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C.–W. Chen, Y. Zhou, M.F. Lou; Investigating the Initial Sites of Redox Signaling in Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1895.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Previously we have shown that the mitogenic action of platelet derived growth factor (PDGF) is mediated via reactive oxygen species (ROS) generation in human lens epithelial cells (Chen et al., 2004). This study is to identify the key protein components that are involved in the initial regulation and transmission of mitogenic signal from PDGF and their association with the spontaneous ROS production. Methods: PDGF was used as a model to stimulate HLE B3 cells. Dominant negative Ras (Ras N17), dominant negative Rac (Rac N17), constitutively active Rac (Rac V12,) or vector alone (as a control) was each transfected into HLE B3. Confluent cells (1.2 millions) were kept quiescent by depriving serum in the medium for 30 min before use. PDGF (1ng/ml)–stimulated ROS production in the wild type or transfected cells was detected by DCFH fluorescein dye and monitored by confocal microscopy with and without the presence of various specific inhibitors in several concentrations. For determining the activation of mitogen activated protein kinases (MAPKs), the cells were treated with PDGF for 10–30 min and analyzed for phosphorylated ERK, JNK or p38. 3H–thymidine incorporation in DNA was measured for cell proliferation. Results: ROS was produced by PDGF stimulation as measured by DCF fluorescence. The ROS generation was visibly elevated in vector transfected cells or in Rac V12 transfected cells, but was dramatically reduced in either Rac N17 or Ras N17 trasnfected cells. AG–1296 (inhibitor to PDGF receptor), PP1 (Src inhibitor), LY294002 (PE3K inhibitor) or pertussis toxin (inhibitor to G protein coupled receptor) each eradicated the ROS generation in wild–type HLE B3 cells. The pattern of transient ROS generation coincided with that of phosphorylated ERK and JNK, but not p38. 3H–thymidine incorporation of DNA was stimulated by PDGF but inhibited in Rac N17 or Ras N17 transfected cells. Conclusions: The results suggest that PDGF receptor, the small GTP–binding proteins (Ras and Rac), Src kinase, PI3K and G protein coupled receptor are all involved in the mitogenic function of PDGF and the process of ROS–stimulated cellular functions.
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