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J. Lee, J.-S. Song, R. E. Smith, E. P. Kay; Different Cell Proliferation Pathways Induced by FGF-2 Through p27 Degradation via ERK1/2 and PI 3-Kinase in Human and Rabbit Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2834.
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© ARVO (1962-2015); The Authors (2016-present)
To induce cell proliferation, the cyclin-dependent kinase inhibitor p27Kip1 (p27) must undergo proteolysis through its phosphorylation mechanism. FGF-2 stimulates cell proliferation of rabbit corneal endothelial cells (CECs) by degrading p27. We investigated whether to be induced the cell proliferation pathway and p27 degradation by FGF-2 stimulation in human CECs and compared with those of rabbit CECs.
Cell proliferation was measured by MTT assay. Expression of ERK1/2, Akt, and FGF-2 was analyzed by immunoblotting. Phosphorylation of Akt, p27, p38 and ERK1/2 was detected by immunoblotting. Intracellular location of p27 and Ki67 was determined by immunofluorescent staining. Pharmacological inhibitors were used to block PI 3-kinase or ERK1/2.
FGF-2 dramatically stimulated cell proliferation in human CECs; the effects were completely blocked by pathway specific inhibitors for PI 3-knase and ERK1/2, respectively. We further defined whether FGF-2 induces degradation of p27 through its phosphorylation. Cells maintained in serum-starved medium were stained for p27 in the nuclei, but not for pp27Thr187. However, FGF-2 abolished the staining of p27, while nuclear staining of pp27Thr187 was observed. Using immunoblotting we defined that FGF-2 induced phosphorylation of p27 at Ser10 site. The effects of FGF-2 on p27 expression and phosphorylation of p27 at Thr187 site were completely blocked by LY294002 treatment. We used immunoblotting to define whether both PI 3-kinase and ERK1/2 pathways are independently involved in cell proliferation induced by FGF-2, as observed in rabbit CECs. ERK1/2 activation was completely blocked by treatment of PI 3-kinase inhibitor in response to FGF-2 stimulation; ERK1/2 activation involves cell proliferation induced by FGF-2 as a downstream molecule to PI 3-kinase, which differs from rabbit CECs in which the ERK1/2 pathway is parallel to PI 3-kinase pathway.
FGF-2 stimulates proliferation of human CECs through PI-3 kinase and its downstream target ERK1/2 pathways, and FGF-2 significantly downregulates p27 through the phosphorylation mechanism.
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