Redox regulation of Nox proteins

The generation of reactive oxygen species (ROS) plays a major role in endothelial signaling and function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH Oxidase (Nox) family of proteins, Nox1, Nox2, Nox4 and Nox5, are major contributors of ROS. Excess generation of ROS contributes to the development and progression of vascular disease. While hyperoxia stimulates ROS production through Nox proteins, hypoxia appears to involve mitochondrial electron transport in the generation of superoxide. ROS generated from Nox proteins and mitochondria are important for oxygen sensing mechanisms. Physiological concentrations of ROS function as signaling molecule in the endothelium; however, excess ROS production leads to pathological disorders like inflammation, atherosclerosis, and lung injury. Regulation of Nox proteins is unclear; however, antioxidants, MAP Kinases, STATs, and Nrf2 regulate Nox under normal physiological and pathological conditions. Studies related to redox regulation of Nox should provide a better understanding of ROS and its role in the pathophysiology of vascular diseases

Sphingosine Kinase 1 Is Required for Mesothelioma Cell Proliferation: Role of Histone Acetylation

Background: Malignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics. Methodology/Principal Findings: In this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK12/2 null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts. Conclusions/Significance: The lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target

S1P stimulates redistribution of coronin 1B and cortactin to lamellipodia in human lung endothelial cells.

<p>HPAECs grown on slide chambers (∼90% confluence) were stimulated with 1 µM S1P for different time interval (2, 5, 15, 30 and 60 min) as indicated. Redistribution of Coronin 1B (<b>B</b>) and Cortactin (<b>C</b>) was visualized by immunocytochemistry and quantified by ImageJ software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown are representative immunofluorescence images from several independent experiments.</p

Role of PKC δ, ε, and ζ isoforms on S1P mediated chemotaxis and lamellipodial localization of Coronin 1B in human lung endothelial cells.

<p>HPAECs grown on slide chambers or 35-mm dishes (∼70% confluence) were infected with empty vector or adenoviral vectors encoding PKC dominant negative (dn) δ, ε, and ζ isoforms (5 MOI) in complete EGM-2 medium for 24 h. (<b>A</b>), Cell lysates (20 µg of protein) were subjected to 10% SDS-PAGE, Western blotting and probed with anti-PKC δ, ε, ζ and actin antibodies. In parallel experiments, the effect of dn PKC δ, ε and ζ isoforms on chemotaxis (<b>B</b>) and lamellipodial localization of coronin 1B and actin (<b>C, D and E</b>) was examined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Values are mean±SEM of three independent experiments. *, p<0.01 compared cells without S1P; **, p<0.005 compared to cells infected with empty vector and stimulated with S1P.</p

Proposed signaling mechanisms involved in S1P-induced lamellipodial localization of Coronin 1B, Cortactin and chemotaxis of human lung endothelial cells.

<p>S1P binding to G-protein coupled S1P1-5 receptors activates PLD2 via PKC δ and ε and activation of PLD2 results in hydrolysis of membrane associated phosphatidylcholine (PC) to phosphatidic acid (PA) and phospho-choline. PA can be converted to DAG by PA-phosphatases or can activate PKC ζ via of phosphatidylinositol-4-phosphate-5kinase activation. Activation of PKC ζ results in redistribution of Coronin 1B and Cortactin to cell periphery and localization in lamellipodia of endothelial cells. PA can directly bind to and activate Rac1 and formation of actin stress fibers. Additionally, PLD2 has guanine nucleotide-exchange factor (GEF) activity for Rho and can regulate actin stress fibers in a manner independent of its lipase activity. S1P-induced activation of PKC δ/ε → PLD2/PA → PKC ζ → Rac1 signaling cascade facilitates recruitment of Coronin 1B, Cortactin and Actin to lamellipodia and chemotaxis of endothelial cells.</p

Coronin 1B localization in human lung endothelial cells.

<p>HPAECs grown to ∼90% confluence on slide chambers were fixed, permeabilized and localization of Coronin 1B, actin and co-localization of Coronin 1B with actin was visualized by immunocytochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown are representative immunofluorescence images from several independent experiments as measured by regular (<b>A</b>) immunofluorescence and (<b>B</b>) confocal microscopy.</p

Role of Rac1 in S1P-induced chemotaxis and redistribution of coronin 1B and cortactin to lamellipodia in human lung endothelial cells.

<p>(<b>A</b>), HPAECs (∼90% confluence) grown on chamber slides were pretreated for 30 min with NSC23766 (50 µM), a Rac1 inhibitor, prior to stimulation with S1P (1 µM) for 15 min. Cells were washed, fixed, permeabilized, probed with antibodies, and redistribution of Coronin 1B and Cortactin was examined by immunofluorescence microscopy using a 60 X oil objective and quantified by ImageJ software (<b>B</b>) as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown is an immunofluorescence micrograph from three independent experiments. (<b>C</b>), In parallel experiments the effect of NSC23766 on chemotaxis was determined by a Boyden chamber-based trans-well assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Values are mean±SEM of three independent experiments. *, p<0.05 compared cells without S1P; **, p<0.005 compared to cells stimulated with S1P in the absence of NSC23766.</p

Coronin 1B siRNA attenuates S1P-induced chemotaxis, wound healing and lamellipodia localization of cortactin in HPAECs.

<p>(<b>A</b>), HPAECs grown on transwell inserts were stimulated with different S1P concentration (0.01, 0.1, 1 and 10 µM) for 15 min and chemotaxis was estimated by a Boyden chamber-based trans-well assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. The values are mean±SEM of three independent experiments. *, p<0.05 compared to cells without S1P. (<b>B</b>) HPAECs were transfected with scrambled (sc) or siRNA for Coronin 1B (50 ng/ml, 72 h), and cell lysates (20 µg of protein) were subjected to 10% SDS-PAGE and probed with Coronin 1B and actin antibodies as indicated. (<b>C</b>) HPAECs grown to 50% confluence in 100-mm dishes were transfected with sc (sc) or Coronin 1B siRNA (50 ng/ml) for 72 h. The cells were trypsinazied and plated on to transwell inserts and S1P-induced chemotaxis was determines as described in (<b>A</b>). The values are mean±SEM of three independent experiments in triplicate. *, p<0.05 compared cells without S1P; **, p<0.001 compared to scrambled siRNA transfected cells plus S1P. (<b>D</b>), HPAECs transfected with scrambled (sc) or Coronin 1B siRNA (50 nM, 72 h) were wounded on the gold electrodes as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Measurement of transendothelial electrical resistance (TER) using an electrical cell substrate impedance-sensing system (ECIS) for 12 h after wounding the cells on the gold electrode and exposure to 1.0 µM S1P was carried out. Shown is a tracing from three independent experiments in triplicate. (<b>E</b>), HPAECs transfected with scrambled (sc) or Coronin 1B siRNA (50 nM, 72 h) were seeded on slide chambers for 24 h prior to stimulation with 1 µM S1P for 15 min. Cells were fixed and Coronin 1B and Cortactin redistribution to cell periphery was visualized by immunocytochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown is a representative immunofluorescence image taken using an X 60 oil objective as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>.</p

Effect of starvation and serum supplementation on Coronin 1B localization in human lung endothelial cells.

<p>HPAECs grown on slide chambers (∼90% confluence) were incubated in EBM-2 medium containing either 0.1% serum for 3 h or in EBM-2 medium containing 5% serum for 3 h and 48 h. Cells were fixed, permeabilized and Coronin 1B localization was visualized by immunocytochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown are representative immunofluorescence images from several independent experiments.</p

Role of PLD2 in S1P-induced chemotaxis, Coronin 1B and actin lamellipodia localization in HPAECs.

<p>HPAECs (∼50% confluence) were transfected with scrambled (sc), PLD1 or PLD2 siRNA (50 ng/ml) for 72 h. (<b>A</b>) Cell lysates (20–40 µg of protein) were subjected to 10% SDS-PAGE, Western blotted and probed with PLD1 and PLD2 antibodies as indicated; (<b>B</b>) chemotaxis of scrambled (sc) or siRNA transfected cells to S1P (1 µM) for 15 min was carried out in a Boyden chamber-based trans-well assay as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Values are mean±SEM of three independent experiments in triplicate. *, p<0.01 compared cells without S1P; **, p<0.005 compared to scrambled siRNA transfected cells plus S1P; HPAECs transfected with sc, PLD1 (<b>C</b>) or PLD2 (<b>E</b>) siRNA in 100-mm dishes as described under (<b>A</b>) were trypsinazied and seeded onto slide chambers prior to stimulation with S1P (1 µM) for 15 min. Cells were washed, fixed, permeabilized, and probed with anti-Coronin 1B and AlexaFluor Phalloidin antibodies, and redistribution of Coronin 1B and actin due to downregulation of PLD1 (<b>D</b>) or PLD2 (<b>F</b>) was examined by immunofluorescence microscopy using a 60 X oil objective and quantified by ImageJ software as described under “Experimental Procedures”. Shown is an immunofluorescence micrograph from three independent experiments.</p