Cytohesin-1 regulates oxidative burst and degranulation in neutrophils

Abstracts / Cytokine 48 (2009) 91–137 that masks true post-transcriptional effect and relied on the use of transcriptional inhibitors that have limitations. We have developed a reporter expression system that is selective for post-transcriptional assessment based on certain ribosomal protein gene promoters that are constitutively expressed but not inducible by external stimuli. Microarray and EST expression data as well as EGFP reporter assays determined that promoters RPS23 and RPS30 are suitable for this purpose. In contrast to viral promoters such as of CMV and SV40, a modified RPS30 promoter (RPS30M1) was not responsive to inflammatory stimuli in HEK293 or Huh7 cell lines. To assess post-transcriptional regulation, cells transfected with RPS30M1-driven reporter constructs that contain TNF0 or IL-80 30 UTRs were treated with cytokines or okadaic acid (OA) or their combination. As expected, reporter activity increased only in the presence of the AREs indicating selective posttranscriptional effect. This was also demonstrated at the RNA level using QPCR. Further, post-transcriptional effects of RNA binding proteins on reporter constructs with 30 UTRs having RNA regulatory sequences were selective as assessed using the RPS30M1 system. Additionally, RPS30M1 can be rendered transcriptionally active by appending specific cis-acting transcriptional response elements using a cloning-free approach. This allows a single signaling event investigation of both transcriptional and post-transcriptional regulation of cytokine expression. The NF-jB response element fused to the RPS30M1 promoter in addition to an ARE-30 UTR increased reporter activity several fold after treatment with IL1-a in an apparent synergetic effect of the two regulatory levels. Thus, the RPS30M1-linked reporter can be used for selective post-transcriptional effects of cytokine gene expression and can provide enhanced versatility and suitability for large scale post-transcriptional analysis. doi:10.1016/j.cyto.2009.07.527

PP2-150 PML positively regulate interferonc signaling Jamila El Bougrini, Laurent Dianoux, Mounira K. Chelbi-Alix, Poster Presentation II Pml Positively regulate interferonc signaling Jamila El Bougrini, Laurent Dianoux, Mounira K. Chelbi-Alix, CNRS FRE 2937, Villejuif, France Promyelocytic leukemia (PML), also known as TRIM19, belongs to the TRIM (TRIpartite Motif) family encoding a characteristic RBCC/TRIM motif comprised of several cystein-rich zinc binding domain (RING and B-boxes) and a Coiled-coil domain.The RBCC domain is required for the localization of PML to subnuclear matrix-associated structures, known as Nuclear Bodies. PML is expressed as a family of cytoplasmic and nuclear isoforms (PML I–VII) as a result of alternative splicing from a single gene. PML is a tumor suppressor gene associated with cell apoptosis, cell proliferation, senescence and antiviral defense. Analysis of PML/ mice provided evidence for a physiological role of PML in apoptosis. Cells derived from these mice are defective in the induction of apoptosis by Fas, TNF, IFN, ceramides and TGF-b. We show that overexpression of all nuclear PML isoforms in human cells increases IFNg-induced STAT1 phosphorylation, resulting in higher STAT1-DNA binding and activation of IFN-stimulated genes (ISGs). These effects, observed with IFNg and not IFNa, require the RING finger of PML and necessitate PML localisation in the nucleus. On the contrary, downregulation of PML by siRNA is accompanied by a decrease of IFNg-induced STAT1 phosphorylation, STAT1-DNA binding and transcription of ISGs. In addition, IFNgmediated STAT1-DNA binding activity was decreased in PML/ cells compared with PML wild-type cells. Taken together these results demonstrate that PML functions as a positive regulator of IFNg signaling. doi:10.1016/j.cyto.2009.07.528

PP2-151 IKKe as an operator of virus-induced signaling Benjamin tenOever, Poster Presentation II IKKe as an operator of virus-induced signaling Sze-Ling Ng, Maria H. Lorini, Sonja Schmid, Mark A. Chua, Benjamin tenOever, Department of Molecular and Cellular Biology, Harvard University; 7 Divinity Ave., Cambridge, MA 02138, USA, Department of Microbiology, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY 10029, USA The ability of a cell to combat an intracellular pathogen requires a mechanism to recognize the threat and elicit a transcriptional response against it. In the context of virus infection, the cell must inhibit replication while additionally sounding the alarm to warn neighboring cells of the imminent threat. This response is predominantly mediated by activation of Interferon Regulatory Factors (IRFs), production of interferon beta (IFNb), and the subsequent activation of JAK/STAT signaling. Activated STATs and IRFs assemble into homo- and hetero-dimeric complexes that bind to IFN gamma (IFNc) activated sequences (GAS) and IFN stimulated response elements (ISREs), respectively. We demonstrate that IKKe acts as a molecular operator in shaping the occupancy of both GAS and ISREs during virus infection. IKKe induces C-ter-

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minal phosphorylation of STAT1 which disrupts the STAT dimerization interface and modifies DNA binding capacity. IKKe-mediated phosphorylation of STAT1 blocks assembly on GAS elements but is tolerated in the context of ISREs. Kinase expression therefore biases ISRE-mediated transcription in response to virus infection to optimize the antiviral transcriptome of the cell. doi:10.1016/j.cyto.2009.07.529

PP2-152 Cytohesin-1 regulates oxidative burst and degranulation in neutrophils Mohammed-Amine El Azreq, Valérie Garceau, Danielle Harbour, Christophe PivotPajot, Sylvain G. Bourgoin, Poster Presentation II Cytohesin-1 regulates oxidative burst and degranulation in neutrophils Mohammed-Amine El Azreq, Valérie Garceau, Danielle Harbour, Christophe PivotPajot, Sylvain G. Bourgoin, Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUQ-CHUL and Département de Microbiologie-Infectiologie et d’Immunologie, Faculté de Médecine, Université Laval, Que., Canada Polymorphonuclear neutrophil (PMN) stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulates small G proteins such as Arfs (Arf1 and Arf6), leading to phospholipase D (PLD) activation and functions such as degranulation and the oxidative burst. However, the molecular links between fMLF receptors and PLD remain unclear. PMNs express cytohesin-1, an Arf-GEF (Guanine Exchange Factor) that activates Arfs, and its expression is strongly induced during the acquisition of the neutrophilic phenotype by neutrophil-like cells. The role of cytohesin-1 in the activation of the fMLF-Arf-PLD signalling axis, and the accomplishment of superoxide anion production, and degranulation was investigated in PMNs using the selective inhibitor of cytohesin, secinH3. Cytohesin-1 inhibition with secinH3 leads to Arf6 but not Arf1 inhibition, demonstrating the specificity for Arf6, and fMLF-mediated activation of PLD and of the oxidative burst as well. We observed a decrease in fMLF-mediated protein secretion and expression of cell surface markers corresponding to primary (CD63/myeloperoxidase), secondary (CD66/lactoferrin) and tertiary (matrix metalloproteinase-9) granules in PMNs incubated with secinH3. Similarly, silencing cytohesin-1 or Arf6 in PLB-985 cells negatively affected fMLF-induced activation of PLD, superoxide production and expression of granule markers on the cell surface. In contrast, stable over-expression of cytohesin-1 in PLB-985 cells enhanced fMLF-induced activation of Arf6, PLD and NADPH oxidase. The results of this study provide evidence for an involvement of cytohesin-1 in the regulation of the functional responses of human PMNs and link these events, in part at least, to the activation of Arf6. doi:10.1016/j.cyto.2009.07.530

PP2-153 Forced homo- and heterodimerization of all gp130-type complexes leads to constitutive ligand independent signaling, and cytokine independent growth Jan Suthaus, Anna Tillmann, Inken Lorenzen, Elena Bulanova, Stefan Rose-John, Jürgen Scheller, Poster Presentation II Forced homo- and heterodimerization of all gp130-type complexes leads to constitutive ligand independent signaling, and cytokine independent growth Jan Suthaus 1, Anna Tillmann 1, Inken Lorenzen 1, Elena Bulanova 2, Stefan Rose-John 1, Jürgen Scheller 1, 1 Department of Biochemistry, Christian-Albrechts-University, Kiel, Germany, 2 Department of Immunology and Cell Biology, Research Centre Borstel, Borstel, Germany The IL-6 family of cytokines consists of IL-6, IL-11, LIF, CNTF, OSM, IL-27, NNT-1 and IL-31. IL-6 and IL-11 induce the formation of a gp130-homodimer, whereas signaling by LIF, CNTF, CT-1, NNT-1 results in the formation of a gp130/LIFR heterodimer. OSM can induce the formation of a dimer of gp130 with LIFR and the related protein OSMR. IL-27 exclusively signals via a heterodimer comprising gp130 and the WSX-1 receptor, whereas IL-31 induces the formation of an OSMR/GPL heterodimer. Interestingly naturally ligand independent constitutively active gp130 variants were described to be responsible for inflammatory hepatocellular adenomas. In the past we have generated constitutively active gp130 variants based on homodimerization of Jun leucine zippers. To address the question whether we can adapt the leucine zipper based technology to heterodimer formation within the gp130 receptor family, we genetically engineered a Fos leucine zipper-gp130 chimera and a Jun leucine zipper-WSX-1 chimera. Fos is believed to form only very weak homodimeric complexes, but preferentially heterodimeric complexes with the protein Jun. Unexpectedly, upon transfection, Fos-gp130 alone was able to induce ligand independent STAT3 phosphorylation and ligand independent growth of murine pre-B-cells. Therefore, we developed a novel IL-15/IL-15Ra based system to generate ligand independent constitutively active heterodimeric complexes for all known gp130-receptor members. In this system, IL-15 is genetically fused to one receptor component