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L. Wang, J. Gao, J. Lu, L. Lu; Mediation of Hypoxia-Induced c-Jun Phosphorylation by Plk3. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2712.
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The purpose of the study is to investigate the role of Polo-like kinase 3 (Plk3) in hypoxia- and reoxygenation-induced stress responses by activating c-Jun in corneal epithelial cells
Human corneal epithelial (HCE) cells were cultured in DMEM/F12 medium containing 10% FBS and 5 µg/ml insulin at 37°C, 5% CO2. Hypoxia experiment was performed by placing culture dishes into 37°C incubator supplemented with 1% O2, 5% CO2 and 94% N2 for 1 to 4 h, and reoxygenation experiment was carried out by continuously exposure of hypoxia-induced cells to the normal culture condition. EMSA was performed to detect the activation of c-Jun. Immunoprecipitation and kinase assays were employed to measure hypoxia-induced Plk3 kinase activity. Protein interactions were detected by using immunofluorescence analysis with specific antibodies.
1) Hypoxia and reoxygenation induced c-Jun phosphorylation in HCE cells. 2)CoCl2 was used to verify the effect of hypoxia on c-Jun phosphorylation. 3) DNA binding activity of c-Jun was increased by hypoxia and reoxygenation detected by using EMSA assay. 4)Hypoxia and reoxygenation induced c-Jun phosphorylation was through activation of Plk3. 5) Overexpression of Plk3 increased the effects of hypoxia- and reoxygenatio on c-Jun phosphorylation. 6)Plk3 and c-Jun interactions were detected by cellular co-localization of these proteins using immunostaining.
Our results provided a new mechanism that hypoxia- and reoxygenation induce PLK3 activation subsequently resulting in activation of c-Jun in HCE cells.
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