A Novel Role of Lactosylceramide in the Regulation of Lipopolysaccharide/Interferon-γ-Mediated Inducible Nitric Oxide Synthase Gene Expression: Implications for Neuroinflammatory Diseases

• DOI: 10.1523/jneurosci.1271-04.2004
• Publication Date: 2004-06-30T16:23:28Z
• Authors: Ravinder Pannu; Je-Seong Won; Mushfiquddin Khan; Avtar K. Singh; Inderjit Singh
• Keywords: Inflammation; Lipopolysaccharides; 0301 basic medicine; Fatty Acids; Drug Evaluation, Preclinical; Lactosylceramides; Apoptosis; Galactosyltransferases; Antioxidants; Acetylcysteine; Interferon-gamma; 03 medical and health sciences; Antigens, CD; Astrocytes; Enzyme Induction; Animals; Female; I-kappa B Proteins; Enzyme Inhibitors; Cells, Cultured; Gait Disorders, Neurologic; Demyelinating 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-γ (IFN-γ) 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-γ induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-γ 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-γ-induced iNOS, TNF-α, and IL-1β 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 IκB phosphorylation, NF-κB DNA binding activity, and NF-κB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidantsN-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC).In vivoadministration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-α, and IL-1β expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. Thein vivostudies 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.