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B Liu, AH Neufeld; Epidermal Growth Factor Receptor Signals Nitric Oxide Synthase-2 Induction In Response To Elevated Hydrostatic Pressure In Human Optic Nerve Head Astrocytes . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2187.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The epidermal growth factor receptor (EGFR) is a key element mediating cellular signal transduction in a variety of normal and pathophysiological phenomena. We hypothesize that tyrosine phosphorylation of EGFR is an early step in the signal pathway for induction of nitric oxide synthase-2 (NOS-2) in human optic nerve head (ONH) astrocytes in response to elevated hydrostatic pressure. Methods: Primary cultures of astrocytes from human ONHs were exposed to elevated hydrostatic pressure (5 cm depth of growth medium per coverslip). Tyrosine phosphorylation of EGFR was studied by immunocytochemistry and Western blot. Tyrosine kinase inhibitors, AG82 or AG18, were added to the medium before and during exposure to elevated hydrostatic pressure. NOS-2 expression was detected by Western blot and quantitative RT-PCR. Results: Immunolabeling for EGFR was present in the cell membrane, cytoplasm and nuclei of control ONH astrocytes and did not change in response to elevated hydrostatic pressure. Immunolabelling of phosphorylated EGFR (p-EGFR) was faintly distributed in the cell bodies of control astrocytes and there was no detectable p-EGFR in the cell nuclei by both immunocytochemistry and Western blot. However, 10 min after ONH astrocytes were exposed to elevated hydrostatic pressure, tyrosine phosphorylation of EGFR was markedly enhanced in both the cell body and nucleus. The intensive staining of p-EGFR in the cell body was filamentous and accumulated in the cell processes. By Western blot, heavy bands of p-EGFR were detected in both non-nuclear and nuclear extracts. Under elevated hydrostatic pressure, pretreatment with tyrosine kinase inhibitors, AG82 or AG18, significantly blocked tyrosine phosphorylation of EGFR, particularly in the cell nucleus. Pretreatment with these tyrosine kinase inhibitors also blocked induction of NOS-2 in response to elevated hydrostatic pressure. Conclusion: Within minutes, elevated hydrostatic pressure causes tyrosine phosphorylation of EGFR in human ONH astrocytes. Phosphorylation of nuclear EGFR is a necessary component pathway for signal transduction to induce NOS-2 in response to elevated hydrostatic pressure.
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