The calcium binding S100A8/A9 complex (MRP8/14; calgranulin) is considered as an important proinflammatory mediator in acute and chronic inflammation and has recently gained attention as a molecular ...marker up-regulated in various human cancers. Here, we report that S100A8/A9 is expressed in breast cancer cell lines and is up-regulated by interleukin-1β and tumor necrosis factor-α in SKBR3 and MCF-7 cells. We identified the phospholipid-binding protein annexin A6 as a potential S100A8/A9 binding protein by affinity chromatography. This finding was verified by Southwestern overlay experiments and by coimmunoprecipitation with the S100A8/A9-specific monoclonal antibody 27E10. Immunocytochemical experiments demonstrated that S100A8/A9 and annexin A6 colocalize in SKBR3 breast cancer cells predominantly in membranous structures. Upon calcium influx both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells. Subcellular fractionation studies suggested that after A23187 stimulation membrane association of S100A8/A9 is not enhanced. However, both S100A8/A9 and annexin A6 are exposed on the cell surface of SKBR3 cells upon calcium influx. Experiments with artificial liposomes indicated that S100A8/A9 is able to associate with membranes independently of both annexin A6 and independently of calcium. Finally, cell surface expression of S100A8/A9 could not be observed in A23187-treated A431 and HaCaT cells. Both cell lines are known to be devoid of annexin A6. Repression of annexin A6 expression by small interfering RNA in SKBR3 cells abolishes the cell surface exposition of S100A8/A9 upon calcium influx, suggesting that annexin A6 contributes to the calcium-dependent cell surface exposition of the membrane associated-S100A8/A9 complex.
EF‐hand proteins are known to translocate to membranes, suggesting that they are involved in signaling events located in the cell membrane. Many proteins involved in signaling events associate ...cholesterol rich membrane domains, so called lipid rafts, which serve as platforms for controlled protein–protein interaction. Here, we demonstrate that the myeloid expressed EF‐hand proteins can be distinguished into three classes with respect to their membrane association. Grancalcin, a myeloid expressed penta EF‐hand protein, is constitutively located in lipid rafts. S100A9 (MRP14) and S100A8 (MRP8) are translocated into detergent resistant lipid structures only after calcium activation of the neutrophils. However, the S100A9/A8 membrane association is cholesterol and sphingolipid independent. On the other hand, the association of S100A12 (EN‐RAGE) and S100A6 (calcyclin) with membranes is detergent sensitive. These diverse affinities to lipid structures of the myeloid expressed EF‐hand proteins most likely reflect their different functions in neutrophils.
The aryl hydrocarbon receptor (AHR) is a ligand‐dependent member of the PAS‐bHLH‐family of nuclear receptors. Anthropogenic ligands include environmental contaminants such as ...2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD). Over‐activation of the AHR causes thymus atrophy and immunosuppression. Signaling via the AHR changes the thymocyte differentiation program at several checkpoints, in particular within the CD4–CD8– double‐negative (DN) thymocyte subset. Here, we show that AHR over‐activation led to the preferential emigration of DN thymocytes to the periphery and accumulation in the spleen. Some of these recent thymic emigrants (RTE) had a novel “activated immature” phenotype (CD3–TCRβ–CD25+/intCD44–CD45RB+/intCD62L+CD69– cells). Gene expression profiling of DN RTE revealed 15 genes that were up‐regulated more than threefold by TCDD, including the S100A9 gene. Exposure of S100A9 null mice to TCDD showed a role for this protein in AHR‐mediated thymic egress.
C-Jun N-terminal kinases (JNK) are activated in course of many viral infections. Here we analyzed the activity of JNK inhibitors on influenza A virus (IAV) amplification. Human lung epithelial cells ...were infected with either the highly pathogenic avian virus strain A/FPV/Bratislava/79 (H7N7) or the pandemic swine-origin influenza virus A/Hamburg/4/09 (H1N1v). The application of the JNK inhibitors SP600125 and AS601245 reduced IAV amplification by suppressing viral protein and RNA synthesis. Although AS601245 appeared to generally block the transcription of newly introduced genes, SP600125 specifically affected viral RNA synthesis. Overexpression of a dominant negative mutant of SEK/MKK4 and siRNA-mediated suppression of JNK2 expression confirmed that specific manipulation of the JNK pathway attenuates virus propagation. An IAV minigenome replication assay revealed that SP600125 did not directly affect the activity of the viral RNA polymerase complex but seems to suppress an anti-influenza nonstructural protein 1-mediated virus supportive function. Finally, when H7N7- or H1N1v-infected mice were treated with SP600125, the viral load is reduced in lungs of treated compared with untreated mice. Our data suggest that this class of ATP competitive inhibitors once optimized for antiviral action potentially represent novel drugs for antiviral intervention.
S100A8, S100A9 and S100A12 proteins are associated with inflammation and tissue remodelling, both processes known to be associated with high protease activity. Here, we report that homo-oligomeric ...forms of S100A8 and S100A9 are readily degraded by proteases, but that the preferred hetero-oligomeric S100A8/A9 complex displays a high resistance even against proteinase K degradation. S100A12 is not as protease resistant as the S100A8/A9 complex. Since specific functions have been assigned to the homo- and heterooligomeric forms of the S100A8 and A9 proteins, this finding may point to a post-translational level of regulation of the various functions of these proteins in inflammation and tissue remodelling.
Expression of the influenza A virus (IAV) nonstructural protein (NS1) results in the activation of c‐Jun N‐terminal kinase (JNK). Both NS1 and JNK are involved in apoptosis induction. To investigate ...their interrelationship, we stably expressed a tamoxifen inducible NS1 oestrogen receptor fusion‐protein (NS1ERT) in mammalian cells. Upon tamoxifen stimulation, NS1ERT‐expressing cells partially rescued the attenuated replication of NS1‐deficient IAVs and also inhibited interferon up‐regulation, confirming the functional competence of NS1ERT. Tamoxifen‐induced NS1ERT created a cytopathic phenotype and led to the activation of JNK and apoptosis. Induction of NS1F103SERT mutant failed to activate JNK, but induced apoptosis, whereas the induction of NS1M106IERT led to JNK phosphorylation, but not apoptosis, indicating that JNK activation and apoptosis induction are not functionally linked. Further mutational analysis highlighted that apoptosis induction is a function of the C‐terminal effector domain of NS1. Finally, IAVs encoding mutant NS1 revealed a modulating effect of NS1 on apoptosis induction in a genuine infection. With respect to apoptogenicity, an NS1 mutant virus that results in a super activation of JNK behaves similarly to the JNK nonactivating virus expressing NS1F103S, thus confirming that NS1‐mediated JNK activation and apoptosis induction are also functionally independent from each other in vivo.
Mechanisms linking innate immunity and autoimmune responses are poorly understood. Myeloid-related protein-8 (Mrp8) and Mrp14 are damage-associated molecular pattern molecules (DAMPs) highly ...upregulated in various autoimmune disorders. We show in a mouse autoimmune model that local Mrp8 and Mrp14 production is essential for the induction of autoreactive CD8+ T cells and the development of systemic autoimmunity. This effect is mediated via Toll-like receptor 4 (TLR4) signaling leading to increased interleukin-17 (IL-17) expression. Notably, expression of Mrp8 and Mrp14 was upregulated in cutaneous lupus erythematosus, and stimulation of CD8+ T cells from individuals with lupus erythematosus with MRP proteins resulted in an upregulation of IL-17, suggesting a key role for MRP8 and MRP14 for the development of autoreactive lymphocytes during human autoimmunity as well. These results demonstrate a link between local expression of DAMP molecules and the development of systemic autoimmunity.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways. They consist of two helix-loop-helix, calcium-binding EF-hand domains. A ...characteristic feature is their tendency to form homo- and/or heterodimeric complexes. This report presents for the first time a functional “in vivo” approach to the analysis of S100 protein dimerization. Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells. It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse. The ability to homodimerize, however, appears to be restricted to the murine MRPs. Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization. Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.
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