c-Abl Inhibition Activates TFEB and Promotes Cellular Clearance in a Lysosomal Disorder.

The transcription factor EB (TFEB) has emerged as a master regulator of lysosomal biogenesis, exocytosis, and autophagy, promoting the clearance of substrates stored in cells. c-Abl is a tyrosine kinase that participates in cellular signaling in physiological and pathophysiological conditions. In this study, we explored the connection between c-Abl and TFEB. Here, we show that under pharmacological and genetic c-Abl inhibition, TFEB translocates into the nucleus promoting the expression of its target genes independently of its well-known regulator, mammalian target of rapamycin complex 1. Active c-Abl induces TFEB phosphorylation on tyrosine and the inhibition of this kinase promotes lysosomal biogenesis, autophagy, and exocytosis. c-Abl inhibition in Niemann-Pick type C (NPC) models, a neurodegenerative disease characterized by cholesterol accumulation in lysosomes, promotes a cholesterol-lowering effect in a TFEB-dependent manner. Thus, c-Abl is a TFEB regulator that mediates its tyrosine phosphorylation, and the inhibition of c-Abl activates TFEB promoting cholesterol clearance in NPC models.

R468A mutation in perfringolysin O destabilizes toxin structure and induces membrane fusion.

Perfringolysin O (PFO) belongs to the family of cholesterol-dependent cytolysins. Upon binding to a cholesterol-containing membrane, PFO undergoes a series of structural changes that result in the formation of a ß-barrel pore and cell lysis. Recognition and binding to cholesterol are mediated by the D4 domain, one of four domains of PFO. The D4 domain contains a conserved tryptophan-rich loop named undecapeptide (E458CTGLAWEWWR468) in which arginine 468 is essential for retaining allosteric coupling between D4 and other domains during interaction of PFO with the membrane. In this report we studied the impact of R468A mutation on the whole protein structure using hydrogen-deuterium exchange coupled with mass spectrometry. We found that in aqueous solution, compared to wild type (PFO), PFOR468A showed increased deuterium uptake due to exposure of internal toxin regions to the solvent. This change reflected an overall structural destabilization of PFOR468A in solution. Conversely, upon binding to cholesterol-containing membranes, PFOR468A revealed a profound decrease of hydrogen-deuterium exchange when compared to PFO. This block of deuterium uptake resulted from PFOR468A-induced aggregation and fusion of liposomes, as found by dynamic light scattering, microscopic observations and FRET measurements. In the result of liposome aggregation and fusion, the entire PFOR468A molecule became shielded from aqueous solution and thereby was protected against proteolytic digestion and deuteration. We have established that structural changes induced by the R468A mutation lead to exposure of an additional cholesterol-independent liposome-binding site in PFO that confers its fusogenic property, altering the mode of the toxin action.

oxLDL and eLDL Induced Membrane Microdomains in Human Macrophages.

BACKGROUND: Extravasation of macrophages and formation of lipid-laden foam cells are key events in the development and progression of atherosclerosis. The degradation of atherogenic lipoproteins subsequently leads to alterations in cellular lipid metabolism that influence inflammatory signaling. Especially sphingolipids and ceramides are known to be involved in these processes. We therefore analyzed monocyte derived macrophages during differentiation and after loading with enzymatically (eLDL) and oxidatively (oxLDL) modified low-density lipoproteins (LDL). METHODS: Primary human monocytes were isolated from healthy, normolipidemic blood donors using leukapheresis and counterflow elutriation. On the fourth day of MCSF-induced differentiation eLDL (40 µg/ml) or oxLDL (80 µg/ml) were added for 48h. Lipid species were analyzed by quantitative tandem mass spectrometry. Taqman qPCR was performed to investigate transcriptional changes in enzymes involved in sphingolipid metabolism. Furthermore, membrane lipids were studied using flow cytometry and confocal microscopy. RESULTS: MCSF dependent phagocytic differentiation of blood monocytes had only minor effects on the sphingolipid composition. Levels of total sphingomyelin and total ceramide remained unchanged, while lactosylceramides, cholesterylesters and free cholesterol decreased. At the species level most ceramide species showed a reduction upon phagocytic differentiation. Loading with eLDL preferentially increased cellular cholesterol while loading with oxLDL increased cellular ceramide content. Activation of the salvage pathway with a higher mRNA expression of acid and neutral sphingomyelinase, neutral sphingomyelinase activation associated factor and glucosylceramidase as well as increased surface expression of SMPD1 were identified as potentially underlying mechanisms. Moreover, flow-cytometric analysis revealed a higher cell-surface-expression of ceramide, lactosylceramide (CDw17), globotriaosylceramide (CD77), dodecasaccharide-ceramide (CD65s) and GM1 ganglioside upon oxLDL loading. ApoE in contrast to apoA-I preferentially bound to the ceramide enriched surfaces of oxLDL loaded cells. Confocal microscopy showed a co-localization of acid sphingomyelinase with ceramide rich membrane microdomains. CONCLUSION: eLDL leads to the formation of lipid droplets and preferentially induces cholesterol/sphingomyelin rich membrane microdomains while oxLDL promotes the development of cholesterol/ceramide rich microdomains via activation of the salvage pathway.

Annexin A1 Tethers Membrane Contact Sites that Mediate ER to Endosome Cholesterol Transport.

Membrane contact sites between the ER and multivesicular endosomes/bodies (MVBs) play important roles in endosome positioning and fission and in neurite outgrowth. ER-MVB contacts additionally function in epidermal growth factor receptor (EGFR) tyrosine kinase downregulation by providing sites where the ER-localized phosphatase, PTP1B, interacts with endocytosed EGFR before the receptor is sorted onto intraluminal vesicles (ILVs). Here we show that these contacts are tethered by annexin A1 and its Ca(2+)-dependent ligand, S100A11, and form a subpopulation of differentially regulated contact sites between the ER and endocytic organelles. Annexin A1-regulated contacts function in the transfer of ER-derived cholesterol to the MVB when low-density lipoprotein-cholesterol in endosomes is low. This sterol traffic depends on interaction between ER-localized VAP and endosomal oxysterol-binding protein ORP1L, and is required for the formation of ILVs within the MVB and thus for the spatial regulation of EGFR signaling.

Ceramide generation during curcumin-induced apoptosis is controlled by crosstalk among Bcl-2, Bcl-xL, caspases and glutathione.

Curcumin exhibits anti-cancer properties manifested by activation of pro-apoptotic signaling. We have demonstrated earlier that apoptosis of HL-60 human leukemia cells induced by curcumin is controlled by ceramide generated by neutral sphingomyelinase (nSMase) which contributes to sphingomyelin synthase (SMS) inhibition favoring accumulation of ceramide in cells. Here we report that the activity of nSMase, ceramide accumulation and death of HL-60 cells are inhibited by overexpression of Bcl-xL or Bcl-2 proteins, while down-regulation of nSMase interferes with degradation of Bcl-2 but not Bcl-xL. Activation of nSMase in curcumin-treated cells requires the activity of apoptosis initiator caspase-8 and executioner caspase-3, whereas nSMase depletion prevents activation of caspase-3, but not caspase-8. These data place nSMase activation downstream of caspase-8 and Bcl-xL and indicate a mutual regulation between nSMase and caspase-3 activity on one hand, and Bcl-2 level on the other hand in curcumin-treated cells. The activation of nSMase and ceramide accumulation also depended on the depletion of glutathione. The depletion of glutathione required the activity of caspase-8 and caspase-3 as well as the down-regulation of Bcl-2 and Bcl-xL. Together, the data indicate a crosstalk among Bcl-2, Bc-xL, caspases and glutathione during curcumin-induced apoptosis and point to the superior role of caspase-8 activity, Bcl-xL down-regulation and glutathione depletion in the pro-apoptotic cascade leading to nSMase activation and generation of ceramide.

Curcumin induces apoptosis of multidrug-resistant human leukemia HL60 cells by complex pathways leading to ceramide accumulation.

Most anti-cancer agents induce apoptosis, however, a development of multidrug resistance in cancer cells and defects in apoptosis contribute often to treatment failure. Here, the mechanism of curcumin-induced apoptosis was investigated in human leukemia HL60 cells and their HL60/VCR multidrug-resistant counterparts. In both cell lines curcumin induced a bi-phasic ceramide generation with a slow phase until 6 h followed by a more rapid one. The level of the ceramide accumulation correlated inversely with the cell viability. We found that the ceramide elevation resulted from multifarious changes of the activity of sphingolipid-modifying enzymes. In both cell lines curcumin induced relatively fast activation of neutral sphingomyelinase (nSMase), which peaked at 3 h, and was followed by inhibition of sphingomyelin synthase activity. In addition, in HL60/VCR cells the glucosylceramide synthase activity was diminished by curcumin. This process was probably due to curcumin-induced down-regulation of P-gp drug transporter, since cyclosporine A, a P-gp blocker, also inhibited the glucosylceramide synthase activity. Inhibition of nSMase activity with GW4869 or silencing ofSMPD3 gene encoding nSMase2 reversed the curcumin-induced inhibition of sphingomyelin synthase without affecting the glucosylceramide synthase activity. The early ceramide generation by nSMase was indispensable for the later lipid accumulation, modulation of Bax, Bcl-2 and caspase 3 levels, and for reduction of cell viability in curcumin-treated cells, as all these events were inhibited by GW4869 or nSMase2 depletion. These data indicate that the early ceramide generation by nSMase2 induced by curcumin intensifies the later ceramide accumulation via inhibition of sphingomyelin synthase, and controls pro-apoptotic signaling.

Crucial role of perfringolysin O D1 domain in orchestrating structural transitions leading to membrane-perforating pores: a hydrogen-deuterium exchange study.

Perfringolysin O (PFO) is a toxic protein that binds to cholesterol-containing membranes, oligomerizes, and forms a ß-barrel transmembrane pore, leading to cell lysis. Previous studies have uncovered the sequence of events in this multistage structural transition to a considerable detail, but the underlying molecular mechanisms are not yet fully understood. By measuring hydrogen-deuterium exchange patterns of peptide bond amide protons monitored by mass spectrometry (MS), we have mapped structural changes in PFO and its variant bearing a point mutation during incorporation to the lipid environment. We have defined all regions that undergo structural changes caused by the interaction with the lipid environment both in wild-type PFO, thus providing new experimental constraints for molecular modeling of the pore formation process, and in a point mutant, W165T, for which the pore formation process is known to be inefficient. We have demonstrated that point mutation W165T causes destabilization of protein solution structure, strongest for domain D1, which interrupts the pathway of structural transitions in other domains necessary for proper oligomerization in the membrane. In PFO, the strongest changes accompanying binding to the membrane focus in D1; the C-terminal part of D4; and strands ß1, ß4, and ß5 of D3. These changes were much weaker for PFO(W165T) lipo where substantial stabilization was observed only in D4 domain. In this study, the application of hydrogen-deuterium exchange analysis monitored by MS provided new insight into conformational changes of PFO associated with the membrane binding, oligomerization, and lytic pore formation.

Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O.

BACKGROUND: Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O. METHODS: To detect cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO-positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified cholesterol in NPC cells. RESULTS: GST-PFO recognized cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of cholesterol with methyl-ß-cyclodextrin. CONCLUSIONS: Our data indicate that a recombinant protein GST-PFO can be used to detect cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing cholesterol deposits in cells and can be useful in diagnostics of NPC disease.

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-ß-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Species differences take shape at nanoparticles: protein corona made of the native repertoire assists cellular interaction.

Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference. We have profiled proteins forming the long-lived corona around silver nanoparticles (75 nm OECD reference materials) and compared the responses of coelomocytes to protein coronas preformed of EfCP or FBS. We find that over time silver nanoparticles can competitively acquire a biological identity native to the cells in situ even in non-native media, and significantly greater cellular accumulation of the nanoparticles was observed with corona complexes preformed of EfCP (p < 0.05). An EfCP-nanoparticle mimicry made with a recombinant protein, lysenin, revealed its critical contribution in the observed cell-nanoparticle response. This confirms the determinant role of the recognizable biological identity during invertebrate in vitro testing of nanoparticles. Our finding shows a case of species-specific formation of biomolecular coronas, and this suggests that the use of representative species may need careful consideration in assessing the risks associated with nanoparticles.

An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS.

Lipopolysaccharide (LPS) activates macrophages by binding to the TLR4/MD-2 complex and triggers two pro-inflammatory signaling pathways: one relies on MyD88 at the plasma membrane, and the other one depends on TRIF in endosomes. When present in high doses, LPS is internalized and undergoes detoxification. We found that the uptake of a high concentration of LPS (1000ng/ml) in macrophage-like J774 cells was upregulated upon inhibition of clathrin- and dynamin-mediated endocytosis which, on the other hand, strongly reduced the production of pro-inflammatory mediators TNF-α and RANTES. The binding and internalization of high amounts of LPS was mediated by scavenger receptor A (SR-A) with participation of CD14 without an engagement of TLR4. Occupation of SR-A by dextran sulfate or anti-SR-A antibodies enhanced LPS-induced production of TNF-α and RANTES by about 70%, with CD14 as a limiting factor. Dextran sulfate also elevated the cell surface levels of TLR4 and CD14, which could have contributed to the upregulation of the pro-inflammatory responses. Silencing of SR-A expression inhibited the LPS-triggered TNF-α production whereas RANTES release was unchanged. These data indicate that SR-A is required for maximal production of TNF-α in cells stimulated with LPS, possibly by modulating the cell surface levels of TLR4 and CD14.

CD14 mediates binding of high doses of LPS but is dispensable for TNF-α production.

Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-α production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-α generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-α release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-α production was less CD14-dependent and could proceed in the absence of serum as an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-α was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF α production induced by high concentrations of sLPS and rLPS can be limited.

Raft coalescence and FcγRIIA activation upon sphingomyelin clustering induced by lysenin.

Activation of immunoreceptor FcγRIIA by cross-linking with antibodies is accompanied by coalescence of sphingolipid/cholesterol-rich membrane rafts leading to the formation of signaling platforms of the receptor. In this report we examined whether clustering of the raft lipid sphingomyelin can reciprocally induce partition of FcγRIIA to rafts. To induce sphingomyelin clustering, cells were exposed to non-lytic concentrations of GST-lysenin which specifically recognizes sphingomyelin. The lysenin/sphingomyelin complexes formed microscale assemblies composed of GST-lysenin oligomers engaging sphingomyelin of rafts. Upon sphingomyelin clustering, non-cross-linked FcγRIIA associated with raft-derived detergent-resistant membrane fractions as revealed by density gradient centrifugation. Pretreatment of cells with GST-lysenin also increased the size of detergent-insoluble molecular complexes of activated FcγRIIA. Sphingomyelin clustering triggered tyrosine phosphorylation of the receptor and its accompanying proteins, Cbl and NTAL, in the absence of receptor ligands and enhanced phosphorylation of these proteins in the ligand presence. These data indicate that clustering of plasma membrane sphingomyelin induces coalescence of rafts and triggers signaling events analogous to those caused by FcγRIIA activation.

Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits.

Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K(+) depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway.

LPS induces phosphorylation of actin-regulatory proteins leading to actin reassembly and macrophage motility.

Upon bacterial infection lipopolysaccharide (LPS) induces migration of monocytes/macrophages to the invaded region and production of pro-inflammatory mediators. We examined mechanisms of LPS-stimulated motility and found that LPS at 100 ng/ml induced rapid elongation and ruffling of macrophage-like J774 cells. A wound-healing assay revealed that LPS also activated directed cell movement that was followed by TNF-α production. The CD14 and TLR4 receptors of LPS translocated to the leading lamella of polarized cells, where they transiently colocalized triggering local accumulation of actin filaments and phosphatidylinositol 4,5-bisphosphate. Fractionation of Triton X-100 cell lysates revealed that LPS induced polymerization of cytoskeletal actin filaments by 50%, which coincided with the peak of cell motility. This microfilament population appeared at the expense of short filaments composing the plasma membrane skeleton of unstimulated cells and actin monomers consisting prior to the LPS stimulation about 60% of cellular actin. Simultaneously with actin polymerization, LPS stimulated phosphorylation of two actin-regulatory proteins, paxillin on tyrosine 118 by 80% and N-WASP on serine 484/485 by 20%, and these events preceded activation of NF-κB. LPS-induced protein phosphorylation and reorganization of the actin cytoskeleton were inhibited by PP2, a drug affecting activity of tyrosine kinases of the Src family. The data indicate that paxillin and N-WASP are involved in the reorganization of actin cytoskeleton driving motility of LPS-stimulated cells. Disturbances of actin organization induced by cytochalasin D did not inhibit TNF-α production suggesting that LPS-induced cell motility is not required for TNF-α release.

Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling.

Lipopolysaccharide (LPS) is recognized by Toll-like receptor 4 (TLR4) of macrophages triggering production of pro-inflammatory mediators. One of the factors determining the magnitude of responses to LPS, which may even lead to life-threatening septic shock, is the cell surface abundance of TLR4. However, quantitation of the surface TLR4 is difficult due to the low level of receptor expression. To develop a method of TLR4 assessment, we labeled the receptor on the cell surface with a rabbit antibody followed by either anti-rabbit immunoglobulin G-fluorescein isothiocyanate (IgG-FITC) for flow cytometry or with anti-rabbit IgG-peroxidase for a cellular enzyme-linked immunosorbent assay (ELISA). Alternatively, the anti-TLR4 antibody was detected by anti-rabbit IgG labeled with (125)I. Flow cytometry did not allow detection of TLR4 on the surface of J774 cells or human macrophages. In contrast, application of cellular ELISA or the radiolabeling technique combined with effective blockage of nonspecific binding of antibodies provided TLR4-specific signals. The level of TLR4 on the surface of J774 cells did not change on treatment with 1-100ng/ml LPS; however, it was reduced by approximately 30-40% after 2 h of treatment with 1 µg/ml LPS. These data indicate that down-regulation of surface TLR4 can serve as a means of negative regulation of cell responses toward high doses of LPS.

Mycobacterium tuberculosis lipoarabinomannan enhances LPS-induced TNF-α production and inhibits NO secretion by engaging scavenger receptors.

Lipoarabinomannan capped with terminal oligomannosides (ManLAM) is a component of mycobacteria cell wall enabling Mycobacterium tuberculosis to infect macrophages. We found that short treatment (3.5h) of macrophage-like J774 cells and thioglycollate-elicited peritoneal murine macrophages with ManLAM and its deacylated form enhanced LPS-stimulated release of tumor necrosis factor-α (TNF-α). In contrast, prolong incubation of J774 cells with ManLAM (16h) led to inhibition of LPS-stimulated TNF-α production. LPS-triggered secretion of nitric oxide (NO) was suppressed by ManLAM and its deacylated form. Effects of ManLAM and its deacylated derivative were mimicked by dextran sulfate, a general ligand of scavenger receptors. The enhancement of LPS-induced TNF-α production by dextran sulfate was partially reversed by an antibody neutralizing scavenger receptor SR-PSOX/CXCL16 while the stimulatory activity of deacylated ManLAM was reversed by an antibody neutralizing class B scavenger receptor CD36. Our data suggest that CD36 mediates the activity of ManLAM and its deacylated form leading to TNF-α release in LPS-stimulated J774 cells and peritoneal murine macrophages, while NO production is modulated by unknown scavenger receptors.

Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide.

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.

Sphingomyelin-rich domains are sites of lysenin oligomerization: implications for raft studies.

Lysenin is a self-assembling, pore-forming toxin which specifically recognizes sphingomyelin. Mutation of tryptophan 20 abolishes lysenin oligomerization and cytolytic activity. We studied the interaction of lysenin WT and W20A with sphingomyelin in membranes of various lipid compositions which, according to atomic force microscopy studies, generated either homo- or heterogeneous sphingomyelin distribution. Liposomes composed of SM/DOPC, SM/DOPC/cholesterol and SM/DPPC/cholesterol could bind the highest amounts of GST-lysenin WT, as shown by surface plasmon resonance analysis. These lipid compositions enhanced the release of carboxyfluorescein from liposomes induced by lysenin WT, pointing to the importance of heterogeneous sphingomyelin distribution for lysenin WT binding and oligomerization. Lysenin W20A bound more weakly to sphingomyelin-containing liposomes than did lysenin WT. The same amounts of lysenin W20A bound to sphingomyelin mixed with either DOPC or DPPC, indicating that the binding was not affected by sphingomyelin distribution in the membranes. The mutant lysenin had a limited ability to penetrate hydrophobic region of the membrane as indicated by measurements of surface pressure changes. When applied to detect sphingomyelin on the cell surface, lysenin W20A formed large conglomerates on the membrane, different from small and regular clusters of lysenin WT. Only lysenin WT recognized sphingomyelin pool affected by formation of raft-based signaling platforms. During fractionation of Triton X-100 cell lysates, SDS-resistant oligomers of lysenin WT associated with membrane fragments insoluble in Triton X-100 while monomers of lysenin W20A partitioned to Triton X-100-soluble membrane fractions. Altogether, the data suggest that oligomerization of lysenin WT is a prerequisite for its docking in raft-related domains.

Contribution of PIP-5 kinase Ialpha to raft-based FcgammaRIIA signaling.

Receptor FcgammaIIA (FcgammaRIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P(2) and PI(4,5)P(2)-synthesizing PIP5-kinase Ialpha to rafts contributes to FcgammaRIIA signaling. A fraction of PIP5-kinase Ialpha was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P(2). PIP5-kinase Ialpha bound PI(4,5)P(2), and depletion of the lipid displaced PIP5-kinase Ialpha from the DRM. Activation of FcgammaRIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P(2). Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After FcgammaRIIA activation, PIP5-kinase Ialpha and PI(4,5)P(2) co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase Ialpha and PI(4,5)P(2) were present at the edges of electron-dense assemblies containing activated FcgammaRIIA in their core. The data suggest that activation of FcgammaRIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase Ialpha and PI(4,5)P(2).