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.
Authors	Ravinder Pannu, Je-Seong Won, Mushfiquddin Khan, Avtar K Singh, Inderjit Singh
Journal	The Journal of neuroscience : the official journal of the Society for Neuroscience (J Neurosci) Vol. 24 Issue 26 Pg. 5942-54 (Jun 30 2004) ISSN: 1529-2401 [Electronic] United States
PMID	15229242 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References	Antigens, CD Antioxidants Enzyme Inhibitors Fatty Acids I-kappa B Proteins Lactosylceramides Lipopolysaccharides Morpholines NF-kappa B Nerve Tissue Proteins Recombinant Proteins Thiocarbamates prolinedithiocarbamate Nitric Oxide CDw17 antigen RV 538 Interferon-gamma Proline Nitric Oxide Synthase Nitric Oxide Synthase Type II Nos2 protein, rat Galactosyltransferases glucosylceramide beta-1-4-galactosyltransferase Acetylcysteine
Topics	Acetylcysteine (pharmacology) Animals Antigens, CD (pharmacology, physiology) Antioxidants (pharmacology) Apoptosis (drug effects) Astrocytes (drug effects, enzymology) Cells, Cultured (drug effects, enzymology) Demyelinating Diseases (drug therapy) Drug Evaluation, Preclinical Enzyme Induction (drug effects) Enzyme Inhibitors (pharmacology) Fatty Acids (metabolism) Female Gait Disorders, Neurologic (etiology, prevention & control) Galactosyltransferases (genetics, metabolism) I-kappa B Proteins (metabolism) Inflammation Interferon-gamma (pharmacology) Lactosylceramides (pharmacology, physiology) Lipopolysaccharides (pharmacology) Morpholines (pharmacology) NF-kappa B (metabolism) Nerve Tissue Proteins (biosynthesis, genetics) Nitric Oxide (metabolism) Nitric Oxide Synthase (biosynthesis, genetics) Nitric Oxide Synthase Type II Oxidation-Reduction Phosphorylation (drug effects) Proline (analogs & derivatives, pharmacology) Protein Processing, Post-Translational (drug effects) Rats Rats, Sprague-Dawley Recombinant Proteins Signal Transduction Spinal Cord Injuries (complications, drug therapy, enzymology) Thiocarbamates (pharmacology) Transfection

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