The regulation of NADPH oxidase and its association with cell proliferation in human lens epithelial cells.
OBJECTIVE
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.
Wang
Yin
Y
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68583, USA.
Lou
Marjorie F
MF
D000368:Aged; D016718:Arachidonic Acid / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D004847:Epithelial Cells / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D020868:Gene Silencing / Q000502:physiology; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D008562:Membrane Glycoproteins; D010982:Platelet-Derived Growth Factor; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016718:Arachidonic Acid; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D017479:Receptors, Platelet-Derived Growth Factor; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4