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Long range genomic cytokine-receptor interaction regulates gene expression
2
Abstracts / Cytokine 48 (2009) 1–2
SLBAW2-A A diffusion barrier in the plasma membrane during the closure stage of macropinocytosis Timothy P. Welliver, Joel A. Swanson, SLB Presidental Award – Student A diffusion barrier in the plasma membrane during the closure stage of macropinocytosis Timothy P. Welliver, Joel A. Swanson, Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, MI, USA
Macropinocytosis is the process by which cells ingest fluid from the surrounding environment. During macropinosome closure, a cup-shaped invagination of plasma membrane constricts at its distal margin, eventually separating from the membrane as a macropinosome inside the cell. Previous studies have identified molecules that participate in closure, including phosphoinositide 30 -kinase (PI3K). PI3K produces high concentrations of phosphatidylinositol trisphosphate (PIP3) in the macropinocytic cup. We hypothesize that PIP3 signal amplification is confined to the cup-shaped domain of plasma membrane by a diffusion barrier at the rim of the cup. To measure molecular diffusion in and near cups, we expressed membrane-tethered, photoactivatable Green Fluorescent Protein (PAGFP-Mem and -FccRIIa) and monomeric Cherry (Cherry-Mem and -FccRIIa) constructs in Cos7 cells and used ratiometric fluorescent microscopy to observe the redistribution of membrane-associated molecules after photoactivation or photobleaching. Photoactivated GFP-Mem and GFP-FccRIIa diffused freely in flat regions of membrane, but were confined within macropinocytic cups. The outer rim of ruffles and the cup margin apparently limited diffusion. Photobleaching experiments demonstrated that this barrier restricted diffusion into and out of cup regions. These barriers might contribute to the PIP3 amplification in cups and the contractile activities that close macropinosomes. These findings therefore indicate the existence of novel diffusion barriers in the inner leaflet of the plasma membrane. The molecular basis of the diffusion barrier remains to be determined. doi:10.1016/j.cyto.2009.07.375
Gene activation SLBAW2-B Long range genomic cytokine-receptor interaction regulates gene expression Chrysoula Deligianni, Charalampos G. Spilianakis, SLB Presidental Award – Student Long range genomic cytokine-receptor interaction regulates gene expression Chrysoula Deligianni 1,2, Charalampos G. Spilianakis 1,3, 1 Laboratory of Molecular Immunology, Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Greece, 2 Department of Medicine, University of Crete, Heraklion, Greece, 3 Department of Biology, University of Crete, Heraklion, Greece
The development of the immune system and the effective host response to microbial infection rely on the recruitment of specialized immune cells and the orchestrated activation and silencing of numerous, differentially expressed genes. Here we report that genes, whose protein products are involved in the same differentiation or developmental pathway, specifically a ligand and its receptors, are co-regulated in the genomic level. Utilizing an in vivo immunological system, namely the differentiation of naïve CD4+ helper T cells in T(helper)H1 and TH2 cells, we focused on the transcriptional regulation of interferon gamma (IFN c), which is a TH1 specific cytokine, implicated in the differentiation and in the action of the above cells in the adaptive immune system, and its receptors, interferon gamma receptor 1 (IFN c R1) and interferon gamma receptor 2 (IFN c R2). In accordance with the notion that the spatial organization of chromosomes plays a crucial role in the proper transcription of
genetic loci, DNA FISH experiments revealed an intrachromosomal monoallelic interaction between IFN c and IFN c R1 gene loci, in cell types that express them such as naïve and TH1 CD4+ cells. The functional significance of the physical approach of the above loci was assessed by single cell analysis of the expression profile of the above genes. Therefore, it was demonstrated that IFN c is expressed biallelically in TH1 cells, whereas IFN c R1 is expressed in naïve and TH1 cells, from the co-localized allele with the IFN c locus. Subsequent chromosome conformation capture experiments verified the aforementioned interactions and revealed a complex pattern of loci proximity in cells that are developmentally predisposed to express these genes. Additional chromatin immunoprecipitation (ChIP) and ChIP-loop methodologies implied the role of CTCF in establishing these phenomena. Conclusively, increasing comprehension of the structure–function of these cellular mechanisms will allow the development of better treatments and diagnostic tools for diseases of the immune system. doi:10.1016/j.cyto.2009.07.376
Immunoregulation SLBAW2-C Soluble human CXCR2: Structure, properties, bioactivity Kanstantsin Katlinski, Sviatlana Akalovich, Yuliya Katlinskaya, Anton Sholukh, Tatyana Doroshenko, Yury Chaly, Nikolai Voitenok, SLB Presidental Award – Student Soluble human CXCR2: Structure, properties, bioactivity Kanstantsin Katlinski 1, Sviatlana Akalovich 1, Yuliya Katlinskaya 1, Anton Sholukh 1, Tatyana Doroshenko 1, Yury Chaly 1, Nikolai N. Voitenok 2, 1 Laboratory of Cellular & Molecular Immunology, The Research Center for Hematology & Transfusiology, Minsk, Belarus, 2 Fund for Molecular Hematology and Immunology, Moscow, Russia
Loss of membrane expression of several cytokine receptors (TNFRI, TNFRII, IL-1RII, IL-2R, IL-6R, IL-15R) occurs via proteolytical cleavage to soluble forms by cell-associated metalloproteinases. We have previously showed that reduction of CXCR2 expression on phagocytosing neutrophils was also mediated by cell-associated metalloproteinase. Here we show that reduction of CXCR2 expression on neutrophils exposed to tumor necrosis factor a was accompanied by the appearance of a soluble CXCR2 (sCXCR2) antigen in cell-culture supernatants. We also detected sCXCR2 in urine from healthy subjects. Leukocyte-derived sCXCR2 (ld-sCXCR2) and urinederived sCXCR2 (ud-sCXCR2) were similarly eluted as a single protein peak of 40 kDa when applied to size-exclusion high performance liquid chromatography and were strongly acidic with isoelectric point of 3.2. sCXCR2 is a glycoprotein sensitive to treatment with N-glycosidase F. Predicted N-glycosylation of the peptide backbone of CXCR2 at Asp22 was demonstrated using monoclonal antibody mapped to 11–22 amino acids of CXCR2 N-terminus that effectively recognized deglycosylated sCXCR2, but weakly interacted with native glycosylated sCXCR2. C-terminal sequencing of native ud-sCXCR2 using carboxypeptidase A and MALDI-TOF MS/MS analysis of deglycosylated ld- and ud-sCXCR2 identified sCXCR2 as N-terminal fragment of CXCR2. Exploring bioactivity of sCXCR2, we observed the induction of interleukin-8 in monocyte culture. The effect was inhibited after treatment of sCXCR2 with N-glycosidase F or proteinase K indicating that both the carbohydrate moiety and peptide backbone are required. sCXCR2 was capable to stimulate immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC), but the stimulation was not effective in PBMC depleted of monocytes, i.e. was monocyte-mediated. Taken together, our results demonstrate for the first that loss of CXCR2 from neutrophils leads to production of soluble acidic glycopeptide CXCR2 which is also generated in vivo and may represent a novel mediator of inflammation. doi:10.1016/j.cyto.2009.07.377
Abstracts / Cytokine 48 (2009) 1–2
SLBAW2-A A diffusion barrier in the plasma membrane during the closure stage of macropinocytosis Timothy P. Welliver, Joel A. Swanson, SLB Presidental Award – Student A diffusion barrier in the plasma membrane during the closure stage of macropinocytosis Timothy P. Welliver, Joel A. Swanson, Department of Microbiology & Immunology, University of Michigan Medical School, Ann Arbor, MI, USA
Macropinocytosis is the process by which cells ingest fluid from the surrounding environment. During macropinosome closure, a cup-shaped invagination of plasma membrane constricts at its distal margin, eventually separating from the membrane as a macropinosome inside the cell. Previous studies have identified molecules that participate in closure, including phosphoinositide 30 -kinase (PI3K). PI3K produces high concentrations of phosphatidylinositol trisphosphate (PIP3) in the macropinocytic cup. We hypothesize that PIP3 signal amplification is confined to the cup-shaped domain of plasma membrane by a diffusion barrier at the rim of the cup. To measure molecular diffusion in and near cups, we expressed membrane-tethered, photoactivatable Green Fluorescent Protein (PAGFP-Mem and -FccRIIa) and monomeric Cherry (Cherry-Mem and -FccRIIa) constructs in Cos7 cells and used ratiometric fluorescent microscopy to observe the redistribution of membrane-associated molecules after photoactivation or photobleaching. Photoactivated GFP-Mem and GFP-FccRIIa diffused freely in flat regions of membrane, but were confined within macropinocytic cups. The outer rim of ruffles and the cup margin apparently limited diffusion. Photobleaching experiments demonstrated that this barrier restricted diffusion into and out of cup regions. These barriers might contribute to the PIP3 amplification in cups and the contractile activities that close macropinosomes. These findings therefore indicate the existence of novel diffusion barriers in the inner leaflet of the plasma membrane. The molecular basis of the diffusion barrier remains to be determined. doi:10.1016/j.cyto.2009.07.375
Gene activation SLBAW2-B Long range genomic cytokine-receptor interaction regulates gene expression Chrysoula Deligianni, Charalampos G. Spilianakis, SLB Presidental Award – Student Long range genomic cytokine-receptor interaction regulates gene expression Chrysoula Deligianni 1,2, Charalampos G. Spilianakis 1,3, 1 Laboratory of Molecular Immunology, Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Greece, 2 Department of Medicine, University of Crete, Heraklion, Greece, 3 Department of Biology, University of Crete, Heraklion, Greece
The development of the immune system and the effective host response to microbial infection rely on the recruitment of specialized immune cells and the orchestrated activation and silencing of numerous, differentially expressed genes. Here we report that genes, whose protein products are involved in the same differentiation or developmental pathway, specifically a ligand and its receptors, are co-regulated in the genomic level. Utilizing an in vivo immunological system, namely the differentiation of naïve CD4+ helper T cells in T(helper)H1 and TH2 cells, we focused on the transcriptional regulation of interferon gamma (IFN c), which is a TH1 specific cytokine, implicated in the differentiation and in the action of the above cells in the adaptive immune system, and its receptors, interferon gamma receptor 1 (IFN c R1) and interferon gamma receptor 2 (IFN c R2). In accordance with the notion that the spatial organization of chromosomes plays a crucial role in the proper transcription of
genetic loci, DNA FISH experiments revealed an intrachromosomal monoallelic interaction between IFN c and IFN c R1 gene loci, in cell types that express them such as naïve and TH1 CD4+ cells. The functional significance of the physical approach of the above loci was assessed by single cell analysis of the expression profile of the above genes. Therefore, it was demonstrated that IFN c is expressed biallelically in TH1 cells, whereas IFN c R1 is expressed in naïve and TH1 cells, from the co-localized allele with the IFN c locus. Subsequent chromosome conformation capture experiments verified the aforementioned interactions and revealed a complex pattern of loci proximity in cells that are developmentally predisposed to express these genes. Additional chromatin immunoprecipitation (ChIP) and ChIP-loop methodologies implied the role of CTCF in establishing these phenomena. Conclusively, increasing comprehension of the structure–function of these cellular mechanisms will allow the development of better treatments and diagnostic tools for diseases of the immune system. doi:10.1016/j.cyto.2009.07.376
Immunoregulation SLBAW2-C Soluble human CXCR2: Structure, properties, bioactivity Kanstantsin Katlinski, Sviatlana Akalovich, Yuliya Katlinskaya, Anton Sholukh, Tatyana Doroshenko, Yury Chaly, Nikolai Voitenok, SLB Presidental Award – Student Soluble human CXCR2: Structure, properties, bioactivity Kanstantsin Katlinski 1, Sviatlana Akalovich 1, Yuliya Katlinskaya 1, Anton Sholukh 1, Tatyana Doroshenko 1, Yury Chaly 1, Nikolai N. Voitenok 2, 1 Laboratory of Cellular & Molecular Immunology, The Research Center for Hematology & Transfusiology, Minsk, Belarus, 2 Fund for Molecular Hematology and Immunology, Moscow, Russia
Loss of membrane expression of several cytokine receptors (TNFRI, TNFRII, IL-1RII, IL-2R, IL-6R, IL-15R) occurs via proteolytical cleavage to soluble forms by cell-associated metalloproteinases. We have previously showed that reduction of CXCR2 expression on phagocytosing neutrophils was also mediated by cell-associated metalloproteinase. Here we show that reduction of CXCR2 expression on neutrophils exposed to tumor necrosis factor a was accompanied by the appearance of a soluble CXCR2 (sCXCR2) antigen in cell-culture supernatants. We also detected sCXCR2 in urine from healthy subjects. Leukocyte-derived sCXCR2 (ld-sCXCR2) and urinederived sCXCR2 (ud-sCXCR2) were similarly eluted as a single protein peak of 40 kDa when applied to size-exclusion high performance liquid chromatography and were strongly acidic with isoelectric point of 3.2. sCXCR2 is a glycoprotein sensitive to treatment with N-glycosidase F. Predicted N-glycosylation of the peptide backbone of CXCR2 at Asp22 was demonstrated using monoclonal antibody mapped to 11–22 amino acids of CXCR2 N-terminus that effectively recognized deglycosylated sCXCR2, but weakly interacted with native glycosylated sCXCR2. C-terminal sequencing of native ud-sCXCR2 using carboxypeptidase A and MALDI-TOF MS/MS analysis of deglycosylated ld- and ud-sCXCR2 identified sCXCR2 as N-terminal fragment of CXCR2. Exploring bioactivity of sCXCR2, we observed the induction of interleukin-8 in monocyte culture. The effect was inhibited after treatment of sCXCR2 with N-glycosidase F or proteinase K indicating that both the carbohydrate moiety and peptide backbone are required. sCXCR2 was capable to stimulate immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC), but the stimulation was not effective in PBMC depleted of monocytes, i.e. was monocyte-mediated. Taken together, our results demonstrate for the first that loss of CXCR2 from neutrophils leads to production of soluble acidic glycopeptide CXCR2 which is also generated in vivo and may represent a novel mediator of inflammation. doi:10.1016/j.cyto.2009.07.377