Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells

Abstract In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22phox compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22phox, but not Nox2 or p47phox, are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2/ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs. Antioxid. Redox Signal. 11, 747-764.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78121/1/ars.2008.2203.pd

Intracellular S1P Generation Is Essential for S1P-Induced Motility of Human Lung Endothelial Cells: Role of Sphingosine Kinase 1 and S1P Lyase

Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility

Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis

Hydroxyalkenals and oxidized phospholipids modulation of endothelial cytoskeleton, focal adhesion and adherens junction proteins in regulating endothelial barrier function

Lipid peroxidation of polyunsaturated fatty acids generates bioactive aldehydes, which exhibit pro- and anti-inflammatory effects in cells and tissues. Accumulating evidence indicates that 4-hydroxynonenal (4-HNE), a major aldehyde derived from lipid peroxidation of n-6 polyunsaturated fatty acids trigger signals that modulates focal adhesion and adherens junction proteins thereby inducing endothelial barrier dysfunction. Similarly, oxidized phospholipids (Ox-PLs) generated by lipid peroxidation of phospholipids with polyunsaturated fatty acids have been implicated in atherogenesis, inflammation and gene expression. Interestingly, physiological concentration of Ox-PLs is anti-inflammatory and protect against endotoxin- and ventilator-associated acute lung injury. Thus, excess generation of bioactive hydroxyalkenals and Ox-PLs during oxidative stress contributes to pathophysiology of various diseases by modulating signaling pathways that regulate pro- and anti-inflammatory responses and barrier regulation. This review summarizes the role of 4-HNE and Ox-PLs affecting cell signaling pathways and endothelial barrier dysfunction through modulation of the activities of proteins/enzymes by Michael adducts formation, enhancing the level of protein tyrosine phosphorylation of the target proteins, and by reorganization of cytoskeletal, focal adhesion, and adherens junction proteins. A better understanding of molecular mechanisms of hydroxyalkenals- and Ox-PLs-mediated pro-and anti-inflammatory responses and barrier function may lead to development of novel therapies to ameliorate oxidative stress related cardio-pulmonary disorders

Involvement of phospholipases D1 and D2 in sphingosine 1-phosphate-induced ERK (extracellular-signal-regulated kinase) activation and interleukin-8 secretion in human bronchial epithelial cells.

Sphingosine 1-phosphate (S1P), a metabolite of sphingomyelin degradation, stimulates interleukin-8 (IL-8) secretion in human bronchial epithelial (Beas-2B) cells. The molecular mechanisms regulating S1P-mediated IL-8 secretion are yet to be completely defined. Here we provide evidence that activation of phospholipases D1 and D2 (PLD1 and PLD2) by S1P regulates the phosphorylation of extracellular-signal-regulated kinase (ERK) and IL-8 secretion in Beas-2B cells. S1P, in a time- and dose-dependent manner, enhanced the threonine/tyrosine phosphorylation of ERK. The inhibition of S1P-induced ERK phosphorylation by pertussis toxin and PD 98059 indicated coupling of S1P receptors to G(i) and the ERK signalling cascade respectively. Treatment of Beas-2B cells with butan-1-ol, but not butan-3-ol, abrogated the S1P-induced phosphorylation of Raf-1 and ERK, suggesting that PLD is involved in this activation. The roles of PLD1 and PLD2 in ERK activation and IL-8 secretion activated by S1P were investigated by infecting cells with adenoviral constructs of wild-type and catalytically inactive mutants of PLD1 and PLD2. Infection of Beas-2B cells with the wild-type constructs resulted in the activation of PLD1 and PLD2 by S1P and PMA. Also, the enhanced production of [(32)P]phosphatidic acid and [(32)P]phosphatidylbutanol in the presence of butan-1-ol and the increased phosphorylation of ERK by S1P were blocked by the catalytically inactive mutants hPLD1-K898R and mPLD2-K758R. Transient transfection of Beas-2B cells with human PLD1 and mouse PLD2 cDNAs potentiated S1P-mediated IL-8 secretion compared with vector controls. In addition, PD 98059 attenuated IL-8 secretion induced by S1P in a dose-dependent fashion. These results demonstrate that both PLD1 and PLD2 participate in S1P stimulation of ERK phosphorylation and IL-8 secretion in bronchial epithelial cells