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Anna Zubilewicz, Christiane Hecquet, Jean-Claude Jeanny, Gisele Soubrane, Yves Courtois, Frederic Mascarelli; Proliferation of CECs Requires Dual Signaling through Both MAPK/ERK and PI 3-K/Akt Pathways. Invest. Ophthalmol. Vis. Sci. 2001;42(2):488-496. doi: https://doi.org/.
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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
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
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
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