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Yin Wang, Marjorie F. Lou; The Regulation of NADPH Oxidase and Its Association with Cell Proliferation in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(5):2291-2300. doi: 10.1167/iovs.08-2568.
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purpose. NADPH oxidase (NOX)–generated reactive oxygen species (ROS) are essential for growth factor–stimulated cell proliferation. In this study, the regulatory role of p22phox, a membrane subunit of NOX, in NOX activity and platelet-derived growth factor (PDGF) mitogenic signaling were examined.
methods. Human lens epithelial B3 (HLE B3) cell lines with p22phox overexpressed (p22-OE) and p22phox knockdown (p22-KD) were used as models. Cells stimulated with PDGF were compared with nonstimulated control cells. The relative NOX activity and intracellular ROS generation were detected by lucigenin-based assay and DCFH fluorescence, respectively. Cell proliferation was measured by BrdU and fluorescent nucleic acid staining assays. p22phox, P-JNK, P-ERK1/2, P-Akt, P-p38, p47phox, and P-PDGF receptor in cell lysates were detected by Western blot analysis with the respective specific antibodies.
results. p22-OE showed higher NOX activity, PDGF-stimulated ROS generation, cell proliferation, and activation of signaling cascades of ERK1/2, JNK, and Akt over the control (vector alone). In contrast, p22-KD displayed opposite results. In addition, PDGF stimulated p47phox and Rac1 translocations and induced binding between p22phox and the cytosolic subunits of p47phox, p67phox, and p40phox. Overexpression of p22phox increased p22phox–p47phox binding, enhanced, and prolonged the phosphorylation of PDGF receptor at Tyr857 with a corresponding inhibition of the activity of the oxidation-sensitive low molecular weight protein tyrosine phosphatase (LMW-PTP). However, p22phox knockdown weakened p22phox–p47phox binding and largely diminished the activation of PDGF receptor with no inhibition of LMW-PTP.
conclusions. PDGF mitogenic action in HLE B3 cells depends on p22phox to regulate NOX activity, which affects PDGF receptor function for cell proliferation.
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