A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases.
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.
Pannu
Ravinder
R
Department of Pediatrics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Won
Je-Seong
JS
Khan
Mushfiquddin
M
Singh
Avtar K
AK
Singh
Inderjit
I
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D015703:Antigens, CD / Q000494:pharmacology / Q000502:physiology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D003711:Demyelinating Diseases / Q000188:drug therapy; D004353:Drug Evaluation, Preclinical; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D020233:Gait Disorders, Neurologic / Q000209:etiology / Q000517:prevention & control; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism; D020672:I-kappa B Proteins / Q000378:metabolism; D007249:Inflammation; D007371:Interferon-gamma / Q000494:pharmacology*; D007790:Lactosylceramides / Q000494:pharmacology / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009025:Morpholines / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010084:Oxidation-Reduction; D010766:Phosphorylation / Q000187:drug effects; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins; D015398:Signal Transduction; D013119:Spinal Cord Injuries / Q000150:complications / Q000188:drug therapy / Q000201:enzymology*; D013859:Thiocarbamates / Q000494:pharmacology; D014162:Transfection
D015703:Antigens, CD; D000975:Antioxidants; D004791:Enzyme Inhibitors; D005227:Fatty Acids; D020672:I-kappa B Proteins; D007790:Lactosylceramides; D008070:Lipopolysaccharides; D009025:Morpholines; D016328:NF-kappa B; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D013859:Thiocarbamates; C066229:prolinedithiocarbamate; D009569:Nitric Oxide; C009744:CDw17 antigen; C033110:RV 538; D007371:Interferon-gamma; D011392:Proline; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D005700:Galactosyltransferases; C023235:glucosylceramide beta-1-4-galactosyltransferase; D000111:Acetylcysteine