The positive feedback role of arachidonic acid in the platelet-derived growth factor-induced signaling in lens epithelial cells.
OBJECTIVE
Platelet-derived growth factor (PDGF)-stimulated cell proliferation has been associated with reactive oxygen species (ROS)-mediated redox signaling. This study examined the role of arachidonic acid (AA) in PDGF-stimulated ROS generation in human lens epithelial B3 cells (HLE B3).


METHODS
PDGF (1 ng/ml)-stimulated ROS generation was examined using dichlorofluorescein (DCFH)-activated fluorescence by laser confocal microscopy while AA (30-150 muM)-stimulated superoxide anion production was measured using lucigenin-amplified chemiluminescence in serum-starved HLE B3 cells. PDGF-stimulated AA release was quantified by cells prelabeled with (3)H-AA with and without the presence of cytosolic phospholipase A(2) (cPLA(2)) inhibitor (AACOCF(3)) and mitogen-activated protein (MAP) kinases (MEK) inhibitor (U0126). Western blot analysis was used to characterize the activated MAP kinase components in cell lysates or protein kinase C (PKC) translocation in isolated cytosolic and membrane fractions. Specific inhibitors to various enzymes were used in the study, including GF109203X for pan protein kinase C (PKC), AACOCF3 for cytosolic phospholipase A2 (cPLA(2)), U0126 for MEK, and DPI for NADPH oxidase. Inhibitors for AA metabolism were also used to examine the role of AA in PDGF-stimulated ROS generation, including CDC and NDGA for pan lipoxygenase, AA861 for 5-lipoxygenase, indomethacin for cycloxygenase, and ketoconazole for cytochrome p450.


RESULTS
We found that PDGF-stimulated ROS was eradicated by inhibitors to MEK, cPLA(2), 5-lipoxygenase, NADPH oxidase, or PKC. PDGF-stimulated AA release depended on both active cPLA(2) and ERK1/2. Exogenous AA showed a concentration-dependent ROS generation via NADPH oxidase activation that was insensitive to MEK inhibitor, but sensitive to PKC inhibitor, and could be attenuated by superoxide dismutase (SOD), mannitol, or DPI. This effect of AA was specific as other long chain fatty acids (leinoleic acid, stearic acid), or AA derivatives (eicosa-11Z, 14Z, 17Z-trienoic acid (20:3) and eicosa-11Z, 14Z-dienoic acid (20:2)) were ineffective. Inhibitor to lipoxygenase, in particular the 5-isoform, but not cycloxygenase or cytochrome p450, could diminish AA-stimulated luminescence generation. Western blot analysis showed that AA-treated cells transiently activated ERK1/2 and JNK, but not p38, in a time- and dose-dependent manner that was similar to that of PDGF. Finally, PDGF-stimulated PKC translocation depended on AA release while AA-stimulated PKC translocation was eradicated by lipoxygenase inhibition.


CONCLUSIONS
We conclude that PDGF signaling in HLE B3 cells is mediated by AA and its lipoxygenase metabolites, which provide a positive feedback loop for PDGF action, as AA and its metabolites can mobilize PKC and other factors needed for NADPH oxidase assembly and activation for ROS generation to facilitate cell proliferation. We further propose the role of AA in PDGF signaling.
Zhang
Wei
W
Beijing Institute of Ophthalmology, Capital University of Medical Sciences affiliated Beijing Tongren Ophthalmology Center, Beijing, China.
Wang
Yin
Y
Chen
Chao-Wei
CW
Xing
Kuiyi
K
Vivekanandan
S
S
Lou
Marjorie F
MF
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D001692:Biological Transport; D002478:Cells, Cultured; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000378:metabolism; D025461:Feedback, Physiological; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010982:Platelet-Derived Growth Factor / Q000378:metabolism* / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism
D000975:Antioxidants; D010982:Platelet-Derived Growth Factor; D017382:Reactive Oxygen Species; D013481:Superoxides; D016718:Arachidonic Acid; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases