Phospholipase D-mediated activation of IQGAP1 through Rac1 regulates hyperoxia-induced p47phox translocation and reactive oxygen species generation in lung endothelial cells.
Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47(phox) to cell periphery, and ROS production. Exposure of [(32)P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O(2) and 5% CO(2)) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [(32)P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O(2)(.)/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [(32)P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47(phox) redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47(phox) translocation and ROS formation in human lung endothelial cells.
Usatyuk
Peter V
PV
Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
Gorshkova
Irina A
IA
He
Donghong
D
Zhao
Yutong
Y
Kalari
Satish K
SK
Garcia
Joe G N
JG
Natarajan
Viswanathan
V
D055162:Biocatalysis; D002462:Cell Membrane / Q000201:enzymology; D051356:Cortactin / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006801:Humans; D018496:Hyperoxia / Q000201:enzymology*; D008168:Lung / Q000166:cytology; D050505:Mutant Proteins / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D051356:Cortactin; D020662:Guanine Nucleotide Exchange Factors; C101203:IQ motif containing GTPase activating protein 1; D050505:Mutant Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human; D020703:ras GTPase-Activating Proteins; D019000:Phosphotyrosine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019061:src-Family Kinases; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D020830:rac1 GTP-Binding Protein