Publications (28) View all
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Article: Cell surface ceramide controls translocation of transferrin receptor to clathrin-coated pits.
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ABSTRACT: 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.Cellular signalling 11/2011; 24(3):677-84. · 4.09 Impact Factor -
Article: Mycobacterium tuberculosis lipoarabinomannan enhances LPS-induced TNF-α production and inhibits NO secretion by engaging scavenger receptors.
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ABSTRACT: 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.Microbial Pathogenesis 03/2011; 50(6):350-9. · 1.94 Impact Factor -
Article: Determination of cell surface expression of Toll-like receptor 4 by cellular enzyme-linked immunosorbent assay and radiolabeling.
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ABSTRACT: 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.Analytical Biochemistry 02/2011; 413(2):185-91. · 3.00 Impact Factor -
Article: One lipid, multiple functions: how various pools of PI(4,5)P(2) are created in the plasma membrane.
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ABSTRACT: Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] is a minor lipid of the inner leaflet of the plasma membrane that controls the activity of numerous proteins and serves as a source of second messengers. This multifunctionality of PI(4,5)P(2) relies on mechanisms ensuring transient appearance of PI(4,5)P(2) clusters in the plasma membrane. One such mechanism involves phosphorylation of PI(4)P to PI(4,5)P(2) by the type I phosphatidylinositol-4-phosphate 5-kinases (PIP5KI) at discrete membrane locations coupled with PI(4)P delivery/synthesis at the plasma membrane. Simultaneously, both PI(4)P and PI(4,5)P(2) participate in anchoring PIP5KI at the plasma membrane via electrostatic bonds. PIP5KI isoforms are also selectively recruited and activated at the plasma membrane by Rac1, talin, or AP-2 to generate PI(4,5)P(2) in ruffles and lamellipodia, focal contacts, and clathrin-coated pits. In addition, PI(4,5)P(2) can accumulate at sphingolipid/cholesterol-based rafts following activation of distinct membrane receptors or be sequestered in a reversible manner due to electrostatic constrains posed by proteins like MARCKS.Cellular and Molecular Life Sciences CMLS 12/2010; 67(23):3927-46. · 6.57 Impact Factor -
Article: Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide.
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ABSTRACT: 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.The Journal of Immunology 11/2010; 185(11):6960-73. · 5.79 Impact Factor
