / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011817:Rabbits; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D015854:Up-Regulation
D000199:Actins; D018797:Cell Cycle Proteins; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; D004791:Enzyme Inhibitors; D016222:Fibroblast Growth Factor 2; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D025521:Tumor Suppressor Proteins