Proliferation of CECs requires dual signaling through both MAPK/ERK and PI3-K/Akt pathways

PURPOSE. TO analyze the intracellular signaling involved in the proliferati on of choroidal endothelial cells (CECs) in vitro. METHODS. Bovine CECs were cultured in endothelial growth medium (EGM) conta ining 20% fetal calf serum (FCS), 10 mug/ml bovine brain extract (BBE), and 10 ng/ml epidermal growth factor (EGF) in fibronectin-coated plates. Cells were treated with various specific pharmacologic inhibitors of the mitogen -activated protein kinase (MAPK) and of the phosphatidylinositol 3-kinase ( PI 3-K) pathways to analyze signaling involved in CEC proliferation. Activa tion of the MAPK and PI 3-K was detected by Western blot analysis, using sp ecific antiphosphosignaling protein antibodies. RESULTS. FCS, EGF, and BBE were all necessary to induce optimal mal CEC pro liferation. Individually, these three components were not mitogenic. EGM-st imulated CEC proliferation involved the activation of the Raf/mitogen extra cellular signal-regulated kinase (MEK)/extracellular signal-regulated kinas e (ERK)/p90(RSK) cascade. Inhibition of Ras resulted in a 92% reduction of CEC proliferation, whereas inhibition of ERK1/2 activity reduced it by only 46%. The PI 3-K/p70(S6K)/Akt pathway was also stimulated during CEC prolif eration, and inhibition of PI 3-K activity resulted in a 94% reduction in C EC proliferation. Inhibition of PI 3-K/p70(S6K) activities also unexpectedl y inhibited ERK activity, whereas the converse was not observed, suggesting that PI S-E; acted upstream from ERK and controlled this pathway for CEC p roliferation. CONCLUSIONS. CEC proliferation involves both ERK and PI 3-K. That PI 3-K si gnaling is a key component in cell proliferation can be demonstrated by con trolling ERK activity. These data on the: molecular mechanism and signaling of CEC proliferation may have major implications fur developing more selec tive methods fur antiangiogenic and antitumoral therapy.