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A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases.

Abstract

In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-gamma caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-gamma-induced iNOS, TNF-alpha, and IL-1beta gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IkappaB phosphorylation, NF-kappaB DNA binding activity, and NF-kappaB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI.

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  • Authors+Show Affiliations

    ,

    Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.

    , , ,

    Source

    MeSH

    Acetylcysteine
    Animals
    Antigens, CD
    Antioxidants
    Apoptosis
    Astrocytes
    Cells, Cultured
    Demyelinating Diseases
    Drug Evaluation, Preclinical
    Enzyme Induction
    Enzyme Inhibitors
    Fatty Acids
    Female
    Gait Disorders, Neurologic
    Galactosyltransferases
    I-kappa B Proteins
    Inflammation
    Interferon-gamma
    Lactosylceramides
    Lipopolysaccharides
    Morpholines
    NF-kappa B
    Nerve Tissue Proteins
    Nitric Oxide
    Nitric Oxide Synthase
    Nitric Oxide Synthase Type II
    Oxidation-Reduction
    Phosphorylation
    Proline
    Protein Processing, Post-Translational
    Rats
    Rats, Sprague-Dawley
    Recombinant Proteins
    Signal Transduction
    Spinal Cord Injuries
    Thiocarbamates
    Transfection

    Pub Type(s)

    Journal Article
    Research Support, U.S. Gov't, P.H.S.

    Language

    eng

    PubMed ID

    15229242

    Citation

    Pannu, Ravinder, et al. "A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/interferon-gamma-mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases." The Journal of Neuroscience : the Official Journal of the Society for Neuroscience, vol. 24, no. 26, 2004, pp. 5942-54.
    Pannu R, Won JS, Khan M, et al. A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases. J Neurosci. 2004;24(26):5942-54.
    Pannu, R., Won, J. S., Khan, M., Singh, A. K., & Singh, I. (2004). A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases. The Journal of Neuroscience : the Official Journal of the Society for Neuroscience, 24(26), pp. 5942-54.
    Pannu R, et al. A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/interferon-gamma-mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases. J Neurosci. 2004 Jun 30;24(26):5942-54. PubMed PMID: 15229242.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases. AU - Pannu,Ravinder, AU - Won,Je-Seong, AU - Khan,Mushfiquddin, AU - Singh,Avtar K, AU - Singh,Inderjit, PY - 2004/7/2/pubmed PY - 2004/12/17/medline PY - 2004/7/2/entrez SP - 5942 EP - 54 JF - The Journal of neuroscience : the official journal of the Society for Neuroscience JO - J. Neurosci. VL - 24 IS - 26 N2 - In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-gamma caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-gamma-induced iNOS, TNF-alpha, and IL-1beta gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IkappaB phosphorylation, NF-kappaB DNA binding activity, and NF-kappaB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI. SN - 1529-2401 UR - https://www.unboundmedicine.com/medline/citation/15229242/A_novel_role_of_lactosylceramide_in_the_regulation_of_lipopolysaccharide/interferon_gamma_mediated_inducible_nitric_oxide_synthase_gene_expression:_implications_for_neuroinflammatory_diseases_ L2 - http://www.jneurosci.org/cgi/pmidlookup?view=long&pmid=15229242 DB - PRIME DP - Unbound Medicine ER -