The role of neutral sphingomyelinase produced ceramide in lipopolysaccharide‐mediated expression of inducible nitric oxide synthase

Abstract

Lipopolysaccharide (LPS) and interferon-c (IFN) treatment of C6 rat glioma cells increased the intracellular ceramide level and the expression of the inducible nitric oxide synthase (iNOS) gene. To delineate the possible role of ceramide in the induction of iNOS, we examined the source of intracellular ceramide and associated signal transduction pathway(s) with the use of inhibitors of intracellular ceramide generation. The inhibitor of neutral sphingomyelinase (3-O-methylsphingomyelin, MSM) inhibited the induction of iNOS, whereas inhibitor of acidic sphingomyelinase (SR33557) or that of ceramide de novo synthesis (fumonisin B1) had no effect on the induction of iNOS. MSM-mediated inhibition of iNOS induction was reversed by the supplementation of exogenous C 8 -ceramide, suggesting that ceramide production by neutral sphingomyelinase (nSMase) is a key mediator in the induction of iNOS. The MSM-mediated inhibition of iNOS gene expression correlated with the decrease in the activity of ras. Inhibition of co-transfected iNOS promoter activity by dominant negative ras supported the role of ras in the nSMase-dependent regulation of iNOS gene. NF-jB DNA binding activity and its transactivity were also reduced by MSM pretreatment, and were completely reversed by the supplementation of C 8 -ceramide. As the dominant negative ras also reduced NF-jB transactivity, NF-jB activation may be downstream of ras. Our results suggest that ceramide generated by nSMase may be a critical mediator in the regulation of iNOS gene expression via ras-mediated NF-jB activation under inflammatory conditions. Keywords: ceramide, inducible nitric oxide synthase, neutral sphingomyelinase, NF-jB, ras. Nitric oxide (NO), a product of inducible nitric oxide synthase (iNOS), is a diffusible gas involved in many physiological and diverse pathological conditions. At low concentrations, NO has been shown to play a role in neurotransmission and vasodilation, while at higher concentrations, it is neurotoxic. This is of particular importance in neurodegenerating conditions such as demyelinating disease and in ischemia and traumatic injuries associated with infiltrating peripheral mononuclear cells and the production of proinflammatory cytokines (Koprowski et al. 1993;Mitrovic et al. 1994;Bonfoco et al. 1995), where astrocytes and microglia-derived NO could contribute to oligodendrocyte degeneration and neuronal death. In the brain, at least three genes encode NO synthase isoforms, with significant differences in their regulation. Neuronal and endothelial NOS are constitutively expressed in astrocytes and in subpopulations of neurons and are regulated predominantly through intracellular calcium/calmodulin signals (Bredt and Snyder 1990;Busse and Mulsch 1990). The third NOS, iNOS that is present in macrophages and astrocytes, is regulated at the transcriptional level in response to stimuli [e.g. cytokine/lipopolysaccharide (LPS)] (Nathan 1992; Jaffrey and Snyder 1995). Abbreviations used: C/EBP, CCAAT box/enhancer binding protein; iNOS, inducib...

Lactosylceramide Mediates the Expression of Adhesion Molecules in TNF-α and IFNγ-stimulated Primary Cultured Astrocytes

“…LacCer is a precursor for complex GSLs, including gangliosides [3]. Previous studies have shown the involvement of sphingolipids such as ceramide and psychosine in the cytokine-mediated inflammatory disease of CNS, such as Krabbe disease and spinal cord injury [4][5][6][7].…”

A Novel Role of Lactosylceramide in the Regulation of Tumor Necrosis Factor α-mediated Proliferation of Rat Primary Astrocytes

“…Although many reasons have been put forward to explain the obvious lack of CNS regeneration following injury/neurotrauma, the robust formation of the glial scar, as a result of astrogliosis, is also known to interfere with subsequent neural repair or axonal regeneration (2, 10). Thus, considerable effort is being directed toward understanding the mechanisms involved in astrocyte proliferation and reactivity in order to design therapeutic approaches to modulate gliosis, which is an impediment to neuronal recovery and axonal regeneration.Studies from our laboratory and others have shown the involvement of sphingolipids such as ceramide and psychosine in the potentiation of cytokine-mediated inflammatory disease (11)(12)(13)(14). In addition, we have recently reported the involvement of lactosylceramide in the regulation of inducible nitricoxide synthase gene expression and the efficacy of the glycosphingolipid biosynthesis inhibitor (PDMP) in attenuating spinal cord injury (SCI)-induced inflammatory disease, demonstrating significantly improved functional outcome post-SCI (15).…”

“…Studies from our laboratory and others have shown the involvement of sphingolipids such as ceramide and psychosine in the potentiation of cytokine-mediated inflammatory disease (11)(12)(13)(14). In addition, we have recently reported the involvement of lactosylceramide in the regulation of inducible nitricoxide synthase gene expression and the efficacy of the glycosphingolipid biosynthesis inhibitor (PDMP) in attenuating spinal cord injury (SCI)-induced inflammatory disease, demonstrating significantly improved functional outcome post-SCI (15).…”

Anti–Dengue Virus Nonstructural Protein 1 Antibodies Cause NO-Mediated Endothelial Cell Apoptosis via Ceramide-Regulated Glycogen Synthase Kinase-3β and NF-κB Activation

“…Ceramideregulated NO production has been reported in vascular smooth muscle cells, astrocytes, microglias, macrophages, and C6 rat glioma cells after TNF-a and LPS/IFN-g stimulation (59,60). Expression of iNOS is required for NO production under inflammatory stimulation.…”