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Y. Wang, M. F. Lou; The Role of Calcium and Cytosolic Phospholipase A2 (cPLA2) in PDGF-Stimulated NADPH Oxidase (NOX) Activation in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2115.
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© ARVO (1962-2015); The Authors (2016-present)
Previously we have found that platelet-derived growth factor (PDGF) mitogenic action depends on its activation of NOX to produce reactive oxygen species (ROS). The purpose of this study was to investigate the regulatory role of cPLA2, an enzyme which governs the release of arachidonic acid (AA), and its correlation with calcium in PDGF-induced ROS, and to examine which NOX isozyme(s) plays an important role in ROS production in human lens epithelial (HLE) cells.
HLE B3 cells and the cells with NOX p22phox subunit knockdown (p22-KD) were used as models. A cPLA2 S228A (S228A) mutant of HLE B3 was made by site-directed mutagenesis of the human cPLA2 gene. AA release was detected in cells pre-incorporated with [3H]-labeled AA by using a scintillation counter. Ca2+ ionophore (ionomycin) and Ca2+ chelator (BAPTA-AM) were used to increase and decrease intracellular Ca2+ levels, respectively. ROS generation induced by PDGF was measured by lucigenin-based luminescence assay with a luminometer. The activation of signaling factors including ERK, JNK, p38, and Akt were detected by Western blotting with corresponding specific antibodies. Expression of NOX isozymes were detected by both RT-PCR and real time PCR.
AA release was drastically reduced in S228A HLE B3 cells. The mutation also decreased PDGF-induced ROS generation and lowered activation of JNK and ERK with no effect on p38 and Akt in comparison with the control (vector alone). Increasing the intracellular Ca2+ concentration with ionophore was able to enhance the level of ROS generation in control cells, either stimulated or unstimulated with PDGF. However, the same condition could only increase the basal level of ROS in unstimulated p22-KD cells but could not enhance ROS production in these cells stimulated with PDGF. In contrast, depleting intracellular calcium with a chelator did not change the level of ROS significantly. Both RT-PCR and realtime PCR analyses indicated that HLE B3 cells express p22phox-dependent NOX2 and NOX4, and p22phox-independent NOX5 isozymes at an approximately 1: 3000: 300 ratio.
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