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.
Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
Peter V
PV
Usatyuk
Irina A
IA
Gorshkova
Donghong
D
He
Yutong
Y
Zhao
Satish K
SK
Kalari
Joe G N
JG
Garcia
Viswanathan
V
Natarajan
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