title: Human corneal endothelial cells employ phosphorylation of p27(Kip1) at both Ser10 and Thr187 sites for FGF-2-mediated cell proliferation via PI 3-kinase.
abstract: OBJECTIVE
  FGF-2 stimulates cell proliferation of rabbit corneal endothelial cells (rCECs) by degrading the cyclin-dependent kinase inhibitor p27(Kip1) (p27) through its phosphorylation mechanism. The authors investigated whether the cell proliferation of human CECs (hCECs) is also induced by FGF-2 stimulation through the p27 phosphorylation pathway.
  
  
  METHODS
  Expression and activation of protein were analyzed by immunoblotting. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Transfection of hCECs with small interference RNA (siRNA) was performed using a transfection reagent.
  
  
  RESULTS
  FGF-2 stimulated cell proliferation in hCECs; the FGF-2 action was completely blocked by pathway-specific inhibitors for PI 3-kinase (LY294002) and MEK1/2 (U0126), respectively. Using immunoblotting, the authors showed that FGF-2 induced phosphorylation of p27 at both serine 10 (Ser10) and threonine 187 (Thr187) sites. These effects were also completely blocked by LY294002 or U0126. The authors then determined cross-talk between PI 3-kinase and extracellular signal-regulated kinase (ERK)1/2; blocking of ERK1/2 activation by LY294002 indicated that in hCECs ERK1/2 works as a downstream effector to PI 3-kinase for cell proliferation induced by FGF-2, whereas the ERK1/2 pathway in rCECs is parallel to the PI 3-kinase pathway. However, the downstream mechanism involved in cell cycle progression in hCECs is identical to that of rCECs: phosphorylation of p27 at Ser10 was mediated by kinase-interacting stathmin (KIS), confirmed with siRNA to KIS, and phosphorylation of p27 at Thr187 was mediated by cell division cycle 25A (Cdc25A), confirmed using Cdc25A inhibitor. CONCLUSIONS; FGF-2 stimulates proliferation of hCECs through PI 3-kinase and its downstream target ERK1/2 pathways. This linear signal transduction significantly downregulates p27 through its phosphorylation at both Ser10 and Thr187 sites mediated by KIS and Cdc25A, respectively.
authors.affiliation: Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, USA.
authors.forename: Jeong Goo
authors.initials: JG
authors.lastname: Lee
authors.forename: Jong-Suk
authors.initials: JS
authors.lastname: Song
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Smith
authors.forename: Eunduck P
authors.initials: EP
authors.lastname: Kay
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002070:Butadienes / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D002867:Chromones / Q000494:pharmacology; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D006801:Humans; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D018613:Microscopy, Confocal; D008875:Middle Aged; D009025:Morpholines / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D012694:Serine / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014019:Tissue Donors; D014162:Transfection; D055815:Young Adult
chemical_list: D002070:Butadienes; D002867:Chromones; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004791:Enzyme Inhibitors; D016222:Fibroblast Growth Factor 2; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D009025:Morpholines; D009570:Nitriles; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D012694:Serine; D013778:Tetrazolium Salts; D013844:Thiazoles; D013912:Threonine; C113580:U 0126; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C482125:MAP2K1 protein, human; C022616:thiazolyl blue