The positive feedback role of arachidonic acid in theplatelet-derived growth factor-induced signaling in lens epithelialcells

[摘要] Purpose: Platelet-derived growth factor (PDGF)-stimulated cellproliferation has been associated with reactive oxygen species(ROS)-mediated redox signaling. This study examined the role ofarachidonic acid (AA) in PDGF-stimulated ROS generation in human lensepithelial B3 cells (HLE B3).Methods: PDGF (1 ng/ml)-stimulated ROS generation was examined usingdichlorofluorescein (DCFH)-activated fluorescence by laser confocalmicroscopy while AA (30-150 μM)-stimulated superoxide anionproduction was measured using lucigenin-amplified chemiluminescence inserum-starved HLE B3 cells. PDGF-stimulated AA release was quantified bycells prelabeled with 3H-AA with and without the presence ofcytosolic phospholipase A2 (cPLA2) inhibitor (AACOCF3)and mitogen-activated protein (MAP) kinases (MEK) inhibitor (U0126).Western blot analysis was used to characterize the activated MAP kinasecomponents in cell lysates or protein kinase C (PKC) translocation inisolated cytosolic and membrane fractions. Specific inhibitors tovarious enzymes were used in the study, including GF109203X for panprotein kinase C (PKC), AACOCF3 for cytosolic phospholipase A2(cPLA2), U0126 for MEK, and DPI for NADPH oxidase. Inhibitors forAA metabolism were also used to examine the role of AA inPDGF-stimulated ROS generation, including CDC and NDGA for panlipoxygenase, AA861 for 5-lipoxygenase, indomethacin for cycloxygenase,and ketoconazole for cytochrome p450.Results: We found that PDGF-stimulated ROS was eradicated byinhibitors to MEK, cPLA2, 5-lipoxygenase, NADPH oxidase, or PKC.PDGF-stimulated AA release depended on both active cPLA2 andERK1/2. Exogenous AA showed a concentration-dependent ROS generation viaNADPH oxidase activation that was insensitive to MEK inhibitor, butsensitive to PKC inhibitor, and could be attenuated by superoxidedismutase (SOD), mannitol, or DPI. This effect of AA was specific asother long chain fatty acids (leinoleic acid, stearic acid), or AAderivatives (eicosa-11Z, 14Z, 17Z-trienoic acid (20:3) and eicosa-11Z,14Z-dienoic acid (20:2)) were ineffective. Inhibitor to lipoxygenase, inparticular the 5-isoform, but not cycloxygenase or cytochrome p450,could diminish AA-stimulated luminescence generation. Western blotanalysis showed that AA-treated cells transiently activated ERK1/2 andJNK, but not p38, in a time- and dose-dependent manner that was similarto that of PDGF. Finally, PDGF-stimulated PKC translocation depended onAA release while AA-stimulated PKC translocation was eradicated bylipoxygenase inhibition.Conclusions: We conclude that PDGF signaling in HLE B3 cells ismediated by AA and its lipoxygenase metabolites, which provide apositive feedback loop for PDGF action, as AA and its metabolites canmobilize PKC and other factors needed for NADPH oxidase assembly andactivation for ROS generation to facilitate cell proliferation. Wefurther propose the role of AA in PDGF signaling.

[学科分类] 生物化学/生物物理