The regulation of NADPH oxidase and its association with cell proliferation in human lens epithelial cells.

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.