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1 From the Department of Ophthalmology, Medical School of Lublin, Poland; and 2 Institut National de la Santé et de la Recherche Médicale, Unité 450, Développement, Vieillissement, et Pathologie de la Rétine, Centre National de la Recherche Scientifique, Association Claude Bernard, Paris, France.
PURPOSE. To analyze the intracellular signaling involved in the proliferation of choroidal endothelial cells (CECs) in vitro.
METHODS. Bovine CECs were cultured in endothelial growth medium (EGM) containing 2% fetal calf serum (FCS), 10 µg/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. Activation of the MAPK and PI 3-K was detected by Western blot analysis, using specific antiphosphosignaling protein antibodies.
RESULTS. FCS, EGF, and BBE were all necessary to induce optimal CEC proliferation. Individually, these three components were not mitogenic. EGM-stimulated CEC proliferation involved the activation of the Raf/mitogen extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90RSK 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/p70S6K/Akt pathway was also stimulated during CEC proliferation, and inhibition of PI 3-K activity resulted in a 94% reduction in CEC proliferation. Inhibition of PI 3-K/p70S6K activities also unexpectedly inhibited ERK activity, whereas the converse was not observed, suggesting that PI 3-K acted upstream from ERK and controlled this pathway for CEC proliferation.
CONCLUSIONS. CEC proliferation involves both ERK and PI 3-K. That PI 3-K signaling is a key component in cell proliferation can be demonstrated by controlling ERK activity. These data on the molecular mechanism and signaling of CEC proliferation may have major implications for developing more selective methods for antiangiogenic and antitumoral therapy.
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