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E.P. Kay, H. Lee; cAMP Regulates Cdk4 and p27 Expression by Inhibiting PI 3-kinase/Akt Pathways in Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4332.
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Purpose: Fibroblast growth factor-2 (FGF-2) is a potent mitogen of corneal endothelial cells (CEC). Cyclic AMP (cAMP) is known to antagonize growth factor activation in a variety of cells by inhibiting PI 3-kinase/Akt pathways and/or extracellular signal-regulated kinases. We investigated whether cAMP regulates Cdk4 and p27Kip1 (p27) expression by inhibiting PI 3-kinase/Akt pathways in CEC. Methods: Cell proliferation was assayed by counting the viable cells. Expression of protein was analyzed by immunoblotting and subcellular localization was determined by immunofluoresecent staining. PI 3-kinase enzyme activity was determined by measuring the level of phosphoinositol 3-phosphate. Transfection was performed using Effectene. Results: We demonstrate here that 8-Br-cAMP, a non-hydrolysable but diffusible cAMP analog, inhibits PI 3-kinase/Akt signaling pathways; 8-Br-cAMP and PI 3-kinase inhibitor (LY294002) produced equivalent stimulation and inhibition, respectively, of p27 and Cdk4 protein levels; they also equally inhibited cell proliferation, nuclear translocation of Cdk4 and phosphorylation of p27. Negative regulation of PI 3-kinase by 8-Br-cAMP is mediated by a direct inhibition of PI 3-kinase enzyme activity, which subsequently blocks phosphorylation of Akt at both the Ser473 and Thr308 sites. In addition, 8-Br-cAMP further promotes a rapid turnover of Akt protein, and interestingly, 8-Br-cAMP markedly reduces the half-life of Cdk4 protein. This inhibitory activity of cAMP is not mediated by PKA; but 8-Br-cAMP inhibits membrane localization of the p85 regulatory subunit of PI 3-kinase as determined by transfecting cells with pCMV6 p85-myc. Conclusions: These data support the hypothesis that cAMP inhibits cell proliferation of CEC by preventing cells from entering S phase by negatively regulating PI 3-kinase
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