Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii

Glycolipids are important components of cellular membranes involved in various biological functions. In this report, we describe the identification of the de novo synthesis of glycosphingolipids by Toxoplasma gondii tachyzoites. Parasite-specific glycolipids were identified by metabolic labelling of parasites with tritiated serine and galactose. These glycolipids were characterised as sphingolipids based on the labelling protocol and their insensitivity towards alkaline treatment. Synthesis of parasite glycosphingolipids were inhibited by threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol and L-cycloserine, two well-established inhibitors of de novo sphingolipid biosynthesis. The identified glycolipids were insensitive towards treatment with endoglycoceramidase II indicating that they might belong to globo-type glycosphingolipids. Taken together, we provide evidence for the first time that T. gondii is capable of synthesising glycosphingolipids de novo.     

The de novo biosynthesis of platelet-activating factor in rat brain

Platelet-Activating Factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is present in nervous tissue and its function is still unknown. We have demonstrated that rat brain is able to synthesize PAF from 1-alkyl-2-acetyl-sn-glycerol and CDP-choline by a "DTT-insensitive" phosphocholine transferase. This represents the last step of the de novo pathway which apparently is the only one existing in the brain for PAF biosynthesis. The enzyme has a microsomal localization, requires Mg++ and is inhibited by Ca++ as reported for phosphocholine transferase utilizing long-chain diradylglycerols as substrates. However, other properties of PAF-synthesizing enzyme (sensitivity to DTT and dependence on pH) are different from those of phosphocholine transferase responsible for the synthesis of diacyl and long-chain alkylacyl glycerophosphocholines. These observations indicate that a specific enzyme for PAF biosynthesis might exist in rat brain.     

Hepatitis C virus core protein activates nuclear factor kappa B-dependent signaling through tumor necrosis factor receptor-associated factor

Hepatitis C virus (HCV) core protein, a viral nucleocapsid, has been shown to affect various intracellular events including the nuclear factor kappaB (NF-kappaB) signaling supposedly associated with inflammatory response, cell proliferation, and apoptosis. In order to elucidate the effect of HCV core protein on the NF-kappaB signaling in HeLa and HepG2 cells, a reporter assay was utilized. HCV core protein significantly activated NF-kappaB signaling in a dose-dependent manner not only in HeLa and HepG2 cells transiently transfected with core protein expression plasmid, but also in HeLa cells induced to express core protein under the control of doxycycline. HCV core protein increased the DNA binding affinity of NF-kappaB in the electrophoretic mobility shift assay. Acetyl salicylic acid, an IKKbeta-specific inhibitor, and dominant negative form of IKKbeta significantly blocked NF-kappaB activation by HCV core protein, suggesting HCV core protein activates the NF-kappaB pathway mainly through IKKbeta. Moreover, the dominant negative forms of TRAF2/6 significantly blocked activation of the pathway by HCV core protein, suggesting HCV core protein mimics proinflammatory cytokine activation of the NF-kappaB pathway through TRAF2/6. In fact, HCV core protein activated interleukin-1beta promoter mainly through NF-kappaB pathway. Therefore, this function of HCV core protein may play an important role in the inflammatory reaction induced by this hepatotropic virus.     

Sphingosine 1-phosphate (S1P) lyase deficiency increases sphingolipid formation via recycling at the expense of de novo biosynthesis in neurons

Sphingosine 1-phosphate lyase (S1P lyase) irreversibly cleaves sphingosine 1-phosphate (S1P) in the final step of sphingolipid catabolism. As sphingoid bases and their 1-phosphate are not only metabolic intermediates but also highly bioactive lipids that modulate a wide range of physiological processes, it would be predicted that their elevation might induce adjustments in other facets of sphingolipid metabolism and/or alter cell behavior. Indeed, we have previously reported that S1P lyase deficiency causes neurodegeneration and other adverse symptoms. We next asked the question whether and how S1P lyase deficiency affects the metabolism of (glyco)sphingolipids and cholesterol, two lipid classes that might be involved in the neurodegenerative processes observed in S1P lyase-deficient mice. As predicted, there was a considerable increase in free and phosphorylated sphingoid bases upon elimination of S1P lyase, but to our surprise, rather than increasing, the mass of (glyco)sphingolipids persisted at wild type levels. This was discovered to be due to reduced de novo sphingoid base biosynthesis and a corresponding increase in the recycling of the backbones via the salvage pathway. There was also a considerable increase in cholesterol esters, although free cholesterol persisted at wild type levels, which might be secondary to the shifts in sphingolipid metabolism. All in all, these findings show that accumulation of free and phosphorylated sphingoid bases by loss of S1P lyase causes an interesting readjustment of the balance between de novo biosynthesis and recycling to maintain (glyco)sphingolipid homeostasis. These changes, and their impact on the metabolism of other cellular lipids, should be explored as possible contributors to the neurodegeneration in S1P lyase deficiency.     

Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression

Cancer progression depends on an accumulation of metastasis supporting cell signaling molecules that target signal transduction pathways and ultimately gene expression. Osteopontin (OPN) is one such chemokine like metastasis gene which plays a key signaling event in regulating the oncogenic potential of various cancers by controlling cell motility, invasiveness and tumor growth. We have reported that OPN stimulates tumor growth and nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkappaBalpha/IKK (IkappaBalpha kinase) signaling pathway in melanoma cells. Urokinase type plasminogen activator (uPA), a widely acting serine protease degrades the ECM components and plays a pivotal role in cancer progression. However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and uPA secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt/PKB (protein kinase B) in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with dominant negative mutant of p85 domain of PI 3'-kinase (Deltap85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECM-invasion. Furthermore, cells transfected with Deltap85 or super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkappaB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN induced PI 3'-kinase dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells.     

The endocannabinoid 2-arachidonoyl-glycerol activates human neutrophils: critical role of its hydrolysis and de novo leukotriene B4 biosynthesis

Although endocannabinoids are important players in nociception and obesity, their roles as immunomodulators remain elusive. The main endocannabinoids described to date, namely 2-arachidonoyl-glycerol (2-AG) and arachidonyl-ethanolamide (AEA), induce an intriguing profile of pro- and anti-inflammatory effects. This could relate to cell-specific cannabinoid receptor expression and/or the action of endocannabinoid-derived metabolites. Importantly, 2-AG and AEA comprise a molecule of arachidonic acid (AA) in their structure and are hydrolyzed rapidly. We postulated the following: 1) the released AA from endocannabinoid hydrolysis would be metabolized into eicosanoids; and 2) these eicosanoids would mediate some of the effects of endocannabinoids. To confirm these hypotheses, experiments were performed in which freshly isolated human neutrophils were treated with endocannabinoids. Unlike AEA, 2-AG stimulated myeloperoxidase release, kinase activation, and calcium mobilization by neutrophils. Although 2-AG did not induce the migration of neutrophils, it induced the release of a migrating activity for neutrophils. 2-AG also rapidly (1 min) induced a robust biosynthesis of leukotrienes, similar to that observed with AA. The effects of 2-AG were not mimicked nor prevented by cannabinoid receptor agonists or antagonists, respectively. Finally, the blockade of either 2-AG hydrolysis, leukotriene (LT) B(4) biosynthesis, or LTB(4) receptor 1 activation prevented all the effects of 2-AG on neutrophil functions. In conclusion, we demonstrated that 2-AG potently activates human neutrophils. This is the consequence of 2-AG hydrolysis, de novo LTB(4) biosynthesis, and an autocrine activation loop involving LTB(4) receptor 1.     

Neuropeptide-inducible upregulation of proteasome activity precedes nuclear factor kappa B activation in androgen-independent prostate cancer cells

Background

Upregulation of nuclear factor kappa B (NF??B) activity and neuroendocrine differentiation are two mechanisms known to be involved in prostate cancer (PC) progression to castration resistance. We have observed that major components of these pathways, including NF??B, proteasome, neutral endopeptidase (NEP) and endothelin 1 (ET-1), exhibit an inverse and mirror image pattern in androgen-dependent (AD) and -independent (AI) states in vitro.

Methods

We have now investigated for evidence of a direct mechanistic connection between these pathways with the use of immunocytochemistry (ICC), western blot analysis, electrophoretic mobility shift assay (EMSA) and proteasome activity assessment.

Results

Neuropeptide (NP) stimulation induced nuclear translocation of NF??B in a dose-dependent manner in AI cells, also evident as reduced total inhibitor ??B (I??B) levels and increased DNA binding in EMSA. These effects were preceded by increased 20?S proteasome activity at lower doses and at earlier times and were at least partially reversed under conditions of NP deprivation induced by specific NP receptor inhibitors, as well as NF??B, I??B kinase (IKK) and proteasome inhibitors. AD cells showed no appreciable nuclear translocation upon NP stimulation, with less intense DNA binding signal on EMSA.

Conclusions

Our results support evidence for a direct mechanistic connection between the NPs and NF??B/proteasome signaling pathways, with a distinct NP-induced profile in the more aggressive AI cancer state.     

Activation of peroxisome proliferator-activated receptor gamma suppresses nuclear factor kappa B-mediated apoptosis induced by Helicobacter pylori in gastric epithelial cells

Helicobacter pylori colonization leads to epithelial cell hyperproliferation within inflamed mucosa, but levels of apoptosis vary, suggesting that imbalances between rates of cell production and loss may contribute to differences in gastric cancer risk among infected populations. Peroxisome proliferator-activated receptor gamma (PPARgamma) regulates inflammatory and growth responses of intestinal epithelial cells. We determined whether activation of PPARgamma modified H. pylori-induced apoptosis in gastric epithelial cells. PPARgamma was expressed and functionally active in gastric epithelial cell lines sensitive to H. pylori-induced apoptosis. PPARgamma ligands 15d-PGJ(2) and BRL-49653 significantly attenuated H. pylomicronri-induced apoptosis, effects that could be reversed by co-treatment with a specific PPARgamma antagonist. Cyclopentanone prostaglandins that do not bind and activate PPARgamma had no effects on H. pylori-induced apoptosis. The ability of H. pylori to activate nuclear factor (NF)-kappaB and increase levels of the NF-kappaB target IL-8 was blocked by co-treatment with PPARgamma agonists, and direct inhibition of NF-kappaB also abolished H. pylori-stimulated apoptosis. These results suggest that activation of the PPARgamma pathway attenuates the ability of H. pylori to induce NF-kappaB-mediated apoptosis in gastric epithelial cells. Because PPARgamma regulates a multitude of host responses, activation of this receptor may contribute to varying levels of cellular turnover as well as the diverse pathologic outcomes associated with chronic H. pylori colonization.     

Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.     

Acute activation of de novo sphingolipid biosynthesis upon heat shock causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins

Recent studies are beginning to implicate sphingolipids in the heat stress response. In the yeast Saccharomyces cerevisiae, heat stress has been shown to activate de novo biosynthesis of sphingolipids, whereas in mammalian cells the sphingolipid ceramide has been implicated in the heat shock responses. In the current study, we found an increase in the ceramide mass of Molt-4 cells in response to heat shock, corroborating findings in HL-60 cells. Increased ceramide was determined to be from de novo biosynthesis by two major lines of evidence. First, the accumulation of ceramide was dependent upon the activities of both ceramide synthase and serine palmitoyltransferase. Second, pulse labeling studies demonstrated increased production of ceramide through the de novo biosynthetic pathway. Significantly, the de novo sphingolipid biosynthetic pathway was acutely induced upon heat shock, which resulted in a 2-fold increased flux in newly made ceramides within 1-2 min of exposure to 42.5 degrees C. Functionally, heat shock induced the dephosphorylation of the SR proteins, and this effect was demonstrated to be dependent upon the accumulation of de novo-produced ceramides. Thus, these studies disclose an evolutionary conserved activation of the de novo pathway in response to heat shock. Moreover, SR dephosphorylation is emerging as a specific downstream target of accumulation of newly made ceramides in mammalian cells.     

Selective substrate supply in the regulation of yeast de novo sphingolipid synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.     

The effect of fumonisin B1 on developing chick embryos: correlation between de novo sphingolipid biosynthesis and gross morphological changes

Fumonisins, mycotoxins produced byFusarium moniliforme and a number of other fungi, are potent inhibitors of the sphinganine-N-acyltransferase, a key enzyme of sphingolipid biosynthesis, and cause neuronal degeneration, liver and renal toxicity, cancer and other injury to animals.In this study we investigated the effect of fumonisin B₁ on the sphingolipids of developing chick embryos. After yolk sac injection of fumonisin B₁ a concentration and time dependent increase of the sphinganine-over-sphingosine ratio of the embryos could be demonstrated. Studies were done to evaluate the effect of fumonisin B₁ on the glycosphingolipid pattern of the chick embryos. In the presence of 72 µg fumonisin B₁ per egg the incorporation of [¹⁴C]galactose and of [¹⁴C]serine into embryonic glycosphingolipids was reduced by about 70%, although the mass of glycosphingolipids was not affected by the toxin. However, a reduction of the wet weight of the treated embryos was observed. Additionally, histological examinations of whole embryo sections of control and fumonisin B₁ treated embryos are presented. Fumonisin B₁ caused haemorrhages under the skin as well as in the liver of treated embyros. A close correlation between disruption of sphingoid metabolism and light microscopic detectable tissue lesions could be observed.Abbreviations Cer ceramide (N-acylsphingosine) - FB₁ fumonisin B₁ - GM3 NeuAc23Gal14Glc11Cer - GD3 NeuAc28NeuAc23Gal14Glc11Cer - GD1a NeuAc23Gal13GalNAc14(NeuAc23)Gal14Glc11Cer - GT1b NeuAc23Gal13GalNAc14(NeuAc28NeuAc23) Gal14Glc11Cer - HPLC high pressure liquid chromatography - PBS phosphate buffered saline - PDMP 1-phenyl-2-dodecanoylamino-3-morpholino-1-propanol - Sa sphinganine - So sphingosine - Sa/So sphinganine-over-sphingosine - TLC thin layer chromatography - Tris Tris(hydroxymethyl)aminomethan Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.