Kazuhisa Iwabuchi

Juntendo University, Edo, Tōkyō, Japan

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Publications (100)379.47 Total impact

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    ABSTRACT: There are a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica labelling immunoelectron microscopy in combination with multiple lipid-binding peptide/proteins to examine both transbilayer and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBC, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine/phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane.
    Journal of Cell Science 02/2015; 128(8). DOI:10.1242/jcs.163105 · 5.33 Impact Factor
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    ABSTRACT: Lactosylceramide (LacCer), which is essential for many cellular processes, is highly expressed on the plasma membranes of human neutrophils and mediates innate immune functions. Less is known, however, about the properties and biological functions of LacCer in mouse neutrophils. This study therefore analyzed the properties of mouse neutrophil LacCer. LacCer was observed on the surface of these cells, with flow cytometry indicating that mouse neutrophil LacCer could be detected by the anti-LacCer mAb T5A7, but not by the anti-LacCer antibodies Huly-m13 and MEM-74. The molecular species of LacCer were nearly identical in mouse and human neutrophils, including C24:0 and C24:1 fatty acid chain-containing species, although the LacCer content in plasma membranes was about 20-fold lower in mouse than in human neutrophils. Surface plasmon resonance analysis revealed that T5A7 bound to a lipid monolayer composed of LacCer, DOPC, cholesterol, and sphingomyelin (molar ratio 0.1:10:10:1), whereas Huly-m13 did not. T5A7 induced neutrophil migration, which was abolished by inhibitors of Src-family kinases, PI-3 kinases, and trimeric G (o/i) proteins. T5A7 also inhibited phagocytosis of non-opsonized zymosans by neutrophils. Taken together, these findings suggest that in mouse neutrophils (i) LacCer is expressed as LacCer-enriched microdomains in cell surface plasma membranes, (ii) these microdomains are recognized by T5A7 but not by other known anti-LacCer antibodies, and (iii) LacCer is involved in cell migration and phagocytosis. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
    Glycobiology 01/2015; 25(6). DOI:10.1093/glycob/cwv008 · 3.75 Impact Factor
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    ABSTRACT: Lactosylceramide (LacCer) has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signalling processes were assessed by synthesizing two molecular species of azide-photoactivable, tritium labelled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium- and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), were incorporated into the lipid rafts of plasma-membranes of neutrophilic differentiated HL-60 (D-HL60) cells. C24-[3H]LacCer-(N3), but not C18-[3H]LacCer-(N3), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[3H]LacCer-(N3) into plasma membranes, followed by illumination, resulted in the formation of several tritium labelled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[3H]LacCer-(N3) to cells, however, did not result in the formation of LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.
    The Journal of Lipid Research 11/2014; 56(1). DOI:10.1194/jlr.M055319 · 4.73 Impact Factor
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    ABSTRACT: Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD). A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase) isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P) stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes"), which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i) 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii) S1P induces the production of TNF-α via S1P receptors, and (iii) released TNF-α stimulates the production of inflammatory mediators such as IL-8.
    PLoS ONE 02/2014; 9(2):e89402. DOI:10.1371/journal.pone.0089402 · 3.53 Impact Factor
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    ABSTRACT: Populations of glycolipids change markedly during leukocyte differentiation, suggesting that these molecules are involved in biological functions. About 70% of the glycosphingolipids in human neutrophils is lactosylceramide, a molecule also expressed on monocytes and dendritic cells, but not on lymphocytes. In contrast, phosphatidylglucoside is mainly expressed on neutrophils. STED microscopic analysis showed that phosphatidylglucoside and lactosylceramide form different domains on plasma membranes of neutrophils, with phosphatidylglucoside preferentially expressed along the neutrophil differentiation pathway. Phosphatidylglucoside was found to induce the differentiation of HL-60 cells into the neutrophilic lineage, and to induce FAS-dependent neutrophil apoptosis. In contrast, lactosylceramide was only expressed on mature neutrophils. Complexes of lactosylceramide and the Src family kinase Lyn form membrane microdomains. . LacCer-enriched membrane microdomains mediate neutrophil innate immune responses; e.g. chemotaxis, phagocytosis, and superoxide generation. C24 fatty acid chains of LacCer are indispensable for the formation of LacCer-Lyn complexes and for LacCer-dependent functions. Moreover, Lyn-coupled LacCer-enriched microdomains serve as signal transduction platforms for αMβ2 integrin-mediated phagocytosis. This review describes the organization and potential functions of glycolipids in phagocytes, as well as the roles of both phosphatidylglucoside and lactosylceramide in neutrophils. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.
    Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 01/2014; 1851(1). DOI:10.1016/j.bbalip.2014.06.009 · 4.50 Impact Factor
  • 43rd Annual Critical Care Congress; 12/2013
  • Journal of dermatological science 06/2013; DOI:10.1016/j.jdermsci.2013.06.006 · 3.34 Impact Factor
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    ABSTRACT: (Objective): We have shown that connective tissue growth factor (CTGF) plays an important role in the pathogenesis of RA. Herein, we evaluated the effects of blockade of CTGF pathway on the development of arthritis in collagen-induced arthritis (CIA) mice. (Methods): Arthritis was induced in DBA/1J mice by immunization with a combination of type II collagen and complete Freund's adjuvant (CFA). We evaluated the efficacy of prevention of development of arthritis in the CIA mice treated with or without neutralizing anti-CTGF monoclonal antibody (mAb). (Results): Inhibition of the CTGF functions in mice treated with neutralizing anti-CTGF mAb significantly ameliorated arthritis compared to that in the non-treated CIA mice. Serum levels of matrix metalloproteinase 3 (MMP-3) were reduced by anti-CTGF mAb treatment. Moreover, blockade of the CTGF decreased interleukin 17 (IL-17) expression on purified CD4+ T lymphocytes. Although the expression of retinoic-acid-receptor-related orphan receptors γt (RORγt) gene was not suppressed by anti-CTGF mAb treatment, those of interferon regulatory factor 4 (IRF4) and IkappaBzeta (Nfkbiz), which are other important molecules for differentiation of Th-17 cells, were suppressed. In addition, blockade of CTGF inhibited pathological proliferation of T lymphocytes against type II collagen restimulation in vitro. Moreover, aberrant osteoclastogenesis in CIA mice was restored by anti-CTGF mAb treatment. (Conclusions): The present study showed that blockade of CTGF prevented progression of arthritis in CIA mice. Anti-CTGF mAb treatment suppressed the pathological T cells function and restored aberrant osteoclastogenesis in CIA mice. CTGF may become a new therapeutic target for treatment of RA. © 2013 American College of Rheumatology.
    Arthritis & Rheumatology 06/2013; 65(6). DOI:10.1002/art.37902 · 7.87 Impact Factor
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    ABSTRACT: Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.
    Archivum Immunologiae et Therapiae Experimentalis 02/2013; 61(3). DOI:10.1007/s00005-013-0221-6 · 2.82 Impact Factor
  • Journal of Dermatological Science 02/2013; 69(2):e17. DOI:10.1016/j.jdermsci.2012.11.349 · 3.34 Impact Factor
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    ABSTRACT: BACKGROUND: /st>Lung ischaemia-reperfusion (I/R) injury is correlated with poor clinical outcome. The inflammatory cytokines interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) are produced by pulmonary epithelial cells during lung transplantation and are considered to be involved in I/R injury. The volatile anaesthetic sevoflurane has been shown to exert a protective effect on I/R injury in various organs. We investigated the effect of sevoflurane on the inflammatory functions of pulmonary epithelial cells in vitro. METHODS: /st>Human normal small airway epithelial cells (SAEC) were incubated under anoxic conditions for 24 h with or without sevoflurane and then stimulated with tumour necrosis factor (TNF)-α under hyperoxic conditions for 5 h with or without sevoflurane. After incubation, IL-6, IL-8, and MCP-1 mRNA expression was analysed by quantitative real-time RT-PCR. The production of IL-6, IL-8, and MCP-1 was assayed by enzyme-linked immunosorbent assay, the effects of sevoflurane on inflammatory gene expression were examined by DNA microarray analysis, and the effects of sevoflurane on NF-κB-mediated inflammatory cytokine production were examined by immunoblotting. RESULTS: /st>Sevoflurane suppressed TNF-α-induced IL-6, IL-8, and MCP-1 gene expression and the production of IL-6 and IL-8 in SAEC under anoxia/reoxygenation conditions. DNA microarray analysis indicated that sevoflurane modulated NF-κB-related gene expression. Sevoflurane significantly inhibited TNF-α-induced translocation of p65 NF-κB into the nucleus. Sevoflurane enhanced TNF-α-induced gene expression of inhibitor κB (IκB) but not of NF-κB. CONCLUSIONS: /st>Sevoflurane suppressed the NF-κB-mediated production of pulmonary epithelial cell-derived inflammatory cytokines, including IL-6 and IL-8, which are capable of causing I/R injury.
    BJA British Journal of Anaesthesia 01/2013; 110(4). DOI:10.1093/bja/aes469 · 4.35 Impact Factor
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    ABSTRACT: To elucidate the role of neuropilin-1 (Nrp-1) and semaphorin 3A (Sema3A) in sinusoidal remodeling during liver regeneration in rats. Male Wistar/ST rats at 7 wk of age, weighing about 200 g, were used for all animal experiments. In vivo, at 24, 48, 72, 96, 144 and 192 h after two-thirds partial hepatectomy (PHx), the remnant livers were removed. Liver tissues were immunohistochemically stained for Nrp-1, Sema3A and SE-1, a liver sinusoidal endothelial cell (SEC) marker. Total RNA of the liver tissue was extracted and reversely transcribed into cDNA. The mRNA expression of Sema3A was analyzed by quantitative real-time polymerase chain reaction and normalized to that of ribosomal protein S18. In vitro, SECs were isolated from rat liver and cultured in endothelial growth medium containing 20 ng/mL vascular endothelial cell growth factor. Migration of SECs in primary culture was assessed by cell transwell assay with or without recombinant Sema3A. Apoptotic cells were determined by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method. In vitro, immunohistochemistry study revealed that Sema3A and Nrp-1 were constitutively expressed in hepatocytes and SECs, respectively, in normal rat liver tissues. Nrp-1 expression in SECs was quantified by the percentage of immunostained area with anti-Nrp-1 antibody in relation to the area stained with SE-1. Between 24 h and 96 h following resection of liver, Nrp-1 expression in SECs was transiently increased. Compared with the baseline (5.2% ± 0.1%), Nrp-1 expression in SECs significantly increased at 24 h (17.3% ± 0.7%, P < 0.05), 48 h (39.1% ± 0.6%, P < 0.01), 72 h (46.9% ± 4.5%, P < 0.01) and 96 h (29.9% ± 3.8%, P < 0.01) after PHx, then returned to the basal level at termination of liver regeneration. Interestingly, the expression of Sema3A was inversely associated with that of Nrp-1 in liver after PHx. Sema3A mRNA expression was significantly reduced by about 75% over the period 24-144 h after PHx (P < 0.05), and returned to basal levels at 192 h after PHx. In vitro, SECs isolated from rats after PHx (PHx-SECs) were observed to migrate to the lower chamber of the cell transwell system after incubation for 24 h, but not cells from normal rats (CONT-SECs), indicating that mobility of PHx-SECs increases as compared with that of CONT-SECs. Moreover, recombinant Sema3A significantly attenuated migration in PHx-SECs in primary culture (vehicle-treated 100% ± 7.9% vs Sema3A-treated 42.6% ± 5.4%, P < 0.01), but not in CONT-SECs. Compared with CONT-SECs, the apoptotic rate of PHx-SECs decreased by 78.3% (P < 0.05). There was no difference in apoptosis between CONT-SECs that were treated with vehicle and Sema3A. However, in PHx-SECs, apoptosis was induced by the presence of 5 nmol Sema3A for 24 h (vehicle-treated 21.7% ± 7.6% vs Sema3A-treated 104.3% ± 8.9%, P < 0.05). In addition, immunohistochemistry confirmed the increased expression of Nrp-1 in PHx-SECs, while it was noted to a lesser extent in CONT-SECs. The interplay of Nrp-1 and Sema3A shown in our results may lead to a better understanding of interaction between sinusoidal remodeling and SECs during liver regeneration.
    World Journal of Gastroenterology 09/2012; 18(36):5034-41. DOI:10.3748/wjg.v18.i36.5034 · 2.43 Impact Factor
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    ABSTRACT: Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.
    BioFactors 07/2012; 38(4):275-83. DOI:10.1002/biof.1017 · 3.00 Impact Factor
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    ABSTRACT: Since gangliosides play many important roles in neural systems, we investigated whether gangliosides are involved in glutamate release from neural cells. Differentiated neruro2a cells were treated with gangliosides, including GM3, GM1, GD1a, GD3, GD1b, or GT1b, for 30 min, and glutamate concentration in the culture media was measured using o-phthalaldehyde derivatization. Among the tested gangliosides, GT1b significantly increased the glutamate concentration when compared with untreated cells. Moreover, GT1b increased the glutamate concentration in the culture media of neuroblastoma × dorsal root ganglion neuron hybrid F11 cells. These results suggested that gangliosides are important in regulating extracellular glutamate concentration in the nervous system.
    Neuroscience Letters 04/2012; 517(2):140-3. DOI:10.1016/j.neulet.2012.04.049 · 2.06 Impact Factor
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    ABSTRACT: To investigate whether di-(2-ethylhexyl) phthalate (DEHP) affects the production of inflammatory cytokines by human macrophages. Differentiated macrophage-like THP-1 cells were exposed to 200 μM DEHP for 3 h, followed by incubation in the presence or absence of opsonized zymosan A, and the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the culture media were determined by ELISA. DNA microarray and quantitative real-time RT-PCR analyses were performed to identify genes that showed changes in expression in response to DEHP. DEHP treatment increased the concentrations of TNF-α, IL-1β, IL-8, and IL-6 in the media, regardless of whether the cells phagocytosed zymosan. DNA microarray analysis showed that DEHP increased the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP3, MMP10, MMP14, and CSF2 mRNA, and real-time RT-PCR showed that DEHP significantly enhanced the levels of expression of IL-8, CXCL1, CXCL2, CXCL3, CXCL6, CCL3, MMP10, CSF2, TNF-α, IL-1β, and IL-6 mRNA in THP-1 cells. DEHP significantly induced translocation of p65 NF-κB into the nucleus. DEHP enhances the production of inflammatory cytokines and chemokines by macrophages, and exacerbates their inflammatory response.
    Agents and Actions 01/2012; 61(1):69-78. DOI:10.1007/s00011-011-0390-x · 2.14 Impact Factor
  • Inflammation and Regeneration 01/2012; 32(5):213-221. DOI:10.2492/inflammregen.32.213
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    ABSTRACT: A protein analysis using mass spectrometry revealed the existence of serum proteins with significant quantitative changes after the administration of infliximab. Among these proteins, regenerating gene (REG) 1α appears to be related to the pathogenesis of rheumatoid arthritis (RA). Therefore, the present study was conducted to examine the mechanism of REG1α in RA disease progression. Serum samples were collected from RA patients and normal healthy controls. REG1α expression was evaluated by ELISA, RT-PCR, and indirect immunofluorescence microscopy. The functions of REG1α on synovial fibroblasts with regard to apoptosis, receptor activator of NF-κB ligand (RANKL) expression, and cellar proliferation were evaluated using siRNA to inhibit the intrinsic REG1α mRNA expression. The serum concentrations of REG1α in RA patients were higher than in normal healthy controls. The high expression of REG1α was also observed in the synovial tissue of RA patients compared to those of osteoarthropathy patients. In addition, tumor necrosis factor-α (TNF-α) upregulated REG1α expression in the synovial fibroblasts cell line (MH7A). Inhibition of REG1α expression suppressed the induction of RANKL expression by TNF-α. Furthermore, exogenous recombinant REG1α protein inhibited apoptosis and promoted cell proliferation in MH7A cells. These effects were abolished in the REG1α-siRNA MH7A cells. The present data suggest that TNF-α induces aberrant REG1α expression and that REG1α plays an important role in aberrant cell proliferation and RANKL expression of synovial fibroblasts, ultimately resulting in pannus formation. Restoration of normal physiological REG1α expression may contribute to disease amelioration.
    Modern Rheumatology 12/2011; 22(2):228-37. DOI:10.1007/s10165-011-0564-y · 2.21 Impact Factor
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    ABSTRACT: We and others have previously demonstrated that p210 Bcr-Abl tyrosine kinase inhibits stromal cell-derived factor-1α/CXCR4 chemokine receptor signaling, contributing to the deficient adhesion of chronic myeloid leukemia (CML) cells to bone marrow stroma. Conversely, exposure of CML cells to a tyrosine kinase inhibitor (TKI) enhances migration of CML cells towards stromal cell layers and promotes non-pharmacological resistance to imatinib. Src-related kinase Lyn is known to interact with CXCL12/CXCR4 signaling and is directly activated by p210 Bcr-Abl. In this study, we demonstrate that TKI treatment promoted CXCR4 redistribution into the lipid raft fraction, in which it co-localized with active phosphorylated form of Lyn (LynTyr396) in CML cells. Lyn inhibition or cholesterol depletion abrogated imatinib-induced migration, and dual Src/Abl kinase inhibitor dasatinib induced fewer CML cells to migrate to the stroma. These findings demonstrate the novel mechanism of microenvironment-mediated resistance through lipid raft modulation, which involves compartmental changes of the multivalent CXCR4 and Lyn complex. We propose that pharmacological targeting of lipid rafts may eliminate bone marrow-resident CML cells through interference with microenvironment-mediated resistance.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 10/2011; 26(5):883-92. DOI:10.1038/leu.2011.291 · 9.38 Impact Factor
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    ABSTRACT: A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.
    The Journal of Immunology 03/2011; 186(9):5323-32. DOI:10.4049/jimmunol.1002100 · 5.36 Impact Factor
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    ABSTRACT: Oxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages. Human monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-β1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment. The present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-β1- and NF-κB-mediated functions of M1 macrophages, but not those of M0 or M2 macrophages. It is likely that M1 macrophages characteristically respond to oxLDL.
    Lipids in Health and Disease 01/2011; 10:1. DOI:10.1186/1476-511X-10-1 · 2.31 Impact Factor

Publication Stats

3k Citations
379.47 Total Impact Points

Institutions

  • 1988–2014
    • Juntendo University
      • • Faculty of Health Care and Nursing
      • • Institute for Environmental and Gender Specific Medicine
      • • Department of Host Defense and Biochemical Research
      • • Department of Medicine
      Edo, Tōkyō, Japan
  • 2008
    • University of Milan
      • Center of Excellence on Neurodegenerative Diseases CEND
      Milano, Lombardy, Italy
  • 2006
    • Seikagaku Corporation
      Edo, Tōkyō, Japan
  • 2004
    • RIKEN
      Вако, Saitama, Japan
  • 1999–2003
    • Pacific Northwest Diabetes Research Institute
      Seattle, Washington, United States
  • 1998–1999
    • University of Washington Seattle
      Seattle, Washington, United States