Address correspondence and reprint requests to Inderjit Singh, Department of Pediatrics, 173 Ashley Ave, Children’s Research Institute, 516, Charleston, SC 29425-5799, USA. E-mail: email@example.com
Inflammatory disease plays a critical role in the pathogenesis of many neurological disorders. Astrogliosis and induction of pro-inflammatory mediators such as chemokines, cytokines and inducible nitric oxide synthase (iNOS) are the ‘hallmarks’ of inflammatory disease. Increased activity of lactosylceramide (LacCer) synthase and increased synthesis of LacCer during glial proliferation, and induction of pro-inflammatory cytokines and iNOS suggests a role for LacCer in these cellular signaling pathways. Studies using complementary techniques of inhibitors and antisense reported that inhibition of LacCer synthesis inhibits glial proliferation, as well as the induction of pro-inflammatory mediators (cytokines and iNOS). This inhibition was bypassed by exogenous LacCer, but not by other related lipids (e.g. glucosylceramide, galactocerebroside, GD1, GM1), indicating a role for LacCer in inflammatory signaling pathways. Furthermore, inhibition of glial proliferation and induction of inflammatory mediators by antisense to Ras GTPase, PI3Kinase and inhibitors of mitogen-activated protein kinase indicate the participation of the phosphoinositide 3-kinase (PI3Kinas)/Ras/mitogen-activated protein kinase/nuclear factor-κB (NF-κB) signaling pathways in LacCer-mediated inflammatory events thus exposing additional targets for therapeutics for inflammatory disease conditions.
Neuroinflammatory disease and the possible role of sphingolipids
Systemic inflammation is a protective response against exogenous organisms and endogenous modified products. It involves the recruitment of many cell types, such as lymphocytes, neutrophils and monocytes, and the production of various inflammatory mediators, such as prostanoids, pro-inflammatory cytokines, chemokines and inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS) and reactive nitrogen species. Similar to the peripheral nervous system, the inflammatory process in the CNS is also mediated by common inflammatory mediators produced by CNS resident glial cells (astrocytes and microglia) as well as infiltrating vascular immune cells. Despite its beneficial role, excessive inflammatory reaction can induce irreversible tissue destruction leading to disease progression which can be the cause of significant morbidity and mortality. Along with induction of such inflammatory mediators, proliferation of astrocytes, i.e. ‘astrogliosis,’ and the subsequent formation of a glial barrier (‘glial scar’) are the ‘hallmarks’ of the pathophysiology of various neurological disorders including stroke, trauma, amyotrophic lateral sclerosis, multiple sclerosis, epilepsy, and Leukodystrophies caused by dysfunction of lysosomes and peroxisomes. Due to these harmful effects, understanding the mechanism whereby signal transduction mediates the inflammatory response is important for formulating therapeutic strategies for the treatment of such inflammatory conditions. In fact, various research groups have unveiled important aspects of the inflammatory signal transduction pathways.
Sphingolipids are a class of lipids derived from the aliphatic amino alcohol sphingosine (Sph). They are generally believed to protect the cell surface against harmful environmental factors by forming a mechanically stable and chemically resistant outer leaflet of plasma membrane. Sphingomyelin (SM) is one of the most abundant sphingolipids found in animals and is densely distributed in white matter of brain where it plays a crucial role in nerve signal conduction by insulating axons through formation of a membranous myelin sheath. Like all sphingolipids, SM consists of Sph bonded to one fatty acid and one polar head group, which, in SM, is either phosphocholine or phosphoethanolamine. Under certain conditions, SM is enzymatically degraded by the action of sphingomyelinase (SMase) to ceramide. Ceramide is one of best characterized bioactive sphingolipids which mediates intracellular signaling cascades. Ceramide can be further converted to various bioactive sphingolipids including ceramide-1-phosphate, Sph, Sph-1-phosphate (SIP), glucosylceramide (GlcCer) and lactosylceramide (LacCer) and complex glycosphingolipids (GSLs) (i.e. gangliosides). Along with ceramide itself, these bioactive sphingolipids have emerged as second messenger molecules and are reported to be involved in differentiation, apoptosis, and cell cycle arrest (Pushkareva et al. 1995; Zhang et al. 2005).
In recent years, the contribution of SM metabolism to neurological disease progression has received considerable attention because of increased levels of ceramide in the CNS under various disease conditions, such as Alzheimer’s disease (Han et al. 2002; Ayasolla et al. 2004), X-adrenoleukodystrophy and multiple sclerosis (Singh et al. 1998), and its role in induction of neural cell death (Singh et al. 1998; Lee et al. 2004), pro-inflammatory gene expression (Pahan et al. 1998; Won et al. 2004b) and oxidative stress (Singh et al. 1998; Lee et al. 2004). Although the ceramide-dependent pathway for signal transduction is not fully understood, present knowledge indicates that ceramide may exert its effects through activation of putative ceramide interacting enzymes, such as Ser/Thr protein kinase (Mathias et al. 1991), kinase suppressor of Ras (KSR) (Zhang et al. 2005), phosphatase (PP) 2A and 1B (Dobrowsky et al. 1993) and PKCζ (Muller et al. 1995). More recently, ceramide is also demonstrated to play a role in clustering of membrane associated proteins through alteration of chemical and physical properties of membrane microdomains. The best example for this is the ceramide-mediated clustering of tumor necrosis factor (TNF) family receptors (i.e. TNF-R1, CD40 and CD95) (Grassme et al. 2001, 2002a,b). Following ligation of these receptors with ligands, the ceramide produced by SMase around TNF receptors generates signaling microdomains. As ceramide has the ability to self-aggregate (Cremesti et al. 2002), subsequent fusion of these small entities into larger membrane domains (ceramide rafts) (Holopainen et al. 1998) has been demonstrated to trigger the clustering of these receptor molecules (Grassme et al. 2001, 2002b). Receptor clustering in the rafts induces close contact of receptors with other signaling molecules (Hanissian and Geha 1997) and exclusion of inhibitory molecules (i.e. CD45 tyrosine phosphatase) (Veiga et al. 1999), thus stabilizing ligand-receptor-signaling protein interactions (Grassme et al. 2001, 2002b). Therefore, the increase in ceramide and the formation of ceramide rafts may enhance inflammatory or death signaling events which are tightly linked to cellular redox potential (Grassme et al. 2001, 2002b).
Emerging evidence also suggests that ceramide may contribute inflammatory signaling cascades via formation of its derivatives, such as S1P and GSLs. S1P is generated from ceramide by the sequential action of neutral or alkaline ceramidases and Sph kinase 1/2. Study of Sph kinase-mediated S1P generation and G protein coupled receptors-mediated intracellular signaling cascade is an active area of investigation and the subject of many recent review articles (Chalfant and Spiegel 2005; Lamour and Chalfant 2005; Rosen and Goetzl 2005; Taha et al. 2006; Tani et al. 2007). Moreover, the recently reported role of S1P in cell proliferation, migration and changes in morphology of astrocytes and microglia suggests a crucial role of S1P in neuroinflammatory disease conditions (Tas and Koschel 1998; Bassi et al. 2006; Kimura et al. 2007). For example, generation of S1P in basic fibroblast growth-factor (bFGF)-induced astrogliosis, and inhibition of gliosis by inhibitors of SK and by pertussis toxin, an inactivator of Gi/Go proteins, document the participation of S1P and G protein coupled receptors signaling in cytokine/bFGF-induced astrocyte proliferation (Anelli et al. 2005; Bassi et al. 2006). As bFGF plays a fundamental role in astrocyte proliferation following injury and in tumorigenesis (Takahashi et al. 1992; Norenberg 1994; Kawamata et al. 1997), S1P is believed to have a role in remodeling neural tissue during the healing process.
GSLs are plasma membrane constituents in mammalian cells, which play an important role in normal cell adhesion, migration, and proliferation as well as in pathologic conditions, such as tumorigenesis and atherosclerosis (Chatterjee 1998; Hakomori Si 2002; Kannagi et al. 2004). GSLs are present mainly in the outer leaflet of the plasma membrane where they interact closely with cholesterol to form lipid microdomains (Brown and London 2000; Simons and Toomre 2000). These GSL-enriched microdomains have been shown to act as organizing centers for some cellular signaling complexes (Simons and Toomre 2000; Hakomori Si 2002) and are also initiation sites for clathrin-independent endocytic events (Simons and Toomre 2000; Hakomori Si 2002; Marks and Pagano 2002). Generally GSLs are divided by two groups; neutral GSL species and acidic GSL (i.e. gangliosides, a complex GSL containing one or more sialic acid residue). The GSL are abundant in the nervous system and defects in their catabolism are known to cause neurodegenerative and neuroinflammatory diseases, such as Gaucher, Fabry, Tay-Sachs, and Sandhoff diseases. In fact, the mouse models of GM1 and GM2 gangliosidosis exhibit progressive inflammatory reactions in the CNS which are characterized by altered blood-brain barrier, induction of pro-inflammatory cytokine expression and apoptosis (Jeyakumar et al. 2003). Recent reports have also demonstrated that LacCer, a common precursor of all types of lactose series of GSL (e.g. the gangliosides and globotriosylceramide), is a bioactive lipid in various cell physiological processes such as smooth muscle cell proliferation (Bhunia et al. 1997), expression of adhesion molecules (Arai et al. 1998; Bhunia et al. 1998), angiogenesis (Rajesh et al. 2005) and in β1-integrin clustering and endocytosis (Ebadi and Sharma 2003). Recently, we have reported that LacCer is an important signaling component for the induction of pro-inflammatory mediators and astrogliosis (Pannu et al. 2004, 2005). Although cellular and molecular mechanisms for LacCer-mediated cellular events are largely unknown, activation of NADPH oxidase and nuclear-factor-κB (NF-κB) signaling cascades are believed to be involved (Arai et al. 1998; Bhunia et al. 1998; Pannu et al. 2004). Because of the limited scope, this review will be limited to the role of LacCer in the inflammatory disease process.
Biosynthesis of LacCer and neutral sphingolipids
Source of ceramide for glucosylceramide and lactosylceramide synthesis
LacCer is not only an intermediate in the synthesis of complex sphingolipids as shown in Fig. 1 but also a bioactive sphingolipid which regulates various cell signaling pathways. Synthesis of LacCer is started by the sequential transfer of glucose and galactose moieties from UDP-glucose and UDP-galactose to ceramide. Ceramide is composed of D-erythro-Sph and fatty acid with C16∼26 carbon atoms acyl-chain. In addition, these fatty acids are saturated or unsaturated which are either non-hydroxylated or hydroxylated at C-1 position. Ceramide synthesized in endoplasmic reticulum (ER) by de novo pathway can be a precursor for the synthesis of LacCer as shown in Fig. 1. However, our recent studies suggest that ceramide generated via hydrolysis of SM by the action of SMases (EC 18.104.22.168) can also serve as a precursor for LacCer synthesis (Martin et al. 2006). In most mammalian cells, two isotypes of SMases are involved in SM hydrolysis and ceramide generation (neutral and acidic SMases). Ceramide synthesis by de novo and hydrolytic pathway may occur in different cellular compartments via different modes of regulation for the synthesis of LacCer (Fig. 2) (Venkataraman and Futerman 2000).
The presence of neutral sphingomyelinase (nSMase) which has neutral pH optimum (∼7.4) and magnesium dependency was firstly described in 1967 by Schneider and Kennedy (Schneider and Kennedy 1967) and subsequently more than one nSMase has been identified. In 1998, nSMase1 was cloned and characterized from mice and humans (Tomiuk et al. 1998). Although nSMase1 showed activity for SM hydrolysis in vitro (Sawai et al. 1999), cell-based studies demonstrated that nSMase1 is 1-Ο-alkyl-lyso-phosphatidylcholine (lyso-platelet activating factor or lyso-PAF) phospholipase C, but not nSMase (Sawai et al. 1999). More recently, another mammalian brain specific magnesium-dependent nSMase was cloned in humans and mice and named ‘nSMase2’ (smpd3) (Hofmann et al. 2000). The nSMase2 exhibited similar properties with previously purified rat brain nSMase and normally present in Golgi and plasma membrane (Hofmann et al. 2000; Marchesini and Hannun 2004). The mechanism of nSMase activation has been intensely studied during the past decade and cellular redox potential as well as caspases and some of bioactive lipids (i.e. arachidonic acid, anionic phospholipids and phosphatidylserine) are known to be crucial activators for nSMase2 (Liu and Hannun 1997; Tomiuk et al. 1998; Bernardo et al. 2000; Hofmann et al. 2000; Kondo et al. 2002; Sumitomo et al. 2002; Marchesini et al. 2003). In 1996, discovery of factor associated with nSMase activation (FAN) protein suggested a new mechanism for nSMase regulation (Adam et al. 1996). Upon ligation of TNF-α, TNF-R1 recruits factor associated with nSMase activation through its nSMase activation domain and activates nSMase (Adam et al. 1996; Kreder et al. 1999; Segui et al. 1999). As nSMase1 does not exhibit SMase activity in vivo, the increased nSMase activity by these activators appears to be mediated by nSMase2.
The anabolic pathway for ceramide generation starts with condensation of palmitoyl-CoA and serine by serine palmitoyl transferase (EC 22.214.171.124) to produce 3-ketosphinganine which is subsequently reduced to sphinganine by 3-ketosphingosine reductase (EC 126.96.36.199). Sphinganine is N-acylated to dihydroceramide by ceramide synthases (Sph N-acyltransferase; EC 188.8.131.52) and then ceramide in turn serves as a precursor for various sphingolipids. So far, eight ceramide synthetases (Lac1, LAS 1–6 and CLN8) have been identified to synthesize ceramides containing different chain length fatty acid (Guillas et al. 2001; Schorling et al. 2001; Futerman and Riezman 2005). Thus these different homologues of ceramide synthetase are reported to control the specificity of fatty acid in ceramides (Riezman and van Meer 2004; Mizutani et al. 2005; Futerman 2006). It is important to determine the fatty acid specificity of ceramide as different classes of glycolipids contain different chain fatty acids, and are suggested to have different biological functions (Koyanagi et al. 2003; Koybasi et al. 2004; Schulz et al. 2006). These synthetic pathways are reversed as a result of enzymatic activities that hydrolyze each product. Thus there is more than one source (synthetic and hydrolytic) for these compounds.
At present the source of ceramide in LacCer synthesis under physiological or pathological conditions is not fully characterized. Recently, Martin et al. reported that LacCer synthesis by stimulation of TNF-α in a human osteosarcoma cell line requires nSMase activity (Martin et al. 2006), suggesting that ceramide generated by nSMase may be a source for LacCer synthesis under inflammatory conditions. In addition to ceramide generated by nSMase, de novo synthesized ceramide may also serve as a precursor for LacCer synthesis. In fact, de novo synthesized ceramide in ER is known to be utilized in various GSLs and SM synthesis. For example, ceramide synthesized on the cytoplasmic side of the ER membrane is translocated to the lumenal side of ER membrane by an unknown mechanism and utilized by galactosylceramide (GalCer) synthetase for GalCer synthesis. The other part of the de novo synthesized ceramide pool in ER is transported to Golgi for the synthesis of GlcCer on the cytoplasmic surface (Ichikawa and Hirabayashi 1998) and for the synthesis of SM on the lumenal surface of the Golgi (Futerman and Riezman 2005). Therefore, the ceramide transported from ER may be readily converted to GlcCer by cytosolic surface localized GlcCer synthase, but not to SM unless it is flipped to the lumenal side of the Golgi membrane. Interestingly, SM and GlcCer are known to have different fatty acid compositions. Several mechanisms may be possible. Ceramides synthesized in ER are transported to the Golgi, possibly as different pools or via different mechanisms. Although ceramide transfer protein has been described to transfer ceramide from ER to Golgi, it is not clear whether it facilitates the transport of pools of ceramides of different fatty acid chain length as precursors for the synthesis of GlcCer versus SM (Hanada et al. 2003; Kumagai et al. 2005). These observations indicate that the pool of ceramide required for synthesis of SM versus GlcCer may be transported from ER to Golgi as different pools via different mechanisms. Ceramide transfer proteins are cytoplasmic proteins that transport ceramide to Golgi in a non-vascular manner (Hanada et al. 2003; Riezman and van Meer 2004; Futerman and Riezman 2005; Kumagai et al. 2005; Perry and Ridgway 2005) for the synthesis of SM, whereas ceramide for the synthesis of GlcCer is reported to be transported from ER to Golgi via an ATP- or cytosol-independent mechanism (Riezman and van Meer 2004).
GlcCer is a product of the first glycosylation step in GSL biosynthesis, which is catalyzed by glucosylceramide synthase (GCS; UDP-glucose:N-acylsphingosine d-glucosyltransferase, EC 184.108.40.206). The cDNA of GCS was cloned by Ichikawa et al. and reported to have an open reading frame encoding 394 amino acids (44.9 kDa) (Ichikawa et al. 1996). In the mean time, another GCS of 60–70 kDa mass was also purified from enriched Golgi membranes from rat liver (Paul et al. 1996). The GCS catalyzes the glucosylation of ceramide on the cytoplasmic surface of Golgi, GlcCer is then trans-located into the lumen of the Golgi for the synthesis of LacCer. However, the mechanism of translocation of glucocerebrosides across the golgi membrane is not well understood (De Rosa et al. 2004; Contreras et al. 2005). The conversion of GlcCer to LacCer is mediated by specific galactosyltransferase called ‘LacCer synthase’ [UDP-Galactose:glucosylceramide β1,4galactosyl transferase (β4GalT)].
Recent studies have shown that LacCer itself is a bioactive lipid involved in various cell signaling cascades. Moreover, LacCer serves as a core for subsequent synthesis of at least three different classes of GSL (e.g. gangliosides, LcOse3Cer, and GbOse3Cer) (Lannert et al. 1998). Synthesis of LacCer is mediated by β-1,4-galactosyltranferases (β-1,4-GalT) which transfers galactose from UDP-galactose to specific acceptor molecules (GlcCer). In 1986, the first β-1,4-GalTs were cloned from bovine kidney and mammary gland (Narimatsu et al. 1986; Shaper et al. 1986). The cloned enzymes transfer galactose to N-acetylglucosamine associated with glycoproteins and glycolipids and named it as GalT-1. In 1998, Sato et al. cloned human cDNA encoding β4GalT (Sato et al. 1998) and named it as β-1,4-GalT II (or GalT-2). Earlier than the cloning of β-1,4-GalT II, Chatterjee et al. also purified 60 and 58 kDa enzyme that had LacCer synthase activity and also named it GalT-2 (Chatterjee et al. 1992). Recently, independent studies based on partial sequences deposited in expressed sequence tags database identified five human genes with 55, 44, 41, 37, and 31% amino acid sequence homologies to β-1,4-GalT I (Almeida et al. 1997; Lo et al. 1998; Schwientek et al. 1998). They are named as β-1,4-GalT (or β4GalT) -II, -III, -IV, -V and -VI according to the homology distances from β-1,4-GalT I. Sequence homology studies indicate that the β-1,4-GalT II cloned by Sato’s group was identical to β4GalT-V and GalT-2 purified by Chatterjee’s group was identical to β4GalT-VI. In fact, β4GalT-V and β4GalT-VI had ∼68% nucleotide sequence homology and were regarded as genes encoding LacCer synthase. However, recent studies using wild type and β4GalT-V mutant Chinese hamster ovary cells suggest that β4GalT-V may not be a LacCer synthase involved in cell signaling cascades (Kolmakova and Chatterjee 2005). Moreover, the constitutive and predominant expression of β4GalT-VI mRNA in human adult brains (Lo et al. 1998) suggests that β4GalT-VI may be a predominant form of LacCer synthase in the brain.
Previous studies have shown that GlcCer is synthesized on the cytoplasmic side of Golgi apparatus (Ichikawa and Hirabayashi 1998). As all other glycosyltransferases including LacCer synthase are localized on the lumenal side of the Golgi apparatus, GlcCer must be flipped to the other side of the membrane for further glycosylation. Although the mechanism for this flipping is not well understood, numerous studies have suggested the presence of ‘flippase’ for GlcCer flipping (Ichikawa and Hirabayashi 1998). Recent studies suggest that pgp (p-glycoprotein, a multidrug transporter) plays a role as a flippase for simple GSLs, such as GlcCer and GalCer, but not for LacCer (Eckford and Sharom 2005). In fact, pgp was previously reported to be involved in the regulation of cellular GlcCer and SM levels (Lavie et al. 1996; Lucci et al. 1998). However, the exact role of pgp in LacCer synthesis is not well understood at present. Further investigation of the route and mechanism of GlcCer translocation is required to shed light on the process of LacCer synthesis.
Induction of astrogliosis by LacCer
We have studied the role of LacCer in astrogliosis both in the in vitro tissue culture model as well as in the animal model of spinal cord injury. In primary astrocyte cultures, TNF-α stimulation induced expression of glial fibrillary acidic protein (GFAP) and astrocyte proliferation. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-HCl (PDMP), inhibitor of GCS and LacCer synthase, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other related glycolipids such as galactocerebrosides, glucocerebrosides, GM1, GD3, and GM3 thus indicating these to be LacCer-specific effects (Pannu et al. 2005). To understand the mechanism of TNF-α-induced astrocyte proliferation, mediated by LacCer, in situ levels of LacCer and activity of LacCer synthase were examined. Increase in LacCer levels, as well as activity of LacCer synthase, were observed within 2–5 min following stimulation with TNF-α. The activity of LacCer synthase and levels of GalCer increased two fold. The role of LacCer synthase, and its product LacCer, in astrocyte proliferation was further confirmed by silencing β4GalT-VI gene using antisense DNA oligomers against rat β4GalT-VI mRNA, and a sequence-scrambler oligomer as a control. The diminished protein levels for β4GalT-VI by antisense oligonucleotide correlated with diminished synthesis of LacCer and expression of GFAP and astrocyte proliferation (Pannu et al. 2005). The inhibition of GTPase by transfection with dominant negative RAS (DNH-Ras N17 mutant) in astrocytes inhibited the expression of glial fibrillary acidic protein (GFAP), activation of extracellular signal regulated kinase (ERK 1/2) and proliferation of astrocytes indicating a role for Ras/ERK 1/2 signaling pathway in TNF-α-induced proliferation of astrocytes. Inhibition of astrocyte proliferation and GFAP expression by inhibition of MAPK/ERK Kinase 1/2, (PD98059) and PI3Kinase (LY294002), and antisense to PI3Kinase, document a role for PI3Kinas-Ras-ERK 1/2 in astrocyte proliferation. Furthermore PDMP treatment attenuated spinal cord injury-induced activation of ERKs and astrogliosis, and improved the functional recovery, indicating that these in vivo studies support the conclusions drawn from cell-culture studies, and provide evidence for LacCer involvement in inflammatory gliosis in a rat model of spinal cord injury (Pannu et al. 2005). The finding that activation of LacCer synthase and increased synthesis of LacCer occurs within 2–5 min following stimulation with cytokines suggests that the enzyme system for the synthesis of LacCer may be present in or near lipid rafts, a site for generation of ceramide by nSMase (Fig. 2). Figure 2 is a schematic representation of LacCer-induced signal transduction pathways in inflammatory gliosis.
Induction of inflammatory mediators by LacCer
SM signaling transduction pathways mediate actions of several extra-cellular stimuli leading to important cellular processes. Cytokines (mediators of various aspects of inflammation), produced under various disease conditions, regulate the metabolism of SM for generation of sphingolipids (Sph) (ceramide, ceramide-1-P, sphingosine, S1P, and LacCer), and some of these metabolites participate in the up-regulation of the inflammatory process. The treatment of cultured astrocytes with exogenous ceramide, or SMase, up-regulated the lipopolysaccharide (LPS)- or cytokine- (TNF-α or IFN-γ or ILβ) induced expression of iNOS and production of nitric oxide (NO) (Pahan et al. 1998). These observations indicate that ceramide generated by hydrolysis of SM participates in the inflammatory signal transduction pathways in astrocytes. Subsequent studies with the use of inhibitors for de novo synthesis of ceramide, or aSMase or nSMase, reported that ceramide generated by neutral SM participates in inflammatory signaling pathways in astrocytes (Won et al. 2004b). Studies with the use of inhibitors of GlcCer and LacCer synthetase reported that induction of pro-inflammatory cytokines, iNOS and production of NO was inhibited by this inhibitor and was reversed by LacCer supplementation, but not by other related sphingolipids, documenting that LacCer, rather than ceramide or other sphingolipids, participate in cytokine-induced expression of iNOS (Pannu et al. 2004).
In the brain, iNOS can be induced in response to numerous pathophysiological processes. In addition, iNOS can be expressed in the brain in response to peripheral events in particular systemic inflammation. Regulation of iNOS is possibly mainly at the transcriptional and translation levels, as once activated, iNOS produces NO until substrate depletion (Hickey et al. 2001). The iNOS gene expression and subsequent mRNA translation is controlled by an ever increasing number of agonists (Pannu et al. 2004; Won and Singh 2006). The most prominent amongst them are endotoxin (LPS) in conjunction with pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) (Won and Singh 2006). In addition, iNOS activity can be modulated post-translationally. Post-translational regulation of iNOS is believed to be through modulation of its catalytic activity via secondary modifications, and/or the availability of critical cofactors such as BH4 (Kunz et al. 1996). A final step for the regulation of iNOS activity is at the point of its turn over. Post-transcriptional regulation of iNOS gene expression is believed to occur predominantly via mechanisms that affect iNOS mRNA stability (Kiemer and Vollmar 1998). The stability of the iNOS protein has been shown to be altered through signaling events (involving cAMP) which can alter protein stability by affecting the rate of ubiquitination-dependent turnover of the iNOS protein (Won et al. 2004a).
Most in vitro studies rely on a combination of endotoxin and/or cytokines to elicit a profound effect on iNOS expression as a single stimulus exhibits only a moderate effect in specific cell types, suggesting the cooperative action of multiple signal transduction pathways for a robust up-regulation of iNOS gene expression. These agonists have been shown to trigger numerous cell-specific signal transduction pathways, out of which activation of the IκB/NF-κB transcription pathway is deemed virtually indispensable for iNOS gene transcription. Alternative signaling pathways include the janus tyrosine kinase-signal transducers and activators of transcription (JAK/STAT) pathway (Kleinert et al. 1998; Bolli et al. 2001), and the mitogen-activated protein kinases (MAPK) pathway, leading to the activation of transcription factors (such as activator protein 1, activating transcription factor 2, cAMP-responsive elements, and the transcription factors from the erythroblastosis virus E26 oncogene homolog protein, family) which are closely linked with iNOS gene expression (Janssen-Heininger et al. 1999).
While studying the mechanisms of cytokine-induced expression of iNOS expression, we observed that treatment of astrocytes, with exogenous ceramide or SMase, up-regulated the expression of iNOS, indicating that metabolites of SMase, such as ceramide, play a role in the induction of iNOS and production of NO (Pahan et al. 1998). This activity of ceramide, but not dihydroceramide, was redox-sensitive as antioxidants (NAC and PDTC) inhibited the induction of iNOS. The role of exogenous ceramide in cellular signaling for iNOS induction was substantiated by inhibition of LPS/ceramide-induced iNOS by inhibitors of small GTPase isoprenylation (FPT inhibitor II) and the inhibitor of MAPK pathway in astrocytes (Pahan et al. 1998).
Pro-inflammatory cytokine (TNF-α, IL-1β) treatment of astrocytes was found to be associated with the generation of ceramide from SM, and this activity was redox-sensitive because NAC was able to inhibit the cytokine-induced conversion of SM to ceramide (Singh et al. 1998). In contrast to ceramide-mediated induction of iNOS in astrocytes (Pahan et al. 1998) the cytokine-induced ceramide generation in oligodendrocytes was associated with cell death of oligodendrocytes in culture (Singh et al. 1998). These findings indicate that ceramide may participate in different signaling pathways in different cell types in CNS (Pahan et al. 1998; Singh et al. 1998). Obviously, ceramide in the cell can be generated via its synthesis and degradation of SM by either aSMase or nSMase. The use of inhibitors for the respective pathways showed that cytokine-induced ceramide generation for induction of iNOS was inhibited by inhibitor of 3MSM (3-o-methylsphingomyelin) but not by inhibitor of aSMase (SR33557) or of ceramide de novo synthesis (furmonisin B1) (Won et al. 2004b). This nSMase-generated ceramide signaling for induction of iNOS was reported to be via Ras-NF-κB pathway.
However, subsequent studies with the inhibitors of glucocerebrosides and lactosylcerebroside synthesis reported that instead of ceramide, LacCer may be the signaling molecule for cytokine-induced gliosis (Pannu et al. 2005) as well as induction of cytokines (TNF-α, IL-1β) and iNOS. The inhibitor of LacCer synthesis (PDMP) inhibited the cytokine-induced expression of iNOS and this inhibition was bypassed with exogenous addition of LacCer but not the addition of glucocerebrosides, galactocerebrosides, GM1, and GD1 (Pannu et al. 2004). This activity of LacCer was mediated via activation of Ras-NF-κB-MAPK signaling pathway (Pannu et al. 2004). These observations suggest that in astrocytes cytokine-mediated ceramide generation is via activation of nSMase and that this pool of ceramide is further converted to GlcCer and in turn into LacCer for astrogliosis (Pannu et al. 2005) and induction of inflammatory cytokines and iNOS (Pannu et al. 2004). Figure 2 is a schematic representation of LacCer-induced signal transduction pathways for the expression of cytokines and iNOS in astrocytes.
Cellular redox and regulation of nSMase
Sphingolipid and cellular redox
Sphingolipids such as ceramide (Reinehr et al. 2005; Zhang et al. 2005) and LacCer and gangliosides (Bhunia et al. 1997, 1998; Arai et al. 1998; Garcia-Ruiz et al. 2000) up-regulate the activity of NADPH oxidase whereas GlcCer suppresses it’s activity (Moskwa et al. 2004). The LacCer-mediated activation of NADPH oxidase and ROS generation is involved in endothelial and neutrophil cell functions through regulation of endothelial cell proliferation, adhesion molecule expression, and phagocytosis (Bhunia et al. 1997, 1998; Arai et al. 1998). Recently, our group also reported the involvement of LacCer-mediated ROS generation in cytokine-induced sequential activation of Ras/NF-κB and inflammatory gene expression in astrocytes (Pannu et al. 2004, 2005). The exact mechanism of LacCer-mediated activation of NADPH oxidase is not clear, but it is believed that LacCer interactions with a Src family kinase (i.e. Lyn) in the lipid rafts may lead to generation of ROS through phosphatidylinositol-3-kinase-, p38 MAPK-, and PKC-dependent signal transduction pathways (Iwabuchi and Nagaoka 2002). Membrane-associated phagocytic NADPH oxidase (Phox; also known as NADPH oxidase-2, Nox2), with its catalytic moiety in gp91phox, in plasma membrane is activated by assembly with regulatory proteins such as p47phox, p67phox, and Rac (Bokoch and Diebold 2002; Nauseef 2004). Recently, other oxidases similar to the Phox complex have also been identified in other cell types and show different expression patterns depending on cell or tissue types (Lambeth 2002). Figure 2 is a schematic representation of participation in LacCer-mediated inflammatory signal transduction pathways in astrocytes.
Cellular redox and iNOS
Inhibition of generation of ceramide (Pahan et al. 1998) and LacCer (Pannu et al. 2004) and induction of iNOS by antioxidants and inhibitor of nSMase indicate a role for cellular redox and nSMase generated ceramide in induction of iNOS. Figure 2 shows a schematic representation of nSMase in generation of LacCer in cytokine-induced expression of inflammatory mediators in astrocytes. Previously, we and other groups have observed that pro-inflammatory cytokines (i.e., IL-1β and TNF-α) or hypoxia induced SM hydrolysis and ceramide generation in a redox-sensitive event (Singh et al. 1998; Yoshimura et al. 1999). Moreover, Aβ1–42 or its synthetic peptide Aβ23–35, which produces pathologies of Alzheimer’s disease, induces ceramide generation by activation of nSMase in a redox-sensitive manner without altering aSMase (Ayasolla et al. 2004; Lee et al. 2004). Therefore, these studies suggest that oxidative stress-mediated nSMase activation and ceramide generation may play a key role(s) in the pathobiology of various disease conditions. However, the mechanism of regulation of nSMase by oxidative stress is not completely known. Liu and Hannun showed that GSH, but not dithiothreitol and β-mercaptoethanol, dose dependently inhibit partially purified nSMase activity (1997). They reported that γ-glutamylcysteine, but not the free sulfohydryl group, in GSH may function as an allosteric regulator of nSMase. Studies from our laboratory reported that cellular redox (GSH) regulates the activity of nSMase (Pahan et al. 1998). GSH is also known to regulate nSMase activity through Bcl-xL or Bcl-2, an anti-apoptotic protein, by inhibiting oxidative stress mediated SM hydrolysis and ceramide generation (El-Assaad et al. 1998; Okamoto et al. 2002). Moreover, tyrosine kinases such as Lyn and PKC are also implicated in oxidative stress-mediated regulation of nSMase activity and ceramide generation (Bezombes et al. 2002; Grazide et al. 2002). In addition to GSH, Takeda et al. reported that sodium nitroprusside, a NO donor, also increases cellular SM hydrolysis and ceramide generation through activation of nSMase (Takeda et al. 1999). However, whether NO mediates nSMase activation through direct interaction, or depletion of cellular GSH levels, is not currently known.
Nitric oxide toxicity
NO released by iNOS and its metabolite peroxinitrite (ONOO−) is believed to play a role in the pathophysiology of many disease processes. Once induced, iNOS expression stays over days and generates copious amounts (nanomolar) of NO for prolonged periods (4–8 days) that has been linked to pathological manifestations observed in inflammatory disease conditions (Pannu et al. 2004; Won and Singh 2006). NO, a diffusible free radical, plays many roles as a signaling and effector mole in diverse biological functions as a neuronal messenger, vasodilator and exhibits antimicrobial and antitumor activities (Nathan 1992; Jaffrey and Snyder 1995) at low concentrations, however, at higher concentrations it is neurotoxic particularly in neurodegenerative conditions of Alzheimer’s disease, multiple sclerosis, stroke, cerebral palsy, brain trauma and leukodystrophies associated with dysfunction of peroxisomes and lysosomes (Pannu et al. 2004). The free radical characteristic of NO makes it reactive with proteins containing the heme-iron prosthetic groups, iron sulfur clusters or reactive thiols. In addition to the direct reaction of NO with protein prosthetic groups, NO is relatively unstable, and in the presence of molecular oxygen can spontaneously auto-oxidize to yield a variety of nitrogen oxides. These are potent nitrosylating agents with ability to N-nitrosylate a variety of primary and secondary amines, and as a result potentially carcinogenic nitrosamines, and/or promote mutagenic deamination of DNA bases (Wink et al. 1991). In addition, NO also reacts with the superoxide anion (O2−) to generate peroxinitirite anion (ONOO−), which further promotes toxicity by nitrosylation and nitration of proteins (Beckman and Koppenol 1996; Dawson and Dawson 1998). The inhibition of induction of iNOS and NO production by inhibitor (PDMP) of LacCer synthesis, decreased neuronal apoptosis/tissue necrosis and demyelination and improved functional recovery in the animal model of spinal cord injury identifies sphingolipid-mediated signaling pathway as targets for therapeutics in neuroinflammatory disease conditions (Pannu et al. 2004, 2005).
The authors are thankful to Mrs Michaela Barno in preparation of this review. These studies were supported in part by grants from the National Institute of Health (NS-22576, NS-34741, NS-37766 and AG25307).