26 research outputs found

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

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    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

    Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

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    In continuation of our recent observations indicating the presence of a lone Calcineurin-dependent Response Element (CDRE) in the −3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45–50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory β-subunit of calcineurin (Ca2+-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression

    Role of acylglycerol kinase in LPA-induced IL-8 secretion and transactivation of epidermal growth factor-receptor in human bronchial epithelial cells

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    LPA (lysophosphatidic acid) is a potent bioactive phospholipid, which regulates a number of diverse cellular responses through G protein-coupled LPA receptors. Intracellular LPA is generated by the phosphorylation of monoacylglycerol by acylglycerol kinase (AGK); however, the role of intracellular LPA in signaling and cellular responses remains to be elucidated. Here, we investigated signaling pathways of IL-8 secretion mediated by AGK and intracellular LPA in human bronchial epithelial cells (HBEpCs). Expression of AGK in HBEpCs was detected by real-time PCR, and overexpressed AGK was mainly localized in mitochondria as determined by immunofluorescence and confocal microscopy. Overexpression of lentiviral AGK wild type increased intracellular LPA production (∼1.8-fold), enhanced LPA-mediated IL-8 secretion, and stimulated tyrosine phosphorylation epidermal growth factor-receptor (EGF-R). Furthermore, downregulation of native AGK by AGK small interfering RNA decreased intracellular LPA levels (∼2-fold) and attenuated LPA-induced p38 MAPK, JNK, and NF-κB activation, tyrosine phosphorylation of EGF-R, and IL-8 secretion. These results suggest that native AGK regulates LPA-mediated IL-8 secretion involving MAPKs, NF-κB, and transactivation of EGF-R. Thus AGK may play an important role in innate immunity and airway remodeling during inflammation

    Protein kinase C-epsilon regulates sphingosine-1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein Kinase C-zeta, and Rac1

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    The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P1 small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P1 to Gi. Overexpression of dominant negative (dn) PKC-ε or -ζ, but not PKC-α or -δ, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-ε, but not PKC-ζ, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-ε, but not PKC-ζ, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-ζ, or treatment with myristoylated PKC-ζ peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P1 and Gi to activate PKC-ε and, subsequently, a PLD2-PKC-ζ-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process

    Antagonist of S1P<sub>1</sub> and S1P<sub>3</sub>, but not S1P<sub>2</sub>, blocks HPAEC migration induced by S1P and S1PL silencing in a wound healing ECIS assay.

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    <p>HPAECs (∼50% confluence) grown on gold electrodes were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. Control and transfected cells were wounded on the gold electrodes as described under “Experimental Procedures” prior to VPC23019 (10 µM for 15 min) and following S1P (1.0 µM) challenge. Transendothelial electrical resistance (TER) was recorded for 16 h. Values are the mean ± S.E.M. for three independent experiments each performed in triplicates.</p

    S1P antibodies decrease S1P- and 4-Deoxypyridoxine-induced HPAEC migration.

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    <p>HPAECs (∼90% confluence) grown on gold electrodes or coverslips were starved for 3 h in 0.1% FBS in EBM-2 without growth factors. <b><i>A</i></b> -Cells were treated with 4-DP (1 mM for 30 min) then wounded on the gold electrodes as described under “Experimental Procedures” prior to the addition of isotype-matched control IgGk1 or anti-S1P antibody (150 µg/ml) and S1P (1 µM) challenge. Transendothelial electrical resistance was recorded for 16 h. Values are the mean ± S.E.M. for three independent experiments each performed in triplicate. <b><i>B,C</i></b> - Cells on cover slips were treated with 4-DP (1 mM) and anti-S1P antibody for 6 h, then stimulated with 0.1% BSA-complexed S1P (1.0 µM for 5 min), washed, fixed, permeabilized, probed with anti-Rac1 antibody (<b><i>B</i></b>) or anti-IQGAP1 antibody (<b><i>C</i></b>), and examined by immunofluorescence microscopy using a ×60 oil objective. Shown is a representative micrograph from several independent experiments.</p

    CII, inhibitor of SphK, decreases both the migration of HPAECs in a scratch assay <i>in vitro</i> and the content of S1P in HPAECs.

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    <p>HPAECs grown to ∼95% confluence in 35 mm dishes were starved for 3 h in 0.1% FBS in EBM-2 without growth factors and treated with 10 µM of CII. Monolayers were scratched, and challenged with medium containing 0.1% BSA or 1.0 µM S1P complexed to 0.1% BSA. <b><i>A</i></b> shows the migration of cells into a “wound” that was scratched and exposed to S1P. <b><i>B</i></b> shows the decrease of S1P content in cells as measured by LC/MS/MS (see Methods) after lipid extraction from harvested cells. The values are mean ± S.E.M for three independent experiments each performed in triplicate (*** - p<0.001 in comparison to T = 0 min).</p

    Silencing of S1PL increases intracellular S1P content in HPAECs and stimulates cell migration in an <i>in vitro</i> scratch and in a wound healing ECIS assay.

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    <p>HPAECs (∼50% confluence) grown on 35-mm dishes or on gold electrodes were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. <b><i>A</i></b> - Cell lysates (20 µg total proteins) were subjected to SDS-PAGE and Western blotted with anti-S1PL antibody as described under “Experimental procedures”. Western blot is representative of three independent experiments. <b><i>B,C</i></b> – S1PL was silenced with siRNA (50 nM, 72 h) then intracellular <i>(</i><b><i>B</i></b><i>)</i> and extracellular <i>(</i><b><i>C</i></b><i>)</i> S1P content was determined by LC/MS/MS. Ortho-vanadate (1 mM) was applied 30 min before lipid extraction. S1P level in the medium was normalized per cellular phospholipid content. <b><i>D</i></b> – HPAECs (∼50% confluence) were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h) then wounded on the gold electrodes as described under “Experimental Procedures” prior to S1P (1.0 µM) challenge. Transendothelial electrical resistance (TER) was recorded using an electrical cell substrate impedance-sensing system (ECIS) for 16 h. Values are the mean ± S.E.M. for three independent experiments in triplicate. <b><i>E -</i></b> HPAECs (∼50% confluence) were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h) prior to scratching the cells for migration assay. Scratched cells were challenged with S1P (1.0 µM) for 16 h. The closure of the wound was evaluated as described under “Experimental Section” 16 h after the wounding of EC monolayer. The values are mean ± S.E.M. for three independent experiments in triplicates.</p

    4-Deoxypyridoxine increases intracellular content of S1P in HPAECs and stimulates cell migration in a wound healing ECIS assay.

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    <p>HPAECs (∼90% confluence) grown on 35-mm dishes or on gold electrodes were starved for 3 h in 0.1% FBS in EBM-2 without growth factors and treated with 1 mM 4-DP for 6 h in the same medium. <b><i>A, B</i></b> – 4-DP increases intracellular content of S1P <i>(</i><b><i>A</i></b><i>)</i> and S1P release into the medium <i>(</i><b><i>B</i></b><i>)</i>. Ortho-vanadate was added 30 min before lipid extraction in a fresh medium. S1P content in cells and medium was determined by LC-MS/MS as described under “Experimental procedures”. <i>(</i><b><i>C, D</i></b><i>)</i> - Control and 4-DP-treated (1 mM for 30 min) cells were wounded on the gold electrodes as described under “Experimental Procedures” prior to S1P (1 µM) challenge. <b><i>D</i></b> shows the changes in TER (ohms) in vehicle and 4-DP or S1P-treated cells at 4 h after wounding. Values are the mean ± S.E.M. for three independent experiments.</p
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