Usatyuk Peter V, Gorshkova Irina A, He Donghong, Zhao Yutong, Kalari Satish K, Garcia Joe G N, Natarajan Viswanathan
Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
J Biol Chem. 2009 May 29;284(22):15339-52. doi: 10.1074/jbc.M109.005439. Epub 2009 Apr 14.
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.
由磷脂酶D（PLD）激活产生的磷脂酸作为第二信使，在细胞信号传导中起重要作用。在此我们证明，PLD依赖的磷脂酸生成对于Rac1/IQGAP1信号转导、p47（phox）向细胞周边的转运以及活性氧（ROS）的产生至关重要。在0.05% 1 - 丁醇而非叔丁醇存在的情况下，将[（32）P]正磷酸盐标记的人肺动脉内皮细胞（HPAECs）暴露于高氧环境（95% O₂和5% CO₂），可刺激PLD，这通过[（32）P]磷脂丁醇的积累得以证明。用PLD1和PLD2野生型的腺病毒构建体感染HPAECs可增强高氧诱导的PLD激活以及超氧阴离子/活性氧（ROS）的积累。相反，催化失活的PLD突变体（hPLD1 - K898R或mPLD2 - K758R）的过表达或用PLD1或PLD2 siRNA下调PLD的表达并不会增强高氧诱导的[（32）P]磷脂丁醇积累和ROS生成。高氧导致Rac1和IQGAP1迅速激活并重新分布到细胞周边，而Rac1和IQGAP1的下调减弱了高氧诱导的Src和皮层肌动蛋白的酪氨酸磷酸化以及ROS生成。此外，PLD1或PLD2小干扰RNA减弱了高氧介导的Rac1和IQGAP1向膜皱褶的重新分布，表明PLD位于Rac1/IQGAP1信号级联的上游。最后，PLD1或PLD2的小干扰RNA减弱了高氧诱导的皮层肌动蛋白酪氨酸磷酸化，并消除了Src、皮层肌动蛋白和p47（phox）向细胞周边的重新分布。这些结果证明了PLD在高氧介导的通过Rac1激活IQGAP1中的作用，这一作用涉及Src和皮层肌动蛋白的酪氨酸磷酸化，以及在人肺内皮细胞中p47（phox）的转运和ROS形成。
