title: Cdk4 and p27Kip1 play a role in PLC-gamma1-mediated mitogenic signaling pathway of 18 kDa FGF-2 in corneal endothelial cells.
abstract: OBJECTIVE
  To determine whether PLC-gamma1 enzyme activity is essential for cell proliferation in response to FGF-2 stimulation and to investigate the effect of PLC-gamma1 activation on cell division and on processes that regulate cell cycle progression.
  
  
  METHODS
  Cell proliferation was assayed using a colorimetric method to determine the number of viable cells. Subcellular localization of proteins was determined by immunocytochemical analysis, and expression of the proteins was analyzed by immunoblotting. PLC activity was determined by measuring the total inositol phosphates.
  
  
  RESULTS
  When CEC were treated with FGF-2, a prolonged and continuous FGF-2 exposure was required for both PLC enzyme activation and cell proliferation. However, there was a long lag period between the PLC enzyme activation and cell proliferation: the maximum enzyme activity was reached 8 h after FGF-2 stimulation, but no cell proliferation was observed in the cells exposed to FGF-2 for 8 h. Using neutralizing anti-PLC-gamma1, PLC-beta1, or PLC-delta1 antibodies, we further demonstrated that PLC-gamma1 accounts for the hydrolysis of total phosphoinositides (PI) and cell proliferation mediated by FGF-2. The role of PLC-gamma1 linking to the cell cycle was then determined by the blockades of FGF-2 action on Cdk4 and p27-Kip1. Interestingly, FGF-2 both upregulates Cdk4 synthesis and facilitates the nuclear import of the molecule from the cytoplasm, whereas it facilitates the nuclear export of p27-Kip1 to the cytoplasm without affecting synthesis of the molecule. The neutralizing anti-PLC-gamma1 antibody completely abolishes the FGF-2 activity on Cdk4, both at the synthetic level and at the level of translocation, and the PLC-gamma1 antibody blocks the nuclear export of p27-Kip1.
  
  
  CONCLUSIONS
  These data indicate that PLC-gamma1 ultimately leads to activation of the cell cycle machinery to induce cell proliferation mediated by FGF-2. It does so by upregulating Cdk4 expression and by facilitating the nuclear import of the molecule and nuclear export of the Cdk inhibitor (p27-Kip1) to the proteolysis site, the cytoplasm.
authors.affiliation: Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA. hyungtaeklee@hotmail.com
authors.forename: Hyung Taek
authors.initials: HT
authors.lastname: Lee
authors.forename: Tae Yon
authors.initials: TY
authors.lastname: Kim
authors.forename: EunDuck P
authors.initials: EP
authors.lastname: Kay
mesh_terms: D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D051358:Cyclin-Dependent Kinase 4; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004789:Enzyme Activation; D004963:Estrenes / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018613:Microscopy, Confocal; D008970:Molecular Weight; D051966:Phospholipase C gamma; D011518:Proto-Oncogene Proteins; D011760:Pyrrolidinones / Q000494:pharmacology; D011817:Rabbits; D015398:Signal Transduction / Q000502:physiology*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D018797:Cell Cycle Proteins; D004963:Estrenes; D007295:Inositol Phosphates; D007527:Isoenzymes; D011518:Proto-Oncogene Proteins; D011760:Pyrrolidinones; D025521:Tumor Suppressor Proteins; D016222:Fibroblast Growth Factor 2; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D050760:Cyclin-Dependent Kinase Inhibitor p27; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma