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Sample records for activated stat1 pias1

  1. SUMO Ligase Protein Inhibitor of Activated STAT1 (PIAS1) Is a Constituent Promyelocytic Leukemia Nuclear Body Protein That Contributes to the Intrinsic Antiviral Immune Response to Herpes Simplex Virus 1

    PubMed Central

    Brown, James R.; Conn, Kristen L.; Wasson, Peter; Charman, Matthew; Tong, Lily; Grant, Kyle; McFarlane, Steven

    2016-01-01

    ABSTRACT Aspects of intrinsic antiviral immunity are mediated by promyelocytic leukemia nuclear body (PML-NB) constituent proteins. During herpesvirus infection, these antiviral proteins are independently recruited to nuclear domains that contain infecting viral genomes to cooperatively promote viral genome silencing. Central to the execution of this particular antiviral response is the small ubiquitin-like modifier (SUMO) signaling pathway. However, the participating SUMOylation enzymes are not fully characterized. We identify the SUMO ligase protein inhibitor of activated STAT1 (PIAS1) as a constituent PML-NB protein. We show that PIAS1 localizes at PML-NBs in a SUMO interaction motif (SIM)-dependent manner that requires SUMOylated or SUMOylation-competent PML. Following infection with herpes simplex virus 1 (HSV-1), PIAS1 is recruited to nuclear sites associated with viral genome entry in a SIM-dependent manner, consistent with the SIM-dependent recruitment mechanisms of other well-characterized PML-NB proteins. In contrast to that of Daxx and Sp100, however, the recruitment of PIAS1 is enhanced by PML. PIAS1 promotes the stable accumulation of SUMO1 at nuclear sites associated with HSV-1 genome entry, whereas the accumulation of other evaluated PML-NB proteins occurs independently of PIAS1. We show that PIAS1 cooperatively contributes to HSV-1 restriction through mechanisms that are additive to those of PML and cooperative with those of PIAS4. The antiviral mechanisms of PIAS1 are counteracted by ICP0, the HSV-1 SUMO-targeted ubiquitin ligase, which disrupts the recruitment of PIAS1 to nuclear domains that contain infecting HSV-1 genomes through mechanisms that do not directly result in PIAS1 degradation. IMPORTANCE Adaptive, innate, and intrinsic immunity cooperatively and efficiently restrict the propagation of viral pathogens. Intrinsic immunity mediated by constitutively expressed cellular proteins represents the first line of intracellular defense against

  2. Novel role and mechanism of protein inhibitor of activated STAT1 in spatial learning

    PubMed Central

    Tai, Derek J C; Hsu, Wei L; Liu, Yen C; Ma, Yun L; Lee, Eminy H Y

    2011-01-01

    By using differential display PCR, we have previously identified 98 cDNA fragments from rat dorsal hippocampus, which are expressed differentially between the fast learners and slow learners from water-maze learning task. One cDNA fragment, which showed a higher expression level in fast learners, encodes the rat protein inhibitor of activated STAT1 (pias1) gene. Spatial training induced a significant increase in PIAS1 expression in rat hippocampus. Transient transfection of the wild-type (WT) PIAS1 plasmid to CA1 neurons facilitated, whereas transfection of PIAS1 siRNA impaired spatial learning in rats. Meanwhile, PIAS1WT increased STAT1 sumoylation, decreased STAT1 DNA binding and decreased STAT1 phosphorylation at Tyr-701 associated with spatial learning facilitation. But PIAS1 siRNA transfection produced an opposite effect on these measures associated with spatial learning impairment. Further, transfection of STAT1 sumoylation mutant impaired spatial acquisition, whereas transfection of STAT1 phosphorylation mutant blocked the impairing effect of PIAS1 siRNA on spatial learning. In this study, we first demonstrate the role of PIAS1 in spatial learning. Both posttranslational modifications (increased sumoylation and decreased phosphorylation) mediate the effect of PIAS1 on spatial learning facilitation. PMID:21102409

  3. Pias1 is essential for erythroid and vascular development in the mouse embryo.

    PubMed

    Constanzo, Jerfiz D; Deng, Mi; Rindhe, Smita; Tang, Ke-Jing; Zhang, Cheng-Cheng; Scaglioni, Pier Paolo

    2016-07-01

    The protein inhibitor of activated STAT-1 (PIAS1) is one of the few known SUMO E3 ligases. PIAS1 has been implicated in several biological processes including repression of innate immunity and DNA repair. However, PIAS1 function during development and tissue differentiation has not been studied. Here, we report that Pias1 is required for proper embryonic development. Approximately 90% of Pias1 null embryos die in utero between E10.5 and E12.5. We found significant apoptosis within the yolk sac (YS) blood vessels and concomitant loss of red blood cells (RBCs) resulting in profound anemia. In addition, Pias1 loss impairs YS angiogenesis and results in defective capillary plexus formation and blood vessel occlusions. Moreover, heart development is impaired as a result of loss of myocardium muscle mass. Accordingly, we found that Pias1 expression in primary myoblasts enhances the induction of cardiac muscle genes MyoD, Myogenin and Myomaker. PIAS1 protein regulation of cardiac gene transcription is dependent on transcription factors Myocardin and Gata-4. Finally, endothelial cell specific inactivation of Pias1 in vivo impairs YS erythrogenesis, angiogenesis and recapitulates loss of myocardium muscle mass. However, these defects are not sufficient to recapitulate the lethal phenotype of Pias1 null embryos. These findings highlight Pias1 as an essential gene for YS erythropoiesis and vasculogenesis in vivo. PMID:27155222

  4. Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

    PubMed Central

    Soares, Iaci N.; Caetano, Fabiana A.; Pinder, Jordan; Rodrigues, Bruna Roz; Beraldo, Flavio H.; Ostapchenko, Valeriy G.; Durette, Chantal; Pereira, Grace Schenatto; Lopes, Marilene H.; Queiroz-Hazarbassanov, Nicolle; Cunha, Isabela W.; Sanematsu, Paulo I.; Suzuki, Sergio; Bleggi-Torres, Luiz F.; Schild-Poulter, Caroline; Thibault, Pierre; Dellaire, Graham; Martins, Vilma R.; Prado, Vania F.; Prado, Marco A. M.

    2013-01-01

    Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity. PMID:23938469

  5. Protein Inhibitors of Activated STAT (Pias1 and Piasy) Differentially Regulate Pituitary Homeobox 2 (PITX2) Transcriptional Activity*

    PubMed Central

    Wang, Jianbo; Sun, Zhao; Zhang, Zichao; Saadi, Irfan; Wang, Jun; Li, Xiao; Gao, Shan; Engle, Jamison J.; Kuburas, Adisa; Fu, Xueyao; Yu, Wenjie; Klein, William H.; Russo, Andrew F.; Amendt, Brad A.

    2013-01-01

    Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and β-catenin. Lef-1, β-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins. PMID:23515314

  6. PIAS-1 Is a Checkpoint Regulator Which Affects Exit from G1 and G2 by Sumoylation of p73

    PubMed Central

    Munarriz, Eliana; Barcaroli, Daniela; Stephanou, Anastasis; Townsend, Paul A.; Maisse, Carine; Terrinoni, Alessandro; Neale, Michael H.; Martin, Seamus J.; Latchman, David S.; Knight, Richard A.; Melino, Gerry; De Laurenzi, Vincenzo

    2004-01-01

    p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications. Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73α, -β, and -γ bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73. PIAS-1 also sumoylates p73α, although not the C-terminally truncated isoforms p73β and -γ, and this requires the RING finger domain of PIAS-1. The ΔNp73α isoform can also bind, and be sumoylated by, PIAS-1. PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax. p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix. PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G1 and cell cycle reentry. Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G2 arrest. These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery. PMID:15572666

  7. Deubiquitinase USP2a Sustains Interferons Antiviral Activity by Restricting Ubiquitination of Activated STAT1 in the Nucleus

    PubMed Central

    Liu, Jin; Yuan, Yukang; Cheng, Qiao; Zuo, Yibo; Qian, Liping; Guo, Tingting; Qian, Guanghui; Li, Lemin; Ge, Jun; Dai, Jianfeng; Xiong, Sidong; Zheng, Hui

    2016-01-01

    STAT1 is a critical transcription factor for regulating host antiviral defenses. STAT1 activation is largely dependent on phosphorylation at tyrosine 701 site of STAT1 (pY701-STAT1). Understanding how pY701-STAT1 is regulated by intracellular signaling remains a major challenge. Here we find that pY701-STAT1 is the major form of ubiquitinated-STAT1 induced by interferons (IFNs). While total STAT1 remains relatively stable during the early stages of IFNs signaling, pY701-STAT1 can be rapidly downregulated by the ubiquitin-proteasome system. Moreover, ubiquitinated pY701-STAT1 is located predominantly in the nucleus, and inhibiting nuclear import of pY701-STAT1 significantly blocks ubiquitination and downregulation of pY701-STAT1. Furthermore, we reveal that the deubiquitinase USP2a translocates into the nucleus and binds to pY701-STAT1, and inhibits K48-linked ubiquitination and degradation of pY701-STAT1. Importantly, USP2a sustains IFNs-induced pY701-STAT1 levels, and enhances all three classes of IFNs- mediated signaling and antiviral activity. To our knowledge, this is the first identified deubiquitinase that targets activated pY701-STAT1. These findings uncover a positive mechanism by which IFNs execute efficient antiviral signaling and function, and may provide potential targets for improving IFNs-based antiviral therapy. PMID:27434509

  8. Analysis of STAT1 expression and biological activity reveals interferon-tau-dependent STAT1-regulated SOCS genes in the bovine endometrium.

    PubMed

    Vitorino Carvalho, A; Eozenou, C; Healey, G D; Forde, N; Reinaud, P; Chebrout, M; Gall, L; Rodde, N; Padilla, A Lesage; Delville, C Giraud; Leveugle, M; Richard, C; Sheldon, I M; Lonergan, P; Jolivet, G; Sandra, O

    2016-03-01

    Signal transducer and activator of transcription (STAT) proteins are critical for the regulation of numerous biological processes. In cattle, microarray analyses identified STAT1 as a differentially expressed gene in the endometrium during the peri-implantation period. To gain new insights about STAT1 during the oestrous cycle and early pregnancy, we investigated STAT1 transcript and protein expression, as well as its biological activity in bovine tissue and cells of endometrial origin. Pregnancy increased STAT1 expression on Day 16, and protein and phosphorylation levels on Day 20. In cyclic and pregnant females, STAT1 was located in endometrial cells but not in the luminal epithelium at Day 20 of pregnancy. The expression of STAT1 during the oestrous cycle was not affected by progesterone supplementation. In vivo and in vitro, interferon-tau (IFNT) stimulated STAT1 mRNA expression, protein tyrosine phosphorylation and nuclear translocation. Using chromatin immunoprecipitation in IFNT-stimulated endometrial cells, we demonstrated an increase of STAT1 binding on interferon regulatory factor 1 (IRF1), cytokine-inducible SH2-containing protein (CISH), suppressor of cytokine signaling 1 and 3 (SOCS1, SOCS3) gene promoters consistent with the induction of their transcripts. Our data provide novel molecular insights into the biological functions of STAT1 in the various cells composing the endometrium during maternal pregnancy recognition and implantation. PMID:25116692

  9. Hes-1 SUMOylation by protein inhibitor of activated STAT1 enhances the suppressing effect of Hes-1 on GADD45α expression to increase cell survival

    PubMed Central

    2014-01-01

    Background Hairy and Enhancer of split 1 (Hes-1) is a transcriptional repressor that plays an important role in neuronal differentiation and development, but post-translational modifications of Hes-1 are much less known. In the present study, we aimed to investigate whether Hes-1 could be SUMO-modified and identify the candidate SUMO acceptors on Hes-1. We also wished to examine the role of the SUMO E3 ligase protein inhibitor of activated STAT1 (PIAS1) in SUMOylation of Hes-1 and the molecular mechanism of Hes-1 SUMOylation. Further, we aimed to identify the molecular target of Hes-1 and examine how Hes-1 SUMOylation affects its molecular target to affect cell survival. Results In this study, by using HEK293T cells, we have found that Hes-1 could be SUMO-modified and Hes-1 SUMOylation was greatly enhanced by the SUMO E3 ligase PIAS1 at Lys8, Lys27 and Lys39. Furthermore, Hes-1 SUMOylation stabilized the Hes-1 protein and increased the transcriptional suppressing activity of Hes-1 on growth arrest and DNA damage-inducible protein alpha (GADD45α) expression. Overexpression of GADD45α increased, whereas knockdown of GADD45αα expression decreased cell apoptosis. In addition, H2O2 treatment increased the association between PIAS1 and Hes-1 and enhanced the SUMOylation of Hes-1 for endogenous protection. Overexpression of Hes-1 decreased H2O2-induced cell death, but this effect was blocked by transfection of the Hes-1 triple sumo-mutant (Hes-1 3KR). Overexpression of PIAS1 further facilitated the anti-apoptotic effect of Hes-1. Moreover, Hes-1 SUMOylation was independent of Hes-1 phosphorylation and vice versa. Conclusions The present results revealed, for the first time, that Hes-1 could be SUMO-modified by PIAS1 and GADD45α is a novel target of Hes-1. Further, Hes-1 SUMOylation mediates cell survival through enhanced suppression of GADD45α expression. These results revealed a novel role of Hes-1 in addition to its involvement in Notch signaling. They also

  10. Activated STAT1 Transcription Factors Conduct Distinct Saltatory Movements in the Cell Nucleus

    PubMed Central

    Speil, Jasmin; Baumgart, Eugen; Siebrasse, Jan-Peter; Veith, Roman; Vinkemeier, Uwe; Kubitscheck, Ulrich

    2011-01-01

    The activation of STAT transcription factors is a critical determinant of their subcellular distribution and their ability to regulate gene expression. Yet, it is not known how activation affects the behavior of individual STAT molecules in the cytoplasm and nucleus. To investigate this issue, we injected fluorescently labeled STAT1 in living HeLa cells and traced them by single-molecule microscopy. We determined that STAT1 moved stochastically in the cytoplasm and nucleus with very short residence times (<0.03 s) before activation. Upon activation, STAT1 mobility in the cytoplasm decreased ∼2.5-fold, indicating reduced movement of STAT1/importinα/β complexes to the nucleus. In the nucleus, activated STAT1 displayed a distinct saltatory mobility, with residence times of up to 5 s and intermittent diffusive motion. In this manner, activated STAT1 factors can occupy their putative chromatin target sites within ∼2 s. These results provide a better understanding of the timescales on which cellular signaling and regulated gene transcription operate at the single-molecule level. PMID:22261046

  11. PIAS1 Promotes Lymphomagenesis Through MYC Upregulation

    PubMed Central

    Rabellino, Andrea; Melegari, Margherita; Tompkins, Van S.; Chen, Weina; Van Ness, Brian G.; Teruya-Feldstein, Julie; Conacci-Sorrell, Maralice; Janz, Siegfried; Scaglioni, Pier Paolo

    2016-01-01

    Summary The MYC proto-oncogene is a transcription factor implicated in a broad range of cancers. MYC is regulated by several post-translational modifications including SUMOylation, but the functional impact of this post-translational modification is still unclear. Here we report that the SUMO E3 ligase PIAS1 SUMOylates MYC. We demonstrate that PIAS1 promotes, in a SUMOylation-dependent manner, MYC phosphorylation at serine 62 and dephosphorylation at threonine 58. These events reduce the MYC turnover leading to increased transcriptional activity. Furthermore, we find that MYC is SUMOylated in primary B-cell lymphomas and that PIAS1 is required for the viability of MYC-dependent B-cell lymphoma cells as well as several cancer cell lines of epithelial origin. Finally, Pias1 null mice display endothelial defects reminiscent of Myc null mice. Taken together these results indicate that PIAS1 is a positive regulator of MYC. PMID:27239040

  12. The DNA replication factor MCM5 is essential for Stat1-mediated transcriptional activation

    PubMed Central

    Snyder, Marylynn; He, Wei; Zhang, J. Jillian

    2005-01-01

    The eukaryotic minichromosome maintenance (MCM) family of proteins (MCM2–MCM7) is evolutionarily conserved from yeast to human. These proteins are essential for DNA replication. The signal transducer and activator of transcription proteins are critical for the signal transduction of a multitude of cytokines and growth factors leading to the regulation of gene expression. We previously identified a strong interaction between Stat1 and MCM5. However, the physiological significance of this interaction was not clear. We show here by chromatin immunoprecipitation (ChIP) analyses that the MCM5 protein, as well as other members of the MCM family, is inducibly recruited to Stat1 target gene promoters in response to cytokine stimulation. Furthermore, the MCM proteins are shown to move along with the RNA polymerase II during transcription elongation. We have also identified an independent domain in MCM5 that mediates the interaction between Stat1 and MCM5; overexpression of this domain can disrupt the interaction between Stat1 and MCM5 and inhibit Stat1 transcriptional activity. Finally, we used the RNA interference technique to show that MCM5 is essential for transcription activation of Stat1 target genes. Together, these results demonstrate that, in addition to their roles in DNA replication, the MCM proteins are also necessary for transcription activation. PMID:16199513

  13. Structural Basis of the Inhibition of STAT1 Activity by Sendai Virus C Protein

    PubMed Central

    Oda, Kosuke; Matoba, Yasuyuki; Irie, Takashi; Kawabata, Ryoko; Fukushi, Masaya; Sugiyama, Masanori

    2015-01-01

    ABSTRACT Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr701 by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the γ-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-γ by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-α/β. IMPORTANCE Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-α/β) and IFN-γ by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr701 residue is potently enhanced, although transcription by STAT1 is

  14. HDAC4-regulated STAT1 activation mediates platinum resistance in ovarian cancer.

    PubMed

    Stronach, Euan A; Alfraidi, Albandri; Rama, Nona; Datler, Christoph; Studd, James B; Agarwal, Roshan; Guney, Tankut G; Gourley, Charlie; Hennessy, Bryan T; Mills, Gordon B; Mai, Antonello; Brown, Robert; Dina, Roberto; Gabra, Hani

    2011-07-01

    Ovarian cancer frequently acquires resistance to platinum chemotherapy, representing a major challenge for improving patient survival. Recent work suggests that resistant clones exist within a larger drug-sensitive cell population prior to chemotherapy, implying that resistance is selected for rather than generated by treatment. We sought to compare clinically derived, intrapatient paired models of initial platinum response and subsequent resistant relapse to define molecular determinants of evolved resistance. Transcriptional analysis of a matched cell line series from three patients with high-grade serous ovarian cancer before and after development of clinical platinum resistance (PEO1/PEO4/PEO6, PEA1/PEA2, PEO14/PEO23) identified 91 up- and 126 downregulated genes common to acquired resistance. Significantly enhanced apoptotic response to platinum treatment in resistant cells was observed following knockdown of histone deacetylase (HDAC) 4, FOLR2, PIK3R1, or STAT1 (P < 0.05). Interestingly, HDAC4 and STAT1 were found to physically interact. Acetyl-STAT1 was detected in platinum-sensitive cells but not in HDAC4 overexpressing platinum-resistant cells from the same patient. In resistant cells, STAT1 phosphorylation/nuclear translocation was seen following platinum exposure, whereas silencing of HDAC4 increased acetyl-STAT1 levels, prevented platinum-induced STAT1 activation, and restored cisplatin sensitivity. Conversely, matched sensitive cells were refractory to STAT1 phosphorylation on platinum treatment. Analysis of 16 paired tumor biopsies taken before and after development of clinical platinum resistance showed significantly increased HDAC4 expression in resistant tumors [n = 7 of 16 (44%); P = 0.04]. Therefore, clinical selection of HDAC4-overexpressing tumor cells upon exposure to chemotherapy promotes STAT1 deacetylation and cancer cell survival. Together, our findings identify HDAC4 as a novel, therapeutically tractable target to counter platinum

  15. HDAC4-regulated STAT1 activation mediates platinum resistance in ovarian cancer

    PubMed Central

    Stronach, Euan A; Alfraidi, Albandri; Rama, Nona; Datler, Christoph; Studd, Jamie; Agarwal, Roshan; Guney, Tankut G; Gourley, Charlie; Hennessy, Bryan T; Mills, Gordon B; Mai, Antonello; Brown, Robert; Dina, Roberto; Gabra, Hani

    2011-01-01

    Ovarian cancer frequently acquires resistance to platinum chemotherapy, representing a major challenge for improving patient survival. Recent work suggests resistant clones exist within a larger drug sensitive cell-population prior to chemotherapy, implying that resistance is selected for rather than generated by treatment. We sought to compare clinically-derived, intra-patient paired models of initial platinum response and subsequent resistant relapse to define molecular determinants of evolved resistance. Transcriptional analysis of a matched cell-line series from three patients with high-grade serous ovarian cancer before and after development of clinical platinum resistance (PEO1/PEO4/PEO6, PEA1/PEA2, PEO14/PEO23) identified 91 up- and 126 down-regulated genes common to acquired resistance. Significantly enhanced apoptotic response to platinum treatment in resistant cells was observed following knockdown of HDAC4, FOLR2, PIK3R1 or STAT1 (p<0.05). Interestingly, HDAC4 and STAT1 were found to physically interact. Acetyl-STAT1 was detected in platinum sensitive but not HDAC4 over-expressing platinum resistant cells from the same patient. In resistant cells, STAT1 phosphorylation/nuclear translocation was seen following platinum exposure, whereas silencing of HDAC4 increased acetyl-STAT1 levels, prevented platinum induced STAT1 activation and restored cisplatin sensitivity. Conversely, matched sensitive cells were refractory to STAT1 phosphorylation on platinum treatment. Analysis of 16 paired tumor biopsies taken before and after development of clinical platinum resistance showed significantly increased HDAC4 expression in resistant tumors (n=7/16[44%]; p=0.04). Therefore, clinical selection of HDAC4 overexpressing tumor cells upon exposure to chemotherapy promotes STAT1 deacetylation and cancer cell survival. Together, our findings identify HDAC4 as a novel, therapeutically tractable target to counter platinum resistance in ovarian cancer. PMID:21571862

  16. PIAS1 is a determinant of poor survival and acts as a positive feedback regulator of AR signaling through enhanced AR stabilization in prostate cancer

    PubMed Central

    Puhr, M; Hoefer, J; Eigentler, A; Dietrich, D; van Leenders, G; Uhl, B; Hoogland, M; Handle, F; Schlick, B; Neuwirt, H; Sailer, V; Kristiansen, G; Klocker, H; Culig, Z

    2016-01-01

    Novel drugs like Abiraterone or Enzalutamide, which target androgen receptor (AR) signaling to improve androgen deprivation therapy (ADT), have been developed during the past years. However, the application of these drugs is limited because of occurrence of inherent or acquired therapy resistances during the treatment. Thus, identification of new molecular targets is urgently required to improve current therapeutic prostate cancer (PCa) treatment strategies. PIAS1 (protein inhibitor of activated STAT1 (signal transducer and activator of transcription-1)) is known to be an important cell cycle regulator and PIAS1-mediated SUMOylation is essential for DNA repair. In this context, elevated PIAS1 expression has already been associated with cancer initiation. Thus, in the present study, we addressed the question of whether PIAS1 targeting can be used as a basis for an improved PCa therapy in combination with anti-androgens. We show that PIAS1 significantly correlates with AR expression in PCa tissue and in cell lines and demonstrate that high PIAS1 levels predict shorter relapse-free survival. Our patient data are complemented by mechanistic and functional in vitro experiments that identify PIAS1 as an androgen-responsive gene and a crucial factor for AR signaling via prevention of AR degradation. Furthermore, PIAS1 knockdown is sufficient to decrease cell proliferation as well as cell viability. Strikingly, Abiraterone or Enzalutamide treatment in combination with PIAS1 depletion is even more effective than single-drug treatment in multiple PCa cell models, rendering PIAS1 as a promising target protein for a combined treatment approach to improve future PCa therapies. PMID:26257066

  17. PIAS1 is a determinant of poor survival and acts as a positive feedback regulator of AR signaling through enhanced AR stabilization in prostate cancer.

    PubMed

    Puhr, M; Hoefer, J; Eigentler, A; Dietrich, D; van Leenders, G; Uhl, B; Hoogland, M; Handle, F; Schlick, B; Neuwirt, H; Sailer, V; Kristiansen, G; Klocker, H; Culig, Z

    2016-05-01

    Novel drugs like Abiraterone or Enzalutamide, which target androgen receptor (AR) signaling to improve androgen deprivation therapy (ADT), have been developed during the past years. However, the application of these drugs is limited because of occurrence of inherent or acquired therapy resistances during the treatment. Thus, identification of new molecular targets is urgently required to improve current therapeutic prostate cancer (PCa) treatment strategies. PIAS1 (protein inhibitor of activated STAT1 (signal transducer and activator of transcription-1)) is known to be an important cell cycle regulator and PIAS1-mediated SUMOylation is essential for DNA repair. In this context, elevated PIAS1 expression has already been associated with cancer initiation. Thus, in the present study, we addressed the question of whether PIAS1 targeting can be used as a basis for an improved PCa therapy in combination with anti-androgens. We show that PIAS1 significantly correlates with AR expression in PCa tissue and in cell lines and demonstrate that high PIAS1 levels predict shorter relapse-free survival. Our patient data are complemented by mechanistic and functional in vitro experiments that identify PIAS1 as an androgen-responsive gene and a crucial factor for AR signaling via prevention of AR degradation. Furthermore, PIAS1 knockdown is sufficient to decrease cell proliferation as well as cell viability. Strikingly, Abiraterone or Enzalutamide treatment in combination with PIAS1 depletion is even more effective than single-drug treatment in multiple PCa cell models, rendering PIAS1 as a promising target protein for a combined treatment approach to improve future PCa therapies. PMID:26257066

  18. STAT1 signaling within macrophages is required for antifungal activity against Cryptococcus neoformans.

    PubMed

    Leopold Wager, Chrissy M; Hole, Camaron R; Wozniak, Karen L; Olszewski, Michal A; Mueller, Mathias; Wormley, Floyd L

    2015-12-01

    Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an opportunistic fungal pathogen that primarily affects AIDS patients and patients undergoing immunosuppressive therapy. In immunocompromised individuals, C. neoformans can lead to life-threatening meningoencephalitis. Studies using a virulent strain of C. neoformans engineered to produce gamma interferon (IFN-γ), denoted H99γ, demonstrated that protection against pulmonary C. neoformans infection is associated with the generation of a T helper 1 (Th1)-type immune response and signal transducer and activator of transcription 1 (STAT1)-mediated classical (M1) macrophage activation. However, the critical mechanism by which M1 macrophages mediate their anti-C. neoformans activity remains unknown. The current studies demonstrate that infection with C. neoformans strain H99γ in mice with macrophage-specific STAT1 ablation resulted in severely increased inflammation of the pulmonary tissue, a dysregulated Th1/Th2-type immune response, increased fungal burden, deficient M1 macrophage activation, and loss of protection. STAT1-deficient macrophages produced significantly less nitric oxide (NO) than STAT1-sufficient macrophages, correlating with an inability to control intracellular cryptococcal proliferation, even in the presence of reactive oxygen species (ROS). Furthermore, macrophages from inducible nitric oxide synthase knockout mice, which had intact ROS production, were deficient in anticryptococcal activity. These data indicate that STAT1 activation within macrophages is required for M1 macrophage activation and anti-C. neoformans activity via the production of NO. PMID:26351277

  19. Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation

    PubMed Central

    Sen, Adrish; Rott, Lusijah; Phan, Nguyen; Mukherjee, Gourab

    2014-01-01

    Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the

  20. Rotavirus NSP1 protein inhibits interferon-mediated STAT1 activation.

    PubMed

    Sen, Adrish; Rott, Lusijah; Phan, Nguyen; Mukherjee, Gourab; Greenberg, Harry B

    2014-01-01

    Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the

  1. STAT1 deficiency redirects IFN signalling toward suppression of TLR response through a feedback activation of STAT3

    PubMed Central

    Kim, Hun Sik; Kim, Dong Chan; Kim, Hong-Mi; Kwon, Hyung-Joon; Kwon, Soon Jae; Kang, Suk-Jo; Kim, Sun Chang; Choi, Go-Eun

    2015-01-01

    Interferons (IFNs) potentiate macrophage activation typically via a STAT1-dependent pathway. Recent studies suggest a functioning of STAT1-independent pathway in the regulation of gene expression by IFN-γ, thus pointing to the diversity in cellular responses to IFNs. Many functions of IFNs rely on cross-regulation of the responses to exogenous inflammatory mediators such as TLR ligands. Here we investigated the contribution of STAT1-independent pathway to macrophage activation and its underlying mechanism in the context of combined stimulation of IFN and TLR. We found that TLR-induced production of inflammatory cytokines (TNF-α, IL-12) was not simply nullified but was significantly suppressed by signaling common to IFN-γ and IFN-β in STAT1-null macrophages. Such a shift in the suppression of TLR response correlated with a sustained STAT3 activation and attenuation of NF-κB signaling. Using a JAK2/STAT3 pathway inhibitor or STAT3-specific siRNA, blocking STAT3 in that context restored TNF-α production and NF-κB signaling, thus indicating a functional cross-regulation among STAT1, STAT3, and NF-κB. Our results suggest that STAT1 deficiency reprograms IFN signaling from priming toward suppression of TLR response via feedback regulation of STAT3, which may provide a new insight into the host defense response against microbial pathogens in a situation of STAT1 deficiency. PMID:26299368

  2. Activation of the JAK/STAT-1 Signaling Pathway by IFN-γ Can Down-Regulate Functional Expression of the MHC Class I-Related Neonatal Fc Receptor for IgG1

    PubMed Central

    Liu, Xindong; Ye, Lilin; Bai, Yu; Mojidi, Habi; Simister, Neil E.; Zhu, Xiaoping

    2009-01-01

    Expression of many MHC genes is enhanced at the transcriptional or posttranscriptional level following exposure to the cytokine IFN-γ. However, in this study we found that IFN-γ down-regulated the constitutive expression of the neonatal Fc receptor (FcRn), an MHC class I-related molecule that functions to transport maternal IgG and protect IgG and albumin from degradation. Epithelial cell, macrophage-like THP-1 cell, and freshly isolated human PBMC exposure to IFN-γ resulted in a significant decrease of FcRn expression as assessed by real-time RT-PCR and Western blotting. The down-regulation of FcRn was not caused by apoptosis or the instability of FcRn mRNA. Chromatin immunoprecipitation and gel mobility shift assays showed that STAT-1 bound to an IFN-γ activation site in the human FcRn promoter region. Luciferase expression from an FcRn promoter-luciferase reporter gene construct was not altered in JAK1- and STAT-1-deficient cells following exposure to IFN-γ, whereas expression of JAK1 or STAT-1 protein restored the IFN-γ inhibitory effect on luciferase activity. The repressive effect of IFN-γ on the FcRn promoter was selectively reversed or blocked by mutations of the core nucleotides in the IFN-γ activation site sequence and by over-expression of the STAT-1 inhibitor PIAS1 or the dominant negative phospho-STAT-1 mutations at Tyr-701 and/or Ser-727 residues. Furthermore, STAT-1 might down-regulate FcRn transcription through sequestering the transcriptional coactivator CREB binding protein/p300. Functionally, IFN-γ stimulation dampened bidirectional transport of IgG across a polarized Calu-3 lung epithelial monolayer. Taken together, our results indicate that the JAK/STAT-1 signaling pathway was necessary and sufficient to mediate the down-regulation of FcRn gene expression by IFN-γ. PMID:18566411

  3. STS-1 promotes IFN-α induced autophagy by activating the JAK1-STAT1 signaling pathway in B cells.

    PubMed

    Dong, Guanjun; You, Ming; Fan, Hongye; Ding, Liang; Sun, Lingyun; Hou, Yayi

    2015-08-01

    Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the overexpression of IFN-α. IFN-α induces autophagy via the JAK1-STAT1 signaling pathway, contributing to the pathogenesis of SLE. Recent studies reported that B cells from patients with SLE and NZB/W F1 mice had enhanced autophagy activity; however, the mechanism still remains unknown. Here, we show that the protein tyrosine phosphatase STS-1 (suppressor of T-cell receptor signaling 1) was significantly overexpressed in B cells from patients with SLE and MRL/lpr mice. Notably, STS-1 promoted IFN-α-induced autophagy in B cells by enhancing the JAK1-STAT1 signaling activation. STS-1 inhibited the phosphorylation of the E3 ubiquitin protein ligase c-cbl, and subsequently promoted IFN-α-induced phosphorylation of tyrosine kinase 2, leading to JAK1-STAT1 signaling activation. Furthermore, STAT1 and JAK1 inhibitors blocked the IFN-α-induced autophagy promoted by STS-1, indicating that STS-1 promotes IFN-α-induced autophagy via the JAK1-STAT1 signaling. Our results demonstrate the importance of STS-1 in regulating IFN-α-induced autophagy in B cells, and this could be used as a therapeutic approach to treat SLE. PMID:25959715

  4. Impaired activation of Stat1 and c-Jun as a possible defect in macrophages of patients with active tuberculosis.

    PubMed

    Esquivel-Solís, H; Quiñones-Falconi, F; Zarain-Herzberg, A; Amieva-Fernández, R I; López-Vidal, Y

    2009-10-01

    Studies of patients with active tuberculosis (TB) and infected healthy individuals have shown that interferon (IFN)-gamma is present in sites of Mycobacterium tuberculosis infection in comparable levels. This suggests that there is a deficiency in the macrophage response to IFN-gamma in TB patients. We used recombinant human IFN-gamma to stimulate adherent monocyte-derived macrophages from three groups of people: patients with active tuberculosis (TBP), their healthy household contacts (HHC) and healthy uninfected controls from the community (CC). We then evaluated the ability of the macrophages to inhibit the growth of M. tuberculosis H37Rv as well as their cytokine profile at early in infection (48 h). After IFN-gamma treatment, macrophages of healthy individuals (HHC and CC) controlled M. tuberculosis growth and produced mainly nitric oxide (NO) and interleukin (IL)-12p70, whereas TBP macrophages did not kill M. tuberculosis. Additionally, TBP macrophages produced low levels of NO and IL-12p70 and high levels of tumour necrosis factor (TNF)-alpha and IL-10. Transforming growth factor (TGF)-beta levels were similar among all three groups. M. tuberculosis infection had little effect on the cytokine response after IFN-gamma stimulus, but infection alone induced more IL-10 and TGF-beta in TBP macrophages. There were no differences in Stat1 nuclear translocation and DNA binding between the groups. However, the phosphorylated Stat1 and c-Jun (AP-1) in nuclear protein extracts was diminished in TBP macrophages compared to macrophages of healthy individuals. These results indicate an impairment of Stat1-dependent and Stat1-independent IFN-gamma signalling in macrophages of people with active tuberculosis, suggesting a different molecular regulation that could impact macrophage functionality and disease outcome. PMID:19737230

  5. Activation of transcription factors STAT1 and STAT5 in the mouse median eminence after systemic ciliary neurotrophic factor administration.

    PubMed

    Severi, Ilenia; Senzacqua, Martina; Mondini, Eleonora; Fazioli, Francesca; Cinti, Saverio; Giordano, Antonio

    2015-10-01

    Exogenously administered ciliary neurotrophic factor (CNTF) causes weight loss in obese rodents and humans through leptin-like activation of the Jak-STAT3 signaling pathway in hypothalamic arcuate neurons. Here we report for the first time that 40min after acute systemic treatment, rat recombinant CNTF (intraperitoneal injection of 0.3mg/kg of body weight) induced nuclear translocation of the tyrosine-phosphorylated forms of STAT1 and STAT5 in the mouse median eminence and other circumventricular organs, including the vascular organ of the lamina terminalis and the subfornical organ. In the tuberal hypothalamus of treated mice, specific nuclear immunostaining for phospo-STAT1 and phospho-STAT5 was detected in ependymal cells bordering the third ventricle floor and lateral recesses, and in median eminence cells. Co-localization studies documented STAT1 and STAT5 activation in median eminence β-tanycytes and underlying radial glia-like cells. A few astrocytes in the arcuate nucleus responded to CNTF by STAT5 activation. The vast majority of median eminence tanycytes and radial glia-like cells showing phospho-STAT1 and phospho-STAT5 immunoreactivity were also positive for phospho-STAT3. In contrast, STAT3 was the sole STAT isoform activated by CNTF in arcuate nucleus and median eminence neurons. Finally, immunohistochemical evaluation of STAT activation 20, 40, 80, and 120min from the injection demonstrated that cell activation was accompanied by c-Fos expression. Collectively, our findings show that CNTF activates STAT3, STAT1, and STAT5 in vivo. The distinctive activation pattern of these STAT isoforms in the median eminence may disclose novel targets and pathways through which CNTF regulates food intake. PMID:26133794

  6. Interferon-gamma regulates nucleoside transport systems in macrophages through signal transduction and activator of transduction factor 1 (STAT1)-dependent and -independent signalling pathways.

    PubMed

    Soler, Concepció; Felipe, Antonio; García-Manteiga, José; Serra, Maria; Guillén-Gómez, Elena; Casado, F Javier; MacLeod, Carol; Modolell, Manuel; Pastor-Anglada, Marçal; Celada, Antonio

    2003-11-01

    The expressions of CNT and ENT (concentrative and equilibrative nucleoside transporters) in macrophages are differentially regulated by IFN-gamma (interferon-gamma). This cytokine controls gene expression through STAT1-dependent and/or -independent pathways (where STAT1 stands for signal transduction and activator of transcription 1). In the present study, the role of STAT1 in the response of nucleoside transporters to IFN-gamma was studied using macrophages from STAT1 knockout mice. IFN-gamma triggered an inhibition of ENT1-related nucleoside transport activity through STAT1-dependent mechanisms. Such inhibition of macrophage growth and ENT1 activity by IFN-gamma is required for DNA synthesis. Interestingly, IFN-gamma led to an induction of the CNT1- and CNT2-related nucleoside transport activities independent of STAT1, thus ensuring the supply of extracellular nucleosides for the STAT1-independent RNA synthesis. IFN-gamma up-regulated CNT2 mRNA and CNT1 protein levels and down-regulated ENT1 mRNA in both wild-type and STAT1 knockout macrophages. This is consistent with a STAT1-independent, long-term-mediated, probably transcription-dependent, regulation of nucleoside transporter genes. Moreover, STAT1-dependent post-transcriptional mechanisms are implicated in the regulation of ENT1 activity. Although nitric oxide is involved in the regulation of ENT1 activity in B-cells at a post-transcriptional level, our results show that STAT1-dependent induction of nitric oxide by IFN-gamma is not implicated in the regulation of ENT1 activity in macrophages. Our results indicate that both STAT1-dependent and -independent pathways are involved in the regulation of nucleoside transporters by IFN-gamma in macrophages. PMID:12868960

  7. Transcription factor Sp3 is silenced through SUMO modification by PIAS1

    PubMed Central

    Sapetschnig, Alexandra; Rischitor, Grigore; Braun, Harald; Doll, Andreas; Schergaut, Marion; Melchior, Frauke; Suske, Guntram

    2002-01-01

    Sp3 is a ubiquitous transcription factor closely related to Sp1. Here we show that Sp3 is a target for SUMO modification in vivo and in vitro. SUMO modification of Sp3 occurs at a single lysine located between the second glutamine-rich activation domain and the DNA-binding domain. Mutational analyses identified the sequence IKXE as essential for SUMO conjugation to Sp3. We identified the protein inhibitor of activated STAT1 (PIAS1) as an interaction partner of Sp3 and Ubc9. Moreover, PIAS1 strongly stimulated SUMO conjugation to Sp3, thus acting as an E3 ligase for SUMO conjugation to Sp3. All mutations that prevented SUMO modification in vitro strongly enhanced the transcriptional activity of Sp3, showing that SUMO modification silences Sp3 activity. SUMO-modified Sp3 bound to DNA with similar specificity and affinity as unmodified Sp3. However, DNA-bound Sp3 did not act as a substrate for SUMO modification. PMID:12356736

  8. The Fanconi Anemia Protein FANCC Binds to and Facilitates the Activation of STAT1 by Gamma Interferon and Hematopoietic Growth Factors

    PubMed Central

    Pang, Qishen; Fagerlie, Sara; Christianson, Tracy A.; Keeble, Winifred; Faulkner, Greg; Diaz, Jane; Rathbun, R. Keaney; Bagby, Grover C.

    2000-01-01

    Hematopoietic progenitor cells from Fanconi anemia (FA) group C (FA-C) patients display hypersensitivity to the apoptotic effects of gamma interferon (IFN-γ) and constitutively express a variety of IFN-dependent genes. Paradoxically, however, STAT1 activation is suppressed in IFN-stimulated FA cells, an abnormality corrected by transduction of normal FANCC cDNA. We therefore sought to define the specific role of FANCC protein in signal transduction through receptors that activate STAT1. Expression and phosphorylation of IFN-γ receptor α chain (IFN-γRα) and JAK1 and JAK2 tyrosine kinases were equivalent in both normal and FA-C cells. However, in coimmunoprecipitation experiments STAT1 did not dock at the IFN-γR of FA-C cells, an abnormality corrected by transduction of the FANCC gene. In addition, glutathione S-transferase fusion genes encoding normal FANCC but not a mutant FANCC bearing an inactivating point mutation (L554P) bound to STAT1 in lysates of IFN-γ-stimulated B cells and IFN-, granulocyte-macrophage colony-stimulating factor- and stem cell factor-stimulated MO7e cells. Kinetic studies revealed that the initial binding of FANCC was to nonphosphorylated STAT1 but that subsequently the complex moved to the receptor docking site, at which point STAT1 became phosphorylated. The STAT1 phosphorylation defect in FA-C cells was functionally significant in that IFN induction of IFN response factor 1 was suppressed and STAT1-DNA complexes were not detected in nuclear extracts of FA-C cells. We also determined that the IFN-γ hypersensitivity of FA-C hematopoietic progenitor cells does not derive from STAT1 activation defects because granulocyte-macrophage CFU and erythroid burst-forming units from STAT1−/− mice were resistant to IFN-γ. However, BFU-E responses to SCF and erythropoietin were suppressed in STAT−/− mice. Consequently, because the FANCC protein is involved in the activation of STAT1 through receptors for at least three hematopoietic

  9. SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin.

    PubMed

    Toropainen, Sari; Malinen, Marjo; Kaikkonen, Sanna; Rytinki, Miia; Jääskeläinen, Tiina; Sahu, Biswajyoti; Jänne, Olli A; Palvimo, Jorma J

    2015-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth. PMID:25552417

  10. SUMO ligase PIAS1 functions as a target gene selective androgen receptor coregulator on prostate cancer cell chromatin

    PubMed Central

    Toropainen, Sari; Malinen, Marjo; Kaikkonen, Sanna; Rytinki, Miia; Jääskeläinen, Tiina; Sahu, Biswajyoti; Jänne, Olli A.; Palvimo, Jorma J.

    2015-01-01

    Androgen receptor (AR) is a ligand-activated transcription factor that plays a central role in the development and growth of prostate carcinoma. PIAS1 is an AR- and SUMO-interacting protein and a putative transcriptional coregulator overexpressed in prostate cancer. To study the importance of PIAS1 for the androgen-regulated transcriptome of VCaP prostate cancer cells, we silenced its expression by RNAi. Transcriptome analyses revealed that a subset of the AR-regulated genes is significantly influenced, either activated or repressed, by PIAS1 depletion. Interestingly, PIAS1 depletion also exposed a new set of genes to androgen regulation, suggesting that PIAS1 can mask distinct genomic loci from AR access. In keeping with gene expression data, silencing of PIAS1 attenuated VCaP cell proliferation. ChIP-seq analyses showed that PIAS1 interacts with AR at chromatin sites harboring also SUMO2/3 and surrounded by H3K4me2; androgen exposure increased the number of PIAS1-occupying sites, resulting in nearly complete overlap with AR chromatin binding events. PIAS1 interacted also with the pioneer factor FOXA1. Of note, PIAS1 depletion affected AR chromatin occupancy at binding sites enriched for HOXD13 and GATA motifs. Taken together, PIAS1 is a genuine chromatin-bound AR coregulator that functions in a target gene selective fashion to regulate prostate cancer cell growth. PMID:25552417

  11. Interferons Induce STAT1-Dependent Expression of Tissue Plasminogen Activator, a Pathogenicity Factor in Puumala Hantavirus Disease.

    PubMed

    Strandin, Tomas; Hepojoki, Jussi; Laine, Outi; Mäkelä, Satu; Klingström, Jonas; Lundkvist, Åke; Julkunen, Ilkka; Mustonen, Jukka; Vaheri, Antti

    2016-05-15

    Hantaviruses are zoonotic viruses that show various degrees of vasculopathy in humans. In this study, we analyzed the regulation of 2 fibrinolytic parameters, tissue plasminogen activator (tPA) and its physiological inhibitor, plasminogen activator inhibitor 1 (PAI-1), in Puumala hantavirus (PUUV)-infected patients and in human microvascular endothelial cells. We detected strong upregulation of tPA in the acute phase of illness and in PUUV-infected macaques and found the tPA level to positively correlate with disease severity. The median levels of PAI-1 during the acute stage did not differ from those during the recovery phase. In concordance, hantaviruses induced tPA but not PAI-1 in microvascular endothelial cells, and the induction was demonstrated to be dependent on type I interferon. Importantly, type I and II interferons directly upregulated tPA through signal transducer and activator of transcription 1 (STAT1), which regulated tPA gene expression via a STAT1-responsive enhancer element. These results suggest that tPA may be a general factor in the immunological response to viruses. PMID:26704613

  12. Histone deacetylase inhibitors block IFNγ-induced STAT1 phosphorylation.

    PubMed

    Ginter, Torsten; Bier, Carolin; Knauer, Shirley K; Sughra, Kalsoom; Hildebrand, Dagmar; Münz, Tobias; Liebe, Theresa; Heller, Regine; Henke, Andreas; Stauber, Roland H; Reichardt, Werner; Schmid, Johannes A; Kubatzky, Katharina F; Heinzel, Thorsten; Krämer, Oliver H

    2012-07-01

    Signal transducer and activator of transcription 1 (STAT1) is important for innate and adaptive immunity. Histone deacetylase inhibitors (HDACi) antagonize unbalanced immune functions causing chronic inflammation and cancer. Phosphorylation and acetylation regulate STAT1 and different IFNs induce phosphorylated STAT1 homo-/heterodimers, e.g. IFNα activates several STATs whereas IFNγ only induces phosphorylated STAT1 homodimers. In transformed cells HDACi trigger STAT1 acetylation linked to dephosphorylation by the phosphatase TCP45. It is unclear whether acetylation differentially affects STAT1 activated by IFNα or IFNγ, and if cellular responses to both cytokines depend on a phosphatase-dependent inactivation of acetylated STAT1. Here, we report that HDACi counteract IFN-induced phosphorylation of a critical tyrosine residue in the STAT1 C-terminus in primary cells and hematopoietic cells. STAT1 mutants mimicking a functionally inactive DNA binding domain (DBD) reveal that the number of acetylation-mimicking sites in STAT1 determines whether STAT1 is recruited to response elements after stimulation with IFNγ. Furthermore, we show that IFNα-induced STAT1 heterodimers carrying STAT1 molecules mimicking acetylation bind cognate DNA and provide innate anti-viral immunity. IFNγ-induced acetylated STAT1 homodimers are though inactive, suggesting that heterodimerization and complex formation can rescue STAT1 lacking a functional DBD. Apparently, the type of cytokine determines how acetylation affects the nuclear entry and DNA binding of STAT1. Our data contribute to a better understanding of STAT1 regulation by acetylation. PMID:22425562

  13. Active components with inhibitory activities on IFN-γ/STAT1 and IL-6/STAT3 signaling pathways from Caulis Trachelospermi.

    PubMed

    Liu, Xiao-Ting; Wang, Zhe-Xing; Yang, Yu; Wang, Lin; Sun, Ruo-Feng; Zhao, Yi-Min; Yu, Neng-Jiang

    2014-01-01

    Initial investigation for new active herbal extract with inhibiting activity on JAK/STAT signaling pathway revealed that the extract of Caulis Trachelospermi, which was separated by 80% alcohol extraction and subsequent HP-20 macroporous resin column chromatography, was founded to strongly inhibit IFN-γ-induced STAT1-responsive luciferase activity (IFN-γ/STAT1) with IC50 value of 2.43 μg/mL as well as inhibiting IL-6-induced STAT3-responsive luciferase activity (IL-6/STAT3) with IC50 value of 1.38 μg/mL. Subsequent study on its active components led to the isolation and identification of two new dibenzylbutyrolactone lignans named 4-demethyltraxillaside (1) and nortrachelogenin 4-O-β-D-glucopyranoside (2), together with six known compounds. The lignan compounds 1-4 together with other lignan compounds isolated in previous study were tested the activities on IFN-γ/STAT1 and IL-6/STAT3 pathways. The following result showed that the main components trachelogenin and arctigenin had corresponding activities on IFN-γ/STAT1 pathway with IC50 values of 3.14 μM and 9.46 μM as well as trachelogenin, arctigenin and matairesinol strongly inhibiting IL-6/STAT3 pathway with IC50 values of 3.63 μM, 6.47 μM and 2.92 μM, respectively. PMID:25100250

  14. Glucolipotoxicity initiates pancreatic β-cell death through TNFR5/CD40-mediated STAT1 and NF-κB activation.

    PubMed

    Bagnati, Marta; Ogunkolade, Babatunji W; Marshall, Catriona; Tucci, Carmen; Hanna, Katie; Jones, Tania A; Bugliani, Marco; Nedjai, Belinda; Caton, Paul W; Kieswich, Julius; Yaqoob, Muhammed M; Ball, Graham R; Marchetti, Piero; Hitman, Graham A; Turner, Mark D

    2016-01-01

    Type 2 diabetes is a chronic metabolic disorder, where failure to maintain normal glucose homoeostasis is associated with, and exacerbated by, obesity and the concomitant-elevated free fatty acid concentrations typically found in these patients. Hyperglycaemia and hyperlipidaemia together contribute to a decline in insulin-producing β-cell mass through activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)-1. There are however a large number of molecules potentially able to modulate NF-κB and STAT1 activity, and the mechanism(s) by which glucolipotoxicity initially induces NF-κB and STAT1 activation is currently poorly defined. Using high-density microarray analysis of the β-cell transcritptome, we have identified those genes and proteins most sensitive to glucose and fatty acid environment. Our data show that of those potentially able to activate STAT1 or NF-κB pathways, tumour necrosis factor receptor (TNFR)-5 is the most highly upregulated by glucolipotoxicity. Importantly, our data also show that the physiological ligand for TNFR5, CD40L, elicits NF-κB activity in β-cells, whereas selective knockdown of TNFR5 ameliorates glucolipotoxic induction of STAT1 expression and NF-κB activity. This data indicate for the first time that TNFR5 signalling has a major role in triggering glucolipotoxic islet cell death. PMID:27512950

  15. LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1.

    PubMed Central

    Weiskirchen, R; Moser, M; Weiskirchen, S; Erdel, M; Dahmen, S; Buettner, R; Gressner, A M

    2001-01-01

    Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells. PMID:11672422

  16. B lymphocytes from patients with chronic lymphocytic leukemia contain signal transducer and activator of transcription (STAT) 1 and STAT3 constitutively phosphorylated on serine residues.

    PubMed

    Frank, D A; Mahajan, S; Ritz, J

    1997-12-15

    To explore the pathogenesis of chronic lymphocytic leukemia (CLL), we examined whether phosphorylation of one or more signal transducer and activator of transcription (STAT) factors was abnormal in cells from CLL patients. No constitutive tyrosine phosphorylation was detected on any STAT in CLL cells. To assess the phosphorylation of serine residues of STAT1 and STAT3 in CLL cells, we raised antibodies that specifically recognize the form of STAT1 phosphorylated on ser-727 and the form of STAT3 phosphorylated on ser-727. We found that in 100% of patients with CLL (n = 32), STAT1 and STAT3 were constitutively phosphorylated on serine. This was in contrast to normal peripheral blood B lymphocytes or CD5+) B cells isolated from tonsils, in which this phosphorylation was absent. Serine phosphorylation of STAT1 and STAT3 was seen occasionally in other leukemias, but it was a universal finding only in CLL. The serine phosphorylation of these STATs was a continuous process, as incubation of CLL cells with the kinase inhibitor H7 led to the dephosphorylation of these serine residues. The STAT serine kinase in CLL cells has not been identified, and appears to be neither mitogen-activated protein kinase nor pp70(s6k). In summary, the constitutive serine phosphorylation of STAT1 and STAT3 is present in all CLL samples tested to date, although the physiologic significance of this modification remains to be determined. PMID:9399961

  17. Development of Th17-associated interstitial kidney inflammation in lupus-prone mice lacking the gene encoding Signal Transduction and Activator of Transcription-1 (STAT-1)

    PubMed Central

    Yiu, Gloria; Rasmussen, Tue Kruse; Ajami, Bahareh; Haddon, David J.; Chu, Alvina D.; Tangsombatvisit, Stephanie; Haynes, Winston A.; Diep, Vivian; Steinman, Larry; Faix, James; Utz, Paul J.

    2016-01-01

    Type I interferon (IFN-I) signaling is a central pathogenic pathway in Systemic Lupus Erythematosus (SLE), and therapeutics targeting IFN-I signaling are in development. Multiple proteins with overlapping function participate in IFN signaling, but the signaling events downstream of receptor engagement are unclear. We employed highly-multiplexed assays to characterize autoantibody production, cytokine/chemokine profiles, and Signal Transduction and Activators of Transcription (STAT) phosphorylation to investigate the individual roles of IFNAR2, IRF9 and STAT1 in MRL/lpr (lpr) mice. Surprisingly, we found that Stat1−/−, but not Irf9−/− or Ifnar2−/− mice, developed interstitial nephritis characterized by infiltration with RORγT+ lymphocytes, macrophages and eosinophils. Despite pronounced interstitial kidney disease and abnormal kidney function, Stat1−/− mice had decreased proteinuria, glomerulonephritis and autoantibody production. Phospho-specific flow cytometry (phosphoflow) revealed shunting of STAT phosphorylation from STAT1 to STAT3/4. In summary, we describe unique contributions of STAT1 to pathology in different kidney compartments, and provide novel insight into tubulointerstitial nephritis (TIN) a poorly understood complication that predicts end-stage kidney disease in SLE patients. PMID:26636548

  18. Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Barchowsky, Aaron

    2009-01-01

    Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

  19. Altered interleukin-12 responsiveness in Th1 and Th2 cells is associated with the differential activation of STAT5 and STAT1.

    PubMed

    Gollob, J A; Murphy, E A; Mahajan, S; Schnipper, C P; Ritz, J; Frank, D A

    1998-02-15

    T-cell activation in response to interleukin-12 (IL-12) is mediated through signaling events that include the tyrosine phosphorylation of STAT4. IL-12 responsiveness and the ability of IL-12 to activate STAT4 is different in T cells induced to differentiate into a Th1 or Th2 phenotype. In this report, we show that STAT5, STAT1alpha, and STAT1beta, in addition to STAT4, are tyrosine phosphorylated in response to IL-12 in phytohemagglutinin (PHA)-activated human T cells. To understand how the activation of these STATs contributes to T-cell IL-12 responsiveness, we analyzed the IL-12-induced activation of STAT5 and STAT1 in T cells stimulated to undergo Th1 or Th2 differentiation. The IL-12-induced tyrosine phosphorylation of STAT5 and STAT1, but not STAT4, is augmented in T cells activated into Th1 cells with PHA + interferon-gamma (IFN-gamma) compared with T cells activated with PHA alone. STAT5 DNA binding induced by IL-12 is also augmented in T cells activated with PHA + IFN-gamma compared with T cells activated with PHA alone, whereas STAT4 DNA binding is not increased. In contrast, the IL-12-induced activation of these STATs is inhibited in T cells activated into Th2 cells with PHA + IL-4. The enhancement of IL-12 signaling by IFN-gamma is not a direct effect of IFN-gamma on T cells, but rather is mediated by IL-12 that is produced by antigen-presenting cells in response to IFN-gamma. This positive autoregulatory effect of IL-12 on the activation of select STATs correlates with an increase in T-cell IFN-gamma production in response to IL-12. These findings suggest that the activation of STAT5 and STAT1 may augment select STAT4-dependent functional responses to IL-12 in Th1 cells. PMID:9454765

  20. ROS-dependent Syk and Pyk2-mediated STAT1 activation is required for 15(S)-Hydroxyeicosatetraenoic acid-induced CD36 expression and foam cell formation

    PubMed Central

    Kotla, Sivareddy; Singh, Nikhlesh K.; Traylor, James G.; Orr, A. Wayne; Rao, Gadiparthi N.

    2014-01-01

    15(S)-Hydroxyeicosatetraenoic acid (15(S)-HETE), the major 15-lipoxygenase 1/2 (15-LO1/2) metabolite of arachidonic acid (AA), induces CD36 expression through xanthine oxidase and NADPH oxidase-dependent ROS production and Syk and Pyk2-dependent STAT1 activation. In line with these observations, 15(S)-HETE also induced foam cell formation involving ROS, Syk, Pyk2 and STAT1-mediated CD36 expression. In addition, peritoneal macrophages from Western diet-fed ApoE−/− mice exhibited elevated levels of xanthine oxidase and NADPH oxidase activities, ROS production, Syk, Pyk2, and STAT1 phosphorylation and CD36 expression compared to those from ApoE−/−:12/15-LO−/− mice and these events correlated with increased lipid deposits, macrophage content and lesion progression in the aortic roots. Human atherosclerotic arteries also showed increased 15-LO1 expression, STAT1 phosphorylation and CD36 levels as compared to normal arteries. Together, these findings suggest that 12/15-LO metabolites of AA, particularly 12/15(S)-HETE might play a crucial role in atherogenesis by enhancing foam cell formation. PMID:25152235

  1. A time- and dose-dependent STAT1 expression system

    PubMed Central

    Leitner, Nicole R; Strobl, Birgit; Bokor, Marion; Painz, Ronald; Kolbe, Thomas; Rülicke, Thomas; Müller, Mathias; Karaghiosoff, Marina

    2006-01-01

    Background The signal transducer and activator of transcription (STAT) family of transcription factors mediates a variety of cytokine dependent gene regulations. STAT1 has been mainly characterized by its role in interferon (IFN) type I and II signaling and STAT1 deficiency leads to high susceptibility to several pathogens. For fine-tuned analysis of STAT1 function we established a dimerizer-inducible system for STAT1 expression in vitro and in vivo. Results The functionality of the dimerizer-induced STAT1 system is demonstrated in vitro in mouse embryonic fibroblasts and embryonic stem cells. We show that this two-vector based system is highly inducible and does not show any STAT1 expression in the absence of the inducer. Reconstitution of STAT1 deficient cells with inducible STAT1 restores IFNγ-mediated gene induction, antiviral responses and STAT1 activation remains dependent on cytokine stimulation. STAT1 expression is induced rapidly upon addition of dimerizer and expression levels can be regulated in a dose-dependent manner. Furthermore we show that in transgenic mice STAT1 can be induced upon stimulation with the dimerizer, although only at low levels. Conclusion These results prove that the dimerizer-induced system is a powerful tool for STAT1 analysis in vitro and provide evidence that the system is suitable for the use in transgenic mice. To our knowledge this is the first report for inducible STAT1 expression in a time- and dose-dependent manner. PMID:17184522

  2. Infectious Bronchitis Coronavirus Inhibits STAT1 Signaling and Requires Accessory Proteins for Resistance to Type I Interferon Activity

    PubMed Central

    Kint, Joeri; Dickhout, Annemiek; Kutter, Jasmin; Maier, Helena J.; Britton, Paul; Koumans, Joseph; Pijlman, Gorben P.; Fros, Jelke J.; Wiegertjes, Geert F.

    2015-01-01

    ABSTRACT The innate immune response is the first line of defense against viruses, and type I interferon (IFN) is a critical component of this response. Similar to other viruses, the gammacoronavirus infectious bronchitis virus (IBV) has evolved under evolutionary pressure to evade and counteract the IFN response to enable its survival. Previously, we reported that IBV induces a delayed activation of the IFN response. In the present work, we describe the resistance of IBV to IFN and the potential role of accessory proteins herein. We show that IBV is fairly resistant to the antiviral state induced by IFN and identify that viral accessory protein 3a is involved in resistance to IFN, as its absence renders IBV less resistant to IFN treatment. In addition to this, we found that independently of its accessory proteins, IBV inhibits IFN-mediated phosphorylation and translocation of STAT1. In summary, we show that IBV uses multiple strategies to counteract the IFN response. IMPORTANCE In the present study, we show that infectious bronchitis virus (IBV) is resistant to IFN treatment and identify a role for accessory protein 3a in the resistance against the type I IFN response. We also demonstrate that, in a time-dependent manner, IBV effectively interferes with IFN signaling and that its accessory proteins are dispensable for this activity. This study demonstrates that the gammacoronavirus IBV, similar to its mammalian counterparts, has evolved multiple strategies to efficiently counteract the IFN response of its avian host, and it identifies accessory protein 3a as multifaceted antagonist of the avian IFN system. PMID:26401035

  3. PIAS1 and STAT-3 impair the tumoricidal potential of IFN-γ-stimulated mouse dendritic cells generated with IL-15.

    PubMed

    Hanke, Neale T; LaCasse, Collin J; Larmonier, Claire B; Alizadeh, Darya; Trad, Malika; Janikashvili, Nona; Bonnotte, Bernard; Katsanis, Emmanuel; Larmonier, Nicolas

    2014-08-01

    Primarily defined by their antigen-presenting property, dendritic cells (DCs) are being implemented as cancer vaccines in immunotherapeutic interventions. DCs can also function as direct tumor cell killers. How DC cytotoxic activity can be efficiently harnessed and the mechanisms controlling this nonconventional property are not fully understood. We report here that the tumoricidal potential of mouse DCs generated from myeloid precursors with GM-CSF and IL-15 (IL-15 DCs) can be triggered with the Toll-like receptor (TLR) 4 ligand lipopolysaccharide to a similar extent compared with that of their counterparts, conventionally generated with IL-4 (IL-4 DCs). The mechanism of tumor cell killing depends on the induction of iNOS expression by DCs. In contrast, interferon (IFN)-γ induces the cytotoxic activity of IL-4 but not IL-15 DCs. Although the IFN-γ-STAT-1 signaling pathway is overall functional in IL-15 DCs, IFN-γ fails to induce iNOS expression in these cells. iNOS expression is negatively controlled in IFN-γ-stimulated IL-15 DCs by the cooperation between the E3 SUMO ligase PIAS1 and STAT-3, and can be partially restored with PIAS1 siRNA and STAT-3 inhibitors. PMID:24777831

  4. Extracellular Vesicles from Neural Stem Cells Transfer IFN-γ via Ifngr1 to Activate Stat1 Signaling in Target Cells

    PubMed Central

    Cossetti, Chiara; Iraci, Nunzio; Mercer, Tim R.; Leonardi, Tommaso; Alpi, Emanuele; Drago, Denise; Alfaro-Cervello, Clara; Saini, Harpreet K.; Davis, Matthew P.; Schaeffer, Julia; Vega, Beatriz; Stefanini, Matilde; Zhao, CongJian; Muller, Werner; Garcia-Verdugo, Jose Manuel; Mathivanan, Suresh; Bachi, Angela; Enright, Anton J.; Mattick, John S.; Pluchino, Stefano

    2015-01-01

    SUMMARY The idea that stem cell therapies work only via cell replacement is challenged by the observation of consistent intercellular molecule exchange between the graft and the host. Here we defined a mechanism of cellular signaling by which neural stem/precursor cells (NPCs) communicate with the microenvironment via extracellular vesicles (EVs), and we elucidated its molecular signature and function. We observed cytokine-regulated pathways that sort proteins and mRNAs into EVs. We described induction of interferon gamma (IFN-γ) pathway in NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We showed that IFN-γ bound to EVs through Ifngr1 activates Stat1 in target cells. Finally, we demonstrated that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-γ/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-γ/Ifngr1 complexes, which grafted stem cells may use to communicate with the host immune system. PMID:25242146

  5. BANK1 Regulates IgG Production in a Lupus Model by Controlling TLR7-Dependent STAT1 Activation

    PubMed Central

    Iida, Ryuji; Bagavant, Harini; Alarcón-Riquelme, Marta E.

    2016-01-01

    The purpose of our study was to investigate the effects of the adaptor Bank1 in TLR7 signaling using the B6.Sle1.yaa mouse, a lupus model that develops disease through exacerbated TLR7 expression. Crosses of B6.Sle1.yaa with Bank1-/- mice maintained several B and myeloid cell phenotypes close to normal wild-type levels. Most striking was the reduction in total serum IgG antibodies, but not of IgM, and reduced serum levels of autoantibodies, IL-6, and BAFF. Bank1 deficiency did modify numbers of MZ B cells and total B cell numbers, as well as expression of CXCR4 by follicular helper T cells. Other T cell changes were not observed. Bank1 deficiency did not modify numbers of germinal center B cells or plasma cells or clinical disease outcomes. Purified B cells from Bank1 deficient mice had strongly reduced Ifnb, Ifna4, Irf7, Aicda and Stat1 gene expression following TLR7 agonist stimulation. Interestingly, phosphorylation of Tyr701, but not of Ser727 of STAT1, was impaired in splenic B cells from B6.Sle1.yaa.Bank1-/- mice, as was the nuclear translocation of IRF7 in response to TLR7 agonist stimulation. Further, Bank1 deficiency in B6.Sle1.yaa mice reduced the production of IgG2c after in vitro TLR7 agonist stimulation. Our results demonstrate that Bank1 controls TLR7-mediated type I interferon production. Combined with the control of the nuclear translocation of IRF7, the modulation of STAT1 transcription and phosphorylation, Bank1 contributes to IgG production during development of autoimmune disease. PMID:27228057

  6. Dose-dependent platelet stimulation and inhibition induced by anti-PIA1 IgG

    SciTech Connect

    Ryu, T.; Davis, J.M.; Schwartz, K.A. )

    1990-07-01

    The PIA1 antibody produces several clinically distinct and severe thrombocytopenias. Investigations have demonstrated divergent effects on platelet function; prior reports demonstrated inhibition, while a conflicting publication showed platelet activation. We have resolved this conflict using anti-PIA1 IgG produced by a patient with posttransfusion purpura. Relatively low concentrations stimulated platelet aggregation and release of adenosine triphosphate (ATP) whereas high concentrations inhibited platelet function, producing a thrombasthenia-like state. The number of molecules of platelet-associated IgG necessary to initiate aggregation and ATP release (2,086 +/- 556) or produce maximum aggregation (23,420 +/- 3,706) or complete inhibition (63,582 +/- 2654) were measured with a quantitative radiometric assay for bound anti-PIA1. Preincubation of platelets with high concentrations of PIA1 antibody inhibited platelet aggregation with 10 mumol/L adenosine diphosphate and blocked 125I-labeled fibrinogen platelet binding. Platelet activation with nonfibrinogen dependent agonist, 1 U/ml thrombin, was not inhibited by this high concentration of PIA1 IgG. In conclusion, anti-PIAI IgG produces (1) stimulation of platelet aggregation and ATP release that is initiated with 2000 molecules IgG per platelet and is associated with an increase of 125I-fibrinogen binding; (2) conversely, inhibition of platelet aggregation is observed with maximum antibody binding, 63,000 molecules IgG per platelet, and is mediated via a blockade of fibrinogen binding.

  7. Reactive Oxygen Species (ROS) Mediate p300-dependent STAT1 Protein Interaction with Peroxisome Proliferator-activated Receptor (PPAR)-γ in CD36 Protein Expression and Foam Cell Formation.

    PubMed

    Kotla, Sivareddy; Rao, Gadiparthi N

    2015-12-18

    Previously, we have demonstrated that 15(S)-hydroxyeicosatetranoic acid (15(S)-HETE) induces CD36 expression involving STAT1. Many studies have shown that peroxisome proliferator-activated receptor (PPAR)-γ mediates CD36 expression. Therefore, we asked the question whether these transcriptional factors interact with each other in the regulation of CD36 expression by 15(S)-HETE. Here, we show that STAT1 interacts with PPARγ in the induction of CD36 expression and foam cell formation by 15(S)-HETE. In addition, using molecular biological approaches such as EMSA, supershift EMSA, ChIP, re-ChIP, and promoter-reporter gene assays, we demonstrate that the STAT1 and PPARγ complex binds to the STAT-binding site at -107 nucleotides in the CD36 promoter and enhances its activity. Furthermore, the interaction of STAT1 with PPARγ depends on STAT1 acetylation, which is mediated by p300. In addition, our findings show that reactive oxygen species-dependent Syk and Pyk2 stimulation is required for p300 tyrosine phosphorylation and activation. Together, these results demonstrate that an interaction between STAT1, p300, and peroxisome proliferator-activated receptor-γ is required for 15(S)-HETE-induced CD36 expression, oxidized low density lipoprotein uptake, and foam cell formation, critical events underlying the pathogenesis of atherosclerosis. PMID:26504087

  8. Shp-2 contributes to anti-RSV activity in human pulmonary alveolar epithelial cells by interfering with the IFN-α-induced Jak/Stat1 pathway

    PubMed Central

    Wang, Saisai; Zheng, Gang; Zhao, Lifang; Xu, Feng; Qian, Jing

    2015-01-01

    Src homology phosphotyrosyl phosphatase 2 (Shp-2) is a ubiquitously expressed protein that is involved in a variety of cellular processes, including antiviral interferon signalling pathways. In this study, we investigated the role of Shp-2 in the host cell interactions of human respiratory syncytial virus (RSV). We report significant changes in the expression of Shp-2 in human pulmonary alveolar epithelial cells (A549) upon RSV infection. We also report that blocking Shp-2 does not affect viral replication or virus-induced interferon-alpha (IFN-α) production. Interestingly, whereas A549 cells were activated by IFN-α, the blocking of Shp-2 resulted in increased viral replication that was associated with the reduced expression of the IFN-stimulated genes of 2′,5′-oligoadenylate synthetases and Mx1, and the concomitant inhibition of Stat1 tyrosine phosphorylation. Our findings suggest that Shp-2 contributes to the control of RSV replication and progeny production in pulmonary alveolar epithelial cells by interfering with IFN-α-induced Jak/Stat1 pathway activation rather than by affecting the production of IFN-α itself. PMID:26119280

  9. AG490 and PF431396 Sensitive Tyrosine Kinase Control the Population Heterogeneity of Basal STAT1 Activity in Ube1l Deficient Cells

    PubMed Central

    Now, Hesung; Yoo, Joo-Yeon

    2016-01-01

    A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l−/− MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity. PMID:27427993

  10. STAT1β Is Not Dominant Negative and Is Capable of Contributing to Gamma Interferon-Dependent Innate Immunity

    PubMed Central

    Semper, Christian; Leitner, Nicole R.; Lassnig, Caroline; Parrini, Matthias; Mahlakõiv, Tanel; Rammerstorfer, Michael; Lorenz, Karin; Rigler, Doris; Müller, Simone; Kolbe, Thomas; Vogl, Claus; Rülicke, Thomas; Staeheli, Peter; Decker, Thomas; Müller, Mathias

    2014-01-01

    The transcription factor STAT1 is essential for interferon (IFN)-mediated immunity in humans and mice. STAT1 function is tightly regulated, and both loss- and gain-of-function mutations result in severe immune diseases. The two alternatively spliced isoforms, STAT1α and STAT1β, differ with regard to a C-terminal transactivation domain, which is absent in STAT1β. STAT1β is considered to be transcriptionally inactive and to be a competitive inhibitor of STAT1α. To investigate the functions of the STAT1 isoforms in vivo, we generated mice deficient for either STAT1α or STAT1β. As expected, the functions of STAT1α and STAT1β in IFN-α/β- and IFN-λ-dependent antiviral activity are largely redundant. In contrast to the current dogma, however, we found that STAT1β is transcriptionally active in response to IFN-γ. In the absence of STAT1α, STAT1β shows more prolonged IFN-γ-induced phosphorylation and promoter binding. Both isoforms mediate protective, IFN-γ-dependent immunity against the bacterium Listeria monocytogenes, although with remarkably different efficiencies. Our data shed new light on the potential contributions of the individual STAT1 isoforms to STAT1-dependent immune responses. Knowledge of STAT1β's function will help fine-tune diagnostic approaches and help design more specific strategies to interfere with STAT1 activity. PMID:24710278

  11. Anti-inflammatory actions of herbal formula Gyejibokryeong-hwan regulated by inhibiting chemokine production and STAT1 activation in HaCaT cells.

    PubMed

    Jeong, Soo-Jin; Lim, Hye-Sun; Seo, Chang-Seob; Jin, Seong-Eun; Yoo, Sae-Rom; Lee, Nari; Shin, Hyeun-Kyoo

    2015-01-01

    Gyejibokryeong-hwan (GJBRH; Keishi-bukuryo-gan in Japan and Guizhi Fuling Wan in China) is a traditional herbal formula comprising five medicinal herbs and is used to treat climacteric syndrome. GJBRH has been shown to exhibit biological activity against diabetes, diabetic nephropathy, atherosclerosis, ischemia, and cancer. However, there is no scientific evidence of its activities against skin inflammation, including atopic dermatitis. We used the HaCaT human keratinocyte cell line to investigate the effects of GJBRH on skin inflammation. No significant cytotoxicity was observed in cells treated with GJBRH up to a concentration of 1000 µg/mL. Exposure to the proinflammatory cytokines tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) significantly increased HaCaT cell production of the following chemokines: macrophage-derived chemokine (MDC)/CCL22; regulated on activation, normal T-cell expressed and secreted (RANTES)/CCL5; and interleukin-8 (IL-8). In contrast, GJBRH significantly reduced the production of MDC, RANTES, and IL-8 compared with control cells simulated with TNF-α and IFN-γ. Consistently, GJBRH suppressed the mRNA expression of MDC, RANTES, and IL-8 in TNF-α and IFN-γ-treated cells. Treatment with GJBRH markedly inhibited phosphorylation of signal transducer and activator of transcription 1 (STAT1) in HaCaT cells stimulated with TNF-α and IFN-γ. Our findings indicate that GJBRH impairs TNF-α and IFN-γ-mediated inflammatory chemokine production and STAT1 phosphorylation in keratinocytes. We suggest that GJBRH may be a potent therapeutic agent for inflammatory skin disorders. PMID:25757924

  12. Radiosensitization by Inhibiting STAT1 in Renal Cell Carcinoma

    SciTech Connect

    Hui Zhouguang; Tretiakova, Maria; Zhang Zhongfa; Li Yan; Wang Xiaozhen; Zhu, Julie Xiaohong; Gao Yuanhong; Mai Weiyuan; Furge, Kyle; Qian Chaonan; Amato, Robert; Butler, E. Brian

    2009-01-01

    Purpose: Renal cell carcinoma (RCC) has been historically regarded as a radioresistant malignancy, but the molecular mechanism underlying its radioresistance is not understood. This study investigated the role of signal transducer and activator of transcription 1 (STAT1), a transcription factor downstream of the interferon-signaling pathway, in radioresistant RCC. Methods and Materials: The expressions of STAT1 and STAT3 in 164 human clear cell RCC samples, 47 papillary RCC samples, and 15 normal kidney tissue samples were examined by microarray expression profiling and immunohistochemistry. Western blotting was performed to evaluate the total and phosphorylated STAT1 expression in CRL-1932 (786-O) (human clear cell RCC), SKRC-39 (human papillary RCC), CCL-116 (human fibroblast), and CRL-1441 (G-401) (human Wilms tumor). STAT1 was reduced or inhibited by fludarabine and siRNA, respectively, and the effects on radiation-induced cell death were investigated using clonogenic assays. Results: STAT1 expression, but not STAT3 expression, was significantly greater in human RCC samples (p = 1.5 x 10{sup -8} for clear cell; and p = 3.6 x 10{sup -4} for papillary). Similarly, the expression of STAT1 was relatively greater in the two RCC cell lines. STAT1 expression was reduced by both fludarabine and siRNA, significantly increasing the radiosensitivity in both RCC cell lines. Conclusion: This is the first study reporting the overexpression of STAT1 in human clear cell and papillary RCC tissues. Radiosensitization in RCC cell lines was observed by a reduction or inhibition of STAT1 signaling, using fludarabine or siRNA. Our data suggest that STAT1 may play a key role in RCC radioresistance and manipulation of this pathway may enhance the efficacy of radiotherapy.

  13. PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status.

    PubMed

    Ledsaak, Marit; Bengtsen, Mads; Molværsmyr, Ann-Kristin; Fuglerud, Bettina Maria; Matre, Vilborg; Eskeland, Ragnhild; Gabrielsen, Odd Stokke

    2016-05-01

    The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a "protein inhibitor of activated c-Myb" in the absence of SUMOylation while, in its presence, PIAS behaves as a "protein activator of repressed c-Myb". PMID:27032383

  14. Comparative proteomic analyses demonstrate enhanced interferon and STAT-1 activation in reovirus T3D-infected HeLa cells

    PubMed Central

    Ezzati, Peyman; Komher, Krysten; Severini, Giulia; Coombs, Kevin M.

    2015-01-01

    As obligate intracellular parasites, viruses are exclusively and intimately dependent upon their host cells for replication. During replication viruses induce profound changes within cells, including: induction of signaling pathways, morphological changes, and cell death. Many such cellular perturbations have been analyzed at the transcriptomic level by gene arrays and recent efforts have begun to analyze cellular proteomic responses. We recently described comparative stable isotopic (SILAC) analyses of reovirus, strain type 3 Dearing (T3D)-infected HeLa cells. For the present study we employed the complementary labeling strategy of iTRAQ (isobaric tags for relative and absolute quantitation) to examine HeLa cell changes induced by T3D, another reovirus strain, type 1 Lang, and UV-inactivated T3D (UV-T3D). Triplicate replicates of cytosolic and nuclear fractions identified a total of 2375 proteins, of which 50, 57, and 46 were significantly up-regulated, and 37, 26, and 44 were significantly down-regulated by T1L, T3D, and UV-T3D, respectively. Several pathways, most notably the Interferon signaling pathway and the EIF2 and ILK signaling pathways, were induced by virus infection. Western blots confirmed that cells were more strongly activated by live T3D as demonstrated by elevated levels of key proteins like STAT-1, ISG-15, IFIT-1, IFIT-3, and Mx1. This study expands our understanding of reovirus-induced host responses. PMID:25905045

  15. Inducible, Dose-Adjustable and Time-Restricted Reconstitution of Stat1 Deficiency In Vivo

    PubMed Central

    Leitner, Nicole R.; Lassnig, Caroline; Rom, Rita; Heider, Susanne; Bago-Horvath, Zsuzsanna; Eferl, Robert; Müller, Simone; Kolbe, Thomas; Kenner, Lukas; Rülicke, Thomas; Strobl, Birgit; Müller, Mathias

    2014-01-01

    Signal transducer and activator of transcription (STAT) 1 is a key player in interferon (IFN) signaling, essential in mediating host defense against viruses and other pathogens. STAT1 levels are tightly regulated and loss- or gain-of-function mutations in mice and men lead to severe diseases. We have generated a doxycycline (dox) -inducible, FLAG-tagged Stat1 expression system in mice lacking endogenous STAT1 (i.e. Stat1ind mice). We show that STAT1 expression depends on the time and dose of dox treatment in primary cells and a variety of organs isolated from Stat1ind mice. In bone marrow-derived macrophages, a fraction of the amount of STAT1 present in WT cells is sufficient for full expression of IFN-induced genes. Dox-induced STAT1 established protection against virus infections in primary cells and mice. The availability of the Stat1ind mouse model will enable an examination of the consequences of variable amounts of STAT1. The model will also permit the study of STAT1 dose-dependent and reversible functions as well as of STAT1's contributions to the development, progression and resolution of disease. PMID:24489749

  16. Despite Increased Type 1 IFN, Autoimmune Nonobese Diabetic Mice Display Impaired Dendritic Cell Response to CpG and Decreased Nuclear Localization of IFN-Activated STAT1.

    PubMed

    Rahman, M Jubayer; Rahir, Gwendoline; Dong, Matthew B; Zhao, Yongge; Rodrigues, Kameron B; Hotta-Iwamura, Chie; Chen, Ye; Guerrero, Alan; Tarbell, Kristin V

    2016-03-01

    Innate immune signals help break self-tolerance to initiate autoimmune diseases such as type 1 diabetes, but innate contributions to subsequent regulation of disease progression are less clear. Most studies have measured in vitro innate responses of GM-CSF dendritic cells (DCs) that are functionally distinct from conventional DCs (cDCs) and do not reflect in vivo DC subsets. To determine whether autoimmune NOD mice have alterations in type 1 IFN innate responsiveness, we compared cDCs from prediabetic NOD and control C57BL/6 (B6) mice stimulated in vivo with the TLR9 ligand CpG, a strong type 1 IFN inducer. In response to CpG, NOD mice produce more type 1 IFN and express higher levels of CD40, and NOD monocyte DCs make more TNF. However, the overall CpG-induced transcriptional response is muted in NOD cDCs. Of relevance the costimulatory proteins CD80/CD86, signals needed for regulatory T cell homeostasis, are upregulated less on NOD cDCs. Interestingly, NOD Rag1(-/-) mice also display a defect in CpG-induced CD86 upregulation compared with B6 Rag1(-/-), indicating this particular innate alteration precedes adaptive autoimmunity. The impaired response in NOD DCs is likely downstream of the IFN-α/β receptor because DCs from NOD and B6 mice show similar CpG-induced CD86 levels when anti-IFN-α/β receptor Ab is added. IFN-α-induced nuclear localization of activated STAT1 is markedly reduced in NOD CD11c(+) cells, consistent with lower type 1 IFN responsiveness. In conclusion, NOD DCs display altered innate responses characterized by enhanced type 1 IFN and activation of monocyte-derived DCs but diminished cDC type 1 IFN response. PMID:26826238

  17. The activating effect of IFN-γ on monocytes/macrophages is regulated by the LIF–trophoblast–IL-10 axis via Stat1 inhibition and Stat3 activation

    PubMed Central

    Dallagi, Angham; Girouard, Julie; Hamelin-Morrissette, Jovane; Dadzie, Rachel; Laurent, Laetitia; Vaillancourt, Cathy; Lafond, Julie; Carrier, Christian; Reyes-Moreno, Carlos

    2015-01-01

    Interferon gamma (IFN-γ) and leukemia inhibitory factor (LIF) are key gestational factors that may differentially affect leukocyte function during gestation. Because IFN-γ induces a pro-inflammatory phenotype in macrophages and because trophoblast cells are principal targets of LIF in the placenta, we investigated whether and how soluble factors from trophoblast cells regulate the effects of IFN-γ on macrophage activation. IFN-γ reduces macrophage motility, but enhances Stat1 activation, pro-inflammatory gene expression and cytotoxic functions. Soluble factors from villous cytotrophoblasts (vCT+LIF cells) and BeWo cells (BW/ST+LIF cells) that were differentiated in the presence of LIF inhibit macrophage Stat1 activation but inversely sustain Stat3 activation in response to IFN-γ. vCT+LIF cells produce soluble factors that induce Stat3 activation; this effect is partially abrogated in the presence of neutralizing anti-interleukin 10 (IL-10) antibodies. Moreover, soluble factors from BW/ST+LIF cells reduce cell proliferation but enhance the migratory responses of monocytes. In addition, these factors reverse the inhibitory effect of IFN-γ on monocyte/macrophage motility. BW/ST+LIF cells also generate IFN-γ-activated macrophages with enhanced IL-10 expression, but reduced tumor-necrosis factor alpha (TNF-α), CD14 and CD40 expression as well as impaired cytotoxic function. Additional assays performed in the presence of neutralizing anti-IL-10 antibodies and exogenous IL-10 demonstrate that reduced macrophage cytotoxicity and proliferation, but increased cell motility result from the ability of trophoblast IL-10 to sustain Stat3 activation and suppress IFN-γ-induced Stat1 activation. These in vitro studies are the first to describe the regulatory role of the LIF–trophoblast–IL-10 axis in the process of macrophage activation in response to pro-inflammatory cytokines. PMID:25027966

  18. The activating effect of IFN-γ on monocytes/macrophages is regulated by the LIF-trophoblast-IL-10 axis via Stat1 inhibition and Stat3 activation.

    PubMed

    Dallagi, Angham; Girouard, Julie; Hamelin-Morrissette, Jovane; Dadzie, Rachel; Laurent, Laetitia; Vaillancourt, Cathy; Lafond, Julie; Carrier, Christian; Reyes-Moreno, Carlos

    2015-05-01

    Interferon gamma (IFN-γ) and leukemia inhibitory factor (LIF) are key gestational factors that may differentially affect leukocyte function during gestation. Because IFN-γ induces a pro-inflammatory phenotype in macrophages and because trophoblast cells are principal targets of LIF in the placenta, we investigated whether and how soluble factors from trophoblast cells regulate the effects of IFN-γ on macrophage activation. IFN-γ reduces macrophage motility, but enhances Stat1 activation, pro-inflammatory gene expression and cytotoxic functions. Soluble factors from villous cytotrophoblasts (vCT+LIF cells) and BeWo cells (BW/ST+LIF cells) that were differentiated in the presence of LIF inhibit macrophage Stat1 activation but inversely sustain Stat3 activation in response to IFN-γ. vCT+LIF cells produce soluble factors that induce Stat3 activation; this effect is partially abrogated in the presence of neutralizing anti-interleukin 10 (IL-10) antibodies. Moreover, soluble factors from BW/ST+LIF cells reduce cell proliferation but enhance the migratory responses of monocytes. In addition, these factors reverse the inhibitory effect of IFN-γ on monocyte/macrophage motility. BW/ST+LIF cells also generate IFN-γ-activated macrophages with enhanced IL-10 expression, but reduced tumor-necrosis factor alpha (TNF-α), CD14 and CD40 expression as well as impaired cytotoxic function. Additional assays performed in the presence of neutralizing anti-IL-10 antibodies and exogenous IL-10 demonstrate that reduced macrophage cytotoxicity and proliferation, but increased cell motility result from the ability of trophoblast IL-10 to sustain Stat3 activation and suppress IFN-γ-induced Stat1 activation. These in vitro studies are the first to describe the regulatory role of the LIF-trophoblast-IL-10 axis in the process of macrophage activation in response to pro-inflammatory cytokines. PMID:25027966

  19. Inborn errors of human STAT1: allelic heterogeneity governs the diversity of immunological and infectious phenotypes

    PubMed Central

    Boisson-Dupuis, Stephanie; Kong, Xiao-Fei; Okada, Satoshi; Cypowyj, Sophie; Puel, Anne; Abel, Laurent; Casanova, Jean-Laurent

    2012-01-01

    The genetic dissection of various human infectious diseases has led to the definition of inborn errors of human STAT1 immunity of four types, including (i) autosomal recessive (AR) complete STAT1 deficiency, (ii) AR partial STAT1 deficiency, (iii) autosomal dominant (AD) STAT1 deficiency, and (iv) AD gain of STAT1 activity. The two types of AR STAT1 defect give rise to a broad infectious phenotype with susceptibility to intramacrophagic bacteria (mostly mycobacteria) and viruses (herpes viruses at least), due principally to the impairment of IFN-γ-mediated and IFN-α/β-mediated immunity, respectively. Clinical outcome depends on the extent to which the STAT1 defect decreases responsiveness to these cytokines. AD STAT1 deficiency selectively predisposes individuals to mycobacterial disease, owing to the impairment of IFN-γ-mediated immunity, as IFN-α/β-mediated immunity is maintained. Finally, AD gain of STAT1 activity is associated with autoimmunity, probably owing to an enhancement of IFN-α/β-mediated immunity. More surprisingly, it is also associated with chronic mucocutaneous candidiasis, through as yet undetermined mechanisms involving an inhibition of the development of IL-17-producing T cells. Thus, germline mutations in human STAT1 define four distinct clinical disorders. Various combinations of viral, mycobacterial and fungal infections are therefore allelic at the human STAT1 locus. These experiments of Nature neatly highlight the clinical and immunological impact of the human genetic dissection of infectious phenotypes. PMID:22651901

  20. STAT1 Pathway Mediates Amplification of Metastatic Potential and Resistance to Therapy

    PubMed Central

    Pitroda, Sean P.; Golden, Daniel W.; Bhayani, Mihir; Shao, Michael Y.; Darga, Thomas E.; Beveridge, Mara G.; Sood, Ravi F.; Sutton, Harold G.; Beckett, Michael A.; Mauceri, Helena J.; Posner, Mitchell C.; Weichselbaum, Ralph R.

    2009-01-01

    Background Traditionally IFN/STAT1 signaling is connected with an anti-viral response and pro-apoptotic tumor-suppressor functions. Emerging functions of a constitutively activated IFN/STAT1 pathway suggest an association with an aggressive tumor phenotype. We hypothesized that tumor clones that constitutively overexpress this pathway are preferentially selected by the host microenvironment due to a resistance to STAT1-dependent cytotoxicity and demonstrate increased metastatic ability combined with increased resistance to genotoxic stress. Methodology/Principal Findings Here we report that clones of B16F1 tumors grown in the lungs of syngeneic C57BL/6 mice demonstrate variable transcriptional levels of IFN/STAT1 pathway expression. Tumor cells that constitutively overexpress the IFN/STAT1 pathway (STAT1H genotype) are selected by the lung microenvironment. STAT1H tumor cells also demonstrate resistance to IFN-gamma (IFNγ), ionizing radiation (IR), and doxorubicin relative to parental B16F1 and low expressors of the IFN/STAT1 pathway (STAT1L genotype). Stable knockdown of STAT1 reversed the aggressive phenotype and decreased both lung colonization and resistance to genotoxic stress. Conclusions Our results identify a pathway activated by tumor-stromal interactions thereby selecting for pro-metastatic and therapy-resistant tumor clones. New therapies targeted against the IFN/STAT1 signaling pathway may provide an effective strategy to treat or sensitize aggressive tumor clones to conventional cancer therapies and potentially prevent distant organ colonization. PMID:19503789

  1. Anti-inflammatory activity of myricetin from Diospyros lotus through suppression of NF-κB and STAT1 activation and Nrf2-mediated HO-1 induction in lipopolysaccharide-stimulated RAW264.7 macrophages.

    PubMed

    Cho, Byoung Ok; Yin, Hong Hua; Park, Sang Hyun; Byun, Eui Baek; Ha, Hun Yong; Jang, Seon Il

    2016-08-01

    Diospyros lotus is traditionally used for the treatment of diabetes, diarrhea, tumor, and hypertension. The purpose of this study was to investigate the anti-inflammatory effect and underlying molecular mechanisms of myricetin in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Myricetin dose-dependently suppressed the production of pro-inflammatory mediators (NO, iNOS, PGE2, and COX-2) in LPS-stimulated RAW264.7 macrophages. Myricetin administration decreased the production of NO, iNOS, TNF-α, IL-6, and IL-12 in mice. Myricetin decreased NF-κB activation by suppressing the degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB DNA binding activity in LPS-stimulated RAW264.7 macrophages. Moreover, myricetin attenuated the phosphorylation of STAT1 and the production of IFN-β in LPS-stimulated RAW264.7 macrophages. Furthermore, myricetin induced the expression of HO-1 through Nrf2 translocation. In conclusion, these results suggest that myricetin inhibits the production of pro-inflammatory mediators through the suppression of NF-κB and STAT1 activation and induction of Nrf2-mediated HO-1 expression in LPS-stimulated RAW264.7 macrophages. PMID:27068250

  2. Infection with the Persistent Murine Norovirus Strain MNV-S99 Suppresses IFN-Beta Release and Activation of Stat1 In Vitro

    PubMed Central

    Niendorf, Sandra; Klemm, Uwe; Mas Marques, Andreas; Bock, C.-Thomas; Höhne, Marina

    2016-01-01

    Norovirus infection is the main cause of epidemic non-bacterial gastroenteritis in humans. Although human norovirus (HuNoV) infection is self-limiting, it can persist for extended periods of time in immune deficient patients. Due to the lack of robust cell culture and small animal systems, little is known about HuNoV pathogenicity. However, murine norovirus (MNV) can be propagated in cell culture and is used as a model to study norovirus infection. Several MNV are known to persist in mice. In this study, we show that the MNV strain MNV-S99 persists in wild type inbred (C57BL/6J) mice over a period of at least 5 weeks post infection. Viral RNA was detectable in the jejunum, ileum, cecum, and colon, with the highest titers in the colon and cecum. To characterize the effect of MNV-S99 on the innate immune response, Stat1 phosphorylation and IFN-β production were analyzed and compared to the non-persistent strain MNV-1.CW3. While MNV-S99 and MNV-1.CW3 showed comparable growth characteristics in vitro, Stat1 phosphorylation and IFN-β release is strongly decreased after infection with MNV-S99 compared to MNV-1.CW3. In conclusion, our results show that unlike MNV-1.CW3, MNV-S99 establishes a persistent infection in mice, possibly due to interfering with the innate immune response. PMID:27294868

  3. Stat1 Nuclear Translocation by Nucleolin upon Monocyte Differentiation

    PubMed Central

    Jerke, Uwe; Tkachuk, Sergey; Kiyan, Julia; Stepanova, Victoria; Kusch, Angelika; Hinz, Michael; Dietz, Rainer; Haller, Hermann; Fuhrman, Bianca; Dumler, Inna

    2009-01-01

    Background Members of the signal transducer and activator of transcription (Stat) family of transcription factors traverse the nuclear membrane through a specialized structure, called the nuclear pore complex (NPC), which represents a selective filter for the import of proteins. Karyophilic molecules can bind directly to a subset of proteins of the NPC, collectively called nucleoporins. Alternatively, the transport is mediated via a carrier molecule belonging to the importin/karyopherin superfamily, which transmits the import into the nucleus through the NPC. Methodology/Principal Findings In this study, we provide evidence for an alternative Stat1 nuclear import mechanism, which is mediated by the shuttle protein nucleolin. We observed Stat1-nucleolin association, nuclear translocation and specific binding to the regulatory DNA element GAS. Using expression of nucleolin transgenes, we found that the nuclear localization signal (NLS) of nucleolin is responsible for Stat1 nuclear translocation. We show that this mechanism is utilized upon differentiation of myeloid cells and is specific for the differentiation step from monocytes to macrophages. Conclusions/Significance Our data add the nucleolin-Stat1 complex as a novel functional partner for the cell differentiation program, which is uniquely poised to regulate the transcription machinery via Stat1 and nuclear metabolism via nucleolin. PMID:20011528

  4. Bcl6 promotes osteoblastogenesis through Stat1 inhibition

    SciTech Connect

    Fujie, Atsuhiro; Funayama, Atsushi; Miyauchi, Yoshiteru; Sato, Yuiko; Kobayashi, Tami; Kanagawa, Hiroya; Katsuyama, Eri; Hao, Wu; Tando, Toshimi; Watanabe, Ryuichi; Morita, Mayu; Miyamoto, Kana; Kanaji, Arihiko; Morioka, Hideo; Matsumoto, Morio; Toyama, Yoshiaki; Miyamoto, Takeshi

    2015-02-13

    Bone mass is tightly controlled by a balance between osteoclast and osteoblast activities. Although these cell types mature via different pathways, some factors reportedly regulate differentiation of both. Here, in a search for factors governing osteoblastogenesis but also expressed in osteoclasts to control both cell types by one molecule, we identified B cell lymphoma 6 (Bcl6) as one of those factors and show that it promotes osteoblast differentiation. Bcl6 was previously shown to negatively regulate osteoclastogenesis. We report that lack of Bcl6 results in significant inhibition of osteoblastogensis in vivo and in vitro and in defects in secondary ossification center formation in vivo. Signal transducer and activator of transcription 1 (Stat1) reportedly attenuates osteoblast differentiation by inhibiting nuclear translocation of runt-related transcription factor 2 (Runx2), which is essential for osteoblast differentiation. We found that lack of Bcl6 resulted in significant elevation of Stat1 mRNA and protein expression in osteoblasts and showed that Stat1 is a direct target of Bcl6 using a chromatin immune-precipitation assay. Mice lacking both Bcl6 and Stat1 (DKO) exhibited significant rescue of bone mass and osteoblastic parameters as well as partial rescue of secondary ossification center formation compared with Bcl6-deficient mice in vivo. Altered osteoblastogenesis in Bcl6-deficient cells was also restored in DKO in vitro. Thus, Bcl6 plays crucial roles in regulating both osteoblast activation and osteoclast inhibition. - Highlights: • Bcl6 is required for osteoblast differentiation. • Bcl6{sup −/−} mice exhibited altered osteoblastogenesis and reduced bone mass in vivo and in vitro. • We identified Stat1 as a direct target of Bcl6 in osteoblasts. • Bcl6 and Stat1 doubly deficient mice exhibited rescued bone phenotypes compared with Bcl6{sup −/−} mice.

  5. STAT1-S727 - the license to kill

    PubMed Central

    Putz, Eva M.; Gotthardt, Dagmar; Sexl, Veronika

    2014-01-01

    Serine phosphorylation has generally been considered indispensable for full transcriptional activity of STAT proteins. Recent data indicate that CDK8-mediated phosphorylation of signal transducer and activator of transcription 1 (STAT1) on S727 inhibits natural killer (NK) cell cytotoxicity and restrains tumor surveillance. These findings implicate CDK8 as a promising target for immunotherapy. PMID:25941617

  6. The role of stat1b in zebrafish hematopoiesis

    PubMed Central

    Song, Hao; Yan, Yi-lin; Titus, Tom; He, Xinjun; Postlethwait, John H.

    2011-01-01

    STAT1 mediates response to interferons and regulates immunity, cell proliferation, apoptosis, and sensitivity of Fanconi Anemia cells to apoptosis after interferon signaling; the roles of STAT1 in embryos, however, are not understood. To explore embryonic functions of STAT1, we investigated stat1b, an unstudied zebrafish co-ortholog of human STAT1. Zebrafish stat1a encodes all five domains of the human STAT1-alpha splice form but, like the human STAT1-beta splice variant, stat1b lacks a complete transactivation domain; thus, two unlinked zebrafish paralogs encode protein forms translated from two splice variants of a single human gene, as expected by subfunctionalization after genome duplication. Phylogenetic and conserved synteny studies showed that stat1b and stat1a arose as duplicates in the teleost genome duplication (TGD) and clarified the evolutionary origin of STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 by tandem and genome duplication. RT-PCR revealed maternal expression of stat1a and stat1b. In situ hybridization detected stat1b but not stat1a expression in embryonic hematopoietic tissues. Morpholino knockdown of stat1b, but not stat1a, decreased expression of the myeloid and granulocyte markers spi and mpo and increased expression of the hematopoietic progenitor marker scl, the erythrocyte marker gata1, and hemoglobin. These results suggest that zebrafish Stat1b promotes myeloid development at the expense of erythroid development. PMID:21914475

  7. The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARα/STAT1 axis.

    PubMed

    Shao, Xuejing; Liu, Yujia; Li, Yangling; Xian, Miao; Zhou, Qian; Yang, Bo; Ying, Meidan; He, Qiaojun

    2016-01-01

    The success of all-trans retinoic acid (ATRA) in differentiation therapy for patients with acute promyelocytic leukemia (APL) highly encourages researches to apply this therapy to other types of acute myeloid leukemia (AML). However, AML, with the exception of APL, fails to respond to differentiation therapy. Therefore, research strategies to further sensitize cells to retinoids and to extend the range of AMLs that respond to retinoids beyond APLs are urgently needed. In this study, we showed that TAK165, a HER2 inhibitor, exhibited a strong synergy with ATRA to promote AML cell differentiation. We observed that TAK165 sensitized the AML cells to ATRA-induced cell growth inhibition, G0/G1 phase arrest, CD11b expression, mature morphologic changes, NBT reduction and myeloid regulator expression. Unexpectedly, HER2 pathway might not be essential for TAK165-enhanced differentiation when combined with ATRA, while the enhanced differentiation was dependent on the activation of the RARα/STAT1 axis. Furthermore, the MEK/ERK cascade regulated the activation of STAT1. Taken together, our study is the first to evaluate the synergy of TAK165 and ATRA in AML cell differentiation and to assess new opportunities for the combination of TAK165 and ATRA as a promising approach for future differentiation therapy. PMID:27074819

  8. A crosstalk triggered by hypoxia and maintained by MCP-1/miR-98/IL-6/p38 regulatory loop between human aortic smooth muscle cells and macrophages leads to aortic smooth muscle cells apoptosis via Stat1 activation.

    PubMed

    Wang, Qing; Shu, Chang; Su, Jing; Li, Xin

    2015-01-01

    Hypoxia and inflammation are central characteristics of the abdominal aortic aneurysm (AAA), but the mechanisms for their relationship and actual role remain far from full understood. Here, we showed MCP-1 (monocyte chemotactic protein-1) induced by hypoxia in primary human Aortic Smooth Muscle Cells (hASMCs) increased the chemotaxis of THP-1 macrophages and MCP-1 induced IL-6 expression in THP-1 cells via downregulating miR-98 which directly targets IL-6. In addition, IL-6 positively feedback regulated MCP-1 expression in hASMCs via p38 signal that is independent on hypoxia, and inhibition of p38 signal blocked the effect of IL-6 on MCP-1 expression regulation. Moreover, IL-6 exposure time-dependently induces phASMCs apoptosis via Stat1 activation. Collectively, our data provide compelling evidence on the association between hypoxia and inflammation triggered by hypoxia and then mediated by MCP-1/miR-98/IL-6/p38 regulatory loop, which leads to hASMCs apoptosis via Stat1 activation to contribute to AAA formation and progression. PMID:26045772

  9. The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARα/STAT1 axis

    PubMed Central

    Shao, Xuejing; Liu, Yujia; Li, Yangling; Xian, Miao; Zhou, Qian; Yang, Bo; Ying, Meidan; He, Qiaojun

    2016-01-01

    The success of all-trans retinoic acid (ATRA) in differentiation therapy for patients with acute promyelocytic leukemia (APL) highly encourages researches to apply this therapy to other types of acute myeloid leukemia (AML). However, AML, with the exception of APL, fails to respond to differentiation therapy. Therefore, research strategies to further sensitize cells to retinoids and to extend the range of AMLs that respond to retinoids beyond APLs are urgently needed. In this study, we showed that TAK165, a HER2 inhibitor, exhibited a strong synergy with ATRA to promote AML cell differentiation. We observed that TAK165 sensitized the AML cells to ATRA-induced cell growth inhibition, G0/G1 phase arrest, CD11b expression, mature morphologic changes, NBT reduction and myeloid regulator expression. Unexpectedly, HER2 pathway might not be essential for TAK165-enhanced differentiation when combined with ATRA, while the enhanced differentiation was dependent on the activation of the RARα/STAT1 axis. Furthermore, the MEK/ERK cascade regulated the activation of STAT1. Taken together, our study is the first to evaluate the synergy of TAK165 and ATRA in AML cell differentiation and to assess new opportunities for the combination of TAK165 and ATRA as a promising approach for future differentiation therapy. PMID:27074819

  10. JLK1486, a Bis 8-Hydroxyquinoline-Substituted Benzylamine, Displays Cytostatic Effects in Experimental Gliomas through MyT1 and STAT1 Activation and, to a Lesser Extent, PPARγ Activation12

    PubMed Central

    Bruyère, Céline; Madonna, Sébastien; Van Goietsenoven, Gwendoline; Mathieu, Véronique; Dessolin, Jean; Kraus, Jean-Louis; Lefranc, Florence; Kiss, Robert

    2011-01-01

    Gliomas account for 5% to 7% of all solid cancers in adults and up to 30% of solid cancers in children; glioblastomas are the most malignant type of glioma and often have dismal prognoses. The alkylating agent temozolomide provides the greatest chemotherapeutic benefits currently available; however, glioblastoma patients cannot be cured. Novel drugs that efficiently combat glioblastomas are therefore of great interest. We report here that JLK1486, an 8-hydroxyquinoline-substituted benzylamine, could represent a novel chemical scaffold to reach this goal. Indeed, JLK1486 mediated anticancer activity in vivo (through intravenous as well as oral routes of administrations) in an orthotopic xenograft model and displayed efficiency similar to that of temozolomide. The therapeutic benefits of JLK1486 seem to relate to its ability to activate various transcription factors (including Myt1, STAT1, and peroxisome proliferator-activated receptor γ) in glioma cells. These transcription factors are implicated in the control of glioma cell proliferation, and the resultant global effect of their activation by JLK1486 was cytostatic, not cytotoxic. Thus, the current study opens the door for the development of novel compounds to combat glioblastoma using 8-hydroxyquinoline benzylamine analogs. PMID:21633668

  11. The measles virus phosphoprotein interacts with the linker domain of STAT1

    SciTech Connect

    Devaux, Patricia Priniski, Lauren; Cattaneo, Roberto

    2013-09-15

    The measles virus (MV) phosphoprotein (P) and V proteins block the interferon (IFN) response by impeding phosphorylation of the signal transducer and activator of transcription 1 (STAT1) by the Janus kinase 1 (JAK1). We characterized how STAT1 mutants interact with P and JAK1 phosphorylation. Certain mutants of the linker, the Src-homology 2 domain (SH2), or the transactivation domain had reduced or abolished phosphorylation through JAK1 after IFN treatment. Other mutants, mainly localized in the linker, failed to interact with P as documented by the lack of interference with nuclear translocation. Thus the functional footprint of P on STAT1 localizes mainly to the linker domain; there is also some overlap with the STAT1 phosphorylation functional footprint on the SH2 domain. Based on these observations, we discuss how the MV-P might operate to inhibit the JAK/STAT pathway. - Highlights: • Residue in the linker and SH2 domains of STAT1 are important for MV-P interaction. • Residue in the linker and SH2 domains of STAT1 are important for STAT1 phosphorylation. • Residues interferring with both functions have similar location on STAT1. • The viral P and V proteins may operate in concert to inhibit the JAK/STAT pathway.

  12. Luteolin inhibits viral-induced inflammatory response in RAW264.7 cells via suppression of STAT1/3 dependent NF-κB and activation of HO-1.

    PubMed

    Liu, Cheng-Wei; Lin, Hui-Wen; Yang, Deng-Jye; Chen, Shih-Yin; Tseng, Jung-Kai; Chang, Tien-Jye; Chang, Yuan-Yen

    2016-06-01

    Luteolin is a common dietary flavonoid present in Chinese herbal medicines that has been reported to have important anti-inflammatory properties. Previous studies have shown that luteolin is an anti-inflammatory and anti-oxidative agent. In this study, the anti-virus inflammatory capacity of luteolin and its molecular mechanisms of action were analyzed. The cytotoxic effects of luteolin were assessed in the presence or absence of pseudorabies virus (PRV) via LDH and MTT assays. The results showed that luteolin (<10μM) had no toxic effects and there were tendencies toward higher cell survival. In PRV-infected RAW264.7 cells, luteolin potently inhibited the production of NO, iNOS, COX-2 and inflammatory cytokine production. Luteolin did not inhibit the phosphorylation of ERK 1/2, p38, and JNK 1/2 either. We found that PRV-induced NF-κB activation is regulated through inhibition of STAT1and STAT3 phosphorylation in response to luteolin. Additionally, luteolin caused the induction of HO-1 via upregulation of Nrf2, both of which are involved in the secretion of proinflammatory mediators. The blockade of HO-1 expression with SnPP, a HO-1 inhibitor, attenuated HO-1 induction by luteolin and thus mitigated its anti-inflammatory effects during PRV-infected RAW264.7 cells. Taken together, our data indicate that luteolin diminishes the proinflammatory mediators NO, inflammatory cytokines and the expression of their regulatory genes, iNOS and COX-2, in PRV-infected RAW264.7 cells by inhibiting STAT1/3 dependent NF-κB activation and inducing Nrf2mediated HO-1 expression. PMID:27016074

  13. Molecular cloning and expression analysis of the STAT1 gene from olive flounder, Paralichthys olivaceus

    PubMed Central

    Park, Eun-Mi; Kang, Jung-Ha; Seo, Jung Soo; Kim, GunDo; Chung, Jongkyeong; Choi, Tae-Jin

    2008-01-01

    Background Signal transducer and activator of transcription 1 (STAT1) is a critical component of interferon (IFN)-alpha/beta and IFN-gamma signaling. Although seven isoforms of STAT proteins have been reported from mammals, limited information is available for the STAT genes in fish. We isolated complementary DNA with high similarity to mammalian STAT1 from the olive flounder, Paralichthys olivaceus. Results A DNA fragment containing the conserved SH2 domain was amplified by RT-PCR using degenerate primers designed based on the highly conserved sequences in the SH2 domains of the zebrafish and mammalian STAT1. The complete cDNA sequence was obtained by 5' and 3' RACE. The flounder STAT1 transcript consisted of 2,909 bp that encoded a polypeptide of 749 amino acids. The overall similarity between flounder STAT1 and other STATs was very high, with the highest amino acid sequence identity to snakehead (89%). Phylogenetic analyses reveal that flounder STAT1 is in the same monophyletic group with snakehead STAT1. Quantitative real time RT-PCR and in situ hybridization revealed that STAT1 was expressed in almost all examined organs and tissues, with high expression in gill, spleen, kidney, and heart. The accumulation of STAT1 mRNA in different developmental stages, as determined by real time RT-PCR, increased with development. Conclusion Recent cloning of various cytokine genes and the STAT1 gene of olive flounder here suggest that fish also use the highly specialized JAK-STAT pathway for cytokine signaling. Identification of other STAT genes will elucidate in detail the signal transduction system in this fish. PMID:18578892

  14. Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling.

    PubMed

    Frank, D A; Mahajan, S; Ritz, J

    1999-04-01

    Fludarabine is a nucleoside analog used in the treatment of hematologic malignancies that can induce severe and prolonged immunosuppression. Although it can be incorporated into the DNA of dividing cells, fludarabine is also a potent inhibitor of cells with a low growth fraction, thus it must have other mechanisms of action. STAT1, which is activated in response to many lymphocyte-activating cytokines including the interferons, is essential for cell-mediated immunity, as the absence of this protein is associated with prominent defects in the ability to control viral infections. Here we show that fludarabine, but not the immunosuppressant cyclosporine A, inhibits the cytokine-induced activation of STAT1 and STAT1-dependent gene transcription in normal resting or activated lymphocytes. Fludarabine caused a specific depletion of STAT1 protein (and mRNA) but not of other STATs. This loss of STAT1 was also seen in cells from patients treated with fludarabine in vivo. Brief exposure to fludarabine led to a sustained loss of STAT1, analogous to the prolonged period of immunosuppression induced by exposure to the drug in vivo. Thus, STAT1 may be a useful target in the development of new immunosuppressive and antineoplastic agents. PMID:10202937

  15. Opposing roles of STAT1 and STAT3 in IL-21 function in CD4+ T cells.

    PubMed

    Wan, Chi-Keung; Andraski, Allison B; Spolski, Rosanne; Li, Peng; Kazemian, Majid; Oh, Jangsuk; Samsel, Leigh; Swanson, Phillip A; McGavern, Dorian B; Sampaio, Elizabeth P; Freeman, Alexandra F; Milner, Joshua D; Holland, Steven M; Leonard, Warren J

    2015-07-28

    IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4(+) T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4(+) T cells. RNA-Seq analysis of CD4(+) T cells from Stat1- and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4(+) T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4(+) T cells from patients with autosomal dominant hyper-IgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions. PMID:26170288

  16. Stat1 negatively regulates immune-mediated injury with Anaplasma phagocytophilum infection1

    PubMed Central

    Choi, Kyoung-Seong; Scorpio, Diana G.; Dumler, J. Stephen

    2014-01-01

    Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium, Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFNγ production through the signal transducer and activation of transcription 1 (Stat1) signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knock out (KO) compared to wild type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated pro-inflammatory cytokine/chemokine responses in serum. CD4+ and CD8+ T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type (WT) mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocytes/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of iNOS in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection. PMID:25305312

  17. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    NASA Astrophysics Data System (ADS)

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-03-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction.

  18. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging.

    PubMed

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-01-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114

  19. Revealing the cellular localization of STAT1 during the cell cycle by super-resolution imaging

    PubMed Central

    Gao, Jing; Wang, Feng; Liu, Yanhou; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Wang, Hongda

    2015-01-01

    Signal transducers and activators of transcription (STATs) can transduce cytokine signals and regulate gene expression. The cellular localization and nuclear trafficking of STAT1, a representative of the STAT family with multiple transcriptional functions, is tightly related with transcription process, which usually happens in the interphase of the cell cycle. However, these priority questions regarding STAT1 distribution and localization at the different cell-cycle stages remain unclear. By using direct stochastic optical reconstruction microscopy (dSTORM), we found that the nuclear expression level of STAT1 increased gradually as the cell cycle carried out, especially after EGF stimulation. Furthermore, STAT1 formed clusters in the whole cell during the cell cycle, with the size and the number of clusters also increasing significantly from G1 to G2 phase, suggesting that transcription and other cell-cycle related activities can promote STAT1 to form more and larger clusters for fast response to signals. Our work reveals that the cellular localization and clustering distribution of STAT1 are associated with the cell cycle, and further provides an insight into the mechanism of cell-cycle regulated STAT1 signal transduction. PMID:25762114

  20. STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes.

    PubMed

    Krejci, Pavel; Salazar, Lisa; Goodridge, Helen S; Kashiwada, Tamara A; Schibler, Matthew J; Jelinkova, Petra; Thompson, Leslie Michels; Wilcox, William R

    2008-02-01

    Activating mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human skeletal dysplasias as a result of attenuation of cartilage growth. It is believed that FGFR3 inhibits chondrocyte proliferation via activation of signal transducers and activators of transcription (STAT) proteins, although the exact mechanism of both STAT activation and STAT-mediated inhibition of chondrocyte growth is unclear. We show that FGFR3 interacts with STAT1 in cells and is capable of activating phosphorylation of STAT1 in a kinase assay, thus potentially serving as a STAT1 kinase in chondrocytes. However, as demonstrated by western blotting with phosphorylation-specific antibodies, imaging of STAT nuclear translocation, STAT transcription factor assays and STAT luciferase reporter assays, FGF does not activate STAT1 or STAT3 in RCS chondrocytes, which nevertheless respond to a FGF stimulus with potent growth arrest. Moreover, addition of active STAT1 and STAT3 to the FGF signal, by means of cytokine treatment, SRC-mediated STAT activation or expression of constitutively active STAT mutants does not sensitize RCS chondrocytes to FGF-mediated growth arrest. Since FGF-mediated growth arrest is rescued by siRNA-mediated downregulation of the MAP kinase ERK1/2 but not STAT1 or STAT3, our data support a model whereby the ERK arm but not STAT arm of FGF signaling in chondrocytes accounts for the growth arrest phenotype. PMID:18198189

  1. Stat1 negatively regulates immune-mediated injury with Anaplasma phagocytophilum infection.

    PubMed

    Choi, Kyoung-Seong; Scorpio, Diana G; Dumler, J Stephen

    2014-11-15

    Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. Our data previously demonstrated that A. phagocytophilum induces an immunopathologic response by activating IFN-γ production through the Stat1 signaling pathway. In this study, we investigated the broader role of Stat1 signaling in the host response to infection with A. phagocytophilum. In Stat1 knockout (KO) compared with wild-type mice, A. phagocytophilum infection was more highly pathogenic as characterized by the unanticipated development of clinical signs in mice including markedly increased splenomegaly, more severe inflammatory splenic and hepatic histopathology, >100-fold higher blood and splenic bacterial loads, and more elevated proinflammatory cytokine/chemokine responses in serum. CD4(+) and CD8(+) T lymphocyte populations were significantly expanded in spleens of A. phagocytophilum-infected Stat1 KO mice compared with wild-type mice. The leukocyte infiltrates in the livers and spleens of A. phagocytophilum-infected Stat1 KO mice also contained expansions in neutrophil and monocyte/macrophage populations. Importantly, A. phagocytophilum-infected Stat1 KO mice did not demonstrate induction of inducible NO synthase in splenocytes. These results show that Stat1 plays an important role in controlling bacterial loads but also by unexpectedly providing an undefined mechanism for dampening of the immunopathologic response observed with A. phagocytophilum infection. PMID:25305312

  2. A novel gain-of-function STAT1 mutation resulting in basal phosphorylation of STAT1 and increased distal IFN-γ-mediated responses in chronic mucocutaneous candidiasis.

    PubMed

    Martinez-Martinez, Laura; Martinez-Saavedra, Maria Teresa; Fuentes-Prior, Pablo; Barnadas, Maria; Rubiales, Maria Victoria; Noda, Judith; Badell, Isabel; Rodríguez-Gallego, Carlos; de la Calle-Martin, Oscar

    2015-12-01

    Gain-of-function STAT1 mutations have recently been associated with autosomal dominant chronic mucocutaneous candidiasis (CMC). The purpose of this study was to characterize the three members of a non-consanguineous family, the father and his two sons, who presented with recurrent oral thrush and ocular candidiasis since early childhood. The three patients had reduced levels of IL-17-producing T cells. This reduction affected specifically IL-17(+)IFN-γ(-) T cells, because the levels of IL-17(+)IFN-γ(+) T cells were similar to controls. We found that PBMC (peripheral blood mononuclear cells) from the patients did not respond to Candida albicans ex vivo. Moreover, after polyclonal activation, patients' PBMC produced lower levels of IL-17 and IL-6 and higher levels of IL-4 than healthy controls. Genetic analyses showed that the three patients were heterozygous for a new mutation in STAT1 (c.894A>C, p.K298N) that affects a highly conserved residue of the coiled-coil domain of STAT1. STAT1 phosphorylation levels were significantly higher in patients' cells than in healthy controls, both in basal conditions and after IFN-γ stimulation, suggesting a permanent activation of STAT1. Cells from the patients also presented increased IFN-γ-mediated responses measured as MIG and IP-10 production. In conclusion, we report a novel gain-of-function mutation in the coiled-coil domain of STAT1, which increases STAT1 phosphorylation and impairs IL-17-mediated immunity. The mutation is responsible for CMC in this family with autosomal dominant inheritance of the disease. PMID:26514428

  3. Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation.

    PubMed

    Mascle, Xavier H; Lussier-Price, Mathieu; Cappadocia, Laurent; Estephan, Patricia; Raiola, Luca; Omichinski, James G; Aubry, Muriel

    2013-12-20

    Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems. PMID:24174529

  4. Identification of a Non-covalent Ternary Complex Formed by PIAS1, SUMO1, and UBC9 Proteins Involved in Transcriptional Regulation*

    PubMed Central

    Mascle, Xavier H.; Lussier-Price, Mathieu; Cappadocia, Laurent; Estephan, Patricia; Raiola, Luca; Omichinski, James G.; Aubry, Muriel

    2013-01-01

    Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems. PMID:24174529

  5. PIAS1 Regulates Mutant Huntingtin Accumulation and Huntington's Disease-Associated Phenotypes In Vivo.

    PubMed

    Ochaba, Joseph; Monteys, Alex Mas; O'Rourke, Jacqueline G; Reidling, Jack C; Steffan, Joan S; Davidson, Beverly L; Thompson, Leslie M

    2016-05-01

    The disruption of protein quality control networks is central to pathology in Huntington's disease (HD) and other neurodegenerative disorders. The aberrant accumulation of insoluble high-molecular-weight protein complexes containing the Huntingtin (HTT) protein and SUMOylated protein corresponds to disease manifestation. We previously identified an HTT-selective E3 SUMO ligase, PIAS1, that regulates HTT accumulation and SUMO modification in cells. Here we investigated whether PIAS1 modulation in neurons alters HD-associated phenotypes in vivo. Instrastriatal injection of a PIAS1-directed miRNA significantly improved behavioral phenotypes in rapidly progressing mutant HTT (mHTT) fragment R6/2 mice. PIAS1 reduction prevented the accumulation of mHTT and SUMO- and ubiquitin-modified proteins, increased synaptophysin levels, and normalized key inflammatory markers. In contrast, PIAS1 overexpression exacerbated mHTT-associated phenotypes and aberrant protein accumulation. These results confirm the association between aberrant accumulation of expanded polyglutamine-dependent insoluble protein species and pathogenesis, and they link phenotypic benefit to reduction of these species through PIAS1 modulation. PMID:27146268

  6. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

    PubMed

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito

    2015-10-15

    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity. PMID:26335395

  7. KAP1 regulates type I interferon/STAT1-mediated IRF-1 gene expression

    SciTech Connect

    Kamitani, Shinya; Ohbayashi, Norihiko; Ikeda, Osamu; Togi, Sumihito; Muromoto, Ryuta; Sekine, Yuichi; Ohta, Kazuhide; Ishiyama, Hironobu; Matsuda, Tadashi

    2008-05-30

    Signal transducers and activators of transcription (STATs) mediate cell proliferation, differentiation, and survival in immune responses, hematopoiesis, neurogenesis, and other biological processes. Recently, we showed that KAP1 is a novel STAT-binding partner that regulates STAT3-mediated transactivation. KAP1 is a universal co-repressor protein for the KRAB zinc finger protein superfamily of transcriptional repressors. In this study, we found KAP1-dependent repression of interferon (IFN)/STAT1-mediated signaling. We also demonstrated that endogenous KAP1 associates with endogenous STAT1 in vivo. Importantly, a small-interfering RNA-mediated reduction in KAP1 expression enhanced IFN-induced STAT1-dependent IRF-1 gene expression. These results indicate that KAP1 may act as an endogenous regulator of the IFN/STAT1 signaling pathway.

  8. Flavanocoumarins from Guazuma ulmifolia bark and evaluation of their affinity for STAT1.

    PubMed

    Maldini, Mariateresa; Di Micco, Simone; Montoro, Paola; Darra, Elena; Mariotto, Sofia; Bifulco, Giuseppe; Pizza, Cosimo; Piacente, Sonia

    2013-02-01

    From the bark of Guazuma ulmifolia, a plant used as anti-inflammatory remedy, the flavanocoumarin epiphyllocoumarin (1) along with epiphyllocoumarin-[4β→8]-(-)-epicatechin (2) and epiphyllocoumarin-[4β→8]-(-)-epicatechin-[4β→8]-(-)-epicatechin (3), never reported before, have been isolated. The structures of the proantocyanidins 2-3 have been elucidated by extensive NMR and ESI-MS analysis. On the basis of the flavane nature of the isolated compounds and considering the strong anti-STAT1 activity reported for epigallocatechin 3-O-gallate (EGCG), the affinity of compounds 1-3 for STAT1 has been evaluated by Surface Plasmon Resonance. Compounds 1-2 showed affinity for STAT1 comparable to that exerted by EGCG. In order to confirm this result, molecular docking studies to evaluate the interactions of compounds 1-3, phyllocoumarin (4), the reference compound EGCG (5), and its related compound (+)-gallocatechin 3-O-gallate (6) with STAT1 have been carried out. Furthermore the ability of compounds 1-3 to inhibit STAT1 activation has been evaluated using a nuclear extract obtained from the human monocytic leukemia cell line TPH-1. Flavanocoumarins 1-2 inhibited STAT1-DNA binding. PMID:23158905

  9. Gain-of-function human STAT1 mutations impair IL-17 immunity and underlie chronic mucocutaneous candidiasis

    PubMed Central

    Liu, Luyan; Okada, Satoshi; Kong, Xiao-Fei; Kreins, Alexandra Y.; Cypowyj, Sophie; Abhyankar, Avinash; Toubiana, Julie; Itan, Yuval; Audry, Magali; Nitschke, Patrick; Masson, Cécile; Toth, Beata; Flatot, Jérome; Migaud, Mélanie; Chrabieh, Maya; Kochetkov, Tatiana; Bolze, Alexandre; Borghesi, Alessandro; Toulon, Antoine; Hiller, Julia; Eyerich, Stefanie; Eyerich, Kilian; Gulácsy, Vera; Chernyshova, Ludmyla; Chernyshov, Viktor; Bondarenko, Anastasia; María Cortés Grimaldo, Rosa; Blancas-Galicia, Lizbeth; Madrigal Beas, Ileana Maria; Roesler, Joachim; Magdorf, Klaus; Engelhard, Dan; Thumerelle, Caroline; Burgel, Pierre-Régis; Hoernes, Miriam; Drexel, Barbara; Seger, Reinhard; Kusuma, Theresia; Jansson, Annette F.; Sawalle-Belohradsky, Julie; Belohradsky, Bernd; Jouanguy, Emmanuelle; Bustamante, Jacinta; Bué, Mélanie; Karin, Nathan; Wildbaum, Gizi; Bodemer, Christine; Lortholary, Olivier; Fischer, Alain; Blanche, Stéphane; Al-Muhsen, Saleh; Reichenbach, Janine; Kobayashi, Masao; Rosales, Francisco Espinosa; Lozano, Carlos Torres; Kilic, Sara Sebnem; Oleastro, Matias; Etzioni, Amos; Traidl-Hoffmann, Claudia; Renner, Ellen D.; Abel, Laurent; Picard, Capucine; Maródi, László; Boisson-Dupuis, Stéphanie

    2011-01-01

    Chronic mucocutaneous candidiasis disease (CMCD) may be caused by autosomal dominant (AD) IL-17F deficiency or autosomal recessive (AR) IL-17RA deficiency. Here, using whole-exome sequencing, we identified heterozygous germline mutations in STAT1 in 47 patients from 20 kindreds with AD CMCD. Previously described heterozygous STAT1 mutant alleles are loss-of-function and cause AD predisposition to mycobacterial disease caused by impaired STAT1-dependent cellular responses to IFN-γ. Other loss-of-function STAT1 alleles cause AR predisposition to intracellular bacterial and viral diseases, caused by impaired STAT1-dependent responses to IFN-α/β, IFN-γ, IFN-λ, and IL-27. In contrast, the 12 AD CMCD-inducing STAT1 mutant alleles described here are gain-of-function and increase STAT1-dependent cellular responses to these cytokines, and to cytokines that predominantly activate STAT3, such as IL-6 and IL-21. All of these mutations affect the coiled-coil domain and impair the nuclear dephosphorylation of activated STAT1, accounting for their gain-of-function and dominance. Stronger cellular responses to the STAT1-dependent IL-17 inhibitors IFN-α/β, IFN-γ, and IL-27, and stronger STAT1 activation in response to the STAT3-dependent IL-17 inducers IL-6 and IL-21, hinder the development of T cells producing IL-17A, IL-17F, and IL-22. Gain-of-function STAT1 alleles therefore cause AD CMCD by impairing IL-17 immunity. PMID:21727188

  10. MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

    SciTech Connect

    Li, Jun; He, Xijing; Wei, Wenzhi; Zhou, Xiaobo

    2015-05-01

    Osteoblast differentiation is a vital process in maintaining bone homeostasis in which various transcriptional factors, signaling molecules, and microRNAs (miRNAs) are involved. Recently, signal transducer and activator of transcription 1 (STAT1) has been found to play an important role in regulating osteoblast differentiation. Here, we identified that STAT1 expression was regulated by miR-194. Using mouse bone mesenchymal stem cells (BMSCs), we found that miR-194 expression was significantly increased following osteoblast differentiation induction. Overexpression of miR-194 by lentivirus-mediated gene transfer markedly increased osteoblast differentiation, whereas inhibition of miR-194 significantly suppressed osteoblast differentiation of BMSCs. Using a dual-luciferase reporter assay, a direct interaction between miR-194 and the 3′-untranslated region (UTR) of STAT1 was confirmed. Additionally, miR-194 regulated mRNA and protein expression of STAT1 in BMSCs. Further analysis showed that miR-194 overexpression promoted the nuclear translocation of runt-related transcription factor 2 (Runx2), which is critical for osteoblast differentiation. In contrast, inhibition of miR-194 blocked the nuclear translocation of Runx2. Moreover, overexpression of STAT1 significantly blocked Runx2 nuclear translocation and osteoblast differentiation mediated by miR-194 overexpression. Taken together, our data suggest that miR-194 regulates osteoblast differentiation through modulating STAT1-mediated Runx2 nuclear translocation. - Highlights: • Overexpression of miR-194 significantly increased osteoblast differentiation. • miR-194 directly targeted the 3′- UTR of STAT1. • miR-194 regulated the expression of STAT1. • Overexpression of miR-194 promoted the nuclear translocation of Runx2.

  11. Modulation of Stat-1 in Human Macrophages Infected with Different Species of Intracellular Pathogenic Bacteria

    PubMed Central

    Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, Mauro

    2016-01-01

    The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr701) after uptake of two Gram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr701Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-γ release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr701Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages. PMID:27437406

  12. STAT2 Mediates Innate Immunity to Dengue Virus in the Absence of STAT1 via the Type I Interferon Receptor

    PubMed Central

    Perry, Stuart T.; Buck, Michael D.; Lada, Steven M.; Schindler, Christian; Shresta, Sujan

    2011-01-01

    Dengue virus (DENV) is a mosquito-borne flavivirus, and symptoms of infection range from asymptomatic to the severe dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). High viral loads correlate with disease severity, and both type I & II interferons (IFNs) are crucial for controlling viral replication. We have previously reported that signal transducer and activator of transcription (STAT) 1-deficient mice are resistant to DENV-induced disease, but little is known about this STAT1-independent mechanism of protection. To determine the molecular basis of the STAT1-independent pathway, mice lacking STAT1, STAT2, or both STAT1 and STAT2 were infected with a virulent mouse-adapted strain of DENV2. In the first 72 hours of infection, the single-deficient mice lacking STAT1 or STAT2 possessed 50–100 fold higher levels of viral RNA than wild type mice in the serum, spleen, and other visceral tissues, but remained resistant to DENV-induced death. In contrast, the double-deficient mice exhibited the early death phenotype previously observed in type I and II IFN receptor knockout mice (AG129), indicating that STAT2 is the mediator of the STAT1-independent host defense mechanism. Further studies demonstrated that this STAT2-dependent STAT1-independent mechanism requires the type I IFN receptor, and contributes to the autocrine amplification of type I IFN expression. Examination of gene expression in the spleen and bone marrow-derived macrophages following DENV infection revealed STAT2-dependent pathways can induce the transcription of a subset of interferon stimulated genes even in the absence of STAT1. Collectively, these results help elucidate the nature of the poorly understood STAT1-independent host defense mechanism against viruses by identifying a functional type I IFN/STAT2 signaling pathway following DENV infection in vivo. PMID:21379341

  13. Human metapneumovirus small hydrophobic (SH) protein downregulates type I IFN pathway signaling by affecting STAT1 expression and phosphorylation.

    PubMed

    Hastings, Andrew K; Amato, Katherine R; Wen, Sherry C; Peterson, Laura S; Williams, John V

    2016-07-01

    Type I interferon (IFN) is a key mediator of antiviral immunity. Human metapneumovirus (HMPV) inhibits IFN signaling, but does not encode homologues of known IFN antagonists. We tested the hypothesis that a specific viral protein prevents type I IFN signaling by targeting signal transducer and activator of transcription-1 (STAT1). We found that human airway epithelial cells (capable of expressing IFNs) became impaired for STAT1 phosphorylation even without direct infection due to intrinsic negative feedback. HMPV-infected Vero cells (incapable of expressing IFN) displayed lower STAT1 expression and impaired STAT1 phosphorylation in response to type I IFN treatment compared to mock-infected cells. Transient overexpression of HMPV small hydrophobic (SH) protein significantly inhibited STAT1 phosphorylation and signaling, and recombinant virus lacking SH protein was unable to inhibit STAT1 phosphorylation. Our results indicate a role for the SH protein of HMPV in the downregulation of type I IFN signaling through the targeting of STAT1. PMID:27131212

  14. STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

    PubMed Central

    Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

    2013-01-01

    The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

  15. Profibrogenic phenotype in caveolin-1 deficiency via differential regulation of STAT-1/3 proteins.

    PubMed

    Ryter, Stefan W; Choi, Augustine M K; Kim, Hong Pyo

    2014-10-01

    Fibrosis underlies the pathogenesis of several human diseases, which can lead to severe injury of vital organs. We previously demonstrated that caveolin-1 expression is reduced in experimental fibrosis and that caveolin-1 exerts antiproliferative and antifibrotic effects in lung fibrosis models. The signal transducers and activators of transcription (STAT) proteins, STAT1 and STAT3, can be activated simultaneously. STAT1 can inhibit cell growth and promote apoptosis while STAT3 inhibits apoptosis. Here, we show that caveolin-1-deficient (cav-1(-/-)) lung fibroblasts display dramatically upregulated STAT3 activation in response to platelet-derived growth factor-BB and transforming growth factor-β stimuli, whereas STAT1 activation is undetectable. Downregulation of protein tyrosine phosphatase-1B played a role in the preferential activation of STAT3 in cav-1(-/-) fibroblasts. Genetic deletion of STAT3 by siRNA modulated the expression of genes involved in cell proliferation and fibrogenesis. Basal expression of α-smooth muscle actin was prominent in cav-1(-/-) liver and kidney, consistent with deposition of collagen in these organs. Collectively, we demonstrate that the antiproliferative and antifibrogenic properties of caveolin-1 in vitro are mediated by the balance between STAT1 and STAT3 activation. Deregulated STAT signaling associated with caveolin-1 deficiency may be relevant to proliferative disorders such as tissue fibrosis. PMID:25263949

  16. IFN-γ induces lipogenesis in mouse mesangial cells via the JAK2/STAT1 pathway.

    PubMed

    Hao, Jun; Zhang, Yu-jun; Lv, Xin; Xu, Ning; Liu, Qin-juan; Zhao, Song; Feng, Xiao-juan; Xing, Ling-ling; Kang, Peng-peng; Li, Gui-ying; Liu, Shu-xia

    2013-04-15

    The objective is to investigate the effect of high mobility group box-1 (HMGB1) on lipid deposition in γ-interferon (IFN-γ)-stimulated mouse mesangial cell line (MMC) and to determine whether the Janus kinase 2 and signal transducer and activator of transcription 1 (JAK2/STAT1) signaling pathway plays an important role in this process. We employed a control group, an IFN-γ stimulation group, and an IFN-γ + AG490 (JAK2 inhibitor) group. RNA interference aimed at sterol regulatory element-binding protein-1 (SREBP-1) or HMGB1 was used to investigate the effect of these proteins on IFN-γ-induced lipid deposition. Western blotting was used to detect phospho (p)-JAK2, JAK2, p-STAT1, STAT1, SREBP-1, fatty acid synthase (FAS), and HMGB1 protein expression. RT-PCR was used to detect SREBP-1, FAS, and HMGB1 mRNA. Oil Red O staining and the triglyceride assay were used to detect lipid deposition and triglyceride content. Results were as follows: 1) IFN-γ increased MMC cell lipid deposition, triglyceride content, and p-JAK2, p-STAT1, SREBP-1, and FAS expression; 2) SREBP-1 inhibition prevented FAS upregulation and attenuated IFN-γ-induced MMC cell lipid deposition and triglyceride content; 3) HMGB1 upregulated SREBP-1 and FAS mRNA and protein levels, which increased lipid deposition in MMC cells. Small interfering RNA-mediated inhibition of HMGB1 decreased SREBP-1 and FAS expression and lipid accumulation; 4) AG490 decreased upregulation of HMGB1 and p-JAK2/p-STAT1, as well as IFN-γ-induced lipogenesis. In conclusion, the JAK2/STAT1 pathway mediates IFN-γ-induced lipogenesis in MMC cells through regulation of HMGB1/SREBP-1/FAS. PMID:23407880

  17. Transcriptional Regulation of Oncogenic Protein Kinase Cϵ (PKCϵ) by STAT1 and Sp1 Proteins*

    PubMed Central

    Wang, HongBin; Gutierrez-Uzquiza, Alvaro; Garg, Rachana; Barrio-Real, Laura; Abera, Mahlet B.; Lopez-Haber, Cynthia; Rosemblit, Cinthia; Lu, Huaisheng; Abba, Martin; Kazanietz, Marcelo G.

    2014-01-01

    Overexpression of PKCϵ, a kinase associated with tumor aggressiveness and widely implicated in malignant transformation and metastasis, is a hallmark of multiple cancers, including mammary, prostate, and lung cancer. To characterize the mechanisms that control PKCϵ expression and its up-regulation in cancer, we cloned an ∼1.6-kb promoter segment of the human PKCϵ gene (PRKCE) that displays elevated transcriptional activity in cancer cells. A comprehensive deletional analysis established two regions rich in Sp1 and STAT1 sites located between −777 and −105 bp (region A) and −921 and −796 bp (region B), respectively, as responsible for the high transcriptional activity observed in cancer cells. A more detailed mutagenesis analysis followed by EMSA and ChIP identified Sp1 sites in positions −668/−659 and −269/−247 as well as STAT1 sites in positions −880/−869 and −793/−782 as the elements responsible for elevated promoter activity in breast cancer cells relative to normal mammary epithelial cells. RNAi silencing of Sp1 and STAT1 in breast cancer cells reduced PKCϵ mRNA and protein expression, as well as PRKCE promoter activity. Moreover, a strong correlation was found between PKCϵ and phospho-Ser-727 (active) STAT1 levels in breast cancer cells. Our results may have significant implications for the development of approaches to target PKCϵ and its effectors in cancer therapeutics. PMID:24825907

  18. Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation

    PubMed Central

    Kaur, T; Mukherjea, D; Sheehan, K; Jajoo, S; Rybak, L P; Ramkumar, V

    2011-01-01

    Cisplatin is widely used for treating various solid tumors. However, this drug produces dose-limiting ototoxicity and nephrotoxicity, which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by diuresis, there is currently no approved treatment for hearing loss. Previous studies show that the ROS and inflammation are major contributors to cisplatin-induced hearing loss. In this study, we show that ROS trigger the inflammatory process in the cochlea by activating signal transducer and activator of transcription-1 (STAT1). Activation of STAT1 activation was dependent on ROS generation through NOX3 NADPH oxidase, knockdown of which by siRNA reduced STAT1 activation. Moreover, STAT1 siRNA protected against activation of p53, reduced apoptosis, reduced damage to OHCs and preserved hearing in rats. STAT1 siRNA attenuated the increase in inflammatory mediators, such as TNF-α, inhibition of which protected cells from cisplatin-mediated apoptosis. Finally, we showed that trans-tympanic administration of etanercept, a TNF-α antagonist, protected against OHC damage and cisplatin-induced hearing loss. These studies suggest that controlling inflammation by inhibition of STAT1-dependent pathways in the cochlea could serve as an effective approach to treat cisplatin ototoxicity and improve the overall quality of life for cancer patients. PMID:21776018

  19. Leishmania donovani amastigotes impair gamma interferon-induced STAT1alpha nuclear translocation by blocking the interaction between STAT1alpha and importin-alpha5.

    PubMed

    Matte, Christine; Descoteaux, Albert

    2010-09-01

    The protozoan parasite Leishmania donovani, the etiological agent of visceral leishmaniasis, is renowned for its capacity to sabotage macrophage functions and signaling pathways stimulated by activators such as gamma interferon (IFN-gamma). Our knowledge of the strategies utilized by L. donovani to impair macrophage responsiveness to IFN-gamma remains fragmentary. In the present study, we investigated the impact of an infection by the amastigote stage of L. donovani on IFN-gamma responses and signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway in mouse bone marrow-derived macrophages. The levels of IFN-gamma-induced expression of major histocompatibility complex class II and inducible nitric oxide synthase (iNOS) were strongly reduced in L. donovani amastigote-infected macrophages. As the expression of those genes is mediated by the transcription factors STAT1alpha and IFN regulatory factor 1 (IRF-1), we investigated their activation in amastigote-infected macrophages treated with IFN-gamma. We found that whereas STAT1alpha protein levels and the levels of phosphorylation on Tyr701 and Ser727 were normal, IRF-1 expression was inhibited in infected macrophages. This inhibition of IRF-1 expression correlated with a defective nuclear translocation of STAT1alpha, and further analyses revealed that the IFN-gamma-induced STAT1alpha association with the nuclear transport adaptor importin-alpha5 was compromised in L. donovani amastigote-infected macrophages. Taken together, our results provide evidence for a novel mechanism used by L. donovani amastigotes to interfere with IFN-gamma-activated macrophage functions and provide a better understanding of the strategies deployed by this parasite to ensure its intracellular survival. PMID:20566692

  20. Androgen receptor- and PIAS1-regulated gene programs in molecular apocrine breast cancer cells.

    PubMed

    Malinen, Marjo; Toropainen, Sari; Jääskeläinen, Tiina; Sahu, Biswajyoti; Jänne, Olli A; Palvimo, Jorma J

    2015-10-15

    We have analyzed androgen receptor (AR) chromatin binding sites (ARBs) and androgen-regulated transcriptome in estrogen receptor negative molecular apocrine breast cancer cells. These analyses revealed that 42% of ARBs and 39% androgen-regulated transcripts in MDA-MB453 cells have counterparts in VCaP prostate cancer cells. Pathway analyses showed a similar enrichment of molecular and cellular functions among AR targets in both breast and prostate cancer cells, with cellular growth and proliferation being among the most enriched functions. Silencing of the coregulator SUMO ligase PIAS1 in MDA-MB453 cells influenced AR function in a target-selective fashion. An anti-apoptotic effect of the silencing suggests involvement of the PIAS1 in the regulation of cell death and survival pathways. In sum, apocrine breast cancer and prostate cancer cells share a core AR cistrome and target gene signature linked to cancer cell growth, and PIAS1 plays a similar coregulatory role for AR in both cancer cell types. PMID:26219822

  1. PIAS1 is a crucial factor for prostate cancer cell survival and a valid target in docetaxel resistant cells

    PubMed Central

    Puhr, Martin; Hoefer, Julia; Neuwirt, Hannes; Eder, Iris E.; Kern, Johann; Schäfer, Georg; Geley, Stephan; Heidegger, Isabel; Klocker, Helmut; Culig, Zoran

    2014-01-01

    Occurrence of an inherent or acquired resistance to the chemotherapeutic drug docetaxel is a major burden for patients suffering from different kinds of malignancies, including castration resistant prostate cancer (PCa). In the present study we address the question whether PIAS1 targeting can be used to establish a basis for improved PCa treatment. The expression status and functional relevance of PIAS1 was evaluated in primary tumors, in metastatic lesions, in tissue of patients after docetaxel chemotherapy, and in docetaxel resistant cells. Patient data were complemented by functional studies on PIAS1 knockdown in vitro as well as in chicken chorioallantoic membrane and mouse xenograft in vivo models. PIAS1 was found to be overexpressed in local and metastatic PCa and its expression was further elevated in tumors after docetaxel treatment as well as in docetaxel resistant cells. Furthermore, PIAS1 knockdown experiments revealed an increased expression of tumor suppressor p21 and declined expression of anti-apoptotic protein Mcl1, which caused diminished cell proliferation and tumor growth in vitro and in vivo. In summary, the presented data indicate that PIAS1 is crucial for parental and docetaxel resistant PCa cell survival and is therefore a promising new target for treatment of primary, metastatic, and chemotherapy resistant PCa. PMID:25474038

  2. PIAS1 is a crucial factor for prostate cancer cell survival and a valid target in docetaxel resistant cells.

    PubMed

    Puhr, Martin; Hoefer, Julia; Neuwirt, Hannes; Eder, Iris E; Kern, Johann; Schäfer, Georg; Geley, Stephan; Heidegger, Isabel; Klocker, Helmut; Culig, Zoran

    2014-12-15

    Occurrence of an inherent or acquired resistance to the chemotherapeutic drug docetaxel is a major burden for patients suffering from different kinds of malignancies, including castration resistant prostate cancer (PCa). In the present study we address the question whether PIAS1 targeting can be used to establish a basis for improved PCa treatment. The expression status and functional relevance of PIAS1 was evaluated in primary tumors, in metastatic lesions, in tissue of patients after docetaxel chemotherapy, and in docetaxel resistant cells. Patient data were complemented by functional studies on PIAS1 knockdown in vitro as well as in chicken chorioallantoic membrane and mouse xenograft in vivo models. PIAS1 was found to be overexpressed in local and metastatic PCa and its expression was further elevated in tumors after docetaxel treatment as well as in docetaxel resistant cells. Furthermore, PIAS1 knockdown experiments revealed an increased expression of tumor suppressor p21 and declined expression of anti-apoptotic protein Mcl1, which caused diminished cell proliferation and tumor growth in vitro and in vivo. In summary, the presented data indicate that PIAS1 is crucial for parental and docetaxel resistant PCa cell survival and is therefore a promising new target for treatment of primary, metastatic, and chemotherapy resistant PCa. PMID:25474038

  3. PI3Kα and STAT1 Interplay Regulates Human Mesenchymal Stem Cell Immune Polarization

    PubMed Central

    Mounayar, Marwan; Kefaloyianni, Eirini; Smith, Brian; Solhjou, Zhabiz; Maarouf, Omar H.; Azzi, Jamil; Chabtini, Lola; Fiorina, Paolo; Kraus, Morey; Briddell, Robert; Fodor, William; Herrlich, Andreas; Abdi, Reza

    2016-01-01

    The immunomodulatory capacity of mesenchymal stem cells (MSCs) is critical for their use in therapeutic applications. MSC response to specific inflammatory cues allows them to switch between a proinflammatory (MSC1) or anti-inflammatory (MSC2) phenotype. Regulatory mechanisms controlling this switch remain to be defined. One characteristic feature of MSC2 is their ability to respond to IFNγ with induction of indoleamine 2,3-dioxygenase (IDO), representing the key immunoregulatory molecule released by human MSC. Here, we show that STAT1 and PI3Kα pathways interplay regulates IFNγ-induced IDO production in MSC. Chemical phosphoinositide 3-kinase (PI3K) pan-inhibition, PI3Kα-specific inhibition or shRNA knockdown diminished IFNγ-induced IDO production. This effect involved PI3Kα-mediated upregulation of STAT1 protein levels and phosphorylation at Ser727. Overexpression of STAT1 or of a constitutively active PI3Kα mutant failed to induce basal IDO production, but shifted MSC into an MSC2-like phenotype by strongly enhancing IDO production in response to IFNγ as compared to controls. STAT1 overexpression strongly enhanced MSC-mediated T-cell suppression. The same effect could be induced using short-term pretreatment of MSC with a chemical inhibitor of the counter player of PI3K, phosphatase and tensin homolog. Finally, downregulation of STAT1 abrogated the immunosuppressive capacity of MSC. Our results for the first time identify critical upstream signals for the induced production of IDO in MSCs that could be manipulated therapeutically to enhance their immunosuppressive phenotype. PMID:25753288

  4. Hepatitis C virus core protein inhibits interferon production by a human plasmacytoid dendritic cell line and dysregulates interferon regulatory factor-7 and signal transducer and activator of transcription (STAT) 1 protein expression.

    PubMed

    Stone, Amy E L; Mitchell, Angela; Brownell, Jessica; Miklin, Daniel J; Golden-Mason, Lucy; Polyak, Stephen J; Gale, Michael J; Rosen, Hugo R

    2014-01-01

    Plasmacytoid Dendritic Cells (pDCs) represent a key immune cell population in the defense against viruses. pDCs detect viral pathogen associated molecular patterns (PAMPs) through pattern recognition receptors (PRR). PRR/PAMP interactions trigger signaling events that induce interferon (IFN) production to initiate local and systemic responses. pDCs produce Type I and Type III (IFNL) IFNs in response to HCV RNA. Extracellular HCV core protein (Core) is found in the circulation in chronic infection. This study defined how Core modulates PRR signaling in pDCs. Type I and III IFN expression and production following exposure to recombinant Core or β-galactosiade was assessed in human GEN2.2 cells, a pDC cell line. Core suppressed type I and III IFN production in response to TLR agonists and the HCV PAMP agonist of RIG-I. Core suppression of IFN induction was linked with decreased IRF-7 protein levels and increased non-phosphorylated STAT1 protein. Circulating Core protein interferes with PRR signaling by pDCs to suppress IFN production. Strategies to define and target Core effects on pDCs may serve to enhance IFN production and antiviral actions against HCV. PMID:24788809

  5. ALASKA GENERAL LAND STATUS (STAT1)

    EPA Science Inventory

    AKSTATUS is a statewide summary of land ownership in Alaska. It includes the major categories of state, native, and federal holdings. Activity on state land is recorded, by section, in DRSs Land Adminstration System (LAS). Information on state land status is extracted from LAS...

  6. STAT1 signaling modulates HIV-1-induced inflammatory responses and leukocyte transmigration across the blood-brain barrier.

    PubMed

    Chaudhuri, Anathbandhu; Yang, Bo; Gendelman, Howard E; Persidsky, Yuri; Kanmogne, Georgette D

    2008-02-15

    The relationship among neuroinflammation, blood-brain barrier (BBB) dysfunction, and progressive HIV-1 infection as they affect the onset and development of neuroAIDS is incompletely understood. One possible link is signal transducers and activators of transcription (STATs) pathways. These respond to proinflammatory and regulatory factors and could affect neuroinflammatory responses induced from infected cells and disease-affected brain tissue. Our previous works demonstrated that HIV-1 activates pro-inflammatory and interferon-alpha-inducible genes in human brain microvascular endothelial cells (HBMECs) and that these genes are linked to the Janus kinase (JAK)/STAT pathway. We now demonstrate that HIV-1 activates STAT1, induces IL-6 expression, and diminishes expression of claudin-5, ZO-1, and ZO-2 in HBMECs. The STAT1 inhibitor, fludarabine, blocked HIV-1-induced IL-6, diminished HIV-1-induced claudin-5 and ZO-1 down-regulation, and blocked HIV-1- and IL-6-induced monocyte migration across a BBB model. Enhanced expression and activation of STAT1 and decreased claudin-5 were observed in microvessels from autopsied brains of patients with HIV-1-associated dementia. These data support the notion that STAT1 plays an integral role in HIV-1-induced BBB damage and is relevant to viral neuropathogenesis. Inhibition of STAT1 activation could provide a unique therapeutic strategy to attenuate HIV-1-induced BBB compromise and as such improve clinical outcomes. PMID:18003888

  7. HIV-1 gp120 induces cytokine expression, leukocyte adhesion, and transmigration across the blood-brain barrier: modulatory effects of STAT1 signaling.

    PubMed

    Yang, Bo; Akhter, Sidra; Chaudhuri, Anathbandhu; Kanmogne, Georgette D

    2009-03-01

    How neuroinflammatory activities affect signaling pathways leading to blood-brain barrier (BBB) injury during HIV/AIDS are currently unknown. Our previous work demonstrated that HIV-1 exposure activates pro-inflammatory genes in human brain microvascular endothelial cells (HBMEC) and showed that these genes are linked to the janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway. Here, we report that HIV-1 gp120 protein activated STAT1 and induced interleukin (IL)-6 and IL-8 secretion in HBMEC. IL-6, IL-8, and gp120 increased monocyte adhesion and migration across in vitro BBB models. The STAT1 inhibitor, fludarabine, prevented gp120-induced IL-6 and IL-8 secretion. Inhibitors of STAT1, mitogen activated protein kinase kinase (MEK) (PD98059), and phosphatidyl inositol 3 kinase (PI3K) (LY294002), blocked gp120-induced STAT1 activation and significantly diminished IL-8-, IL-6-, and gp120-induced monocyte adhesion and migration across in vitro BBB models. These data support the notion that STAT1 plays an important role in gp120-induced inflammation and BBB dysfunction associated with viral infection. Results also suggest crosstalk between STAT1, MEK, and PI3K pathways in gp120-induced BBB dysfunction. Inhibition of STAT1 activation could provide a unique therapeutic strategy to decrease neuroinflammation and BBB dysfunction in HIV/AIDS. PMID:19103208

  8. EphrinB2 controls vessel pruning through STAT1-JNK3 signaling

    PubMed Central

    Salvucci, Ombretta; Ohnuki, Hidetaka; Maric, Dragan; Hou, Xu; Li, Xuri; Yoon, Sung Ok; Segarra, Marta; Eberhart, Charles G.; Acker-Palmer, Amparo; Tosato, Giovanna

    2015-01-01

    Angiogenesis produces primitive vascular networks that need pruning to yield hierarchically organized and functional vessels. Despite the critical importance of vessel pruning to vessel patterning and function, the mechanisms regulating this process are not clear. Here we show that EphrinB2, a well-known player in angiogenesis, is an essential regulator of endothelial cell death and vessel pruning. This regulation depends upon phosphotyrosine-EphrinB2 signaling repressing JNK3 activity via STAT1. JNK3 activation causes endothelial cell death. In the absence of JNK3, hyaloid vessel physiological pruning is impaired, associated with abnormal persistence of hyaloid vessels, defective retinal vasculature and microphthalmia. This syndrome closely resembles human persistent hyperplastic primary vitreus (PHPV), attributed to failed involution of hyaloid vessels. Our results provide evidence that EphrinB2/STAT1/JNK3 signaling is essential for vessel pruning, and that defects in this pathway may contribute to PHPV. PMID:25807892

  9. TYK2-STAT1-BCL2 Pathway Dependence in T-Cell Acute Lymphoblastic Leukemia

    PubMed Central

    Sanda, Takaomi; Tyner, Jeffrey W.; Gutierrez, Alejandro; Ngo, Vu N.; Glover, Jason; Chang, Bill H.; Yost, Arla; Ma, Wenxue; Fleischman, Angela G.; Zhou, Wenjun; Yang, Yandan; Kleppe, Maria; Ahn, Yebin; Tatarek, Jessica; Kelliher, Michelle A.; Neuberg, Donna S.; Levine, Ross L.; Moriggl, Richard; Müller, Mathias; Gray, Nathanael S.; Jamieson, Catriona H. M.; Weng, Andrew P.; Staudt, Louis M.; Druker, Brian J.; Look, A. Thomas

    2013-01-01

    Targeted molecular therapy has yielded remarkable outcomes in certain cancers, but specific therapeutic targets remain elusive for many others. As a result of two independent RNA interference (RNAi) screens, we identified pathway dependence on a member of the JAK tyrosine kinase family, TYK2, and its downstream effector STAT1 in T-cell acute lymphoblastic leukemia (T-ALL). Gene knockdown experiments consistently demonstrated TYK2 dependence in both T-ALL primary specimens and cell lines, and a small-molecule inhibitor of JAK kinase activity induced T-ALL cell death. Activation of this TYK2-STAT1 pathway i n T-ALL cell lines occurs by gain-of-function TYK2 mutations or activation of IL-10 receptor signaling, and this pathway mediates T-ALL cell survival through upregulation of the anti-apoptotic protein BCL2. These findings indicate that in many T-ALL cases, the leukemic cells are dependent upon the TYK2-STAT1-BCL2 pathway for continued survival, supporting the development of molecular therapies targeting TYK2 and other components of this pathway. PMID:23471820

  10. Liver X receptor and STAT1 cooperate downstream of Gas6/Mer to induce anti-inflammatory arginase 2 expression in macrophages

    PubMed Central

    Kim, Si-Yoon; Lim, Eun-Jin; Yoon, Young-So; Ahn, Young-Ho; Park, Eun-Mi; Kim, Hee-Sun; Kang, Jihee Lee

    2016-01-01

    Mer signaling increases the transcriptional activity of liver X receptor (LXR) to promote the resolution of acute sterile inflammation. Here, we aimed to understand the pathway downstream of Mer signaling after growth arrest-specific protein 6 (Gas6) treatment that leads to LXR expression and transcriptional activity in mouse bone-marrow derived macrophages (BMDM). Gas6-induced increases in LXRα and LXRβ and expression of their target genes were inhibited in BMDM from STAT1−/− mice or by the STAT1-specific inhibitor fludarabine. Gas6-induced STAT1 phosphorylation, LXR activation, and LXR target gene expression were inhibited in BMDM from Mer−/− mice or by inhibition of PI3K or Akt. Gas6-induced Akt phosphorylation was inhibited in BMDM from STAT1−/− mice or in the presence of fludarabine. Gas6-induced LXR activity was enhanced through an interaction between LXRα and STAT1 on the DNA promoter of Arg2. Additionally, we found that Gas6 inhibited lipopolysaccharide (LPS)-induced nitrite production in a STAT1 and LXR pathway-dependent manner in BMDM. Additionally, Mer-neutralizing antibody reduced LXR and Arg2 expression in lung tissue and enhanced NO production in bronchoalveolar lavage fluid in LPS-induced acute lung injury. Our data suggest the possibility that the Gas6-Mer-PI3K/Akt-STAT1-LXR-Arg2 pathway plays an essential role for resolving inflammatory response in acute lung injury. PMID:27406916

  11. Lupus Risk Variant Increases pSTAT1 Binding and Decreases ETS1 Expression

    PubMed Central

    Lu, Xiaoming; Zoller, Erin E.; Weirauch, Matthew T.; Wu, Zhiguo; Namjou, Bahram; Williams, Adrienne H.; Ziegler, Julie T.; Comeau, Mary E.; Marion, Miranda C.; Glenn, Stuart B.; Adler, Adam; Shen, Nan; Nath, Swapan K.; Stevens, Anne M.; Freedman, Barry I.; Tsao, Betty P.; Jacob, Chaim O.; Kamen, Diane L.; Brown, Elizabeth E.; Gilkeson, Gary S.; Alarcón, Graciela S.; Reveille, John D.; Anaya, Juan-Manuel; James, Judith A.; Sivils, Kathy L.; Criswell, Lindsey A.; Vilá, Luis M.; Alarcón-Riquelme, Marta E.; Petri, Michelle; Scofield, R. Hal; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Joo, Young Bin; Choi, Jeongim; Bae, Sang-Cheol; Boackle, Susan A.; Graham, Deborah Cunninghame; Vyse, Timothy J.; Guthridge, Joel M.; Gaffney, Patrick M.; Langefeld, Carl D.; Kelly, Jennifer A.; Greis, Kenneth D.; Kaufman, Kenneth M.; Harley, John B.; Kottyan, Leah C.

    2015-01-01

    Genetic variants at chromosomal region 11q23.3, near the gene ETS1, have been associated with systemic lupus erythematosus (SLE), or lupus, in independent cohorts of Asian ancestry. Several recent studies have implicated ETS1 as a critical driver of immune cell function and differentiation, and mice deficient in ETS1 develop an SLE-like autoimmunity. We performed a fine-mapping study of 14,551 subjects from multi-ancestral cohorts by starting with genotyped variants and imputing to all common variants spanning ETS1. By constructing genetic models via frequentist and Bayesian association methods, we identified 16 variants that are statistically likely to be causal. We functionally assessed each of these variants on the basis of their likelihood of affecting transcription factor binding, miRNA binding, or chromatin state. Of the four variants that we experimentally examined, only rs6590330 differentially binds lysate from B cells. Using mass spectrometry, we found more binding of the transcription factor signal transducer and activator of transcription 1 (STAT1) to DNA near the risk allele of rs6590330 than near the non-risk allele. Immunoblot analysis and chromatin immunoprecipitation of pSTAT1 in B cells heterozygous for rs6590330 confirmed that the risk allele increased binding to the active form of STAT1. Analysis with expression quantitative trait loci indicated that the risk allele of rs6590330 is associated with decreased ETS1 expression in Han Chinese, but not other ancestral cohorts. We propose a model in which the risk allele of rs6590330 is associated with decreased ETS1 expression and increases SLE risk by enhancing the binding of pSTAT1. PMID:25865496

  12. Exploring dual inhibitors for STAT1 and STAT5 receptors utilizing virtual screening and dynamics simulation validation.

    PubMed

    Raj, Utkarsh; Kumar, Himansu; Gupta, Saurabh; Varadwaj, Pritish Kumar

    2016-10-01

    Signal transducer and activator of transcription (STAT) proteins are latent cytoplasmic transcription factors that transduce signals from cytokines and growth factors to the nucleus and thereby regulate the expression of a variety of target genes. Although mutations of STATs have not been reported in human tumors but the activity of several members of the family, such as STAT1 and STAT5, is deregulated in a variety of human carcinoma. STAT1 and STAT5 share a structural similarity with a highly conserved SH2 domain which is responsible for the activation of STAT proteins on interaction with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The purpose of this study is to identify domain-specific dual inhibitors for both STAT1 and STAT5 proteins from a database of natural products and natural product-like compounds comprising of over 90,000 compounds. Virtual screening-based molecular docking was performed in order to find novel natural dual inhibitors. Further, the study was supported by the 50-ns molecular dynamics simulation for receptor-ligand complexes (STAT1-STOCK-1N-69677 and STAT5-STOCK-1N-69677). Analysis of molecular interactions in the SH2 domains of both STAT1 and STAT5 proteins with the ligand revealed few conserved amino acid residues which are responsible to stabilize the ligands within the binding pocket through bonded and non-bonded interactions. This study suggested that compound STOCK-1N-69677 might putatively act as a dual inhibitor of STAT1 and STAT5 receptors, through its binding to the SH2 domain. PMID:26471877

  13. Genkwadaphnin Induces IFN-γ via PKD1/NF-κB/STAT1 Dependent Pathway in NK-92 Cells

    PubMed Central

    Kang, Ho-Bum; Ahn, Kyung-Seop; Oh, Sei-Ryang; Kim, Jae Wha

    2014-01-01

    The flower buds of Daphne genkwa Sieb. et Zucc. have been used as a traditional Chinese medicine although their functional mechanisms have not been discovered yet. We have studied the potential effects of the plant extracts on natural killer (NK) cell activation, and isolated an active fraction. Genkwadaphnin (GD-1) displayed a potent efficacy to induce IFN-γ transcription in NK cells with concentration- and time-dependent manners. GD-1 treatment triggered the phosphorylation of PKD1, a member of PKC family, MEK and ERK, resulting in IKK activation to induce IκB degradation, and the nuclear localization of p65, an NF-κB subunit, which regulates IFN-γ transcription. GD-1 effect on IFN-γ production was blocked by the addition of Rottlerin, a PKC inhibitor, CID 755673, a PKD inhibitor, or Bay11-7082, an IKKα inhibitor. The nuclear localization of p65 was also inhibited by the kinase inhibitors. Secreted IFN-γ activates STAT1 phosphorylation as autocrine-loops to sustain its secretion. GD-1 induced the phosphorylation of STAT1 probably through the increase of IFN-γ. STAT1 inhibitor also abrogated the sustained IFN-γ secretion. These results suggest that GD-1 is involved in the activation of PKD1 and/or ERK pathway, which activate NK-κB triggering IFN-γ production. As positive feedback loops, secreted IFN-γ activates STAT1 and elongates its production in NK-92 cells. PMID:25517939

  14. c-Myc is targeted to the proteasome for degradation in a SUMOylation-dependent manner, regulated by PIAS1, SENP7 and RNF4.

    PubMed

    González-Prieto, Román; Cuijpers, Sabine Ag; Kumar, Ramesh; Hendriks, Ivo A; Vertegaal, Alfred Co

    2015-01-01

    c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway. Currently, the relevant SUMO target proteins in this pathway are unknown. Here we show that c-Myc is a target protein for SUMOylation, and that SUMOylated c-Myc is subsequently ubiquitylated and degraded by the proteasome. SUMO chains appeared to be dispensable for this process, polymerization-deficient SUMO mutants supported proteolysis of SUMOylated c-Myc. These results indicate that multiple SUMO monomers conjugated to c-Myc could be sufficient to direct SUMOylated c-Myc to the ubiquitin-proteasome pathway. Knocking down the SUMO-targeted ubiquitin ligase RNF4 enhanced the levels of SUMOylated c-Myc, indicating that RNF4 could recognize a multi-SUMOylated protein as a substrate in addition to poly-SUMOylated proteins. Knocking down the SUMO E3 ligase PIAS1 resulted in reduced c-Myc SUMOylation and increased c-Myc transcriptional activity, indicating that PIAS1 mediates c-Myc SUMOylation. Increased SUMOylation of c-Myc was noted upon knockdown of the SUMO protease SENP7, indicating that it also could regulate a multi-SUMOylated protein in addition to poly-SUMOylated proteins. C-Myc lacks KxE-type SUMOylation consensus motifs. We used mass spectrometry to identify 10 SUMO acceptor lysines: K52, K148, K157, K317, K323, K326, K389, K392, K398 and K430. Intriguingly, mutating all 10 SUMO acceptor lysines did not reduce c-Myc SUMOylation, suggesting that SUMO acceptor lysines in c-Myc act promiscuously. Our results provide novel insight into the complexity of c-Myc post-translational regulation. PMID:25895136

  15. STAT1: A Novel Target of miR-150 and miR-223 Is Involved in the Proliferation of HTLV-I–Transformed and ATL Cells1

    PubMed Central

    Moles, Ramona; Bellon, Marcia; Nicot, Christophe

    2015-01-01

    We have previously reported on the deregulation of cellular microRNAs involved in hematopoiesis and inflammation in human T-cell lymphotropic virus type 1 (HTLV-I)–transformed cells. In this study, we demonstrate that miR-150 and miR-223 specifically target the signal transducer and activator of transcription 1 (STAT1) 3′ untranslated region, reducing STAT1 expression and dampening STAT1-dependent signaling in human T cells. The effects of miR-150 and miR-223 on endogenous STAT1 were confirmed using inducible cell lines. Our studies also showed that miR-150 expression is upregulated by interleukin-2 signaling in adult T cell leukemia/lymphoma (ATL) cells. HTLV-I–transformed and ATL-derived cells have reduced levels of miR150 and miR223 expression, which coincide with increased STAT1 expression and STAT1-dependent signaling. Knockdown of STAT1 by short hairpin RNA demonstrated that the constitutive activation of STAT1 is required for the continuous proliferation of HTLV-I–transformed cells. Our studies further demonstrate that increased expression of STAT1 in ATL cells is associated with higher levels of major histocompatibility complex class I expression. Previous studies have demonstrated that the pressure exerted by natural killer (NK) cells in vivo can edit leukemic tumor cells by forcing an increased expression of major histocompatibility complex class I to escape immune clearance. STAT1-expressing tumor cells produce more aggressive tumors because they cannot be eliminated by NK cells. Our results suggest that therapeutic approaches using combined targeting of STAT1 and MHC class I may be an effective approach to activate NK cell–mediated clearance of ATL tumor cells. PMID:26025667

  16. Measles virus V protein blocks Jak1-mediated phosphorylation of STAT1 to escape IFN-{alpha}/{beta} signaling

    SciTech Connect

    Caignard, Gregory; Guerbois, Mathilde; Labernardiere, Jean-Louis; Jacob, Yves; Jones, Louis M.; Wild, Fabian; Tangy, Frederic Vidalain, Pierre-Olivier

    2007-11-25

    Viruses have evolved various strategies to escape the antiviral activity of type I interferons (IFN-{alpha}/{beta}). For measles virus, this function is carried by the polycistronic gene P that encodes, by an unusual editing strategy, for the phosphoprotein P and the virulence factor V (MV-V). MV-V prevents STAT1 nuclear translocation by either sequestration or phosphorylation inhibition, thereby blocking IFN-{alpha}/{beta} pathway. We show that both the N- and C-terminal domains of MV-V (PNT and VCT) contribute to the inhibition of IFN-{alpha}/{beta} signaling. Using the two-hybrid system and co-affinity purification experiments, we identified STAT1 and Jak1 as interactors of MV-V and demonstrate that MV-V can block the direct phosphorylation of STAT1 by Jak1. A deleterious mutation within the PNT domain of MV-V (Y110H) impaired its ability to interact and block STAT1 phosphorylation. Thus, MV-V interacts with at least two components of IFN-{alpha}/{beta} receptor complex to block downstream signaling.

  17. CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells.

    PubMed

    Suyama, Kumi; Onishi, Hideya; Imaizumi, Akira; Shinkai, Kentaro; Umebayashi, Masayo; Kubo, Makoto; Mizuuchi, Yusuke; Oda, Yoshinao; Tanaka, Masao; Nakamura, Masafumi; Katano, Mitsuo

    2016-04-28

    Hedgehog (Hh) signaling has been found to be activated in breast cancer stem cells (BCSCs). However, the precise role of the BCSCs marker, CD24, remains unclear. Here, we describe a relationship between CD24 and Sonic Hedgehog (SHH), and reveal a role for this relationship in the induction of a malignant phenotype of breast cancer. CD24 siRNA-transfected breast cancer cells (BCCs) demonstrated higher expression of SHH and GLI1, increased anchorage-independent proliferation, and enhanced invasiveness and superior tumorigenicity compared with control. Conversely, CD24 forced-expressing BCCs possessed decreased SHH and GLI1 expression, anchorage-independent proliferation, and invasiveness. Suppression of SHH decreased invasiveness through inhibition of matrix metalloproteinase (MMP)-2 expression, GLI1 expression, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo in CD24 siRNA transfected BCCs. DNA microarray analysis identified STAT1 as a relationship between CD24 and SHH. CD24 siRNA-transfected BCCs with concurrent STAT1 inhibition exhibited decreased SHH expression, invasiveness, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo. These results suggest that CD24 suppresses development of a malignant phenotype by down-regulating SHH transcription through STAT1 inhibition. CD24 gene transfer or STAT1 inhibition may represent new effective therapeutic strategies to target refractory breast cancer. PMID:26797459

  18. Combination Treatment with Apricoxib and IL-27 Enhances Inhibition of Epithelial-Mesenchymal Transition in Human Lung Cancer Cells through a STAT1 Dominant Pathway

    PubMed Central

    Lee, Mi-Heon; Kachroo, Puja; Pagano, Paul C; Yanagawa, Jane; Wang, Gerald; Walser, Tonya C; Krysan, Kostyantyn; Sharma, Sherven; John, Maie St.; Dubinett, Steven M; Lee, Jay M

    2015-01-01

    Background The cyclooxygenase 2 (COX-2) pathway has been implicated in the molecular pathogenesis of many malignancies, including lung cancer. Apricoxib, a selective COX-2 inhibitor, has been described to inhibit epithelial-mesenchymal transition (EMT) in human malignancies. The mechanism by which apricoxib may alter the tumor microenvironment by affecting EMT through other important signaling pathways is poorly defined. IL-27 has been shown to have anti-tumor activity and our recent study showed that IL-27 inhibited EMT through a STAT1 dominant pathway. Objective The purpose of this study is to investigate the role of apricoxib combined with IL-27 in inhibiting lung carcinogenesis by modulation of EMT through STAT signaling. Methods and Results Western blot analysis revealed that IL-27 stimulation of human non-small cell lung cancer (NSCLC) cell lines results in STAT1 and STAT3 activation, decreased Snail protein and mesenchymal markers (N-cadherin and vimentin) and a concomitant increase in expression of epithelial markers (E-cadherin, β-and γ-catenins), and inhibition of cell migration. The combination of apricoxib and IL-27 resulted in augmentation of STAT1 activation. However, IL-27 mediated STAT3 activation was decreased by the addition of apricoxib. STAT1 siRNA was used to determine the involvement of STAT1 pathway in the enhanced inhibition of EMT and cell migration by the combined IL-27 and apricoxib treatment. Pretreatment of cells with STAT1 siRNA inhibited the effect of combined IL-27 and apricoxib in the activation of STAT1 and STAT3. In addition, the augmented expression of epithelial markers, decreased expression mesenchymal markers, and inhibited cell migration by the combination treatment were also inhibited by STAT1 siRNA, suggesting that the STAT1 pathway is important in the enhanced effect from the combination treatment. Conclusion Combined apricoxib and IL-27 has an enhanced effect in inhibition of epithelial-mesenchymal transition and cell

  19. PIAS1-mediated sumoylation promotes STUbL-dependent proteasomal degradation of the human telomeric protein TRF2.

    PubMed

    Her, Joonyoung; Jeong, Yu Young; Chung, In Kwon

    2015-10-24

    The human telomeric protein TRF2 protects chromosome ends by facilitating their organization into the protective capping structure. Here we show that the stability of TRF2 is regulated via modification by the small ubiquitin-like modifiers (SUMO). TRF2 specifically interacts with and is sumoylated by PIAS1 in mammalian cells. The proteasome inhibitor stabilizes SUMO-conjugated TRF2 without affecting the level of unmodified TRF2, suggesting that SUMO conjugation is required for proteasomal degradation of TRF2. We also show that RNF4, a mammalian SUMO-targeted ubiquitin ligase, interacts with TRF2 in a SUMO-dependent manner and preferentially targets SUMO-conjugated TRF2 for ubiquitination. Collectively, our data demonstrate that the PIAS1-mediated sumoylation status of TRF2 serves as a molecular switch that controls the level of TRF2 at telomeres. PMID:26450775

  20. Constitutive and IFN-gamma-induced nuclear import of STAT1 proceed through independent pathways.

    PubMed

    Meyer, Thomas; Begitt, Andreas; Lödige, Inga; van Rossum, Marleen; Vinkemeier, Uwe

    2002-02-01

    STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine-phosphorylated transcription factor. Here, we identify and characterize a non-transferable nuclear targeting sequence in the STAT1 DNA-binding domain. This conserved signal is critical for the interferon-gamma (IFN-gamma)-induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN-gamma stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive towards wheat germ agglutinin and to occur independently of the import receptor p97. Loss-of-function mutations of the dimer-specific import signal block nuclear entry of tyrosine-phosphorylated STAT1, which in turn also prevents induction of cytokine-inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the tumor necrosis factor-alpha-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes. PMID:11823427

  1. Potential role of 8-oxoguanine DNA glycosylase 1 as a STAT1 coactivator in endotoxin-induced inflammatory response.

    PubMed

    Kim, Hong Sook; Kim, Byung-Hak; Jung, Joo Eun; Lee, Chang Seok; Lee, Hyun Gyu; Lee, Jung Weon; Lee, Kun Ho; You, Ho Jin; Chung, Myung-Hee; Ye, Sang-Kyu

    2016-04-01

    Human 8-oxoguanine DNA glycosylase 1 (OGG1) is the major DNA repair enzyme that plays a key role in excision of oxidative damaged DNA bases such as 8-oxoguainine (8-oxoG). Recent studies suggest another function of OGG1, namely that it may be involved in the endotoxin- or oxidative stress-induced inflammatory response. In this study, we investigated the role of OGG1 in the inflammatory response. OGG1 expression is increased in the organs of endotoxin-induced or myelin oligodendrocyte glycoprotein (MOG)-immunized mice and immune cells, resulting in induction of the expression of pro-inflammatory mediators at the transcriptional levels. Biochemical studies showed that signal transducer and activator of transcription 1 (STAT1) plays a key role in endotoxin-induced OGG1 expression and inflammatory response. STAT1 regulates the transcriptional activity of OGG1 through recruiting and binding to the gamma-interferon activation site (GAS) motif of the OGG1 promoter region, and chromatin remodeling by acetylation and dimethylation of lysine-14 and -4 residues of histone H3. In addition, OGG1 acts as a STAT1 coactivator and has transcriptional activity in the presence of endotoxin. The data presented here identifies a novel mechanism, and may provide new therapeutic strategies for the treatment of endotoxin-mediated inflammatory diseases. PMID:26496208

  2. Identification of STAT1 and STAT3 Specific Inhibitors Using Comparative Virtual Screening and Docking Validation

    PubMed Central

    Szelag, Malgorzata; Czerwoniec, Anna; Wesoly, Joanna; Bluyssen, Hans A. R.

    2015-01-01

    Signal transducers and activators of transcription (STATs) facilitate action of cytokines, growth factors and pathogens. STAT activation is mediated by a highly conserved SH2 domain, which interacts with phosphotyrosine motifs for specific STAT-receptor contacts and STAT dimerization. The active dimers induce gene transcription in the nucleus by binding to a specific DNA-response element in the promoter of target genes. Abnormal activation of STAT signaling pathways is implicated in many human diseases, like cancer, inflammation and auto-immunity. Searches for STAT-targeting compounds, exploring the phosphotyrosine (pTyr)-SH2 interaction site, yielded many small molecules for STAT3 but sparsely for other STATs. However, many of these inhibitors seem not STAT3-specific, thereby questioning the present modeling and selection strategies of SH2 domain-based STAT inhibitors. We generated new 3D structure models for all human (h)STATs and developed a comparative in silico docking strategy to obtain further insight into STAT-SH2 cross-binding specificity of a selection of previously identified STAT3 inhibitors. Indeed, by primarily targeting the highly conserved pTyr-SH2 binding pocket the majority of these compounds exhibited similar binding affinity and tendency scores for all STATs. By comparative screening of a natural product library we provided initial proof for the possibility to identify STAT1 as well as STAT3-specific inhibitors, introducing the ‘STAT-comparative binding affinity value’ and ‘ligand binding pose variation’ as selection criteria. In silico screening of a multi-million clean leads (CL) compound library for binding of all STATs, likewise identified potential specific inhibitors for STAT1 and STAT3 after docking validation. Based on comparative virtual screening and docking validation, we developed a novel STAT inhibitor screening tool that allows identification of specific STAT1 and STAT3 inhibitory compounds. This could increase our

  3. Pretreatment of LPS inhibits IFN-β-induced STAT1 phosphorylation through SOCS3 induced by LPS.

    PubMed

    Ando, Takashi; Komatsu, Takayuki; Naiki, Yoshikazu; Yokochi, Takashi; Watanabe, Daisuke; Koide, Naoki

    2015-12-01

    It has been known that LPS activates macrophages and induces IFN-β production from macrophages. The endogenous IFN-β produced by LPS stimulates the cells, which plays a role in innate immune. However, it was not elucidated yet if the signaling by exogenous IFN-β was influenced by LPS stimulation. In this study, it was found pretreatment of LPS interrupted IFN-β-induced JAK1/STAT1 phosphorylation. LPS pretreatment also reduced IFN-β-induced ISG54, one of IFN-β-inducible genes. Pretreatment with LPS for more than 2h shows inhibitory effect on IFN-β-induced STAT1 phosphorylation but simultaneous treatment or post-treatment of LPS with IFN-β did not show the inhibitory effect. The study using a neutralizing antibody to IFN-β indicated that IFN-β produced by LPS does not take part in the inhibitory effect of LPS. Furthermore, LPS did not affect the expression of IFN αβ receptor. A previous report has shown that LPS-induced SOCS3 inhibited IFN-γ-induced STAT1 phosphorylation, likewise, it was also shown in this study that LPS induced SOCS3 expression and its expression inhibited IFN-β-induced STAT1 phosphorylation which was confirmed by the knockdown study by the siRNA of SOCS3. The real-time PCR and immune-blot studies of SOCS3 indicated that LPS induced SOCS3 is independent of IL-6, IL-10, TNF-α and STAT3, and might depend on p38 activation by LPS. It was suggested that bacterial LPS rather interfere with IFN-β actions, dependent on the timing of LPS stimulation. PMID:26653542

  4. Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis

    PubMed Central

    Mizoguchi, Yoko; Tsumura, Miyuki; Okada, Satoshi; Hirata, Osamu; Minegishi, Shizuko; Imai, Kohsuke; Hyakuna, Nobuyuki; Muramatsu, Hideki; Kojima, Seiji; Ozaki, Yusuke; Imai, Takehide; Takeda, Sachiyo; Okazaki, Tetsuya; Ito, Tsuyoshi; Yasunaga, Shin'ichiro; Takihara, Yoshihiro; Bryant, Vanessa L.; Kong, Xiao-Fei; Cypowyj, Sophie; Boisson-Dupuis, Stéphanie; Puel, Anne; Casanova, Jean-Laurent; Morio, Tomohiro; Kobayashi, Masao

    2014-01-01

    CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patients' leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patients' leukocytes showed abnormally high levels of pSTAT1 following IFN-γ stimulation. Based on this finding, we performed a flow cytometry-based functional analysis of STAT1 GOF alleles using IFN-γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14+ cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry-based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations. PMID:24343863

  5. New and recurrent gain-of-function STAT1 mutations in patients with chronic mucocutaneous candidiasis from Eastern and Central Europe

    PubMed Central

    Soltész, Beáta; Tóth, Beáta; Shabashova, Nadejda; Bondarenko, Anastasia; Okada, Satoshi; Cypowyj, Sophie; Abhyankar, Avinash; Csorba, Gabriella; Taskó, Szilvia; Sarkadi, Adrien Katalin; Méhes, Leonóra; Rozsíval, Pavel; Neumann, David; Chernyshova, Liudmyla; Tulassay, Zsolt; Puel, Anne; Casanova, Jean-Laurent; Sediva, Anna; Litzman, Jiri; Maródi, László

    2013-01-01

    Background Chronic mucocutaneous candidiasis disease (CMCD) may result from various inborn errors of interleukin (IL)-17-mediated immunity. Twelve of the 13 causal mutations described to date affect the coiled-coil domain (CCD) of STAT1. Several mutations, including R274W in particular, are recurrent, but the underlying mechanism is unclear. Objective To investigate and describe nine patients with CMCD in Eastern and Central Europe, to assess the biochemical impact of STAT1 mutations, to determine cytokines in supernatants of Candida-exposed blood cells, to determine IL-17-producing T cell subsets and to determine STAT1 haplotypes in a family with the c.820C>T (R274W) mutation. Results The novel c.537C>A (N179K) STAT1 mutation was gain-of-function (GOF) for γ-activated factor (GAF)-dependent cellular responses. In a Russian patient, the cause of CMCD was the newly identified c.854 A>G (Q285R) STAT1 mutation, which was also GOF for GAF-dependent responses. The c.1154C>T (T385M) mutation affecting the DNA-binding domain (DBD) resulted in a gain of STAT1 phosphorylation in a Ukrainian patient. Impaired Candida-induced IL-17A and IL-22 secretion by leucocytes and lower levels of intracellular IL-17 and IL-22 production by T cells were found in several patients. Haplotype studies indicated that the c.820C>T (R274W) mutation was recurrent due to a hotspot rather than a founder effect. Severe clinical phenotypes, including intracranial aneurysm, are presented. Conclusions The c.537C>A and c.854A>G mutations affecting the CCD and the c.1154C>T mutation affecting the DBD of STAT1 are GOF. The c.820C>T mutation of STAT1 in patients with CMCD is recurrent due to a hotspot. Patients carrying GOF mutations of STAT1 may develop multiple intracranial aneurysms by hitherto unknown mechanisms. PMID:23709754

  6. Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

    SciTech Connect

    Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana; Springfeld, Christoph; Cattaneo, Roberto . E-mail: cattaneo.roberto@mayo.edu

    2007-03-30

    The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Our study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.

  7. The non-pathogenic Henipavirus Cedar paramyxovirus phosphoprotein has a compromised ability to target STAT1 and STAT2.

    PubMed

    Lieu, Kim G; Marsh, Glenn A; Wang, Lin-Fa; Netter, Hans J

    2015-12-01

    Immune evasion by the lethal henipaviruses, Hendra (HeV) and Nipah virus, is mediated by its interferon (IFN) antagonist P gene products, phosphoprotein (P), and the related V and W proteins, which can target the signal transducer and activator of transcription 1 (STAT1) and STAT2 proteins to inhibit IFN/STAT signaling. However, it is not clear if the recently identified non-pathogenic Henipavirus, Cedar paramyxovirus (CedPV), is also able to antagonize the STAT proteins. We performed comparative studies between the HeV P gene products (P/V/W) and CedPV-P (CedPV does not encode V or W) and demonstrate that differences exist in their ability to engage the STAT proteins using immunoprecipitation and quantitative confocal microscopic analysis. In contrast to HeV-P gene encoded proteins, the ability of CedPV-P to interact with and relocalize STAT1 or STAT2 is compromised, correlating with a reduced capacity to inhibit the mRNA synthesis of IFN-inducible gene MxA. Furthermore, infection studies with HeV and CedPV demonstrate that HeV is more potent than CedPV in inhibiting the IFN-α-mediated nuclear accumulation of STAT1. These results strongly suggest that the ability of CedPV to counteract the IFN/STAT response is compromised compared to HeV. PMID:26526590

  8. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    PubMed Central

    Putz, Eva Maria; Gotthardt, Dagmar; Hoermann, Gregor; Csiszar, Agnes; Wirth, Silvia; Berger, Angelika; Straka, Elisabeth; Rigler, Doris; Wallner, Barbara; Jamieson, Amanda M.; Pickl, Winfried F.; Zebedin-Brandl, Eva Maria; Müller, Mathias; Decker, Thomas; Sexl, Veronika

    2013-01-01

    Summary The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance. PMID:23933255

  9. Newcastle Disease Virus V Protein Targets Phosphorylated STAT1 to Block IFN-I Signaling

    PubMed Central

    Qiu, Xusheng; Fu, Qiang; Meng, Chunchun; Yu, Shengqing; Zhan, Yuan; Dong, Luna; Song, Cuiping; Sun, Yingjie; Tan, Lei; Hu, Shunlin; Wang, Xiaoquan; Liu, Xiaowen; Peng, Daxin; Liu, Xiufan; Ding, Chan

    2016-01-01

    Newcastle disease virus (NDV) V protein is considered as an effector for IFN antagonism, however, the mechanism remains unknown. In this study, the expression of STAT1 and phospho-STAT1 in cells infected with NDV or transfected with V protein-expressing plasmids were analyzed. Our results showed that NDV V protein targets phospho-STAT1 reduction in the cells depends on the stimulation of IFN-α. In addition, a V-deficient genotype VII recombinant NDV strain rZJ1-VS was constructed using reverse genetic technique to confirm the results. The rZJ1-VS lost the ability to reduce phospho-STAT1 and induced higher expression of IFN-responsive genes in infected cells. Furthermore, treatment with an ubiquitin E1 inhibitor PYR-41 demonstrated that phospho-STAT1 reduction was caused by degradation, but not de-phosphorylation. We conclude that NDV V protein targets phospho-STAT1 degradation to block IFN-α signaling, which adds novel knowledge to the strategies used by paramyxoviruses to evade IFN. PMID:26859759

  10. Toxoplasma Effector Recruits the Mi-2/NuRD Complex to Repress STAT1 Transcription and Block IFN-γ-Dependent Gene Expression.

    PubMed

    Olias, Philipp; Etheridge, Ronald D; Zhang, Yong; Holtzman, Michael J; Sibley, L David

    2016-07-13

    Interferon gamma (IFN-γ) is an essential mediator of host defense against intracellular pathogens, including the protozoan parasite Toxoplasma gondii. However, prior T. gondii infection blocks IFN-γ-dependent gene transcription, despite the downstream transcriptional activator STAT1 being activated and bound to cognate nuclear promoters. We identify the parasite effector that blocks STAT1-dependent transcription and show it is associated with recruitment of the Mi-2 nucleosome remodeling and deacetylase (NuRD) complex, a chromatin-modifying repressor. This secreted effector, toxoplasma inhibitor of STAT1-dependent transcription (TgIST), translocates to the host cell nucleus, where it recruits Mi-2/NuRD to STAT1-dependent promoters, resulting in altered chromatin and blocked transcription. TgIST is conserved across strains, underlying their shared ability to block IFN-γ-dependent transcription. TgIST deletion results in increased parasite clearance in IFN-γ-activated cells and reduced mouse virulence, which is restored in IFN-γ-receptor-deficient mice. These findings demonstrate the importance of both IFN-γ responses and the ability of pathogens to counteract these defenses. PMID:27414498

  11. Elevated interleukin-27 levels in human neonatal macrophages regulate indoleamine dioxygenase in a STAT-1 and STAT-3-dependent manner.

    PubMed

    Jung, Joo-Yong; Gleave Parson, Madeline; Kraft, Jennifer D; Lyda, Logan; Kobe, Brianna; Davis, Celestia; Robinson, Jembber; Peña, Maria Marjorette O; Robinson, Cory M

    2016-09-01

    Microbial infections are a major cause of infant mortality as a result of limitations in immune defences. Interleukin-27 (IL-27) is a heterodimeric cytokine produced primarily by leucocytes and is immunosuppressive toward lymphocytes and leucocytes. Our laboratory demonstrated that human neonatal macrophages express IL-27 more abundantly than adult macrophages. Similarly in mice, IL-27 expression is elevated early in life and maintained through infancy. To determine IL-27-regulated mechanisms that may limit immunity, we evaluated the expression of a number of genes in response to this cytokine in primary human neonatal macrophages. Indoleamine 2,3-dioxygenase (IDO) gene expression was increased dose-responsively by IL-27. We have previously demonstrated inhibition of T-cell proliferation and cytokine production by neonatal macrophage-generated IL-27, and IDO is often implicated in this negative regulation. An increase in IDO protein was demonstrated by immunofluorescence microscopy and was consistent with increased enzyme activity following treatment with IL-27. Inclusion of a soluble receptor to neutralize endogenous IL-27, decreased IDO expression and activity compared with untreated macrophages. In response to IL-27, neonatal macrophages phosphorylate signal transdcuer and activator of transcription 1 (STAT-1) and STAT-3. Both transcription factors are recruited to the IDO regulatory region. STAT-3 dominates during steady-state regulation by lower levels of endogenous IL-27 production. A shift to enhanced STAT-1 recruitment occurs during increased levels of exogenously supplied IL-27. These data suggest an interesting interplay of STAT-1 and STAT-3 to regulate IDO activity and immunosuppression in response to different levels of IL-27 in the microenvironment of the immune response that may further our understanding of this interesting cytokine. PMID:27238498

  12. The HIV Matrix Protein p17 Subverts Nuclear Receptors Expression and Induces a STAT1-Dependent Proinflammatory Phenotype in Monocytes

    PubMed Central

    Renga, Barbara; Francisci, Daniela; D'Amore, Claudio; Schiaroli, Elisabetta; Mencarelli, Andrea; Cipriani, Sabrina

    2012-01-01

    Background Long-term remission of HIV-1 disease can be readily achieved by combinations of highly effective antiretroviral therapy (HAART). However, a residual persistent immune activation caused by circulating non infectious particles or viral proteins is observed under HAART and might contribute to an higher risk of non-AIDS pathologies and death in HIV infected persons. A sustained immune activation supports lipid dysmetabolism and increased risk for development of accelerated atehrosclerosis and ischemic complication in virologically suppressed HIV-infected persons receiving HAART. Aim While several HIV proteins have been identified and characterized for their ability to maintain immune activation, the role of HIV-p17, a matrix protein involved in the viral replication, is still undefined. Results Here, we report that exposure of macrophages to recombinant human p17 induces the expression of proinflammatory and proatherogenic genes (MCP-1, ICAM-1, CD40, CD86 and CD36) while downregulating the expression of nuclear receptors (FXR and PPARγ) that counter-regulate the proinflammatory response and modulate lipid metabolism in these cells. Exposure of macrophage cell lines to p17 activates a signaling pathway mediated by Rack-1/Jak-1/STAT-1 and causes a promoter-dependent regulation of STAT-1 target genes. These effects are abrogated by sera obtained from HIV-infected persons vaccinated with a p17 peptide. Ligands for FXR and PPARγ counteract the effects of p17. Conclusions The results of this study show that HIV p17 highjacks a Rack-1/Jak-1/STAT-1 pathway in macrophages, and that the activation of this pathway leads to a simultaneous dysregulation of immune and metabolic functions. The binding of STAT-1 to specific responsive elements in the promoter of PPARγ and FXR and MCP-1 shifts macrophages toward a pro-atherogenetic phenotype characterized by high levels of expression of the scavenger receptor CD36. The present work identifies p17 as a novel target in HIV

  13. Two Domains of the V Protein of Virulent Canine Distemper Virus Selectively Inhibit STAT1 and STAT2 Nuclear Import▿

    PubMed Central

    Röthlisberger, Anne; Wiener, Dominique; Schweizer, Matthias; Peterhans, Ernst; Zurbriggen, Andreas; Plattet, Philippe

    2010-01-01

    Canine distemper virus (CDV) causes in dogs a severe systemic infection, with a high frequency of demyelinating encephalitis. Among the six genes transcribed by CDV, the P gene encodes the polymerase cofactor protein (P) as well as two additional nonstructural proteins, C and V; of these V was shown to act as a virulence factor. We investigated the molecular mechanisms by which the P gene products of the neurovirulent CDV A75/17 strain disrupt type I interferon (IFN-α/β)-induced signaling that results in the establishment of the antiviral state. Using recombinant knockout A75/17 viruses, the V protein was identified as the main antagonist of IFN-α/β-mediated signaling. Importantly, immunofluorescence analysis illustrated that the inhibition of IFN-α/β-mediated signaling correlated with impaired STAT1/STAT2 nuclear import, whereas the phosphorylation state of these proteins was not affected. Coimmunoprecipitation assays identified the N-terminal region of V (VNT) responsible for STAT1 targeting, which correlated with its ability to inhibit the activity of the IFN-α/β-mediated antiviral state. Conversely, while the C-terminal domain of V (VCT) could not function autonomously, when fused to VNT it optimally interacted with STAT2 and subsequently efficiently suppressed the IFN-α/β-mediated signaling pathway. The latter result was further supported by a single mutation at position 110 within the VNT domain of CDV V protein, resulting in a mutant that lost STAT1 binding while retaining a partial STAT2 association. Taken together, our results identified the CDV VNT and VCT as two essential modules that complement each other to interfere with the antiviral state induced by IFN-α/β-mediated signaling. Hence, our experiments reveal a novel mechanism of IFN-α/β evasion among the morbilliviruses. PMID:20427537

  14. Common variable immunodeficiency, impaired neurological development and reduced numbers of T regulatory cells in a 10-year-old boy with a STAT1 gain-of-function mutation.

    PubMed

    Kobbe, Robin; Kolster, Manuela; Fuchs, Sebastian; Schulze-Sturm, Ulf; Jenderny, Jutta; Kochhan, Lothar; Staab, Julia; Tolosa, Eva; Grimbacher, Bodo; Meyer, Thomas

    2016-07-25

    Recently, gain-of-function (GOF) mutations in the gene encoding signal transducer and activator of transcription 1 (STAT1) have been associated with chronic mucocutaneous candidiasis (CMC). This case report describes a 10-year-old boy presenting with signs of common variable immunodeficiency (CVID), failure to thrive, impaired neurological development, and a history of recurrent mucocutaneous Candida infections. Sequencing of the STAT1 gene identified a heterozygous missense mutation in exon 7 encoding the STAT1 coiled-coil domain (c.514T>C, p.Phe172Leu). In addition to hypogammaglobulinemia with B-cell deficiency, and a low percentage of Th17 cells, immunological analysis of the patient revealed a marked depletion of forkhead-box P3(+)-expressing regulatory T cells (Tregs). In vitro stimulation of T cells from the patient with interferon-α (IFNα) and/or IFNɣ resulted in a significantly increased expression of STAT1-regulated target genes such as MIG1, IRF1, MX1, MCP1/CCL2, IFI-56K, and CXCL10 as compared to IFN-treated cells from a healthy control, while no IFNα/ɣ-mediated up-regulation of the FOXP3 gene was found. These data demonstrate that the STAT1 GOF mutation F172L, which results in impaired stability of the antiparallel STAT1 dimer conformation, is associated with inhibited Treg cell development and neurological symptoms. PMID:27063510

  15. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response

    PubMed Central

    Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C.; Paulus, Christina

    2016-01-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  16. Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response.

    PubMed

    Harwardt, Thomas; Lukas, Simone; Zenger, Marion; Reitberger, Tobias; Danzer, Daniela; Übner, Theresa; Munday, Diane C; Nevels, Michael; Paulus, Christina

    2016-07-01

    The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. PMID:27387064

  17. Structural basis for recruitment of CBP/p300 coactivators by STAT1 and STAT2 transactivation domains

    PubMed Central

    Wojciak, Jonathan M; Martinez-Yamout, Maria A; Dyson, H Jane; Wright, Peter E

    2009-01-01

    CBP/p300 transcriptional coactivators mediate gene expression by integrating cellular signals through interactions with multiple transcription factors. To elucidate the molecular and structural basis for CBP-dependent gene expression, we determined structures of the CBP TAZ1 and TAZ2 domains in complex with the transactivation domains (TADs) of signal transducer and activator of transcription 2 (STAT2) and STAT1, respectively. Despite the topological similarity of the TAZ1 and TAZ2 domains, subtle differences in helix packing and surface grooves constitute major determinants of target selectivity. Our results suggest that TAZ1 preferentially binds long TADs capable of contacting multiple surface grooves simultaneously, whereas smaller TADs that are restricted to a single contiguous binding surface form complexes with TAZ2. Complex formation for both STAT TADs involves coupled folding and binding, driven by intermolecular hydrophobic and electrostatic interactions. Phosphorylation of S727, required for maximal transcriptional activity of STAT1, does not enhance binding to any of the CBP domains. Because the different STAT TADs recognize different regions of CBP/p300, there is a potential for multivalent binding by STAT heterodimers that could enhance the recruitment of the coactivators to promoters. PMID:19214187

  18. STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-{alpha}2b peptide

    SciTech Connect

    Blank, Viviana C.; Pena, Clara; Roguin, Leonor P.

    2010-02-15

    In the search of mimetic peptides of the interferon-{alpha}2b molecule (IFN-{alpha}2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-{alpha}2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-{alpha}2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

  19. The TATA-containing core promoter of the type II collagen gene (COL2A1) is the target of interferon-gamma-mediated inhibition in human chondrocytes: requirement for Stat1 alpha, Jak1 and Jak2.

    PubMed Central

    Osaki, Makoto; Tan, Lujian; Choy, Bob K; Yoshida, Yasuhiro; Cheah, Kathryn S E; Auron, Philip E; Goldring, Mary B

    2003-01-01

    Interferon-gamma (IFN-gamma) inhibits the synthesis of the cartilage-specific extracellular matrix protein type II collagen, and suppresses the expression of the type II collagen gene ( COL2A1 ) at the transcriptional level. To further examine this mechanism, the responses of COL2A1 regulatory sequences to IFN-gamma and the role of components of the Janus kinase/signal transducer and activators of transcription (JAK/STAT) pathway were examined in the immortalized human chondrocyte cell line, C-28/I2. IFN-gamma inhibited the mRNA levels of COL2A1 and aggrecan, but not Sox9, L-Sox5 and Sox6, all of which were expressed by these cells as markers of the differentiated phenotype. IFN-gamma suppressed the expression of luciferase reporter constructs containing sequences of the COL2A1 promoter spanning -6368 to +125 bp in the absence and presence of the intronic enhancer and stimulated activity of the gamma-interferon-activated site (GAS) luciferase reporter vector, associated with induction of Stat1 alpha-binding activity in nuclear extracts. These responses to IFN-gamma were blocked by overexpression of the JAK inhibitor, JAK-binding protein (JAB), or reversed by dominant-negative Stat1 alpha Y701F containing a mutation at Tyr-701, the JAK phosphorylation site. IFN-gamma had no effect on COL2A1 promoter expression in Jak1 (U4A)-, Jak2 (gamma 2A)- and Stat1 alpha (U3A)-deficient cell lines. In the U3A cell line, the response to IFN-gamma was rescued by overexpression of Stat1 alpha, but not by either Stat1 alpha Y701F or Stat1 beta. Functional analysis using deletion constructs showed that the IFN-gamma response was retained in the COL2A1 core promoter region spanning -45 to +11 bp, containing the TATA-box and GC-rich sequences but no Stat1-binding elements. Inhibition of COL2A1 promoter activity by IFN-gamma persisted in the presence of multiple deletions within the -45/+11 bp region. Our results indicate that repression of COL2A1 gene transcription by IFN

  20. GAIN OF FUNCTION STAT1 MUTATION-RELATED PRIMARY IMMUNODEFICIENCY IS ASSOCIATED WITH DISSEMINATED MUCORMYCOSIS

    PubMed Central

    Kumar, Nilay; Hanks, Mary E.; Chandrasekaran, Prabha; Davis, Brian C.; Hsu, Amy P.; Van Wagoner, Nicholas J.; Merlin, Jessica S.; Spalding, Christine; La Hoz, Ricardo M.; Holland, Steven M.; Zerbe, Christa S.; Sampaio, Elizabeth P.

    2014-01-01

    SUMMARY We identified a novel gain of function mutation in STAT1 in a patient with disseminated Apophysomyces trapeziformis infection who had never had mucocutaneous candidiasis or autoimmunity. To our knowledge this is the first report of a genetic predisposition associated with mucormycosis. PMID:24709374

  1. The Inflammatory Transcription Factors NFκB, STAT1 and STAT3 Drive Age-Associated Transcriptional Changes in the Human Kidney

    PubMed Central

    O’Brown, Zach K.; Van Nostrand, Eric L.; Higgins, John P.; Kim, Stuart K.

    2015-01-01

    Human kidney function declines with age, accompanied by stereotyped changes in gene expression and histopathology, but the mechanisms underlying these changes are largely unknown. To identify potential regulators of kidney aging, we compared age-associated transcriptional changes in the human kidney with genome-wide maps of transcription factor occupancy from ChIP-seq datasets in human cells. The strongest candidates were the inflammation-associated transcription factors NFκB, STAT1 and STAT3, the activities of which increase with age in epithelial compartments of the renal cortex. Stimulation of renal tubular epithelial cells with the inflammatory cytokines IL-6 (a STAT3 activator), IFNγ (a STAT1 activator), or TNFα (an NFκB activator) recapitulated age-associated gene expression changes. We show that common DNA variants in RELA and NFKB1, the two genes encoding subunits of the NFκB transcription factor, associate with kidney function and chronic kidney disease in gene association studies, providing the first evidence that genetic variation in NFκB contributes to renal aging phenotypes. Our results suggest that NFκB, STAT1 and STAT3 underlie transcriptional changes and chronic inflammation in the aging human kidney. PMID:26678048

  2. IRF9 and STAT1 are required for IgG autoantibody production and B cell expression of TLR7 in mice.

    PubMed

    Thibault, Donna L; Chu, Alvina D; Graham, Kareem L; Balboni, Imelda; Lee, Lowen Y; Kohlmoos, Cassidy; Landrigan, Angela; Higgins, John P; Tibshirani, Robert; Utz, Paul J

    2008-04-01

    A hallmark of SLE is the production of high-titer, high-affinity, isotype-switched IgG autoantibodies directed against nucleic acid-associated antigens. Several studies have established a role for both type I IFN (IFN-I) and the activation of TLRs by nucleic acid-associated autoantigens in the pathogenesis of this disease. Here, we demonstrate that 2 IFN-I signaling molecules, IFN regulatory factor 9 (IRF9) and STAT1, were required for the production of IgG autoantibodies in the pristane-induced mouse model of SLE. In addition, levels of IgM autoantibodies were increased in pristane-treated Irf9 -/- mice, suggesting that IRF9 plays a role in isotype switching in response to self antigens. Upregulation of TLR7 by IFN-alpha was greatly reduced in Irf9 -/- and Stat1 -/- B cells. Irf9 -/- B cells were incapable of being activated through TLR7, and Stat1 -/- B cells were impaired in activation through both TLR7 and TLR9. These data may reveal a novel role for IFN-I signaling molecules in both TLR-specific B cell responses and production of IgG autoantibodies directed against nucleic acid-associated autoantigens. Our results suggest that IFN-I is upstream of TLR signaling in the activation of autoreactive B cells in SLE. PMID:18340381

  3. Ebola Virus VP24 Binds Karyopherin α1 and Blocks STAT1 Nuclear Accumulation

    PubMed Central

    Reid, St. Patrick; Leung, Lawrence W.; Hartman, Amy L.; Martinez, Osvaldo; Shaw, Megan L.; Carbonnelle, Caroline; Volchkov, Viktor E.; Nichol, Stuart T.; Basler, Christopher F.

    2006-01-01

    Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-α/β) and the ability of cells to respond to IFN-α/β or IFN-γ. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-α/β production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-α/β and IFN-γ signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-α/β and IFN-γ signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with karyopherin α1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin α2, α3, or α4. Overexpression of VP24 results in a loss of karyopherin α1-PY-STAT1 interaction, indicating that the VP24-karyopherin α1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs. PMID:16698996

  4. Toxoplasma gondii TgIST co-opts host chromatin repressors dampening STAT1-dependent gene regulation and IFN-γ-mediated host defenses.

    PubMed

    Gay, Gabrielle; Braun, Laurence; Brenier-Pinchart, Marie-Pierre; Vollaire, Julien; Josserand, Véronique; Bertini, Rose-Laurence; Varesano, Aurélie; Touquet, Bastien; De Bock, Pieter-Jan; Coute, Yohann; Tardieux, Isabelle; Bougdour, Alexandre; Hakimi, Mohamed-Ali

    2016-08-22

    An early hallmark of Toxoplasma gondii infection is the rapid control of the parasite population by a potent multifaceted innate immune response that engages resident and homing immune cells along with pro- and counter-inflammatory cytokines. In this context, IFN-γ activates a variety of T. gondii-targeting activities in immune and nonimmune cells but can also contribute to host immune pathology. T. gondii has evolved mechanisms to timely counteract the host IFN-γ defenses by interfering with the transcription of IFN-γ-stimulated genes. We now have identified TgIST (T. gondii inhibitor of STAT1 transcriptional activity) as a critical molecular switch that is secreted by intracellular parasites and traffics to the host cell nucleus where it inhibits STAT1-dependent proinflammatory gene expression. We show that TgIST not only sequesters STAT1 on dedicated loci but also promotes shaping of a nonpermissive chromatin through its capacity to recruit the nucleosome remodeling deacetylase (NuRD) transcriptional repressor. We found that during mice acute infection, TgIST-deficient parasites are rapidly eliminated by the homing Gr1(+) inflammatory monocytes, thus highlighting the protective role of TgIST against IFN-γ-mediated killing. By uncovering TgIST functions, this study brings novel evidence on how T. gondii has devised a molecular weapon of choice to take control over a ubiquitous immune gene expression mechanism in metazoans, as a way to promote long-term parasitism. PMID:27503074

  5. STAT1-Induced HLA class I Upregulation Enhances Immunogenicity and Clinical Response to anti-EGFR mAb Cetuximab Therapy in HNC Patients

    PubMed Central

    Srivastava, Raghvendra M.; Trivedi, Sumita; Concha-Benavente, Fernando; Hyun-bae, Jie; Wang, Lin; Seethala, Raja R.; Branstetter, Barton F.; Ferrone, Soldano; Ferris, Robert L.

    2015-01-01

    The goal of this study was to characterize the molecular mechanisms underlying cetuximab-mediated upregulation of HLA class I antigen-processing machinery components in head and neck cancer (HNC) cells and to determine the clinical significance of these changes in cetuximab-treated HNC patients. Flow cytometry, signaling studies and chromatin immunoprecipitation (ChIP) assays were performed using HNC cells treated with cetuximab alone or with Fcγ receptor (FcγR)-bearing lymphocytes to establish the mechanism of EGFR-dependent regulation of HLA APM expression. A prospective phase II clinical trial of neoadjuvant cetuximab was utilized to correlate HLA class I expression with clinical response in HNC patients. EGFR blockade triggered STAT1 activation and HLA upregulation, in a src homology-containing protein (SHP)-2-dependent fashion, more prominently in HLA-B/C than in HLA-A alleles. EGFR signaling blockade also enhanced IFNγ receptor 1 (IFNAR) expression, augmenting induction of HLA class I and TAP1/2 expression by IFNγ, which was abrogated in STAT1−/− cells. Cetuximab enhanced HNC cell recognition by EGFR853–861-specific CTLs, and notably enhanced surface presentation of a non-EGFR peptide (MAGE-3271–279). HLA class I upregulation was significantly associated with clinical response in cetuximab-treated HNC patients. EGFR induces HLA downregulation through SHP-2/STAT1 suppression. Reversal of HLA class I downregulation was more prominent in clinical responders to cetuximab therapy, supporting an important role for adaptive immunity in cetuximab antitumor activity. Abrogating EGFR-induced immune escape mechanisms and restoring STAT1 signaling to reverse HLA downregulation using cetuximab should be combined with strategies to enhance adaptive cellular immunity. PMID:25972070

  6. Inhibition of integrin β1 decreases the malignancy of ovarian cancer cells and potentiates anticancer therapy via the FAK/STAT1 signaling pathway

    PubMed Central

    ZHANG, LEI; ZOU, WEN

    2015-01-01

    Integrin β1 (ITGB1) is frequently upregulated in ovarian cancer, and promotes ovarian tumorigenesis and cancer progression. However, the effects of ITGB1 inhibition on ovarian cancer progression and anticancer therapy remain to be elucidated. The results of the present study indicated that ITGB1 was upregulated in HO-8910 and HO-8910PM ovarian cancer cell lines, and knockdown of ITGB1 using short hairpin RNA markedly increased tumor cell apoptosis, decreased tumor cell adhesion and migration, and reduced tumor cell invasion by suppressing matrix metalloproteinase (MMP)-2 and MMP-9 expression. Furthermore, the results of the present study provided evidence regarding the role of ITGB1 inhibition in bevacizumab anticancer therapy. The activation of signal transducer and activator of transcription 1 (STAT1) by focal adhesion kinase (FAK) is involved in integrin-mediated cell migration and adhesion. In the present study, the expression levels of FAK were markedly upregulated in ovarian cancer. The adherence and migratory potentials of ovarian cancer cells were significantly reduced when the FAK/STAT1 signaling pathway was inhibited by fludarabine. The results of the present study demonstrated that ITGB1 inhibition effectively reduced tumorigenesis and disease exacerbation, and contributed to bevacizumab anticancer therapy via the FAK/STAT1 signaling pathway, suggesting that inhibition of ITGB1 is a potential novel therapeutic strategy for ovarian carcinogenesis. PMID:26497667

  7. Tagging of Genomic STAT3 and STAT1 with Fluorescent Proteins and Insertion of a Luciferase Reporter in the Cyclin D1 Gene Provides a Modified A549 Cell Line to Screen for Selective STAT3 Inhibitors

    PubMed Central

    Samsonov, Andrey; Zenser, Nathan; Zhang, Fan; Zhang, Hongyi; Fetter, John; Malkov, Dmitry

    2013-01-01

    Signal transducer and activator of transcription 3 (STAT3) is an oncogenic protein that is constitutively activated in numerous cancer cell lines and human cancers. Another STAT family member, STAT1, possesses cancer-inhibitory properties and can promote apoptosis in tumor cells upon activation. To better characterize these important cancer related genes, we tagged STAT3 and STAT1 loci with fluorescent protein (FP) sequences (RFP and GFP respectively) by targeted integration via zinc finger nuclease (ZFN) - mediated homologous recombination in A549 cells that express aberrantly activated STAT3. We inserted the FP transgenes at the N-terminus of the STAT3 locus and at the C-terminus of the STAT1 locus. The integration resulted in endogenous expression of fluorescent STAT3 and STAT1 chimeric fusion proteins. When stimulated with IL-6 or IFN-γ, the cells showed robust nuclear translocation of RFP-STAT3 or STAT1-GFP, respectively. Pre-incubation of cells with a known specific STAT3 inhibitor showed that IFN-γ-induced translocation of STAT1-GFP was not impaired. STAT3 activates multiple downstream targets such as genes involved in cell cycle progression - e.g. cyclin D1. To detect changes in expression of endogenous cyclin D1, we used ZFN technology to insert a secreted luciferase reporter behind the cyclin D1 promoter and separated the luciferase and cyclin D1 coding regions by a 2A sequence to induce a translational skip. The luciferase insertion was made in the RFP-STAT3/STAT1-GFP cell line to have all three reporters in a single cell line. Addition of a STAT3 inhibitor led to suppression of cyclin D1 promoter activity and cell growth arrest. The triple-modified cell line provides a simple and convenient method for high-content screening and pre-clinical testing of potential STAT3 inhibitors in live cells while ensuring that the STAT1 pathway is not affected. This approach of reporting endogenous gene activities using ZFN technology could be applied to other cancer

  8. Murine Norovirus 1 Infection Is Associated with Histopathological Changes in Immunocompetent Hosts, but Clinical Disease Is Prevented by STAT1-Dependent Interferon Responses▿

    PubMed Central

    Mumphrey, Shannon M.; Changotra, Harish; Moore, Tara N.; Heimann-Nichols, Ellen R.; Wobus, Christiane E.; Reilly, Michael J.; Moghadamfalahi, Mana; Shukla, Deepti; Karst, Stephanie M.

    2007-01-01

    Human noroviruses are the major cause of nonbacterial epidemic gastroenteritis worldwide. However, little is known regarding their pathogenesis or the immune responses that control them because until recently there has been no small animal model or cell culture system of norovirus infection. We recently reported the discovery of the first murine norovirus, murine norovirus 1 (MNV-1), and its cultivation in macrophages and dendritic cells in vitro. We further defined interferon receptors and the STAT-1 molecule as critical in both resistance to MNV-1-induced disease in vivo and control of virus growth in vitro. To date, neither histopathological changes upon infection nor viral replication in wild-type mice has been shown. Here we extend our studies to demonstrate that MNV-1 replicates and rapidly disseminates to various tissues in immunocompetent mice and that infection is restricted by STAT1-dependent interferon responses at the levels of viral replication and virus dissemination. Infection of wild-type mice is associated with histopathological alterations in the intestine (mild inflammation) and the spleen (red pulp hypertrophy and white pulp activation); viral dissemination to the spleen, liver, lung, and lymph nodes; and low-level persistent infection in the spleen. STAT-1 inhibits viral replication in the intestine, prevents virus-induced apoptosis of intestinal cells and splenocytes, and limits viral dissemination to peripheral tissues. These findings demonstrate that murine norovirus infection of wild-type mice is associated with initial enteric seeding and subsequent extraintestinal spread, and they provide mechanistic evidence of the role of STAT-1 in controlling clinical norovirus-induced disease. PMID:17229692

  9. Molecular characterization of RIG-I, STAT-1 and IFN-beta in the horseshoe bat.

    PubMed

    Li, Jinju; Zhang, Guangxu; Cheng, Dalong; Ren, Hua; Qian, Min; Du, Bing

    2015-04-25

    Wild Chinese horseshoe bats have been proven to be natural reservoirs of SARS-like coronaviruses. However, the molecular characterization of key proteins in bats still needs to be explored further. In this study, we used cloning and bioinformatics to analyze the sequence of RIG-I, STAT-1 and IFN-β in the immortalized cell lines from Rhinolophus affinis and Rhinolophus sinicus. Then, we treated different bat cells, mouse embryonic fibroblasts (MEF) and splenocytes with polyinosinic-polycytidylic acid (polyI:C) and vesicular stomatitis virus (VSV) to assess and compare antiviral immune responses between bats and mice. Our results demonstrated that bat RIG-I, STAT-1 and IFN-β showed close homology with human, mouse, pig and rhesus monkey. RIG-I and STAT-1 were both highly expressed in bat spleen. Furthermore, IFN-β was induced by polyI:C and VSV in both bat and mouse cells. These findings have provided new insight into the potential characteristics of the bat innate immune system against viral infection. PMID:25680291

  10. Selective inhibition of JAK2/STAT1 signaling and iNOS expression mediates the anti-inflammatory effects of coniferyl aldehyde.

    PubMed

    Akram, Muhammad; Kim, Kyeong-A; Kim, Eun-Sun; Shin, Young-Jun; Noh, Dabi; Kim, Eunji; Kim, Jeong-Hyeon; Majid, Arshad; Chang, Sun-Young; Kim, Jin-Ki; Bae, Ok-Nam

    2016-08-25

    Urgent needs still exist for selective control of excessive inflammation. Despite the therapeutic potential of natural compounds against inflammation-associated chronic conditions, lack of specific molecular targets renders these bioactive compounds difficult for further development. Here we examined the bioactivity of coniferyl aldehyde (CA), a natural phenolic compound found in several dietary substances and medicinal plants, elucidating its efficacy both in vivo and in vitro with underlying molecular mechanisms. IFN-γ/TNF-α-stimulated human keratinocytes and lipopolysaccharide (LPS)-stimulated murine macrophages were used to examine the effect of CA in vitro and to elucidate the underlying mechanisms. In vivo models of phorbol 12-myristate 13-acetate (TPA)-induced ear edema and carrageenan (CRG)-induced paw edema were employed to investigate the topical and systemic anti-inflammatory effects of CA, respectively. CA significantly reduced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in LPS-stimulated macrophages. While nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) pathways, the representative cellular pathways for iNOS induction, were not affected by CA, phosphorylation of Janus kinase 2 (JAK2) and signal Transducers and Activators of Transcription 1 (STAT1) and subsequent nuclear translocation of p-STAT1 were significantly decreased by CA. The effect of CA on JAK2-STAT1-iNOS axis was also observed in human keratinocytes stimulated with IFN-γ/TNF-α. Topical application of CA to mice produced significant protection against TPA-induced ear edema along with suppressed epidermal hyperproliferation and leucocyte infiltration. Systemic administration of CA significantly reduced CRG-induced paw edema in rats, where CRG-induced iNOS expression and STAT1 phosphorylation were decreased by CA. In summary, CA has significant anti-inflammatory properties both in vitro and in vivo, mediated by

  11. Biflorin, Isolated from the Flower Buds of Syzygium aromaticum L., Suppresses LPS-Induced Inflammatory Mediators via STAT1 Inactivation in Macrophages and Protects Mice from Endotoxin Shock.

    PubMed

    Lee, Hwi-Ho; Shin, Ji-Sun; Lee, Woo-Seok; Ryu, Byeol; Jang, Dae Sik; Lee, Kyung-Tae

    2016-04-22

    Two chromone C-glucosides, biflorin (1) and isobiflorin (2), were isolated from the flower buds of Syzygium aromaticum L. (Myrtaceae). Here, inhibitory effects of 1 and 2 on lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 macrophages were evaluated, and 1 (IC50 = 51.7 and 37.1 μM, respectively) was more potent than 2 (IC50 > 60 and 46.0 μM). The suppression of NO and PGE2 production by 1 correlated with inhibition of iNOS and COX-2 protein expression. Compound 1 reduced inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression via inhibition of their promoter activities. Compound 1 inhibited the LPS-induced production and mRNA expression of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6. Furthermore, 1 reduced p-STAT1 and p-p38 expression but did not affect the activity of nuclear factor κ light-chain enhancer of activated B cells (NF-κB) or activator protein 1 (AP-1). In a mouse model of LPS-induced endotoxemia, 1 reduced the mRNA levels of iNOS, COX-2, and TNF-α, and the phosphorylation-mediated activation of the signal transducer and activator of transcription 1 (STAT1), consequently improving the survival rates of mice. Compound 1 showed a significant anti-inflammatory effect on carrageenan-induced paw edema and croton-oil-induced ear edema in rats. The collective data indicate that the suppression of pro-inflammatory gene expression via p38 mitogen-activated protein kinase and STAT1 inactivation may be a mechanism for the anti-inflammatory activity of 1. PMID:26977531

  12. Induced eosinophilia and proliferation in Angiostrongylus cantonensis-infected mouse brain are associated with the induction of JAK/STAT1, IAP/NF-kappaB and MEKK1/JNK signals.

    PubMed

    Lan, K P; Wang, C J; Hsu, J D; Chen, K M; Lai, S C; Lee, H H

    2004-12-01

    Eosinophilic meningitis or meningoencephalitis caused by Angiostrongylus cantonensis is endemic to the Pacific area of Asia, especially Taiwan, Thailand, and Japan. Although eosinophilia is an important clinical manifestation of A. cantonensis infection, the role of eosinophils in the progress of the infection remains to be elucidated. In this experiment, we show that A. cantonensis-induced eosinophilia and inflammation might lead to the induction of IAP/NF-kappaB, JAK/STAT1 and MEKK1/JNK signals. The phosphorylation levels of JAK and JNK, STAT1, IAP, NF-kappaB and MEKK1 protein products were significantly increased after 12 days or 15 days of A. cantonensis infection. However, no significant differences in MAPKs such as Raf, MEK-1, ERK1/2 and p38 expression were found between control and infected mice. The activation potency of JAK/STAT1, IAP/NF-kappaB and MEKK1/JNK started increasing on day 3, with significant induction on day 12 or day 15 after A. cantonensis infection. Consistent results were noted in the pathological observations, including eosinophilia, leukocyte infiltration, granulomatous reactions, and time responses in the brain tissues of infected mice. These data suggest that the development of brain injury by eosinophilia of A. cantonensis infection is associated with activation of JAK/STAT1 signals by cytokines, and/or activation of MEKK1/JNK by oxidant stress, and/or activation of NF-kappaB by increasing IAP expression. PMID:15575987

  13. 17β-estradiol ameliorates age-associated loss of fibroblast function by attenuating IFN-γ/STAT1-dependent miR-7 upregulation.

    PubMed

    Midgley, Adam C; Morris, Glyn; Phillips, Aled O; Steadman, Robert

    2016-06-01

    Age-related defects in fibroblast differentiation and functionality were previously shown to be associated with impaired hyaluronan (HA) synthase 2 (HAS2) and epidermal growth factor receptor (EGFR) function, as a result of upregulated microRNA-7 (miR-7) expression. In aging fibroblasts, inhibiting miR-7 prevented the dysregulation of the HA-mediated CD44/EGFR signaling pathway. Here, we investigated transcriptional upregulation of miR-7 and implicated the age-associated over-activation of JAK/STAT1 as a primary candidate. STAT1 binding sites were identified on the putative miR-7 promoter and stimulation of fibroblasts with the inflammatory cytokine, interferon-γ (IFN-γ), significantly increased miR-7 transcriptional activity and resulted in upregulated miR-7 and loss of EGFR. Additionally, we demonstrated a role for the anti-inflammatory steroid, 17β-estradiol (E2), in the attenuation of miR-7 expression. E2 stimulation promoted estrogen receptor (ER) interactions with the miR-7 putative promoter and suppressed miR-7 expression. E2 also attenuated STAT1 expression and activity. Furthermore, treatments with E2 restored fibroblast functionality, including proliferation, migration and differentiation, key events in effective wound healing. In light of our findings, we propose that the regulation of miR-7 by pro- and anti-inflammatory mediators plays a wider role than previously thought. The modulation of fibroblast functions and ultimately wound healing by miR-7 activators or inhibitors could provide realistic targets for the restoration of chronic wound healing capabilities in the elderly. PMID:26931423

  14. Rotavirus antagonizes cellular antiviral responses by inhibiting the nuclear accumulation of STAT1, STAT2, and NF-kappaB.

    PubMed

    Holloway, Gavan; Truong, Thanhmai T; Coulson, Barbara S

    2009-05-01

    A vital arm of the innate immune response to viral infection is the induction and subsequent antiviral effects of interferon (IFN). Rotavirus reduces type I IFN induction in infected cells by the degradation of IFN regulatory factors. Here, we show that the monkey rotavirus RRV and human rotavirus Wa also block gene expression induced by type I and II IFNs through a mechanism allowing signal transducer and activator of transcription 1 (STAT1) and STAT2 activation but preventing their nuclear accumulation. In infected cells, this may allow rotavirus to block the antiviral actions of IFN produced early in infection or by activated immune cells. As the intracellular expression of rotavirus nonstructural proteins NSP1, NSP3, and NSP4 individually did not inhibit IFN-stimulated gene expression, their involvement in this process is unlikely. RRV and Wa rotaviruses also prevented the tumor necrosis factor alpha-stimulated nuclear accumulation of NF-kappaB and NF-kappaB-driven gene expression. In addition, NF-kappaB was activated by rotavirus infection, confirming earlier findings by others. As NF-kappaB is important for the induction of IFN and other cytokines during viral infection, this suggests that rotavirus prevents cellular transcription as a means to evade host responses. To our knowledge, this is the first report of the use of this strategy by a double-stranded RNA virus. PMID:19244315

  15. Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes

    PubMed Central

    Holcar, Marija; Goropevšek, Aleš; Ihan, Alojz; Avčin, Tadej

    2015-01-01

    The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161− subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence. PMID:26525134

  16. Age-Related Differences in Percentages of Regulatory and Effector T Lymphocytes and Their Subsets in Healthy Individuals and Characteristic STAT1/STAT5 Signalling Response in Helper T Lymphocytes.

    PubMed

    Holcar, Marija; Goropevšek, Aleš; Ihan, Alojz; Avčin, Tadej

    2015-01-01

    The dynamic process of the development of the immune system can in itself result in age-related immune malfunctions. In this study, we analysed lymphocyte subsets in the peripheral blood of 60 healthy donors, divided into groups of children, adolescents, and adults, focusing on effector (Teff) and regulatory (Treg) T lymphocytes and STAT1/STAT5 signalling response in helper T lymphocytes (Th) in adults, using flow cytometry. Our results demonstrate a decrease in the percentage of total Tregs and an increase in the percentage of total Teffs with age and a consequential immense increase in the Teff/Treg ratio. The increase of Teffs was most apparent in Th1, Th1Th17, and Th17CD161- subsets. Significant Th lymphocyte STAT1 expression differences were observed between children and adolescents, which were associated with the decrease in activated Tregs. Higher expression of STAT1 was found in FoxP3hi than in FoxP3low Th lymphocytes, while significant IL-2 induced STAT5 phosphorylation differences were found among the subsets of Th lymphocytes in adults. Our study demonstrates age-related changes in circulating Teff and Treg, as well as significant differences in STAT5/STAT1 signalling among FoxP3+ Th lymphocytes, providing new advances in the understanding of immunosenescence. PMID:26525134

  17. Impaired Chromatin Remodelling at STAT1-Regulated Promoters Leads to Global Unresponsiveness of Toxoplasma gondii-Infected Macrophages to IFN-γ

    PubMed Central

    Lang, Christine; Hildebrandt, Anke; Brand, Franziska; Opitz, Lennart; Dihazi, Hassan; Lüder, Carsten G. K.

    2012-01-01

    Intracellular pathogens including the apicomplexan and opportunistic parasite Toxoplasma gondii profoundly modify their host cells in order to establish infection. We have shown previously that intracellular T. gondii inhibit up-regulation of regulatory and effector functions in murine macrophages (MΦ) stimulated with interferon (IFN)-γ, which is the cytokine crucial for controlling the parasites' replication. Using genome-wide transcriptome analysis we show herein that infection with T. gondii leads to global unresponsiveness of murine macrophages to IFN-γ. More than 61% and 89% of the transcripts, which were induced or repressed by IFN-γ in non-infected MΦ, respectively, were not altered after stimulation of T. gondii-infected cells with IFN-γ. These genes are involved in a variety of biological processes, which are mostly but not exclusively related to immune responses. Analyses of the underlying mechanisms revealed that IFN-γ-triggered nuclear translocation of STAT1 still occurred in Toxoplasma-infected MΦ. However, STAT1 bound aberrantly to oligonucleotides containing the IFN-γ-responsive gamma-activated site (GAS) consensus sequence. Conversely, IFN-γ did not induce formation of active GAS-STAT1 complexes in nuclear extracts from infected MΦ. Mass spectrometry of protein complexes bound to GAS oligonucleotides showed that T. gondii-infected MΦ are unable to recruit non-muscle actin to IFN-γ-responsive DNA sequences, which appeared to be independent of stimulation with IFN-γ and of STAT1 binding. IFN-γ-induced recruitment of BRG-1 and acetylation of core histones at the IFN-γ-regulated CIITA promoter IV, but not β-actin was diminished by >90% in Toxoplasma-infected MΦ as compared to non-infected control cells. Remarkably, treatment with histone deacetylase inhibitors restored the ability of infected macrophages to express the IFN-γ regulated genes H2-A/E and CIITA. Taken together, these results indicate that Toxoplasma-infected MΦ are

  18. Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype

    PubMed Central

    Toubiana, Julie; Okada, Satoshi; Hiller, Julia; Oleastro, Matias; Lagos Gomez, Macarena; Aldave Becerra, Juan Carlos; Ouachée-Chardin, Marie; Fouyssac, Fanny; Girisha, Katta Mohan; Etzioni, Amos; Van Montfrans, Joris; Camcioglu, Yildiz; Kerns, Leigh Ann; Belohradsky, Bernd; Blanche, Stéphane; Bousfiha, Aziz; Rodriguez-Gallego, Carlos; Meyts, Isabelle; Kisand, Kai; Reichenbach, Janine; Renner, Ellen D.; Rosenzweig, Sergio; Grimbacher, Bodo; van de Veerdonk, Frank L.; Traidl-Hoffmann, Claudia; Picard, Capucine; Marodi, Laszlo; Morio, Tomohiro; Kobayashi, Masao; Lilic, Desa; Milner, Joshua D.; Holland, Steven; Casanova, Jean-Laurent

    2016-01-01

    Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guérin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism (22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms (6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A–producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis. PMID:27114460

  19. Heterozygous STAT1 gain-of-function mutations underlie an unexpectedly broad clinical phenotype.

    PubMed

    Toubiana, Julie; Okada, Satoshi; Hiller, Julia; Oleastro, Matias; Lagos Gomez, Macarena; Aldave Becerra, Juan Carlos; Ouachée-Chardin, Marie; Fouyssac, Fanny; Girisha, Katta Mohan; Etzioni, Amos; Van Montfrans, Joris; Camcioglu, Yildiz; Kerns, Leigh Ann; Belohradsky, Bernd; Blanche, Stéphane; Bousfiha, Aziz; Rodriguez-Gallego, Carlos; Meyts, Isabelle; Kisand, Kai; Reichenbach, Janine; Renner, Ellen D; Rosenzweig, Sergio; Grimbacher, Bodo; van de Veerdonk, Frank L; Traidl-Hoffmann, Claudia; Picard, Capucine; Marodi, Laszlo; Morio, Tomohiro; Kobayashi, Masao; Lilic, Desa; Milner, Joshua D; Holland, Steven; Casanova, Jean-Laurent; Puel, Anne

    2016-06-23

    Since their discovery in patients with autosomal dominant (AD) chronic mucocutaneous candidiasis (CMC) in 2011, heterozygous STAT1 gain-of-function (GOF) mutations have increasingly been identified worldwide. The clinical spectrum associated with them needed to be delineated. We enrolled 274 patients from 167 kindreds originating from 40 countries from 5 continents. Demographic data, clinical features, immunological parameters, treatment, and outcome were recorded. The median age of the 274 patients was 22 years (range, 1-71 years); 98% of them had CMC, with a median age at onset of 1 year (range, 0-24 years). Patients often displayed bacterial (74%) infections, mostly because of Staphylococcus aureus (36%), including the respiratory tract and the skin in 47% and 28% of patients, respectively, and viral (38%) infections, mostly because of Herpesviridae (83%) and affecting the skin in 32% of patients. Invasive fungal infections (10%), mostly caused by Candida spp. (29%), and mycobacterial disease (6%) caused by Mycobacterium tuberculosis, environmental mycobacteria, or Bacille Calmette-Guérin vaccines were less common. Many patients had autoimmune manifestations (37%), including hypothyroidism (22%), type 1 diabetes (4%), blood cytopenia (4%), and systemic lupus erythematosus (2%). Invasive infections (25%), cerebral aneurysms (6%), and cancers (6%) were the strongest predictors of poor outcome. CMC persisted in 39% of the 202 patients receiving prolonged antifungal treatment. Circulating interleukin-17A-producing T-cell count was low for most (82%) but not all of the patients tested. STAT1 GOF mutations underlie AD CMC, as well as an unexpectedly wide range of other clinical features, including not only a variety of infectious and autoimmune diseases, but also cerebral aneurysms and carcinomas that confer a poor prognosis. PMID:27114460

  20. The Ets-1 transcription factor is required for Stat1-mediated T-bet expression and IgG2a class switching in mouse B cells.

    PubMed

    Nguyen, Hai Vu; Mouly, Enguerran; Chemin, Karine; Luinaud, Romain; Despres, Raymonde; Fermand, Jean-Paul; Arnulf, Bertrand; Bories, Jean-Christophe

    2012-05-01

    In response to antigens and cytokines, mouse B cells undergo class-switch recombination (CSR) and differentiate into Ig-secreting cells. T-bet, a T-box transcription factor that is up-regulated in lymphocytes by IFN-γ or IL-27, was shown to regulate CSR to IgG2a after T cell-independent B-cell stimulations. However, the molecular mechanisms controlling this process remain unclear. In the present study, we show that inactivation of the Ets-1 transcription factor results in a severe decrease in IgG2a secretion in vivo and in vitro. No T-bet expression was observed in Ets-1-deficient (Ets-1(-/-)) B cells stimulated with IFN-γ and lipopolysaccharide, and forced expression of T-bet in these cells rescued IgG2a secretion. Furthermore, we identified a transcriptional enhancer in the T-bet locus with an activity in B cells that relies on ETS-binding sites. After IFN-γ stimulation of Ets-1(-/-) B cells, activated Stat1, which forms a complex with Ets-1 in wild-type cells, no longer binds to the T-bet enhancer or promotes histone modifications at this site. These results demonstrate that Ets-1 is critical for IgG2a CSR and acts as an essential cofactor for Stat1 in the regulation of T-bet expression in B cells. PMID:22438254

  1. Interaction of mumps virus V protein variants with STAT1-STAT2 heterodimer: experimental and theoretical studies

    PubMed Central

    2010-01-01

    Background Mumps virus V protein has the ability to inhibit the interferon-mediated antiviral response by inducing degradation of STAT proteins. Two virus variants purified from Urabe AM9 mumps virus vaccine differ in their replication and transcription efficiency in cells primed with interferon. Virus susceptibility to IFN was associated with insertion of a non-coded glycine at position 156 in the V protein (VGly) of one virus variant, whereas resistance to IFN was associated with preservation of wild-type phenotype in the V protein (VWT) of the other variant. Results VWT and VGly variants of mumps virus were cloned and sequenced from Urabe AM9 vaccine strain. VGly differs from VWT protein because it possesses an amino acid change Gln103Pro (Pro103) and the Gly156 insertion. The effect of V protein variants on components of the interferon-stimulated gene factor 3 (ISGF3), STAT1 and STAT2 proteins were experimentally tested in cervical carcinoma cell lines. Expression of VWT protein decreased STAT1 phosphorylation, whereas VGly had no inhibitory effect on either STAT1 or STAT2 phosphorylation. For theoretical analysis of the interaction between V proteins and STAT proteins, 3D structural models of VWT and VGly were predicted by comparing with simian virus 5 (SV5) V protein structure in complex with STAT1-STAT2 heterodimer. In silico analysis showed that VWT-STAT1-STAT2 complex occurs through the V protein Trp-motif (W174, W178, W189) and Glu95 residue close to the Arg409 and Lys415 of the nuclear localization signal (NLS) of STAT2, leaving exposed STAT1 Lys residues (K85, K87, K296, K413, K525, K679, K685), which are susceptible to proteasome degradation. In contrast, the interaction between VGly and STAT1-STAT2 heterodimer occurs in a region far from the NLS of STAT2 without blocking of Lys residues in both STAT1 and STAT2. Conclusions Our results suggest that VWT protein of Urabe AM9 strain of mumps virus may be more efficient than VGly to inactivate both the IFN

  2. STAT1-regulated lung MDSC-like cells produce IL-10 and efferocytose apoptotic neutrophils with relevance in resolution of bacterial pneumonia.

    PubMed

    Poe, S L; Arora, M; Oriss, T B; Yarlagadda, M; Isse, K; Khare, A; Levy, D E; Lee, J S; Mallampalli, R K; Chan, Y R; Ray, A; Ray, P

    2013-01-01

    Bacterial pneumonia remains a significant burden worldwide. Although an inflammatory response in the lung is required to fight the causative agent, persistent tissue-resident neutrophils in non-resolving pneumonia can induce collateral tissue damage and precipitate acute lung injury. However, little is known about mechanisms orchestrated in the lung tissue that remove apoptotic neutrophils to restore tissue homeostasis. In mice infected with Klebsiella pneumoniae, a bacterium commonly associated with hospital-acquired pneumonia, we show that interleukin (IL)-10 is essential for resolution of lung inflammation and recovery of mice after infection. Although IL-10(-/-) mice cleared bacteria, they displayed increased morbidity with progressive weight loss and persistent lung inflammation in the later phase after infection. A source of tissue IL-10 was found to be resident CD11b(+)Gr1(int)F4/80(+) cells resembling myeloid-derived suppressor cells (MDSCs) that appeared with a delayed kinetics after infection. These cells efficiently efferocytosed apoptotic neutrophils, which was aided by IL-10. The lung neutrophil burden was attenuated in infected signal transducer and activator of transcription 1 (STAT1)(-/-) mice with concomitant increase in the frequency of the MDSC-like cells and lung IL-10 levels. Thus, inhibiting STAT1 in combination with antibiotics may be a novel therapeutic strategy to address inefficient resolution of bacterial pneumonia. PMID:22785228

  3. IFNγ Induces DNA Methylation-Silenced GPR109A Expression via pSTAT1/p300 and H3K18 Acetylation in Colon Cancer.

    PubMed

    Bardhan, Kankana; Paschall, Amy V; Yang, Dafeng; Chen, May R; Simon, Priscilla S; Bhutia, Yangzom D; Martin, Pamela M; Thangaraju, Muthusamy; Browning, Darren D; Ganapathy, Vadivel; Heaton, Christopher M; Gu, Keni; Lee, Jeffrey R; Liu, Kebin

    2015-07-01

    Short-chain fatty acids, metabolites produced by colonic microbiota from fermentation of dietary fiber, act as anti-inflammatory agents in the intestinal tract to suppress proinflammatory diseases. GPR109A is the receptor for short-chain fatty acids. The functions of GPR109A have been the subject of extensive studies; however, the molecular mechanisms underlying GPR109A expression is largely unknown. We show that GPR109A is highly expressed in normal human colon tissues, but is silenced in human colon carcinoma cells. The GPR109A promoter DNA is methylated in human colon carcinoma. Strikingly, we observed that IFNγ, a cytokine secreted by activated T cells, activates GPR109A transcription without altering its promoter DNA methylation. Colon carcinoma grows significantly faster in IFNγ-deficient mice than in wild-type mice in an orthotopic colon cancer mouse model. A positive correlation was observed between GPR109A protein level and tumor-infiltrating T cells in human colon carcinoma specimens, and IFNγ expression level is higher in human colon carcinoma tissues than in normal colon tissues. We further demonstrated that IFNγ rapidly activates pSTAT1 that binds to the promoter of p300 to activate its transcription. p300 then binds to the GPR109A promoter to induce H3K18 hyperacetylation, resulting in chromatin remodeling in the methylated GPR109A promoter. The IFNγ-activated pSTAT1 then directly binds to the methylated but hyperacetylated GPR109 promoter to activate its transcription. Overall, our data indicate that GPR109A acts as a tumor suppressor in colon cancer, and the host immune system might use IFNγ to counteract DNA methylation-mediated GPR109A silencing as a mechanism to suppress tumor development. PMID:25735954

  4. microRNA let-7c is essential for the anisomycin-elicited apoptosis in Jurkat T cells by linking JNK1/2 to AP-1/STAT1/STAT3 signaling.

    PubMed

    Zhou, Zhiwei; Lu, Xijian; Wang, Jin; Xiao, Jia; Liu, Jing; Xing, Feiyue

    2016-01-01

    Anisomycin, an antibiotic produced by Streptomyces griseolus, strongly induces apoptosis in various tumor cells in vitro, superior dramatically to adriamycin. The present study aims to elucidate its detailed mechanistic process. The results showed that anisomycin sufficiently promoted the apoptosis in human leukemic Jurkat T cells at a quite low dose. microRNA let-7c (let-7c) contributed to the anisomycin-induced apoptosis, which could be abrogated by the inactivation of JNK signaling. The let-7c over-expression and the addition of its mimics facilitated the activation of AP-1, STAT1 and Bim by linking JNK1/2 to AP-1/STAT1, but rather inhibited the activation of STAT3 and Bcl-xL by connecting JNK1/2 to STAT3, followed by the augmented apoptosis in the cells. The let-7c deficiency reduced the AP-1, STAT1 and Bim activities, and enhanced the STAT3 and Bcl-xL, alleviating the anisomycin-induced apoptosis. The knockdown of the bim gene repressed the anisomycin-boosted apoptosis through the attenuation of the active Bak and Bax. The findings indicate for the first time that miR let-7c is essential for the anisomycin-triggered apoptosis by linking JNK1/2 to AP-1/STAT1/STAT3/Bim/Bcl-xL/Bax/Bak signaling. This provides a novel insight into the mechanism by which anisomycin leads to the tumor cell apoptosis, potentially laying the foundations for its development and clinical application. PMID:27087117

  5. microRNA let-7c is essential for the anisomycin-elicited apoptosis in Jurkat T cells by linking JNK1/2 to AP-1/STAT1/STAT3 signaling

    PubMed Central

    Zhou, Zhiwei; Lu, Xijian; Wang, Jin; Xiao, Jia; Liu, Jing; Xing, Feiyue

    2016-01-01

    Anisomycin, an antibiotic produced by Streptomyces griseolus, strongly induces apoptosis in various tumor cells in vitro, superior dramatically to adriamycin. The present study aims to elucidate its detailed mechanistic process. The results showed that anisomycin sufficiently promoted the apoptosis in human leukemic Jurkat T cells at a quite low dose. microRNA let-7c (let-7c) contributed to the anisomycin-induced apoptosis, which could be abrogated by the inactivation of JNK signaling. The let-7c over-expression and the addition of its mimics facilitated the activation of AP-1, STAT1 and Bim by linking JNK1/2 to AP-1/STAT1, but rather inhibited the activation of STAT3 and Bcl-xL by connecting JNK1/2 to STAT3, followed by the augmented apoptosis in the cells. The let-7c deficiency reduced the AP-1, STAT1 and Bim activities, and enhanced the STAT3 and Bcl-xL, alleviating the anisomycin-induced apoptosis. The knockdown of the bim gene repressed the anisomycin-boosted apoptosis through the attenuation of the active Bak and Bax. The findings indicate for the first time that miR let-7c is essential for the anisomycin-triggered apoptosis by linking JNK1/2 to AP-1/STAT1/STAT3/Bim/Bcl-xL/Bax/Bak signaling. This provides a novel insight into the mechanism by which anisomycin leads to the tumor cell apoptosis, potentially laying the foundations for its development and clinical application. PMID:27087117

  6. Dieckol, a Component of Ecklonia cava, Suppresses the Production of MDC/CCL22 via Down-Regulating STAT1 Pathway in Interferon-γ Stimulated HaCaT Human Keratinocytes

    PubMed Central

    Kang, Na-Jin; Koo, Dong-Hwan; Kang, Gyeoung-Jin; Han, Sang-Chul; Lee, Bang-Won; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2015-01-01

    Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 μM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation. PMID:25995822

  7. Dieckol, a Component of Ecklonia cava, Suppresses the Production of MDC/CCL22 via Down-Regulating STAT1 Pathway in Interferon-γ Stimulated HaCaT Human Keratinocytes.

    PubMed

    Kang, Na-Jin; Koo, Dong-Hwan; Kang, Gyeoung-Jin; Han, Sang-Chul; Lee, Bang-Won; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2015-05-01

    Macrophage-derived chemokine, C-C motif chemokine 22 (MDC/CCL22), is one of the inflammatory chemokines that controls the movement of monocytes, monocyte-derived dendritic cells, and natural killer cells. Serum and skin MDC/CCL22 levels are elevated in atopic dermatitis, which suggests that the chemokines produced from keratinocytes are responsible for attracting inflammatory lymphocytes to the skin. A major signaling pathway in the interferon-γ (IFN-γ)-stimulated inflammation response involves the signal transducers and activators of transcription 1 (STAT1). In the present study, we investigated the anti-inflammatory effect of dieckol and its possible action mechanisms in the category of skin inflammation including atopic dermatitis. Dieckol inhibited MDC/CCL22 production induced by IFN-γ (10 ng/mL) in a dose dependent manner. Dieckol (5 and 10 μM) suppressed the phosphorylation and the nuclear translocation of STAT1. These results suggest that dieckol exhibits anti-inflammatory effect via the down-regulation of STAT1 activation. PMID:25995822

  8. Interferon-stimulated gene (ISG) 60, as well as ISG56 and ISG54, positively regulates TLR3/IFN-β/STAT1 axis in U373MG human astrocytoma cells.

    PubMed

    Imaizumi, Tadaatsu; Yoshida, Hidemi; Hayakari, Ryo; Xing, Fei; Wang, Lian; Matsumiya, Tomoh; Tanji, Kunikazu; Kawaguchi, Shogo; Murakami, Manabu; Tanaka, Hiroshi

    2016-04-01

    Treatment of cells with interferons (IFNs) induces the phosphorylation of signal transducer and activator of transcription 1 (STAT1), leading to the expression of hundreds of IFN-stimulated genes (ISGs). ISGs exert various antiviral and pro-inflammatory reactions. We have previously reported that ISG56 and ISG54 are induced by polyinosinic-polycytidylic acid (poly IC), an authentic agonist for Toll-like receptor 3 (TLR3), in U373MG human astrocytoma cells. ISG56 and ISG54 are also named as IFN-induced proteins with tetratricopeptide repeats (IFIT) 1 and IFIT2, respectively. In the present study, we demonstrated that poly IC induces the expression of ISG60, also named as IFIT3, in U373MG cells. RNA interference experiments showed that the induction of ISG60 by poly IC was mediated by TLR3, IFN-β, ISG56 and ISG54, whereas ISG60 is involved in poly IC-induced expression of ISG56, ISG54 and a chemokine CXCL10. The level of phosphorylated STAT1 was enhanced by poly IC, and it was inhibited by knockdown of ISG56, ISG54 or ISG60. These results suggest that there is a positive feedback loop between phosphorylated STAT1 and these ISGs. PMID:26423178

  9. The Ebola Virus Interferon Antagonist VP24 Directly Binds STAT1 and Has a Novel, Pyramidal Fold

    PubMed Central

    Zhang, Adrianna P. P.; Bornholdt, Zachary A.; Liu, Tong; Abelson, Dafna M.; Lee, David E.; Li, Sheng; Woods, Virgil L.; Saphire, Erica Ollmann

    2012-01-01

    Ebolaviruses cause hemorrhagic fever with up to 90% lethality and in fatal cases, are characterized by early suppression of the host innate immune system. One of the proteins likely responsible for this effect is VP24. VP24 is known to antagonize interferon signaling by binding host karyopherin α proteins, thereby preventing them from transporting the tyrosine-phosphorylated transcription factor STAT1 to the nucleus. Here, we report that VP24 binds STAT1 directly, suggesting that VP24 can suppress at least two distinct branches of the interferon pathway. Here, we also report the first crystal structures of VP24, derived from different species of ebolavirus that are pathogenic (Sudan) and nonpathogenic to humans (Reston). These structures reveal that VP24 has a novel, pyramidal fold. A site on a particular face of the pyramid exhibits reduced solvent exchange when in complex with STAT1. This site is above two highly conserved pockets in VP24 that contain key residues previously implicated in virulence. These crystal structures and accompanying biochemical analysis map differences between pathogenic and nonpathogenic viruses, offer templates for drug design, and provide the three-dimensional framework necessary for biological dissection of the many functions of VP24 in the virus life cycle. PMID:22383882

  10. Induction of an Inflammatory Loop by Interleukin-1β and Tumor Necrosis Factor-α Involves NF-kB and STAT-1 in Differentiated Human Neuroprogenitor Cells

    PubMed Central

    Pugazhenthi, Subbiah; Zhang, Yuji; Bouchard, Ron; Mahaffey, Gregory

    2013-01-01

    Proinflammatory cytokines secreted from microglia are known to induce a secondary immune response in astrocytes leading to an inflammatory loop. Cytokines also interfere with neurogenesis during aging and in neurodegenerative diseases. The present study examined the mechanism of induction of inflammatory mediators at the transcriptional level in human differentiated neuroprogenitor cells (NPCs). Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) induced the expression of cytokines and chemokines in differentiated human NPCs as shown by an immune pathway-specific array. Network motif (NM) analysis of these genes revealed 118 three-node NMs, suggesting complex interactions between inflammatory mediators and transcription factors. Immunofluorescent staining showed increases in the levels of IL-8 and CXCL10 proteins in neurons and glial cells. Findings from Taqman low density array suggested the synergistic actions of IL-1β and TNF-α in the induction of a majority of inflammatory genes by a mechanism involving NF-kB and STAT-1. Nuclear localization of these transcription factors in differentiated NPCs was observed following exposure to IL-1α and TNF-α. Further studies on CXCL10, a chemokine known to be elevated in the Alzheimer's brain, showed that TNF-α is a stronger inducer of CXCL10 promoter when compared to IL-1β. The synergy between these cytokines was lost when ISRE or kB elements in CXCL10 promoter were mutated. Our findings suggest that the activation of inflammatory pathways in neurons and astrocytes through transcription factors including NF-kB and STAT-1 play important roles in neuroglial interactions and in sustaining the vicious cycle of inflammatory response. PMID:23922745

  11. Isorhapontigenin (ISO) Inhibits Invasive Bladder Cancer Formation In Vivo and Human Bladder Cancer Invasion In Vitro by Targeting STAT1/FOXO1 Axis.

    PubMed

    Jiang, Guosong; Wu, Amy D; Huang, Chao; Gu, Jiayan; Zhang, Liping; Huang, Haishan; Liao, Xin; Li, Jingxia; Zhang, Dongyun; Zeng, Xingruo; Jin, Honglei; Huang, Haojie; Huang, Chuanshu

    2016-07-01

    Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1-FOXO1-MMP-2 axis. Cancer Prev Res; 9(7); 567-80. ©2016 AACR. PMID:27080594

  12. STAT1 Hyperphosphorylation and Defective IL12R/IL23R Signaling Underlie Defective Immunity in Autosomal Dominant Chronic Mucocutaneous Candidiasis

    PubMed Central

    Heinhuis, Bas; Hoischen, Alexander; Joosten, Leo A. B.; Arkwright, Peter D.; Gennery, Andrew; Kullberg, Bart Jan; Veltman, Joris A.; Lilic, Desa; van der Meer, Jos W. M.; Netea, Mihai G.

    2011-01-01

    We recently reported the genetic cause of autosomal dominant chronic mucocutaneous candidiasis (AD-CMC) as a mutation in the STAT1 gene. In the present study we show that STAT1 Arg274Trp mutations in the coiled-coil (CC) domain is the genetic cause of AD-CMC in three families of patients. Cloning and transfection experiments demonstrate that mutated STAT1 inhibits IL12R/IL-23R signaling, with hyperphosphorylation of STAT1 as the likely underlying molecular mechanism. Inhibition of signaling through the receptors for IL-12 and IL-23 leads to strongly diminished Th1/Th17 responses and hence to increased susceptibility to fungal infections. The challenge for the future is to translate this knowledge into novel strategies for the treatment of this severe immunodeficiency. PMID:22195034

  13. ICP0 antagonizes Stat 1-dependent repression of herpes simplex virus: implications for the regulation of viral latency

    PubMed Central

    Halford, William P; Weisend, Carla; Grace, Jennifer; Soboleski, Mark; Carr, Daniel JJ; Balliet, John W; Imai, Yumi; Margolis, Todd P; Gebhardt, Bryan M

    2006-01-01

    Background The herpes simplex virus type 1 (HSV-1) ICP0 protein is an E3 ubiquitin ligase, which is encoded within the HSV-1 latency-associated locus. When ICP0 is not synthesized, the HSV-1 genome is acutely susceptible to cellular repression. Reciprocally, when ICP0 is synthesized, viral replication is efficiently initiated from virions or latent HSV-1 genomes. The current study was initiated to determine if ICP0's putative role as a viral interferon (IFN) antagonist may be relevant to the process by which ICP0 influences the balance between productive replication versus cellular repression of HSV-1. Results Wild-type (ICP0+) strains of HSV-1 produced lethal infections in scid or rag2-/- mice. The replication of ICP0- null viruses was rapidly repressed by the innate host response of scid or rag2-/- mice, and the infected animals remained healthy for months. In contrast, rag2-/- mice that lacked the IFN-α/β receptor (rag2-/- ifnar-/-) or Stat 1 (rag2-/- stat1-/-) failed to repress ICP0- viral replication, resulting in uncontrolled viral spread and death. Thus, the replication of ICP0- viruses is potently repressed in vivo by an innate immune response that is dependent on the IFN-α/β receptor and the downstream transcription factor, Stat 1. Conclusion ICP0's function as a viral IFN antagonist is necessary in vivo to prevent an innate, Stat 1-dependent host response from rapidly repressing productive HSV-1 replication. This antagonistic relationship between ICP0 and the host IFN response may be relevant in regulating whether the HSV-1 genome is expressed, or silenced, in virus-infected cells in vivo. These results may also be clinically relevant. IFN-sensitive ICP0- viruses are avirulent, establish long-term latent infections, and induce an adaptive immune response that is highly protective against lethal challenge with HSV-1. Therefore, ICP0- viruses appear to possess the desired safety and efficacy profile of a live vaccine against herpetic disease. PMID

  14. The effect of glycoprotein IIIa PIA 1/A2 polymorphism on the PFA-100 response to GP IIb IIa receptor inhibitors-the importance of anticoagulants used.

    PubMed

    Aalto-Setälä, Katriina; Karhunen, Pekka J; Mikkelsson, Jussi; Niemelä, Kari

    2005-08-01

    Antithrombotic drugs including glycoprotein (GP) IIb IIIa receptor inhibitors have significantly reduced ischaemic events in coronary disease. Variability in the response to GP receptor inhibitors has been observed both with healthy individuals and in clinical studies. One single nucleotide polymorphism on GP IIIa (PI(A1/A2)) correlates with increased risk for cardiovascular events in many studies. In this study we investigated whether this polymorphism associates with individual differences in the response to GP IIb IIIa receptor inhibitors in healthy individuals. Fresh blood samples were collected randomly from individuals without a history of coronary disease. Blood samples were anticoagulated with either sodium citrate or with PPACK. The ability of different GP IIb IIIa receptor inhibitors (tirofiban, eptifibatide and abciximab) to inhibit platelet aggregation was investigated using a commercial PFA-100 analyser. At baseline, the function of platelets with different PI(A) genotypes did not differ from each other. With sodium citrate anticoagulated samples, tirofiban prolonged the closure time slightly more rapidly when platelets with PI(A 2 A 2) genotype were used than with other genotypes (p<0.05) both on epinephrine-collagen and ADP-collagen coated membranes. With eptifibatide or abciximab no differences were observed. If an anticoagulant not affecting Ca(2+) concentration (PPACK) was used, no differences were observed between different GP IIIa genotypes and the ability of any of the GP IIb IIIa receptor inhibitors to prolong the closure time. The effect of tirofiban and eptifibatide was significantly affected by the anticoagulant used (p<0.001), whereas abciximab functioned equally regardless of the anticoagulant. Glycoprotein IIIa PI(A2) allele has been found in many studies to associate with risk of thrombosis. In healthy controls the function of GP IIb IIIa receptor inhibitors on platelets with different PI(A) genotypes was modified by anticoagulants used. PI(A) polymorphism does not explain the interindividual variance in the response to GP IIb IIIa receptor inhibitors in healthy individuals. The study, however, demonstrates the importance of the anticoagulant used in in vitro studies and the important role of Ca(2+) concentration with tirofiban and eptifibatide, but not with abciximab. PMID:16133898

  15. A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71.

    PubMed

    Liou, An-Ting; Wu, Szu-Yao; Liao, Chun-Che; Chang, Ya-Shu; Chang, Chih-Shin; Shih, Chiaho

    2016-01-01

    Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2(+/+)/stat-1(-/-)) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis. PMID:27499235

  16. A new animal model containing human SCARB2 and lacking stat-1 is highly susceptible to EV71

    PubMed Central

    Liou, An-Ting; Wu, Szu-Yao; Liao, Chun-Che; Chang, Ya-Shu; Chang, Chih-Shin; Shih, Chiaho

    2016-01-01

    Enterovirus 71 (EV71) is a major threat to children worldwide. Children infected with EV71 could develop subclinical infection and hand-foot-and -mouth disease (HFMD). In severe cases, patients could develop encephalitis, paralysis, pulmonary edema, and death. A more user-friendly and robust animal model is essential to investigating EV71 pathogenesis. Here, we established a hybrid (hSCARB2+/+/stat-1−/−) mouse strain from crossbreeding SCARB2 transgenic and stat-1 KO mice, and compared the susceptibilities to EV71 infection and pathogenesis between parental and hybrid mice. Virus-encoded VP1 protein can be detected in the streaking nerve fibers in brain and spinal cord. This hybrid mouse strain at 2-week-old age can still be infected with different genotypes of EV71 at 1000-fold lower titer via an ip route. Infected hybrid mice developed earlier onset of CNS disease, paralysis, and death at a higher incidence. These advantages of this novel model meet the urgent need from the scientific community in basic and preclinical research in therapeutics and pathogenesis. PMID:27499235

  17. Absent in Melanoma 2 (AIM2) limits pro-inflammatory cytokine transcription in cardiomyocytes by inhibiting STAT1 phosphorylation.

    PubMed

    Furrer, Antonia; Hottiger, Michael O; Valaperti, Alan

    2016-06-01

    Interferon (IFN)-γ is highly upregulated during heart inflammation and enhances the production of pro-inflammatory cytokines. Absent in Melanoma 2 (AIM2) is an IFN-inducible protein implicated as a component of the inflammasome. Here we seek to determine the role of AIM2 during inflammation in cardiac cells. We found that the presence of AIM2, but not of the other inflammasome components Nod-like receptor (NLR) NLRP3 or NLRC4, specifically limited the transcription of the pro-inflammatory cytokines interleukin (IL)-6, IP-10, and tumor necrosis factor (TNF)-α in HL-1 mouse cardiomyocytes stimulated with IFN-γ and lipopolysaccharides (LPS). Similarly, AIM2 reduced pro-inflammatory cytokine transcription in primary mouse neonatal cardiomyocytes (MNC), but not in primary mouse neonatal cardiac fibroblasts (MNF). Interestingly, AIM2-dependent reduction of pro-inflammatory cytokines in cardiomyocytes was independent of Caspase-1. Mechanistically, AIM2 reduced pro-inflammatory cytokine transcription in cardiomyocytes by interacting with and inhibiting the phosphorylation of STAT1. In AIM2-depleted cardiomyocytes, increased STAT1 phosphorylation enhanced the NF-κB pathway by promoting NF-κB p65 phosphorylation and acetylation. These results show for the first time that AIM2 plays an important anti-inflammatory, yet inflammasome-independent function in cardiomyocytes. Our findings will help to further understand how the various heart cell types differently react to inflammatory stimuli. PMID:27148820

  18. GYF-17, a chloride substituted 2-(2-phenethyl)-chromone, suppresses LPS-induced inflammatory mediator production in RAW264.7 cells by inhibiting STAT1/3 and ERK1/2 signaling pathways.

    PubMed

    Zhu, Zhixiang; Gu, Yufan; Zhao, Yunfang; Song, Yuelin; Li, Jun; Tu, Pengfei

    2016-06-01

    GYF-17, a 2-(2-phenethyl)-chromone derivative, was isolated from agarwood and showed superior activity of inhibiting NO production of RAW264.7 cells induced by LPS in our preliminary pharmacodynamic screening. In order to develop novel therapeutic drug for acute and chronic inflammatory disorders, the anti-inflammatory activity and underlying mechanism of GYF-17 were investigated in LPS-induced RAW264.7 cells. The results showed that GYF-17 could reduce LPS-induced expression of iNOS and then result in the decrement of NO production. More meaningful, the expression and secretion of key pro-inflammatory factors, including TNF-α, IL-6 and IL-1β, were intensively inhibited by GYF-17. Furthermore, GYF-17 also down regulated the expression of COX2 and the production of PGE2 which plays important role in causing algesthesia during inflammatory response. In mechanism study, GYF-17 selectively suppressed phosphorylation of STAT1/3 and ERK1/2 during the activation of NF-κB, MAPK and STAT signaling pathways induced by LPS. Collectively, GYF-17 can intensively suppress the production of LPS-induced inflammatory mediators in RAW264.7 cells by inhibiting STAT1/3 and ERK1/2 signaling pathways and thereby shows great potential to be developed into therapeutic drug for inflammatory diseases. PMID:27064545

  19. Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation

    PubMed Central

    Eberle, Kirsten C.; McGill, Jodi L.; Reinhardt, Timothy A.

    2015-01-01

    ABSTRACT Parainfluenza viruses are known to inhibit type I interferon (IFN) production; however, there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, IFN-λR1/interleukin-10R2 (IL-10R2), which is primarily expressed by epithelial cells. Parainfluenza virus 3 (PIV-3) infection is highly restricted to the airway epithelium. We therefore sought to examine type III IFN signaling pathways during PIV-3 infection of epithelial cells. We used three strains of PIV-3: human PIV-3 (HPIV-3), bovine PIV-3 (BPIV-3), and dolphin PIV-1 (Tursiops truncatus PIV-1, or TtPIV-1). Here, we show that message levels of IL-29 are significantly increased during PIV-3 infection, yet downstream antiviral signaling molecules are not upregulated to levels similar to those of the positive control. Furthermore, in Vero cells infected with PIV-3, stimulation with recombinant IL-29/-28A/-28B does not cause upregulation of downstream antiviral molecules, suggesting that PIV-3 interferes with the JAK/STAT pathway downstream of the IFN-λR1/IL-10R2 receptor. We used Western blotting to examine the phosphorylation of Stat1 and Stat2 in Vero cells and the bronchial epithelial cell line BEAS-2B. In Vero cells, we observed reduced phosphorylation of the serine 727 (S727) site on Stat1, while in BEAS-2B cells Stat1 phosphorylation was decreased at the tyrosine 701 (Y701) site during PIV-3 infection. PIV-3 therefore interferes with the phosphorylation of Stat1 downstream of the type III IFN receptor. These data provide new evidence regarding strategies employed by parainfluenza viruses to effectively circumvent respiratory epithelial cell-specific antiviral immunity. IMPORTANCE Parainfluenza virus (PIV) in humans is associated with bronchiolitis and pneumonia and can be especially problematic in infants and the elderly. Also seen in cattle, bovine PIV-3 causes respiratory infections in young calves. In addition

  20. Upregulation of the Suppressors of Cytokine Signaling 1 and 3 Is Associated with Arrest of Phosphorylated-STAT1 Nuclear Importation and Reduced Innate Response in Denguevirus-Infected Macrophages.

    PubMed

    Estrada-Jiménez, Tania; Millán-Pérez Peña, Lourdes; Flores-Mendoza, Lilian; Sedeño-Monge, Virginia; Santos-López, Gerardo; Rosas-Murrieta, Nora; Reyes-Carmona, Sandra; Terán-Cabanillas, Eli; Hernández, Jesus; Herrera-Camacho, Irma; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio

    2016-03-01

    To clarify whether the suppressors of cytokine signaling (SOCS) are associated with denguevirus (DENV) evasion of the antiviral response, we analyzed the expression kinetics of SOCS1 and SOCS3 and of the antiviral genes MxA and OAS during DENV infection of U937 macrophages that were or not treated with interferon (IFN)-α. DENV infection produced a viral titer three times higher in untreated than in IFN-α-treated cells (p < 0.001 at 72 h postinfection [p.i.]). Partial inhibition of DENV replication was associated with reduced expression of MxA and OAS antiviral genes as well as higher SOCS1 and SOCS3 expression in DENV-infected cells than in cells treated only with IFN-α. Complete loss of phosphorylated-signal transducer and activator of transcription (p-STAT)2 and reduced nuclear importation of p-STAT1 were observed in DENV-infected cells compared to IFN-α treatment that induced p-STAT1 and p-STAT2. Our data thus suggest that overexpression of SOCS1 and SOCS3 induced by DENV infection leads to impairment of antiviral response through the inhibition of STAT functionality. PMID:26709547

  1. STAT1-dependent innate immunity to a Norwalk-like virus.

    PubMed

    Karst, Stephanie M; Wobus, Christiane E; Lay, Margarita; Davidson, John; Virgin, Herbert W

    2003-03-01

    Norwalk-like caliciviruses (Noroviruses) cause over 90% of nonbacterial epidemic gastroenteritis worldwide, but the pathogenesis of norovirus infection is poorly understood because these viruses do not grow in cultured cells and there is no small animal model. Here, we report a previously unknown murine norovirus. Analysis of Murine Norovirus 1 infection revealed that signal transducer and activator of transcription 1-dependent innate immunity, but not T and B cell-dependent adaptive immunity, is essential for norovirus resistance. The identification of host molecules essential for murine norovirus resistance may provide targets for prevention or control of an important human disease. PMID:12624267

  2. IFN-γ Directly Controls IL-33 Protein Level through a STAT1- and LMP2-dependent Mechanism*

    PubMed Central

    Kopach, Pavel; Lockatell, Virginia; Pickering, Edward M.; Haskell, Ronald E.; Anderson, Richard D.; Hasday, Jeffrey D.; Todd, Nevins W.; Luzina, Irina G.; Atamas, Sergei P.

    2014-01-01

    IL-33 contributes to disease processes in association with Th1 and Th2 phenotypes. IL-33 mRNA is rapidly regulated, but the fate of synthesized IL-33 protein is unknown. To understand the interplay among IL-33, IFN-γ, and IL-4 proteins, recombinant replication-deficient adenoviruses were produced and used for dual expression of IL-33 and IFN-γ or IL-33 and IL-4. The effects of such dual gene delivery were compared with the effects of similar expression of each of these cytokines alone. In lung fibroblast culture, co-expression of IL-33 and IFN-γ resulted in suppression of the levels of both proteins, whereas co-expression of IL-33 and IL-4 led to mutual elevation. In vivo, co-expression of IL-33 and IFN-γ in the lungs led to attenuation of IL-33 protein levels. Purified IFN-γ also attenuated IL-33 protein in fibroblast culture, suggesting that IFN-γ controls IL-33 protein degradation. Specific inhibition of caspase-1, -3, and -8 had minimal effect on IFN-γ-driven IL-33 protein down-regulation. Pharmacological inhibition, siRNA-mediated silencing, or gene deficiency of STAT1 potently up-regulated IL-33 protein expression levels and attenuated the down-regulating effect of IFN-γ on IL-33. Stimulation with IFN-γ strongly elevated the levels of the LMP2 proteasome subunit, known for its role in IFN-γ-regulated antigen processing. siRNA-mediated silencing of LMP2 expression abrogated the effect of IFN-γ on IL-33. Thus, IFN-γ, IL-4, and IL-33 are engaged in a complex interplay. The down-regulation of IL-33 protein levels by IFN-γ in pulmonary fibroblasts and in the lungs in vivo occurs through STAT1 and non-canonical use of the LMP2 proteasome subunit in a caspase-independent fashion. PMID:24619410

  3. BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge

    PubMed Central

    Crump, Ryan W.; Doronin, Konstantin; Hembrador, Edguardo; Pompilio, Daniela; Tebaldi, Giulia; Estep, Ryan D.; Wong, Scott W.; Buller, Mark R.; Donofrio, Gaetano

    2015-01-01

    Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against

  4. BoHV-4-Based Vector Single Heterologous Antigen Delivery Protects STAT1(-/-) Mice from Monkeypoxvirus Lethal Challenge.

    PubMed

    Franceschi, Valentina; Parker, Scott; Jacca, Sarah; Crump, Ryan W; Doronin, Konstantin; Hembrador, Edguardo; Pompilio, Daniela; Tebaldi, Giulia; Estep, Ryan D; Wong, Scott W; Buller, Mark R; Donofrio, Gaetano

    2015-06-01

    Monkeypox virus (MPXV) is the etiological agent of human (MPX). It is an emerging orthopoxvirus zoonosis in the tropical rain forest of Africa and is endemic in the Congo-basin and sporadic in West Africa; it remains a tropical neglected disease of persons in impoverished rural areas. Interaction of the human population with wildlife increases human infection with MPX virus (MPXV), and infection from human to human is possible. Smallpox vaccination provides good cross-protection against MPX; however, the vaccination campaign ended in Africa in 1980, meaning that a large proportion of the population is currently unprotected against MPXV infection. Disease control hinges on deterring zoonotic exposure to the virus and, barring that, interrupting person-to-person spread. However, there are no FDA-approved therapies against MPX, and current vaccines are limited due to safety concerns. For this reason, new studies on pathogenesis, prophylaxis and therapeutics are still of great interest, not only for the scientific community but also for the governments concerned that MPXV could be used as a bioterror agent. In the present study, a new vaccination strategy approach based on three recombinant bovine herpesvirus 4 (BoHV-4) vectors, each expressing different MPXV glycoproteins, A29L, M1R and B6R were investigated in terms of protection from a lethal MPXV challenge in STAT1 knockout mice. BoHV-4-A-CMV-A29LgD106ΔTK, BoHV-4-A-EF1α-M1RgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK were successfully constructed by recombineering, and their capacity to express their transgene was demonstrated. A small challenge study was performed, and all three recombinant BoHV-4 appeared safe (no weight-loss or obvious adverse events) following intraperitoneal administration. Further, BoHV-4-A-EF1α-M1RgD106ΔTK alone or in combination with BoHV-4-A-CMV-A29LgD106ΔTK and BoHV-4-A-EF1α-B6RgD106ΔTK, was shown to be able to protect, 100% alone and 80% in combination, STAT1(-/-) mice against

  5. Lead ions abrogate lipopolysaccharide-induced nitric monoxide toxicity by reducing the expression of STAT1 and iNOS.

    PubMed

    Dörpinghaus, Michael; Brieger, Anne; Panichkina, Olga; Rink, Lothar; Haase, Hajo

    2016-09-01

    Lead is a widespread environmental pollutant and the highly poisonous metal compromises multiple organs in the body. Among other tissues and cells, lead ions (Pb(2+)) can affect macrophages and microglia cells. The present study observed a concentration-dependent protection of BV-2 microglia and RAW 264.7 macrophages by Pb(2+) against lipopolysaccharide (LPS)-induced toxicity. Both cell lines are potent producers of two substances that have previously been shown to mediate cytotoxic effects of LPS. These are the pro-inflammatory cytokine tumor necrosis factor (TNF)-α and nitric monoxide (NO), which creates nitrosative stress, hampering the distribution of invading pathogens and tumor cells. While the expression of TNF-α was unaffected by Pb(2+), the production of NO was significantly inhibited. Moreover, blocking NO synthesis by low molecular weight inhibitors prevented LPS-mediated toxicity, confirming the role of NO in these events. Pb(2+) exposure led to a downregulation of LPS-induced expression of the transcription factor STAT1, which is involved in iNOS transcription. Moreover, iNOS mRNA and protein levels were reduced in the presence of Pb(2+), explaining the reduced formation of NO and a subsequent increase of cellular viability in vitro. In vivo, the effect might limit collateral damage caused by excessive NO production, but also impair the efficiency of NO as a central mediator of the defense against various pathogens. PMID:27134082

  6. A STAT1-gain-of-function mutation causing Th17 deficiency with chronic mucocutaneous candidiasis, psoriasiform hyperkeratosis and dermatophytosis.

    PubMed

    Nielsen, Jakob; Kofod-Olsen, Emil; Spaun, Eva; Larsen, Carsten S; Christiansen, Mette; Mogensen, Trine Hyrup

    2015-01-01

    During recent years, inborn errors of human IL-17 immunity have been demonstrated to underlie primary immunodeficiencies with chronic mucocutaneous candidiasis (CMC). Various defects in receptors responsible for sensing of Candida albicans or downstream signalling to IL-17 may lead to susceptibility to Candida infection. While CMC is common in patients with profound T cell immunodeficiencies, CMC is also recognised as part of other immunodeficiencies in syndromic CMC, or as relatively isolated CMC disease. We describe a 40-year-old woman with a clinical picture involving cutaneous bacterial abscesses, chronic oral candidiasis and extensive dermatophytic infection of the feet. By whole exome sequencing, we identified a STAT1-gain-of-function mutation. Moreover, the patient's peripheral blood mononuclear cells displayed severely impaired Th17 responses. The patient was treated with antifungals and prophylactic antibiotics, which led to resolution of the infection. We discuss the current knowledge within the field of Th17 deficiency and the pathogenesis and treatment of CMC. PMID:26494717

  7. Proposed Standards for Variable Harmonization Documentation and Referencing: A Case Study Using QuickCharmStats 1.1.

    PubMed

    Winters, Kristi; Netscher, Sebastian

    2016-01-01

    Comparative statistical analyses often require data harmonization, yet the social sciences do not have clear operationalization frameworks that guide and homogenize variable coding decisions across disciplines. When faced with a need to harmonize variables researchers often look for guidance from various international studies that employ output harmonization, such as the Comparative Survey of Election Studies, which offer recoding structures for the same variable (e.g. marital status). More problematically there are no agreed documentation standards or journal requirements for reporting variable harmonization to facilitate a transparent replication process. We propose a conceptual and data-driven digital solution that creates harmonization documentation standards for publication and scholarly citation: QuickCharmStats 1.1. It is free and open-source software that allows for the organizing, documenting and publishing of data harmonization projects. QuickCharmStats starts at the conceptual level and its workflow ends with a variable recording syntax. It is therefore flexible enough to reflect a variety of theoretical justifications for variable harmonization. Using the socio-demographic variable 'marital status', we demonstrate how the CharmStats workflow collates metadata while being guided by the scientific standards of transparency and replication. It encourages researchers to publish their harmonization work by providing researchers who complete the peer review process a permanent identifier. Those who contribute original data harmonization work to their discipline can now be credited through citations. Finally, we propose peer-review standards for harmonization documentation, describe a route to online publishing, and provide a referencing format to cite harmonization projects. Although CharmStats products are designed for social scientists our adherence to the scientific method ensures our products can be used by researchers across the sciences. PMID:26859494

  8. Proposed Standards for Variable Harmonization Documentation and Referencing: A Case Study Using QuickCharmStats 1.1

    PubMed Central

    Winters, Kristi; Netscher, Sebastian

    2016-01-01

    Comparative statistical analyses often require data harmonization, yet the social sciences do not have clear operationalization frameworks that guide and homogenize variable coding decisions across disciplines. When faced with a need to harmonize variables researchers often look for guidance from various international studies that employ output harmonization, such as the Comparative Survey of Election Studies, which offer recoding structures for the same variable (e.g. marital status). More problematically there are no agreed documentation standards or journal requirements for reporting variable harmonization to facilitate a transparent replication process. We propose a conceptual and data-driven digital solution that creates harmonization documentation standards for publication and scholarly citation: QuickCharmStats 1.1. It is free and open-source software that allows for the organizing, documenting and publishing of data harmonization projects. QuickCharmStats starts at the conceptual level and its workflow ends with a variable recording syntax. It is therefore flexible enough to reflect a variety of theoretical justifications for variable harmonization. Using the socio-demographic variable ‘marital status’, we demonstrate how the CharmStats workflow collates metadata while being guided by the scientific standards of transparency and replication. It encourages researchers to publish their harmonization work by providing researchers who complete the peer review process a permanent identifier. Those who contribute original data harmonization work to their discipline can now be credited through citations. Finally, we propose peer-review standards for harmonization documentation, describe a route to online publishing, and provide a referencing format to cite harmonization projects. Although CharmStats products are designed for social scientists our adherence to the scientific method ensures our products can be used by researchers across the sciences. PMID

  9. Nicotine-Mediated Regulation of Nicotinic Acetylcholine Receptors in Non-Small Cell Lung Adenocarcinoma by E2F1 and STAT1 Transcription Factors

    PubMed Central

    Schaal, Courtney; Chellappan, Srikumar

    2016-01-01

    Cigarette smoking is the major risk factor for non-small cell lung cancer (NSCLC), which accounts for 80% of all lung cancers. Nicotine, the addictive component of tobacco smoke, can induce proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), angiogenesis, and survival in NSCLC cell lines, as well as growth and metastasis of NSCLC in mice. This nicotine-mediated tumor progression is facilitated through activation of nicotinic acetylcholine receptors (nAChRs), specifically the α7 subunit; however, how the α7 nAChR gene is regulated in lung adenocarcinoma is not fully clear. Here we demonstrate that the α7 nAChR gene promoter is differentially regulated by E2F and STAT transcription factors through a competitive interplay; E2F1 induces the promoter, while STAT transcription factors repress it by binding to an overlapping site at a region -294 through -463bp upstream of the transcription start site. Treatment of cells with nicotine induced the mRNA and protein levels of α7 nAChR; this could be abrogated by treatment with inhibitors targeting Src, PI3K, MEK, α7 nAChR, CDK4/6 or a disruptor of the Rb-Raf-1 interaction. Further, nicotine–mediated induction of α7 nAChR was reduced when E2F1 was depleted and in contrast elevated when STAT1 was depleted by siRNAs. Interestingly, extracts from e-cigarettes, which have recently emerged as healthier alternatives to traditional cigarette smoking, can also induce α7 nAChR expression in a manner similar to nicotine. These results suggest an autoregulatory feed-forward loop that induces the levels of α7 nAChR upon exposure to nicotine, which enhances the strength of the signal. It can be imagined that such an induction of α7 nAChR contributes to the tumor-promoting functions of nicotine. PMID:27228072

  10. Cell-intrinsic role for IFN-α–STAT1 signals in regulating murine Peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response

    PubMed Central

    Li, Haiyan S.; Gelbard, Alexander; Martinez, Gustavo J.; Esashi, Eiji; Zhang, Huiyuan; Nguyen-Jackson, Hoainam; Liu, Yong-Jun; Overwijk, Willem W.

    2011-01-01

    Plasmacytoid dendritic cells (pDCs) reside in bone marrrow and lymphoid organs in homeostatic conditions and typically secrete abundant quantities of type I interferons (IFNs) on Toll-like receptor triggering. Recently, a pDC population was identified within Peyer patches (PPs) of the gut that is distinguished by its lack of IFN production; however, the relationship of PP pDCs to pDCs in other organs has been unclear. We report that PP pDCs are derived from common DC progenitors and accumulate in response to Fms-like tyrosine kinase 3 ligand, yet appear divergent in transcription factor profile and surface marker phenotype, including reduced E2-2 and CCR9 expression. Type I IFN signaling via STAT1 has a cell-autonomous role in accrual of PP pDCs in vivo. Moreover, IFN-α enhances pDC generation from DC progenitors by a STAT1-dependent mechanism. pDCs that have been developed in the presence of IFN-α resemble PP pDCs, produce inflammatory cytokines, stimulate Th17 cell generation, and fail to secrete IFN-α on Toll-like receptor engagement. These results indicate that IFN-α influences the development and function of pDCs by inducing emergence of an inflammatory (Th17-inducing) antigen-presenting subset, and simultaneously regulating accumulation of pDCs in the intestinal microenvironment. PMID:21828128

  11. Phagosomes induced by cytokines function as anti-Listeria vaccines: novel role for functional compartmentalization of STAT-1 protein and cathepsin-D.

    PubMed

    Carrasco-Marín, Eugenio; Rodriguez-Del Rio, Estela; Frande-Cabanes, Elisabet; Tobes, Raquel; Pareja, Eduardo; Lecea-Cuello, M Jesús; Ruiz-Sáez, Marta; Madrazo-Toca, Fidel; Hölscher, Christoph; Alvarez-Dominguez, Carmen

    2012-04-27

    Phagosomes are critical compartments for innate immunity. However, their role in the protection against murine listeriosis has not been examined. We describe here that listericidal phago-receptosomes are induced by the function of IFN-γ or IL-6 as centralized compartments for innate and adaptive immunity because they are able to confer protection against murine listeriosis. These phago-receptosomes elicited LLO(91-99)/CD8(+)- and LLO(189-201)/CD4(+)-specific immune responses and recruited mature dendritic cells to the vaccination sites controlled by T cells. Moreover, they present exceptional features as efficient vaccine vectors. First, they compartmentalize a novel listericidal STAT-1-mediated signaling pathway that confines multiple innate immune components to the same environment. Second, they show features of MHC class II antigen-loading competent compartments for cathepsin-D-mediated LLO processing. Third, murine cathepsin-D deficiencies fail to develop protective immunity after vaccination with listericidal phago-receptosomes induced by IFN-γ or IL-6. Therefore, it appears that the connection of STAT-1 and cathepsin-D in a single compartment is relevant for protection against listeriosis. PMID:22337873

  12. Disruption of Type I Interferon Signaling by the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus via the Hijacking of STAT2 and STAT1 into Inclusion Bodies

    PubMed Central

    Ning, Yun-Jia; Feng, Kuan; Min, Yuan-Qin; Cao, Wu-Chun; Wang, Manli; Deng, Fei; Hu, Zhihong

    2015-01-01

    ABSTRACT The type I interferon (IFN) system, including IFN induction and signaling, is the critical component of the host defense line against viral infection, which, in turn, is also a vulnerable target for viral immune evasion. Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging bunyavirus. Previous data have shown that SFTSV can interfere with the early induction of type I IFNs through targeting host kinases TBK1/IKKε. In this study, we demonstrated that SFTSV also can suppress type I IFN-triggered signaling and interferon-stimulated gene (ISG) expression. Interestingly, we observed the significant inhibition of IFN signaling in cells transfected with the plasmids encoding the nonstructural protein (NSs) but not the nucleocapsid protein (NP), indicating the role of NSs as an antagonist of IFN signaling. Furthermore, coimmunoprecipitation (Co-IP) and pulldown assays indicated that NSs interacts with the cellular signal transducer and activator of transcription 2 (STAT2), and the DNA-binding domain of STAT2 may contribute to the NSs-STAT2 interaction. Combined with confocal microscopy analyses, we demonstrated that NSs sequesters STAT2 and STAT1 into viral inclusion bodies (IBs) and impairs IFN-induced STAT2 phosphorylation and nuclear translocation of both STATs, resulting in the inhibition of IFN signaling and ISG expression. SFTSV NSs-mediated hijacking of STATs in IBs represents a novel mechanism of viral suppression of IFN signaling, highlighting the role of viral IBs as the virus-built “jail” sequestering some crucial host factors and interfering with the corresponding cellular processes. IMPORTANCE SFTSV is an emerging bunyavirus which can cause a severe hemorrhagic fever-like disease with high case fatality rates in humans, posing a serious health threat. However, there are no specific antivirals available, and the pathogenesis and virus-host interactions are largely unclear. Here, we demonstrated that SFTSV can inhibit type I IFN

  13. IL-7 Promotes CD95-Induced Apoptosis in B Cells via the IFN-γ/STAT1 Pathway

    PubMed Central

    Sammicheli, Stefano; Dang Vu Phuong, Linh; Ruffin, Nicolas; Pham Hong, Thang; Lantto, Rebecka; Vivar, Nancy; Chiodi, Francesca; Rethi, Bence

    2011-01-01

    Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals. PMID:22194871

  14. Role of signal transducer and activator of transcription 1 in murine allergen-induced airway remodeling and exacerbation by carbon nanotubes.

    PubMed

    Thompson, Elizabeth A; Sayers, Brian C; Glista-Baker, Ellen E; Shipkowski, Kelly A; Ihrie, Mark D; Duke, Katherine S; Taylor, Alexia J; Bonner, James C

    2015-11-01

    Asthma is characterized by a T helper type 2 phenotype and by chronic allergen-induced airway inflammation (AAI). Environmental exposure to air pollution ultrafine particles (i.e., nanoparticles) exacerbates AAI, and a concern is possible exacerbation posed by engineered nanoparticles generated by emerging nanotechnologies. Signal transducer and activator of transcription (STAT) 1 is a transcription factor that maintains T helper type 1 cell development. However, the role of STAT1 in regulating AAI or exacerbation by nanoparticles has not been explored. In this study, mice with whole-body knockout of the Stat1 gene (Stat1(-/-)) or wild-type (WT) mice were sensitized to ovalbumin (OVA) allergen and then exposed to multiwalled carbon nanotubes (MWCNTs) by oropharygneal aspiration. In Stat1(-/-) and WT mice, OVA increased eosinophils in bronchoalveolar lavage fluid, whereas MWCNTs increased neutrophils. Interestingly, OVA sensitization prevented MWCNT-induced neutrophilia and caused only eosinophilic inflammation. Stat1(-/-) mice displayed increased IL-13 in bronchoalveolar lavage fluid at 1 day compared with WT mice after treatment with OVA or OVA and MWCNTs. At 21 days, the lungs of OVA-sensitized Stat1(-/-) mice displayed increased eosinophilia, goblet cell hyperplasia, airway fibrosis, and subepithelial apoptosis. MWCNTs further increased OVA-induced goblet cell hyperplasia, airway fibrosis, and apoptosis in Stat1(-/-) mice at 21 days. These changes corresponded to increased levels of profibrogenic mediators (transforming growth factor-β1, TNF-α, osteopontin) but decreased IL-10 in Stat1(-/-) mice. Finally, fibroblasts isolated from the lungs of Stat1(-/-) mice produced significantly more collagen mRNA and protein in response to transforming growth factor-β1 compared with WT lung fibroblasts. Our results support a protective role for STAT1 in chronic AAI and exacerbation of remodeling caused by MWCNTs. PMID:25807359

  15. Hyaluronan-Mediated Leukocyte Adhesion and Dextran Sulfate Sodium-Induced Colitis Are Attenuated in the Absence of Signal Transducer and Activator of Transcription 1

    PubMed Central

    Bandyopadhyay, Sudip K.; de la Motte, Carol A.; Kessler, Sean P.; Hascall, Vincent C.; Hill, David R.; Strong, Scott A.

    2008-01-01

    Inflammatory bowel disease is a chronic inflammatory condition of the intestinal mucosa whose etiology is unclear but is likely to be multifactorial. We have shown previously that an increased amount of hyaluronan (HA) is present both in the inflamed mucosa of inflammatory bowel disease patients and in isolated human cells after polyI:C treatment. The signal transducer and activator of transcription (STAT)1 protein plays an important role in many signaling pathways that are associated with inflammation. We therefore investigated the role of STAT1 in adhesive interactions that occur between leukocytes and polyI:C-induced mucosal smooth muscle cells (M-SMCs). Activation of STAT1 was observed after the polyI:C treatment of M-SMCs. Specific phosphorylation of tyrosine and serine residues of STAT1 was observed in polyI:C-treated, but not untreated, M-SMC cultures. To evaluate further the role of STAT1, a corresponding STAT-1-null mouse was used. PolyI:C-induced, HA-mediated leukocyte adhesion to colon SMCs from STAT1-null mice was significantly decreased compared with that from wild-type control mice. In vivo, using the dextran sulfate sodium-induced model of colon inflammation, both tissue damage and HA deposition were attenuated in STAT1-null mice compared with that in wild-type control mice. Additionally, the inter-α-trypsin inhibitor (IαI), a proteoglycan essential for facilitating leukocyte binding to the HA matrix, was reduced in STAT1-null mice. Together, these results demonstrate that STAT1 plays an important role in HA-mediated inflammatory processes. PMID:18818378

  16. Polyomavirus T Antigens Activate an Antiviral State

    PubMed Central

    Giacobbi, Nicholas S.; Gupta, Tushar; Coxon, Andrew; Pipas, James M.

    2014-01-01

    Ectopic expression of Simian Virus 40 (SV40) large T antigen (LT) in mouse embryonic fibroblasts (MEFs) increased levels of mRNAs encoding interferon stimulated genes (ISGs). The mechanism by which T antigen increases levels of ISGs in MEFs remains unclear. We present evidence that expression of T antigen from SV40, Human Polyomaviruses BK (BKV) or JC (JCV) upregulate production of ISGs in MEFs, and subsequently result in an antiviral state, as determined by inhibition of VSV or EMCV growth. The first 136 amino acids of LT are sufficient for these activities. Furthermore, increased ISG expression and induction of the antiviral state requires STAT1. Finally, the RB binding motif of LT is necessary for activation of STAT1. We conclude that the induction of the STAT1 mediated innate immune response in MEFs is a common feature shared by SV40, BKV and JCV. PMID:25589241

  17. Regulation and activity of secretory leukoprotease inhibitor (SLPI) is altered in smokers.

    PubMed

    Meyer, Megan; Bauer, Rebecca N; Letang, Blanche D; Brighton, Luisa; Thompson, Elizabeth; Simmen, Rosalia C M; Bonner, James; Jaspers, Ilona

    2014-02-01

    A hallmark of cigarette smoking is a shift in the protease/antiprotease balance, in favor of protease activity. However, it has recently been shown that smokers have increased expression of a key antiprotease, secretory leukoprotease inhibitor (SLPI), yet the mechanisms involved in SLPI transcriptional regulation and functional activity of SLPI remain unclear. We examined SLPI mRNA and protein secretion in differentiated nasal epithelial cells (NECs) and nasal lavage fluid (NLF) from nonsmokers and smokers and demonstrated that SLPI expression is increased in NECs and NLF from smokers. Transcriptional regulation of SLPI expression was confirmed using SLPI promoter reporter assays followed by chromatin immunoprecipitation. The role of STAT1 in regulating SLPI expression was further elucidated using WT and stat1(-/-) mice. Our data demonstrate that STAT1 regulates SLPI transcription in epithelial cells and slpi protein in the lungs of mice. Additionally, we reveal that NECs from smokers have increased STAT1 mRNA/protein expression. Finally, we demonstrate that SLPI contained in the nasal mucosa of smokers is proteolytically cleaved but retains functional activity against neutrophil elastase. These results demonstrate that smoking enhances expression of SLPI in NECs in vitro and in vivo, and that this response is regulated by STAT1. In addition, despite posttranslational cleavage of SLPI, antiprotease activity against neutrophil elastase is enhanced in smokers. Together, our findings show that SLPI regulation and activity is altered in the nasal mucosa of smokers, which could have broad implications in the context of respiratory inflammation and infection. PMID:24285265

  18. A Co-Culture Model of the Developing Small Intestine Offers New Insight in the Early Immunomodulation of Enterocytes and Macrophages by Lactobacillus spp. through STAT1 and NF-kB p65 Translocation

    PubMed Central

    Trapecar, Martin; Goropevsek, Ales; Gorenjak, Mario; Gradisnik, Lidija; Slak Rupnik, Marjan

    2014-01-01

    The early establishment of a complete microbiome has been shown to play an integral part in the development and maintenance of an intact intestine and its immune system, although much remains unknown about the specific mechanisms of immune modulation in newborns. In our study we show in a co-culture model of the undeveloped small intestine that members of Lactobacillus spp. influence STAT1 and NF-kB p65 nuclear translocation in both intestinal epithelial cells as well as underlying macrophages. Moreover, by using imaging flow cytometry we were able to monitor each individual cell and create a framework of the percentage of cells in which translocation occurred in challenged versus control cell populations. We also observed a significant difference in baseline translocation in intestinal cells when cultured alone versus those in a co-culture model, underpinning the importance of 3D models over monolayer set-ups in epithelial in vitro research. In conclusion, our work offers new insights into the potential routes by which the commensal microbiome primes the early immune system to fight pathogens, and shows how strain-specific these mechanisms really are. PMID:24454965

  19. NOX3 NADPH Oxidase Couples Transient Receptor Potential Vanilloid 1 to Signal Transducer and Activator of Transcription 1-Mediated Inflammation and Hearing Loss

    PubMed Central

    Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P.

    2011-01-01

    Abstract Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss. Antioxid. Redox Signal. 14, 999–1010. PMID:20712533

  20. NOX3 NADPH oxidase couples transient receptor potential vanilloid 1 to signal transducer and activator of transcription 1-mediated inflammation and hearing loss.

    PubMed

    Mukherjea, Debashree; Jajoo, Sarvesh; Sheehan, Kelly; Kaur, Tejbeer; Sheth, Sandeep; Bunch, Jennifer; Perro, Christopher; Rybak, Leonard P; Ramkumar, Vickram

    2011-03-15

    Transient receptor potential vanilloid 1 (TRPV1) is implicated in cisplatin ototoxicity. Activation of this channel by cisplatin increases reactive oxygen species generation, which contribute to loss of outer hair cells in the cochlea. Knockdown of TRPV1 by short interfering RNA protected against cisplatin ototoxicity. In this study, we examined the mechanism underlying TRPV1-mediated ototoxicity using cultured organ of Corti transformed cells (UB/OC-1) and rats. Trans-tympanic injections of capsaicin produced transient hearing loss within 24 h, which recovered by 72 h. In UB/OC-1 cells, capsaicin increased NOX3 NADPH oxidase activity and activation of signal transducer and activator of transcription 1 (STAT1). Intratympanic administration of capsaicin transiently increased STAT1 activity and expression of downstream proinflammatory molecules. Capsaicin produced a transient increase in CD14-positive inflammatory cells into the cochlea, which mimicked the temporal course of STAT1 activation but did not alter the expression of apoptotic genes or damage to outer hair cells. In addition, trans-tympanic administration of STAT1 short interfering RNA protected against capsaicin-induced hearing loss. These data suggest that activation of TRPV1 mediates temporary hearing loss by initiating an inflammatory process in the cochlea via activation of NOX3 and STAT1. Thus, these proteins represent reasonable targets for ameliorating hearing loss. PMID:20712533

  1. The C-terminal tail of polycystin-1 regulates complement factor B expression by signal transducer and activator of transcription 1.

    PubMed

    Wu, Ming; Chen, Meihan; Jing, Ying; Gu, Junhui; Mei, Shuqin; Yao, Qing; Zhou, Jie; Yang, Ming; Sun, Lijun; Wang, Wutao; Hu, Huimin; Wüthrich, Rudolf P; Mei, Changlin

    2016-06-01

    Inhibition of the overactivated alternative complement pathway in autosomal dominant polycystic kidney disease (ADPKD) retards disease progression in animal models; however, it remains unknown how complement factor B (CFB) is upregulated in ADPKD. Here, we showed that the overexpression of CFB in cystic kidneys is associated with increased JAK2/STAT1 activity and enhanced expression of the polycystin-1 C-terminal tail (PC1-CTT). Overexpression or blockage of STAT1 increased or decreased CFB expression and CFB promoter activity. Moreover, overexpression of PC1-CTT induced JAK2/STAT1 activation and CFB upregulation in renal tubular epithelial cells. Furthermore, PC1-CTT overexpression increased human CFB promoter activity, whereas dominant negative STAT1 plasmids or mutation of putative STAT1 responsive elements decreased PC1-CTT-induced CFB promoter activity. The effect of CFB on macrophage differentiation was tested on a mouse macrophage cell line. Bioactive CFB dose dependently promoted macrophage M2 phenotype conversion. In addition, conditioned media from renal epithelial cells promoted macrophage M2 phenotype conversion which was blocked by STAT1 inhibition in a dose-dependent manner. Conditioned media from PC1-CTT-transfected renal epithelial cells further promoted macrophage M2 phenotype conversion, which was suppressed by fludarabine or a CFB antibody. In addition, we show that NF-κB acts downstream of PC1-CTT and may partly mediate PC1-CTT-induced CFB expression. In conclusion, our study reveals possible mechanisms of CFB upregulation in ADPKD and a novel role of PC1-CTT in ADPKD-associated inflammation. Furthermore, our study suggests that targeting STAT1 may be a new strategy to prevent inflammation in the kidney of patients with ADPKD. PMID:26984954

  2. IL-6 blockade reverses the abnormal STAT activation of peripheral blood leukocytes from rheumatoid arthritis patients.

    PubMed

    Ortiz, M A; Diaz-Torné, C; Hernández, M V; Reina, D; de la Fuente, D; Castellví, I; Moya, P; Ruiz, J M; Corominas, H; Zamora, C; Cantó, E; Sanmartí, R; Juarez, C; Vidal, S

    2015-06-01

    Considering the interplay of multiple STATs in response to cytokines, we investigated how IL-6 and its blocking affect STAT signaling in rheumatoid arthritis (RA). Leukocytes obtained from RA patients before and after tocilizumab treatment and healthy donors (HDs) were cytokine-stimulated and STAT phosphorylation was analyzed by cytometry. RA patients had significantly fewer pSTAT1+, pSTAT3+, and pSTAT6+ monocytes and pSTAT5+ lymphocytes than HDs. After 24weeks of treatment, percentages of IFNγ-induced pSTAT1+ and IL-10-induced pSTAT3+ monocytes in RA patients increased, reaching levels comparable to HDs. pSTAT1+ and pSTAT3+ cells correlated inversely with RA disease activity index and levels of pSTAT+ cells at baseline were higher in patients with good EULAR response to tocilizumab. IFNγ-induced pSTAT1+ cells correlated inversely with memory T cells and anti-CCP levels. IL-10-induced pSTAT3+ cells correlated with Treg/Teff ratio. Our findings suggest that IL-6 blocking reduces the inflammatory mechanisms through the correction of STAT1 and STAT3 activation status. PMID:25847223

  3. Chronic mucocutaneous candidiasis: characterization of a family with STAT-1 gain-of-function and development of an ex-vivo assay for Th17 deficiency of diagnostic utility.

    PubMed

    Dhalla, F; Fox, H; Davenport, E E; Sadler, R; Anzilotti, C; van Schouwenburg, P A; Ferry, B; Chapel, H; Knight, J C; Patel, S Y

    2016-05-01

    Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent and persistent superficial infections, with Candida albicans affecting the mucous membranes, skin and nails. It can be acquired or caused by primary immune deficiencies, particularly those that impair interleukin (IL)-17 and IL-22 immunity. We describe a single kindred with CMC and the identification of a STAT1 GOF mutation by whole exome sequencing (WES). We show how detailed clinical and immunological phenotyping of this family in the context of WES has enabled revision of disease status and clinical management. Together with analysis of other CMC cases within our cohort of patients, we used knowledge arising from the characterization of this family to develop a rapid ex-vivo screening assay for the detection of T helper type 17 (Th17) deficiency better suited to the routine diagnostic setting than established in-vitro techniques, such as intracellular cytokine staining and enzyme-linked immunosorbent assay (ELISA) using cell culture supernatants. We demonstrate that cell surface staining of unstimulated whole blood for CCR6(+) CXCR3(-) CCR4(+) CD161(+) T helper cells generates results that correlate with intracellular cytokine staining for IL-17A, and is able to discriminate between patients with molecularly defined CMC and healthy controls with 100% sensitivity and specificity within the cohort tested. Furthermore, removal of CCR4 and CD161 from the antibody staining panel did not affect assay performance, suggesting that the enumeration of CCR6(+) CXCR3(-) CD4(+) T cells is sufficient for screening for Th17 deficiency in patients with CMC and could be used to guide further investigation aimed at identifying the underlying molecular cause. PMID:26621323

  4. Lipoteichoic Acid-Induced Nitric Oxide Production Depends on the Activation of Platelet-Activating Factor Receptor and Jak21

    PubMed Central

    Han, Seung Hyun; Kim, Je Hak; Seo, Ho Seong; Martin, Michael H.; Chung, Gook-Hyun; Michalek, Suzanne M.; Nahm, Moon H.

    2006-01-01

    NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-α and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-α. Jak2 tyrosine kinase blocked LTA-induced production of NO but not TNF-α. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-β expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-α production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-β, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production. PMID:16365452

  5. Co-repressor activity of scaffold attachment factor B1 requires sumoylation

    SciTech Connect

    Garee, Jason P.; Meyer, Rene; Systems Biology of Signal Transduction, German Cancer Research Center , INF 280, 69120 Heidelberg ; Oesterreich, Steffi

    2011-05-20

    Highlights: {yields} SAFB1 is sumoylated to two lysine residues K231 and K294. {yields} SAFB1 sumoylation is regulated by PIAS1 and SENP1. {yields} Sumoylation of SAFB1 regulates its transcriptional repressor activity. {yields} Mutation of sumoylation sites leads to decreased SAFB1 binding to HDAC3. -- Abstract: Sumoylation is an emerging modification associated with a variety of cellular processes including the regulation of transcriptional activities of nuclear receptors and their coregulators. As SUMO modifications are often associated with transcriptional repression, we examined if sumoylation was involved in modulation of the transcriptional repressive activity of scaffold attachment factor B1. Here we show that SAFB1 is modified by both the SUMO1 and SUMO2/3 family of proteins, on lysine's K231 and K294. Further, we demonstrate that SAFB1 can interact with PIAS1, a SUMO E3 ligase which mediates SAFB1 sumoylation. Additionally, SENP1 was identified as the enzyme desumoylating SAFB1. Mutation of the SAFB1 sumoylation sites lead to a loss of transcriptional repression, at least in part due to decreased interaction with HDAC3, a known transcriptional repressor and SAFB1 binding partner. In summary, the transcriptional repressor SAFB1 is modified by both SUMO1 and SUMO2/3, and this modification is necessary for its full repressive activity.

  6. Enterohemorrhagic Escherichia coli O157:H7 Shiga Toxins Inhibit Gamma Interferon-Mediated Cellular Activation

    PubMed Central

    Ho, Nathan K.; Ossa, Juan C.; Silphaduang, Uma; Johnson, Roger; Johnson-Henry, Kathene C.

    2012-01-01

    Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 is a food-borne pathogen that causes significant morbidity and mortality in developing and industrialized nations. EHEC infection of host epithelial cells is capable of inhibiting the gamma interferon (IFN-γ) proinflammatory pathway through the inhibition of Stat-1 phosphorylation, which is important for host defense against microbial pathogens. The aim of this study was to determine the bacterial factors involved in the inhibition of Stat-1 tyrosine phosphorylation. Human HEp-2 and Caco-2 epithelial cells were challenged directly with either EHEC or bacterial culture supernatants and stimulated with IFN-γ, and then the protein extracts were analyzed by immunoblotting. The data showed that IFN-γ-mediated Stat-1 tyrosine phosphorylation was inhibited by EHEC secreted proteins. Using two-dimensional difference gel electrophoresis, EHEC Shiga toxins were identified as candidate inhibitory factors. EHEC Shiga toxin mutants were then generated and complemented in trans, and mutant culture supernatant was supplemented with purified Stx to confirm their ability to subvert IFN-γ-mediated cell activation. We conclude that while other factors are likely involved in the suppression of IFN-γ-mediated Stat-1 tyrosine phosphorylation, E. coli-derived Shiga toxins represent a novel mechanism by which EHEC evades the host immune system. PMID:22526675

  7. IL-17A induces signal transducers and activators of transcription-6-independent airway mucous cell metaplasia.

    PubMed

    Newcomb, Dawn C; Boswell, Madison G; Sherrill, Taylor P; Polosukhin, Vasiliy V; Boyd, Kelli L; Goleniewska, Kasia; Brody, Steven L; Kolls, Jay K; Adler, Kenneth B; Peebles, R Stokes

    2013-06-01

    Mucous cell metaplasia is a hallmark of asthma, and may be mediated by signal transducers and activators of transcription (STAT)-6 signaling. IL-17A is increased in the bronchoalveolar lavage fluid of patients with severe asthma, and IL-17A also increases mucus production in airway epithelial cells. Asthma therapeutics are being developed that inhibit STAT6 signaling, but the role of IL-17A in inducing mucus production in the absence of STAT6 remains unknown. We hypothesized that IL-17A induces mucous cell metaplasia independent of STAT6, and we tested this hypothesis in two murine models in which increased IL-17A protein expression is evident. In the first model, ovalbumin (OVA)-specific D011.10 Th17 cells were adoptively transferred into wild-type (WT) or STAT6 knockout (KO) mice, and the mice were challenged with OVA or PBS. WT-OVA and STAT6 KO-OVA mice demonstrated increased airway IL-17A and IL-13 protein expression and mucous cell metaplasia, compared with WT-PBS or STAT6 KO-PBS mice. In the second model, WT, STAT1 KO, STAT1/STAT6 double KO (DKO), or STAT1/STAT6/IL-17 receptor A (RA) triple KO (TKO) mice were challenged with respiratory syncytial virus (RSV) or mock viral preparation, and the mucous cells were assessed. STAT1 KO-RSV mice demonstrated increased airway mucous cell metaplasia compared with WT-RSV mice. STAT1 KO-RSV and STAT1/STAT6 DKO-RSV mice also demonstrated increased mucous cell metaplasia, compared with STAT1/STAT6/IL17RA TKO-RSV mice. We also treated primary murine tracheal epithelial cells (mTECs) from WT and STAT6 KO mice. STAT6 KO mTECs showed increased periodic acid-Schiff staining with IL-17A but not with IL-13. Thus, asthma therapies targeting STAT6 may increase IL-17A protein expression, without preventing IL-17A-induced mucus production. PMID:23392574

  8. Controlling nuclear JAKs and STATs for specific gene activation by IFNγ

    PubMed Central

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-01-01

    We previously showed that gamma interferon (IFNγ) and its receptor subunit, IFNGR1, interacted with the promoter region of IFNγ-activated genes along with transcription factor STAT1α. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFNγ. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFNγ treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The β-actin gene, which is not activated by IFNγ, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFNγ treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFNγ treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFNγ treatment resulted in its disassociation and then re-association as pSTAT1. The results suggest a novel role for activated JAKs in epigenetic events for specific gene activation. PMID:21689637

  9. FSH-induced p38-MAPK-mediated dephosphorylation at serine 727 of the signal transducer and activator of transcription 1 decreases Cyp1b1 expression in mouse granulosa cells.

    PubMed

    Du, Xue-Hai; Zhou, Xiao-Long; Cao, Rui; Xiao, Peng; Teng, Yun; Ning, Cai-Bo; Liu, Hong-Lin

    2015-01-01

    Most mammalian follicles undergo atresia at various stages before ovulation, and granulosa cell apoptosis is a major cause of antral follicular atresia. Estradiol is an essential mitogen for granulosa cell proliferation in vivo and inhibition of apoptosis. The estradiol-producing capacity and metabolism levels are important for follicle health, and sufficient estradiol is necessary for follicle development and ovulation. Cyp1b1, a member of the cytochrome P450 1 subfamily, is responsible for the metabolism of a wide variety of halogenated and polycyclic aromatic hydrocarbons in diverse tissues. In mouse follicles, Cyp1b1 converts estradiol to 4-hydroxyestradiol. We investigated mouse granulosa cells (MGCs) in vivo and in vitro and found that Cyp1b1 played a crucial role in estradiol metabolism in dominant follicles. Follicle-stimulating hormone (FSH) decreased estrogen metabolism by reducing Cyp1b1 mRNA and protein levels in MGCs. Furthermore, FSH regulated signal transducer and activator of transcription 1 (STAT1), a significant transcription factor of Cyp1b1, by mediating the dephosphorylation of STAT1 on serine 727 (Ser(727)) in MGCs. p38 mitogen-activated protein kinase (MAPK) may be involved in the FSH-induced dephosphorylation of STAT1 on Ser(727) in MGCs. These results suggested that FSH functions via p38 MAPK-induced dephosphorylation at Ser(727) of STAT1 to downregulate Cyp1b1 expression and maintain the estradiol levels in mouse dominant follicles. PMID:25315223

  10. Ubiquitin-SUMO circuitry controls activated fanconi anemia ID complex dosage in response to DNA damage.

    PubMed

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T; Passmore, Lori A; Patel, Ketan J; Olsen, Jesper V; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage. PMID:25557546

  11. Molecules Altering the Intracellular Thiol Content Modulate NF-kB and STAT-1/IRF-1 Signalling Pathways and IL-12 p40 and IL-27 p28 Production in Murine Macrophages

    PubMed Central

    Fraternale, Alessandra; Crinelli, Rita; Casabianca, Anna; Paoletti, Maria Filomena; Orlandi, Chiara; Carloni, Elisa; Smietana, Michaël; Palamara, Anna Teresa; Magnani, Mauro

    2013-01-01

    Background The aim of this study was to investigate the molecular mechanisms involved in the production of Th1 cytokines, namely IL-12 and IL-27, when the intra-macrophage redox state was altered by different chemical entities such as GSH-C4, which is reduced glutathione carrying an aliphatic chain, or I-152, a pro-drug of N-acetyl-cysteine (NAC) and beta-mercaptoethylamine. We had already demonstrated that GSH-C4 and I-152 could shift the immune response towards Th1 in Ovalbumin-immunized mice as well as enhance Th1 response in HIV-1 Tat-immunized mice. Methodology/Principal Findings By a new high performance liquid chromatography method, we found that 20 mM GSH-C4 provided a number of thiol species in the form of GSH, while 20 mM I-152 decreased GSH and increased the thiols in the form of NAC and I-152. Under these experimental conditions, GSH-C4 and I-152 enhanced and suppressed respectively the mRNA expression levels of IL-12 p40 induced by LPS/IFN-γ as assessed by Real-Time PCR. The protein production of IL-12 p40 was increased by GSH-C4 and decreased by I-152 as determined by Enzyme-linked immunosorbent assay. Western immunoblot and electrophoretic mobility shift assays revealed that Nuclear Factor -kB (NF-kB) activation was inhibited by I-152 and prolonged by GSH-C4. Twenty mM I-152 stimulated IL-27 p28 gene expression and sustained Signal Transducer and Activator of Transcription (STAT)-mediated interferon regulator factor 1 (IRF-1) de novo synthesis. By contrast, 20 mM GSH-C4 did not exert any effect on IL-27 p28 gene expression. Conclusions and Significance an increase in the intra-macrophage redox state by GSH-C4 and I-152 enhances Th1 cytokine production although the chemical structure and the intra-cellular metabolism influence differently signalling pathways involved in IL-27 or IL-12 production. GSH-C4 and I-152 may be used as Th1 immunomodulators in some pathologies and in ageing where GSH depletion may contribute to the Th1/Th2 imbalance, and in

  12. Controlling nuclear JAKs and STATs for specific gene activation by IFN{gamma}

    SciTech Connect

    Noon-Song, Ezra N.; Ahmed, Chulbul M.; Dabelic, Rea; Canton, Johnathan; Johnson, Howard M.

    2011-07-08

    Highlights: {yields} Gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interact with the promoter region of IFN{gamma}-associated genes along with transcription factor STAT1{alpha}. {yields} We show that activated Janus kinases pJAK2 and pJAK1 also associate with IFNGR1 in the nucleus. {yields} The activated Janus kinases are responsible for phosphorylation of tyrosine 41 on histone H3, an important epigenetic event for specific gene activation. -- Abstract: We previously showed that gamma interferon (IFN{gamma}) and its receptor subunit, IFNGR1, interacted with the promoter region of IFN{gamma}-activated genes along with transcription factor STAT1{alpha}. Recent studies have suggested that activated Janus kinases pJAK2 and pJAK1 also played a role in gene activation by phosphorylation of histone H3 on tyrosine 41. This study addresses the question of the role of activated JAKs in specific gene activation by IFN{gamma}. We carried out chromatin immunoprecipitation (ChIP) followed by PCR in IFN{gamma} treated WISH cells and showed association of pJAK1, pJAK2, IFNGR1, and STAT1 on the same DNA sequence of the IRF-1 gene promoter. The {beta}-actin gene, which is not activated by IFN{gamma}, did not show this association. The movement of activated JAK to the nucleus and the IRF-1 promoter was confirmed by the combination of nuclear fractionation, confocal microscopy and DNA precipitation analysis using the biotinylated GAS promoter. Activated JAKs in the nucleus was associated with phosphorylated tyrosine 41 on histone H3 in the region of the GAS promoter. Unphosphorylated JAK2 was found to be constitutively present in the nucleus and was capable of undergoing activation in IFN{gamma} treated cells, most likely via nuclear IFNGR1. Association of pJAK2 and IFNGR1 with histone H3 in IFN{gamma} treated cells was demonstrated by histone H3 immunoprecipitation. Unphosphorylated STAT1 protein was associated with histone H3 of untreated cells. IFN

  13. A novel c-Jun-dependent signal transduction pathway necessary for the transcriptional activation of interferon gamma response genes.

    PubMed

    Gough, Daniel J; Sabapathy, Kanaga; Ko, Enoch Yi-No; Arthur, Helen A; Schreiber, Robert D; Trapani, Joseph A; Clarke, Christopher J P; Johnstone, Ricky W

    2007-01-12

    The biological effects of interferon gamma (IFNgamma) are mediated by interferon-stimulated genes (ISGs), many of which are activated downstream of Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling. Herein we have shown that IFNgamma rapidly activated AP-1 DNA binding that required c-Jun but was independent of JAK1 and STAT1. IFNgamma-induced c-Jun phosphorylation and AP-1 DNA binding required the MEK1/2 and ERK1/2 signaling pathways, whereas the JNK1/2 and p38 mitogen-activated protein kinase pathways were dispensable. The induction of several ISGs, including ifi-205 and iNOS, was impaired in IFNgamma-treated c-Jun-/- cells, but others, such as IP-10 and SOCS3, were unaffected, and chromatin immunoprecipitation demonstrated that c-Jun binds to the iNOS promoter following treatment with IFNgamma. Thus, IFNgamma induced JAK1- and STAT1-independent activation of the ERK mitogen-activated protein kinase pathway, phosphorylation of c-Jun, and activation of AP-1 DNA binding, which are important for the induction of a subset of ISGs. This represents a novel signal transduction pathway induced by IFNgamma that proceeds in parallel with conventional JAK/STAT signaling to activate ISGs. PMID:17105733

  14. Interleukin-6-Specific Activation of the C/EBPδ Gene in Hepatocytes Is Mediated by Stat3 and Sp1

    PubMed Central

    Cantwell, Carrie A.; Sterneck, Esta; Johnson, Peter F.

    1998-01-01

    C/EBPδ (CCAAT/enhancer binding protein δ) has been implicated as a regulator of acute-phase response (APR) genes in hepatocytes. Its expression increases dramatically in liver during the APR and can be induced in hepatic cell lines by interleukin-6 (IL-6), an acute-phase mediator that activates transcription of many APR genes. Here we have investigated the mechanism by which C/EBPδ expression is regulated by IL-6 in hepatoma cells. C/EBPδ promoter sequences to −125 bp are sufficient for IL-6 inducibility of a reporter gene and include an APR element (APRE) that is essential for IL-6 responsiveness. DNA binding experiments and transactivation assays demonstrate that Stat3, but not Stat1, interacts with this APRE. Two Sp1 sites, one of which is adjacent to the APRE, are required for IL-6 induction and transactivation by Stat3. Thus, Stat3 and Sp1 function cooperatively to activate the C/EBPδ promoter. Replacement of the APRE with Stat binding elements (SBEs) from the ICAM-1 or C/EBPβ promoter, both of which recognize both Stat1 and Stat3, confers responsiveness to gamma interferon, a cytokine that selectively activates Stat1. Sequence comparisons suggest that the distinct Stat binding specificities of the C/EBPδ and C/EBPβ SBEs are determined primarily by a single base pair difference. Our findings indicate that the cytokine specificity of C/EBPδ gene expression is governed by the APRE sequence. PMID:9528783

  15. Sleep Loss Activates Cellular Inflammation and Signal Transducer and Activator of Transcription (STAT) Family Proteins in Humans

    PubMed Central

    Irwin, Michael R.; Witarama, Tuff; Caudill, Marissa; Olmstead, Richard; Breen, Elizabeth Crabb

    2014-01-01

    Sleep disturbance and short sleep duration are associated with inflammation and related disorders including cardiovascular disease, arthritis, diabetes mellitus, and certain cancers. This study was undertaken to test the effects of experimental sleep loss on spontaneous cellular inflammation and activation of signal transducer and activator of transcription (STAT) family proteins, which together promote an inflammatory microenvironment. In 24 healthy adults (16 females; 8 males), spontaneous production of IL-6 and TNF in monocytes and spontaneous intranuclear expression of activated STAT1, STAT3, and STAT5 in peripheral blood mononuclear cells (PBMC), monocyte-, and lymphocyte populations were measured in the morning after uninterrupted baseline sleep, partial sleep deprivation (PSD, sleep period from 3 a.m. to 7 a.m.), and recovery sleep. Relative to baseline, spontaneous monocytic expression of IL-6 and TNF-α was significantly greater after PSD (P<0.02) and after recovery sleep (P<0.01). Relative to baseline, spontaneous monocytic expression of activated STAT 1 and STAT 5 was significantly greater after recovery sleep (P<0.007P<0.02, respectively) but not STAT 3 (P=0.09). No changes in STAT1, STAT3, or STAT5 were found in lymphocyte populations. Sleep loss induces activation of spontaneous cellular innate immunity and of STAT family proteins, which together map the dynamics of sleep loss on the molecular signaling pathways that regulate inflammatory and other immune responses. Treatments that target short sleep duration have the potential to constrain inflammation and reduce the risk for inflammatory disorders and some cancers in humans. PMID:25451613

  16. Interferon γ Attenuates Insulin Signaling, Lipid Storage, and Differentiation in Human Adipocytes via Activation of the JAK/STAT Pathway*

    PubMed Central

    McGillicuddy, Fiona C.; Chiquoine, Elise H.; Hinkle, Christine C.; Kim, Roy J.; Shah, Rachana; Roche, Helen M.; Smyth, Emer M.; Reilly, Muredach P.

    2009-01-01

    Recent reports demonstrate T-cell infiltration of adipose tissue in early obesity. We hypothesized that interferon (IFN) γ, a major T-cell inflammatory cytokine, would attenuate human adipocyte functions and sought to establish signaling mechanisms. Differentiated human adipocytes were treated with IFNγ ± pharmacological inhibitors prior to insulin stimulation. [3H]Glucose uptake and AKT phosphorylation were assessed as markers of insulin sensitivity. IFNγ induced sustained loss of insulin-stimulated glucose uptake in human adipocytes, coincident with reduced Akt phosphorylation and down-regulation of the insulin receptor, insulin receptor substrate-1, and GLUT4. Loss of adipocyte triglyceride storage was observed with IFNγ co-incident with reduced expression of peroxisome proliferator-activated receptor γ, adiponectin, perilipin, fatty acid synthase, and lipoprotein lipase. Treatment with IFNγ also blocked differentiation of pre-adipocytes to the mature phenotype. IFNγ-induced robust STAT1 phosphorylation and SOCS1 mRNA expression, with modest, transient STAT3 phosphorylation and SOCS3 induction. Preincubation with a non-selective JAK inhibitor restored glucose uptake and Akt phosphorylation while completely reversing IFNγ suppression of adipogenic mRNAs and adipocyte differentiation. Specific inhibition of JAK2 or JAK3 failed to block IFNγ effects suggesting a predominant role for JAK1-STAT1. We demonstrate that IFNγ attenuates insulin sensitivity and suppresses differentiation in human adipocytes, an effect most likely mediated via sustained JAK-STAT1 pathway activation. PMID:19776010

  17. Constitutive STAT5 Activation Correlates With Better Survival in Cervical Cancer Patients Treated With Radiation Therapy

    SciTech Connect

    Chen, Helen H.W.; Chou, Cheng-Yang; Wu, Yuan-Hua; Hsueh, Wei-Ting; Hsu, Chiung-Hui; Guo, How-Ran; Lee, Wen-Ying; Su, Wu-Chou

    2012-02-01

    Purpose: Constitutively activated signal transducers and activators of transcription (STAT) factors, in particular STAT1, STAT3, and STAT5, have been detected in a wide variety of human primary tumors and have been demonstrated to directly contribute to oncogenesis. However, the expression pattern of these STATs in cervical carcinoma is still unknown, as is whether or not they have prognostic significance. This study investigated the expression patterns of STAT1, STAT3, and STAT5 in cervical cancer and their associations with clinical outcomes in patients treated with radical radiation therapy. Methods and Materials: A total of 165 consecutive patients with International Federation of Gynecology and Obstetrics (FIGO) Stages IB to IVA cervical cancer underwent radical radiation therapy, including external beam and/or high-dose-rate brachytherapy between 1989 and 2002. Immunohistochemical studies of their formalin-fixed, paraffin-embedded tissues were performed. Univariate and multivariate analyses were performed to identify and to evaluate the effects of these factors affecting patient survival. Results: Constitutive activations of STAT1, STAT3, and STAT5 were observed in 11%, 22%, and 61% of the participants, respectively. While STAT5 activation was associated with significantly better metastasis-free survival (p < 0.01) and overall survival (p = 0.04), STAT1 and STAT3 activation were not. Multivariate analyses showed that STAT5 activation, bulky tumor ({>=}4 cm), advanced stage (FIGO Stages III and IV), and brachytherapy (yes vs. no) were independent prognostic factors for cause-specific overall survival. None of the STATs was associated with local relapse. STAT5 activation (odds ratio = 0.29, 95% confidence interval = 0.13-0.63) and advanced stage (odds ratio = 2.54; 95% confidence interval = 1.03-6.26) were independent predictors of distant metastasis. Conclusions: This is the first report to provide the overall expression patterns and prognostic significance of

  18. Regulation of Signal Transducer and Activator of Transcription and Suppressor of Cytokine-Signaling Gene Expression in the Brain of Mice with Astrocyte-Targeted Production of Interleukin-12 or Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Maier, Joachim; Kincaid, Carrie; Pagenstecher, Axel; Campbell, Iain L.

    2002-01-01

    Interleukin (IL)-12 and interferon (IFN)-γ are implicated in the pathogenesis of immune disorders of the central nervous system (CNS). To define the basis for the actions of these cytokines in the CNS, we examined the temporal and spatial regulation of key signal transducers and activators of transcription (STATs) and suppressors of cytokine signaling (SOCS) in the brain of transgenic mice with astrocyte production of IL-12 or in mice with experimental autoimmune encephalomyelitis (EAE). In healthy mice, with the exception of STAT4 and STAT6, the expression of a number of STAT and SOCS genes was detectable. However, in symptomatic transgenic mice and in EAE significant up-regulation of STAT1, STAT2, STAT3, STAT4, IRF9, and SOCS1 and SOCS3 RNA transcripts was observed. Although the increased expression of STAT1 RNA was widely distributed and included neurons, astrocytes, and microglia, STAT4 and STAT3 and SOCS1 and SOCS3 RNA was primarily restricted to the infiltrating mononuclear cell population. The level and location of the STAT1, STAT3, and STAT4 proteins overlapped with their corresponding RNA and additionally showed nuclear localization indicative of activation of these molecules. Thus, in both the glial fibrillary acidic protein-IL-12 mice and in EAE the CNS expression of key STAT and SOCS genes that regulate IL-12 (STAT4) and IFN-γ (STAT1, SOCS1, and SOCS3) receptor signaling is highly regulated and compartmentalized. We conclude the interaction between these positive and negative signaling circuits and their distinct cellular locations likely play a defining role in coordinating the actions of IL-12 and IFN-γ during the pathogenesis of type 1 immune responses in the CNS. PMID:11786421

  19. Role of Reactive Oxygen Species in the Abrogation of Oxaliplatin Activity by Cetuximab in Colorectal Cancer

    PubMed Central

    Santoro, Valeria; Jia, Ruochen; Thompson, Hannah; Nijhuis, Anke; Jeffery, Rosemary; Kiakos, Konstantinos; Silver, Andrew R.; Hartley, John A.

    2016-01-01

    Background: The antibody cetuximab, targeting epidermal growth factor receptor (EGFR), is used to treat metastatic colorectal cancer (mCRC). Clinical trials suggest reduced benefit from the combination of cetuximab with oxaliplatin. The aim of this study was to investigate potential negative interactions between cetuximab and oxaliplatin. Methods: Thiazolyl blue tetrazolium bromide (MTT) assay and Calcusyn software were used to characterize drug interactions. Reactive oxygen species (ROS) were measured by flow cytometry and real-time polymerase chain reaction oxidative stress arrays identified genes regulating ROS production. Chromatin immunoprecipitation (ChIP) measured signal transducer and activator of transcription 1 (STAT-1) binding to dual oxidase 2 (DUOX2) promoter. SW48, DLD-1 KRAS wild-type cell lines and DLD-1 xenograft models exposed to cetuximab, oxaliplatin, or oxaliplatin + cetuximab (control [saline]; n = 3 mice per treatment group) were used. Statistical tests were two-sided. Results: Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, P = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, P < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity. Conclusions: Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR

  20. The Rabies Virus Interferon Antagonist P Protein Interacts with Activated STAT3 and Inhibits Gp130 Receptor Signaling

    PubMed Central

    Lieu, Kim G.; Brice, Aaron; Wiltzer, Linda; Hirst, Bevan; Jans, David A.; Blondel, Danielle

    2013-01-01

    Immune evasion by rabies virus depends on targeting of the signal transducers and activator of transcription 1 (STAT1) and STAT2 proteins by the viral interferon antagonist P protein, but targeting of other STAT proteins has not been investigated. Here, we find that P protein associates with activated STAT3 and inhibits STAT3 nuclear accumulation and Gp130-dependent signaling. This is the first report of STAT3 targeting by the interferon antagonist of a virus other than a paramyxovirus, indicating that STAT3 antagonism is important to a range of human-pathogenic viruses. PMID:23698294

  1. Tofacitinib regulates synovial inflammation in psoriatic arthritis, inhibiting STAT activation and induction of negative feedback inhibitors

    PubMed Central

    Gao, W; McGarry, T; Orr, C; McCormick, J; Veale, D J; Fearon, U

    2016-01-01

    Background Psoriatic arthritis (PsA) is a chronic inflammatory disease, characterised by synovitis and destruction of articular cartilage/bone. Janus-kinase and signal transducer and activator of transcription (JAK-STAT) signalling pathway is implicated in the pathogenesis of PsA. Objectives To examine the effect of tofacitinib (JAK inhibitor) on proinflammatory mechanisms in PsA. Methods Primary PsA synovial fibroblasts (PsAFLS) and ex vivo PsA synovial explants were cultured with tofacitinib (1 µM). PhosphoSTAT3 (pSTAT3), phosphoSTAT1 (pSTAT1), suppressor of cytokine signaling-3 (SOCS3), protein inhibitor of activated Stat3 (PIAS3) and nuclear factor kappa B cells (NFκBp65) were quantified by western blot. The effect of tofacitinib on PsAFLS migration, invasion, Matrigel network formation and matrix metallopeptidase (MMP)2/9 was quantified by invasion/migration assays and zymography. Interleukin (IL)-6, IL-8, IFN-gamma-inducible protein 10 (IP-10) monocyte chemoattractant protein (MCP)-1, IL-17, IL-10, MMP3 and tissue inhibitor of metalloproteinases 3 (TIMP3) were assessed by ELISA. Results Tofacitinib significantly decreased pSTAT3, pSTAT1, NFκBp65 and induced SOCS3 and PIAS3 expression in PsAFLS and synovial explant cultures (p<0.05). Functionally, PsAFLS invasion, network formation and migration were inhibited by tofacitinib (all p<0.05). In PsA explant, tofacitinib significantly decreased spontaneous secretion of IL-6, IL-8, MCP-1, MMP9/MMP2, MMP3 (all p<0.05) and decreased the MMP3/TIMP3 ratio (p<0.05), with no effect observed for IP-10 or IL-10. Conclusions This study further supports JAK-STAT inhibition as a therapeutic target for the treatment of PsA. PMID:26353790

  2. Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

    PubMed Central

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E.; Sha, Quan; Siebert, Jason; Gupta, Dave S.; Schroeder, James W.

    2015-01-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ–induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  3. Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

    PubMed

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E; Sha, Quan; Siebert, Jason; Gupta, Dave S; Schroeder, James W; Schleimer, Robert P

    2015-07-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  4. SUMO-1 modification of the C-terminal KVEKVD of Axin is required for JNK activation but has no effect on Wnt signaling.

    PubMed

    Rui, Hong-Liang; Fan, Ernest; Zhou, Hai-Meng; Xu, Zhen; Zhang, Yi; Lin, Sheng-Cai

    2002-11-01

    Axin is a multifunctional protein, regulating Wnt signaling and the c-Jun N-terminal/stress-activated protein kinase (JNK/SAPK) pathway as well as tumorigenesis. In the present study, we found that Axin interacts with three SUMO-1 (small ubiquitin-related modifier) conjugating enzymes 3 (E3), PIAS1, PIASxbeta, and PIASy. The extreme C-terminal six amino acid residues of Axin are critical for the Axin/E3 interaction as deletion of the six residues (AxinDeltaC6) completely abolished the ability of Axin to interact with E3 enzymes. AxinDeltaC6 also failed to activate JNK, although it was intact in both its interaction with MEKK1 and homodimerization. Consistent with the presence of a doublet of the KV(E/D) sumoylation consensus motif at the C-terminal end (KVEKVD), we found that Axin is heavily sumoylated. Deletion of the C-terminal six amino acids drastically reduced sumoylation, indicating that the C-terminal six amino acids stretch is the main sumoylation site for Axin. Sumoylation-defective mutants failed to activate JNK but effectively destabilized beta-catenin and attenuated LEF1 transcriptional activity. In addition, we show that dominant negative Axin mutants blocked PIAS-mediated JNK activation, in accordance with the requirement of sumoylation for Axin-mediated JNK activation. Taken together, we demonstrate that sumoylation plays a role for Axin to function in the JNK pathway. PMID:12223491

  5. Activity.

    ERIC Educational Resources Information Center

    Clearing: Nature and Learning in the Pacific Northwest, 1984

    1984-01-01

    Presents three activities: (1) investigating succession in a schoolground; (2) investigating oak galls; and (3) making sun prints (photographs made without camera or darkroom). Each activity includes a list of materials needed and procedures used. (JN)

  6. Ubiquitin-SUMO Circuitry Controls Activated Fanconi Anemia ID Complex Dosage in Response to DNA Damage

    PubMed Central

    Gibbs-Seymour, Ian; Oka, Yasuyoshi; Rajendra, Eeson; Weinert, Brian T.; Passmore, Lori A.; Patel, Ketan J.; Olsen, Jesper V.; Choudhary, Chunaram; Bekker-Jensen, Simon; Mailand, Niels

    2015-01-01

    Summary We show that central components of the Fanconi anemia (FA) DNA repair pathway, the tumor suppressor proteins FANCI and FANCD2 (the ID complex), are SUMOylated in response to replication fork stalling. The ID complex is SUMOylated in a manner that depends on the ATR kinase, the FA ubiquitin ligase core complex, and the SUMO E3 ligases PIAS1/PIAS4 and is antagonized by the SUMO protease SENP6. SUMOylation of the ID complex drives substrate selectivity by triggering its polyubiquitylation by the SUMO-targeted ubiquitin ligase RNF4 to promote its removal from sites of DNA damage via the DVC1-p97 ubiquitin segregase complex. Deregulation of ID complex SUMOylation compromises cell survival following replication stress. Our results uncover a regulatory role for SUMOylation in the FA pathway, and we propose that ubiquitin-SUMO signaling circuitry is a mechanism that contributes to the balance of activated ID complex dosage at sites of DNA damage. PMID:25557546

  7. Activities.

    ERIC Educational Resources Information Center

    Moody, Mally

    1992-01-01

    A series of four activities are presented to enhance students' abilities to appreciate and use trigonometry as a tool in problem solving. Activities cover problems applying the law of sines, the law of cosines, and matching equivalent trigonometric expressions. A teacher's guide, worksheets, and answers are provided. (MDH)

  8. Activated STING in a Vascular and Pulmonary Syndrome

    PubMed Central

    Yang, D.; Ramsey, S.E.; Sanchez, G.A. Montealegre; Tenbrock, K.; Wittkowski, H.; Jones, O.Y.; Kuehn, H.S.; Lee, C.-C.R.; DiMattia, M.A.; Cowen, E.W.; Gonzalez, B.; Palmer, I.; DiGiovanna, J.J.; Biancotto, A.; Kim, H.; Tsai, W.L.; Trier, A.M.; Huang, Y.; Stone, D.L.; Hill, S.; Kim, H.J.; Hilaire, C. St.; Gurprasad, S.; Plass, N.; Chapelle, D.; Horkayne-Szakaly, I.; Foell, D.; Barysenka, A.; Candotti, F.; Holland, S.M.; Hughes, J.D.; Mehmet, H.; Issekutz, A.C.; Raffeld, M.; McElwee, J.; Fontana, J.R.; Minniti, C.P.; Moir, S.; Kastner, D.L.; Gadina, M.; Steven, A.C.; Wingfield, P.T.; Brooks, S.R.; Rosenzweig, S.D.; Fleisher, T.A.; Goldbach-Mansky, R.

    2014-01-01

    BACKGROUND The study of autoinflammatory diseases has uncovered mechanisms underlying cytokine dysregulation and inflammation. METHODS We analyzed the DNA of an index patient with early-onset systemic inflammation, cutaneous vasculopathy, and pulmonary inflammation. We sequenced a candidate gene, TMEM173, encoding the stimulator of interferon genes (STING), in this patient and in five unrelated children with similar clinical phenotypes. Four children were evaluated clinically and immunologically. With the STING ligand cyclic guanosine monophosphate–adenosine monophosphate (cGAMP), we stimulated peripheral-blood mononuclear cells and fibroblasts from patients and controls, as well as commercially obtained endothelial cells, and then assayed transcription of IFNB1, the gene encoding interferon-β, in the stimulated cells. We analyzed IFNB1 reporter levels in HEK293T cells cotransfected with mutant or nonmutant STING constructs. Mutant STING leads to increased phosphorylation of signal transducer and activator of transcription 1 (STAT1), so we tested the effect of Janus kinase (JAK) inhibitors on STAT1 phosphorylation in lymphocytes from the affected children and controls. RESULTS We identified three mutations in exon 5 of TMEM173 in the six patients. Elevated transcription of IFNB1 and other gene targets of STING in peripheral-blood mono-nuclear cells from the patients indicated constitutive activation of the pathway that cannot be further up-regulated with stimulation. On stimulation with cGAMP, fibro-blasts from the patients showed increased transcription of IFNB1 but not of the genes encoding interleukin-1 (IL1), interleukin-6 (IL6), or tumor necrosis factor (TNF). HEK293T cells transfected with mutant constructs show elevated IFNB1 reporter levels. STING is expressed in endothelial cells, and exposure of these cells to cGAMP resulted in endothelial activation and apoptosis. Constitutive up-regulation of phosphorylated STAT1 in patients’ lymphocytes was

  9. The bone marrow microenvironment enhances multiple myeloma progression by exosome-mediated activation of myeloid-derived suppressor cells

    PubMed Central

    Wang, Jinheng; De Veirman, Kim; De Beule, Nathan; Maes, Ken; De Bruyne, Elke; Van Valckenborgh, Els

    2015-01-01

    Exosomes, extracellular nanovesicles secreted by various cell types, modulate the bone marrow (BM) microenvironment by regulating angiogenesis, cytokine release, immune response, inflammation, and metastasis. Interactions between bone marrow stromal cells (BMSCs) and multiple myeloma (MM) cells play crucial roles in MM development. We previously reported that BMSC-derived exosomes directly promote MM cell growth, whereas the other possible mechanisms for supporting MM progression by these exosomes are still not clear. Here, we investigated the effect of BMSC-derived exosomes on the MM BM cells with specific emphasis on myeloid-derived suppressor cells (MDSCs). BMSC-derived exosomes were able to be taken up by MM MDSCs and induced their expansion in vitro. Moreover, these exosomes directly induced the survival of MDSCs through activating STAT3 and STAT1 pathways and increasing the anti-apoptotic proteins Bcl-xL and Mcl-1. Inhibition of these pathways blocked the enhancement of MDSC survival. Furthermore, these exosomes increased the nitric oxide release from MM MDSCs and enhanced their suppressive activity on T cells. Taken together, our results demonstrate that BMSC-derived exosomes activate MDSCs in the BM through STAT3 and STAT1 pathways, leading to increased immunosuppression which favors MM progression. PMID:26556857

  10. The synergistic interaction between the calcineurin B subunit and IFN-γ enhances macrophage antitumor activity

    PubMed Central

    Su, Z; Yang, R; Zhang, W; Xu, L; Zhong, Y; Yin, Y; Cen, J; DeWitt, J P; Wei, Q

    2015-01-01

    Macrophages are involved in tumor growth and progression. They infiltrate into tumors and cause inflammation, which creates a microenvironment favoring tumor growth and metastasis. However, certain stimuli may induce macrophages to act as tumor terminators. Here we report that the calcineurin B subunit (CnB) synergizes with IFN-γ to make macrophages highly cytotoxic to cancer cells. Furthermore, CnB and IFN-γ act synergistically to polarize mouse tumor-associated macrophages, as well as human monocyte-derived macrophages to an M1-like phenotype. This synergy is mediated by the crosstalk between CnB-engaged integrin αM-p38 MAPK signaling and IFN-γ-initiated p38/PKC-δ/Jak2 signaling. Interestingly, the signal transducer and activator of transcription 1 (STAT1) is a key factor that orchestrates the synergy of CnB and IFN-γ, and the phosphorylation status at Ser727 and Tyr701 of STAT1 is directly regulated by CnB and IFN-γ. PMID:25950470

  11. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  12. Type 1 IFN-independent activation of a subset of interferon stimulated genes in West Nile virus Eg101-infected mouse cells

    PubMed Central

    Pulit-Penaloza, Joanna A.; Scherbik, Svetlana V.; Brinton, Margo A.

    2012-01-01

    Although infection of mouse embryofibroblasts (MEFs) with WNV Eg101 induced interferon (IFN) beta production and STAT1 and STAT2 phosphorylation, these transcription factors (TFs) were not detected in the nucleus or on the promoters of four IRF-3-independent interferon stimulated genes (ISGs): Oas1a and Irf7 (previously characterized as IFN/ISGF3-dependent), Oas1b and Irf1. These ISGs were upregulated in WNV Eg101-infected STAT1−/−, STAT2−/−, and IFN alpha/beta receptor −/− MEFs. Although either IRF-3 or IRF-7 could amplify/sustain Oas1a and Oas1b upregulation at later times after infection, these factors were not required for the initial gene activation. The lack of upregulation of these ISGs in WNV Eg101-infected IRF-3/9−/− MEFs suggested the involvement of IRF-9. Activation of Irf1 in infected MEFs did not depend on any of these IRFs. The data indicate that additional alternative activation mechanisms exist for subsets of ISGs when a virus infection has blocked ISG activation by the canonical IFN-mediated pathway. PMID:22305622

  13. Type 1 IFN-independent activation of a subset of interferon stimulated genes in West Nile virus Eg101-infected mouse cells

    SciTech Connect

    Pulit-Penaloza, Joanna A.; Scherbik, Svetlana V.; Brinton, Margo A.

    2012-04-10

    Although infection of mouse embryofibroblasts (MEFs) with WNV Eg101 induced interferon (IFN) beta production and STAT1 and STAT2 phosphorylation, these transcription factors (TFs) were not detected in the nucleus or on the promoters of four IRF-3-independent interferon stimulated genes (ISGs): Oas1a and Irf7 (previously characterized as IFN/ISGF3-dependent), Oas1b and Irf1. These ISGs were upregulated in WNV Eg101-infected STAT1-/-, STAT2-/-, and IFN alpha/beta receptor -/- MEFs. Although either IRF-3 or IRF-7 could amplify/sustain Oas1a and Oas1b upregulation at later times after infection, these factors were not required for the initial gene activation. The lack of upregulation of these ISGs in WNV Eg101-infected IRF-3/9-/- MEFs suggested the involvement of IRF-9. Activation of Irf1 in infected MEFs did not depend on any of these IRFs. The data indicate that additional alternative activation mechanisms exist for subsets of ISGs when a virus infection has blocked ISG activation by the canonical IFN-mediated pathway.

  14. IL-2 induces STAT4 activation in primary NK cells and NK cell lines, but not in T cells.

    PubMed

    Wang, K S; Ritz, J; Frank, D A

    1999-01-01

    IL-2 exerts potent but distinct functional effects on two critical cell populations of the immune system, T cells and NK cells. Whereas IL-2 leads to proliferation in both cell types, it enhances cytotoxicity primarily in NK cells. In both T cells and NK cells, IL-2 induces the activation of STAT1, STAT3, and STAT5. Given this similarity in intracellular signaling, the mechanism underlying the distinct response to IL-2 in T cells and NK cells is not clear. In this study, we show that in primary NK cells and NK cell lines, in addition to the activation of STAT1 and STAT5, IL-2 induces tyrosine phosphorylation of STAT4, a STAT previously reported to be activated only in response to IL-12 and IFN-alpha. This activation of STAT4 in response to IL-2 is not due to the autocrine production of IL-12 or IFN-alpha. STAT4 activated in response to IL-2 is able to bind to a STAT-binding DNA sequence, suggesting that in NK cells IL-2 is capable of activating target genes through phosphorylation of STAT4. IL-2 induces the activation of Jak2 uniquely in NK cells, which may underlie the ability of IL-2 to activate STAT4 only in these cells. Although the activation of STAT4 in response to IL-2 occurs in primary resting and activated NK cells, it does not occur in primary resting T cells or mitogen-activated T cells. The unique activation of the STAT4-signaling pathway in NK cells may underlie the distinct functional effect of IL-2 on this cell population. PMID:9886399

  15. Signal Transducer and Activator of Transcription-5 Mediates Neuronal Apoptosis Induced by Inhibition of Rac GTPase Activity*

    PubMed Central

    Stankiewicz, Trisha R.; Loucks, F. Alexandra; Schroeder, Emily K.; Nevalainen, Marja T.; Tyler, Kenneth L.; Aktories, Klaus; Bouchard, Ron J.; Linseman, Daniel A.

    2012-01-01

    In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase. PMID:22378792

  16. Signal transducer and activator of transcription-5 mediates neuronal apoptosis induced by inhibition of Rac GTPase activity.

    PubMed

    Stankiewicz, Trisha R; Loucks, F Alexandra; Schroeder, Emily K; Nevalainen, Marja T; Tyler, Kenneth L; Aktories, Klaus; Bouchard, Ron J; Linseman, Daniel A

    2012-05-11

    In several neuronal cell types, the small GTPase Rac is essential for survival. We have shown previously that the Rho family GTPase inhibitor Clostridium difficile toxin B (ToxB) induces apoptosis in primary rat cerebellar granule neurons (CGNs) principally via inhibition of Rac GTPase function. In the present study, incubation with ToxB activated a proapoptotic Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, and a pan-JAK inhibitor protected CGNs from Rac inhibition. STAT1 expression was induced by ToxB; however, CGNs from STAT1 knock-out mice succumbed to ToxB-induced apoptosis as readily as wild-type CGNs. STAT3 displayed enhanced tyrosine phosphorylation following treatment with ToxB, and a reputed inhibitor of STAT3, cucurbitacin (JSI-124), reduced CGN apoptosis. Unexpectedly, JSI-124 failed to block STAT3 phosphorylation, and CGNs were not protected from ToxB by other known STAT3 inhibitors. In contrast, STAT5A tyrosine phosphorylation induced by ToxB was suppressed by JSI-124. In addition, roscovitine similarly inhibited STAT5A phosphorylation and protected CGNs from ToxB-induced apoptosis. Consistent with these results, adenoviral infection with a dominant negative STAT5 mutant, but not wild-type STAT5, significantly decreased ToxB-induced apoptosis of CGNs. Finally, chromatin immunoprecipitation with a STAT5 antibody revealed increased STAT5 binding to the promoter region of prosurvival Bcl-xL. STAT5 was recruited to the Bcl-xL promoter region in a ToxB-dependent manner, and this DNA binding preceded Bcl-xL down-regulation, suggesting transcriptional repression. These data indicate that a novel JAK/STAT5 proapoptotic pathway significantly contributes to neuronal apoptosis induced by the inhibition of Rac GTPase. PMID:22378792

  17. Activation of different Stat5 isoforms contributes to cell-type-restricted signaling in response to interferons.

    PubMed Central

    Meinke, A; Barahmand-Pour, F; Wöhrl, S; Stoiber, D; Decker, T

    1996-01-01

    Tyrosine phosphorylation and activation of the transcription factor Stat5 occur in response to stimuli like granulocyte-macrophage colony-stimulating factor, interleukin-3, or erythropoietin that stimulate both proliferation and differentiation of hematopoietic cells. It is unclear whether Stat5 is part of a proliferative response or part of the events leading to cellular differentiation. Here we report that agents promoting differentiation but not proliferation of hematopoietic cells, like phorbol ester or both types of interferons (IFNs), activate Stat5 in promonocytic U937 cells. Both IFN types caused tyrosine phosphorylation and DNA binding of predominantly one Stat5 isoform (Stat5a) despite expression of both Stat5a and Stat5b proteins. Monocytic differentiation of U937 cells led to a strong decrease in IFN-gamma-mediated activation of Stat5 but not of Stat1. Transactivation of Stat5-target genes occurred in response to IFN-gamma, which activates both Stat5 and Stat1, but not in response to granulocyte-macrophage colony-stimulating factor, which activates only Stat5. Tyrosine phosphorylation of Stat5 is not generally part of the IFN response. IFN-gamma did not cause Stat5 activation in HeLa cells, despite the expression of both Stat5 isoforms at similar levels. By contrast, IFN-alpha caused tyrosine phosphorylation and DNA binding of exclusively the b isoform of Stat5, and activated Stat5b formed a DNA binding activity previously found in HeLa cells and designated IFN-alpha activation factor 2. Taken together, our results demonstrate that ligand binding of IFN receptors leads to an isoform-specific activation of Stat5 in a restricted number of cell lineages. Moreover, they suggest that Stat5 might be part of the differentiation response of myeloid cells. PMID:8943349

  18. Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload.

    PubMed

    Huang, Su-Hua; Lien, Jin-Cherng; Chen, Chao-Jung; Liu, Yu-Ching; Wang, Ching-Ying; Ping, Chia-Fong; Lin, Yu-Fong; Huang, An-Cheng; Lin, Cheng-Wen

    2016-01-01

    Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Time-of-addition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca(2+) overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents. PMID:27563890

  19. Different pathways of macrophage activation and polarization.

    PubMed

    Juhas, Ulana; Ryba-Stanisławowska, Monika; Szargiej, Patryk; Myśliwska, Jolanta

    2015-01-01

    Monocytes are short-lived cells and undergo spontaneous apoptosis every day. Inflammatory responses may induce dramatic up-regulation of monocyte survival and differentiation. When monocytes are recruited to an area of infection they may differentiate into macrophages. In different microenvironments macrophages polarize into two types. The M1 or classically activated macrophages are characterized by the high ability to produce pro-inflammatory cytokines and the production of NO through the induced synthesis of iNOS. The M2 or alternatively activated macrophages are divided into 3 subtypes, M2 a, b and c, and they have anti-inflammatory properties. Mediators of M1 macrophage TLR-dependent polarization include transcription factors such as NF-κB, AP-1, PU.1, CCAAT/enhancer-binding protein α (C/EBP-α), STAT1 as well as interferon regulatory factor 5 (IRF5), while the transcription factors which promote M2 activation include IRF4, C/EBP-β, Krüppel-like factor 4 (KLF4), STAT6 and PPARγ receptor. PMID:25983288

  20. Identification of Tyrosine Residues in Constitutively Activated Fibroblast Growth Factor Receptor 3 Involved in Mitogenesis, Stat Activation, and Phosphatidylinositol 3-Kinase Activation

    PubMed Central

    Hart, Kristen C.; Robertson, Scott C.; Donoghue, Daniel J.

    2001-01-01

    Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine rendered this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restored its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3. PMID:11294897

  1. BaeR protein acts as an activator of nuclear factor-kappa B and Janus kinase 2 to induce inflammation in murine cell lines.

    PubMed

    Lee, Seung-Jin; Birhanu, Biruk Tesfaye; Awji, Elias Gebru; Kim, Myung Hee; Park, Ji-Yong; Suh, Joo-Won; Park, Seung-Chun

    2016-09-01

    BaeR, a response regulator protein, takes part in multidrug efflux, bacterial virulence activity, and other biological functions. Recently, BaeR was shown to induce inflammatory responses by activating the mitogen-activated protein kinases (MAPKs). In this study, we investigated additional pathways used by BaeR to induce an inflammatory response. BaeR protein was purified from Salmonella enterica Paratyphi A and subcloned into a pPosKJ expression vector. RAW 264.7 cells were treated with BaeR, and RNA was extracted by TRIzol reagent for RT-PCR. Cytokine gene expression was analyzed by using the comparative cycle threshold method, while western blotting and ELISA were used to assess protein expression. We confirmed that BaeR activates nuclear factor-kappa B (NF-κB), thereby inducing an inflammatory response and increases the production of interleukins (IL-)1β and IL-6. During this process, the Janus kinase 2 (JAK2)-STAT1 signaling pathway was activated, resulting in an increase in the release of interferons I and II. Additionally, COX-2 was activated and its expression increased with time. In conclusion, BaeR induced an inflammatory response through activation of NF-κB in addition to the MAPKs. Furthermore, activation of the JAK2-STAT1 pathway and COX-2 facilitated the cytokine binding activity, suggesting an additional role for BaeR in the modulation of the immune system of the host and the virulence activity of the pathogen. PMID:27374640

  2. The proinflammatory role of HECTD2 in innate immunity and experimental lung injury

    PubMed Central

    Coon, Tiffany A.; McKelvey, Alison C.; Lear, Travis; Rajbhandari, Shristi; Dunn, Sarah R.; Connelly, William; Zhao, Joe Y.; Han, SeungHye; Liu, Yuan; Weathington, Nathaniel M.; McVerry, Bryan J.; Zhang, Yingze; Chen, Bill B.

    2015-01-01

    Invading pathogens may trigger overactivation of the innate immune system, which results in the release of large amounts of proinflammatory cytokines (cytokine storm) and leads to the development of pulmonary edema, multiorgan failure, and shock. PIAS1 is a multifunctional and potent anti-inflammatory protein that negatively regulates several key inflammatory pathways such as Janus kinase (JAK)–signal transducer and activator of transcription (STAT) and nuclear factor κB (NF-κB). We discovered a ubiquitin E3 ligase, HECTD2, which ubiquitinated and mediated the degradation of PIAS1, thus increasing inflammation in an experimental pneumonia model. We found that GSK3b phosphorylation of PIAS1 provided a phosphodegron for HECTD2 targeting. We also identified a mislocalized HECTD2 polymorphism, HECTD2A19P, that was present in 8.5% of the population and functioned to reduce inflammation. This polymorphism prevented HECTD2/PIAS1 nuclear interaction, thus preventing PIAS1 degradation. The HECTD2A19P polymorphism was also protective toward acute respiratory distress syndrome (ARDS). We then developed a small-molecule inhibitor, BC-1382, that targeted HECTD2 and attenuated lipopolysaccharide (LPS)– and Pseudomonas aeruginosa–induced lung inflammation. These studies describe an unreported innate immune pathway and suggest that mutation or antagonism of the E3 ligase HECTD2 results in reduced severity of lung inflammation by selectively modulating the abundance of the anti-inflammatory protein PIAS1. PMID:26157031

  3. The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation.

    PubMed

    Gollob, J A; Schnipper, C P; Murphy, E A; Ritz, J; Frank, D A

    1999-04-15

    IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways. PMID:10201984

  4. cIAP2 represses IKKα/β-mediated activation of MDM2 to prevent p53 degradation.

    PubMed

    Lau, Rosanna; Niu, Min Ying; Pratt, M A Christine

    2012-11-01

    Cellular inhibitor of apoptosis proteins (cIAP1 and cIAP2) function to prevent apoptosis and are often overexpressed in various cancers. However, mutations in cIAP1/2 can activate the alternative NFκB pathway through IκBα-kinase-α (IKKα) and are associated with hematopoetic malignancies. In the current study, we found that knockdown of cIAP2 in human mammary epithelial cells resulted in activation of MDM2 through increased SUMOylation and profound reduction of the pool of MDM2 not phosphorylated at Ser166. cIAP2 siRNA markedly decreased p53 levels, which were rescued by addition of the MDM2 inhibitor, Nutlin3a. An IAP antagonist, which induces cIAP degradation, transiently increased MDM2 mRNA. Simultaneous transfection of siRNA for cIAP2 and IKKα reduced MDM2 protein, while expression of a kinase-dead IKKβ strongly increased non-Ser166 P-MDM2. Inhibition of either IKKα or -β partially rescued p53 levels, while concomitant IKKα/β inhibition fully rescued p53 after cIAP2 knockdown. Surprisingly, IKKα knockdown alone increased SUMO-MDM2, suggesting that in the absence of activation, IKKα can prevent MDM2 SUMOylation. cIAP2 knockdown disrupted the interaction between the MDM2 SUMO ligase, PIAS1 and IKKα. Partial knockdown of cIAP2 cooperated with (V12) H-ras-transfected mammary epithelial cells to enhance colony formation. In summary, our data identify a novel role for cIAP2 in maintaining wild-type p53 levels by preventing both an NFκB-mediated increase and IKKα/-β-dependent transcriptional and post-translational modifications of MDM2. Thus, mutations or reductions in cIAP2 could contribute to cancer promotion, in part, through downregulation of p53. PMID:23032264

  5. Suppression of microRNA activity amplifies IFN-γ-induced macrophage activation and promotes anti-tumour immunity.

    PubMed

    Baer, Caroline; Squadrito, Mario Leonardo; Laoui, Damya; Thompson, Danielle; Hansen, Sarah K; Kiialainen, Anna; Hoves, Sabine; Ries, Carola H; Ooi, Chia-Huey; De Palma, Michele

    2016-07-01

    Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeutic implications. PMID:27295554

  6. STAT-5 is activated constitutively in T cells, B cells and monocytes from patients with primary Sjögren's syndrome

    PubMed Central

    Pertovaara, M; Silvennoinen, O; Isomäki, P

    2015-01-01

    The expression and phosphorylation of signal transducer and activator of transcription-1 (STAT-1) have been shown to be markedly increased in the salivary gland epithelial cells of patients with primary Sjögren's syndrome (pSS). The present aim was to investigate the activation status of different STAT proteins in peripheral blood (PB) lymphocytes and monocytes, and their correlations with clinical parameters in patients with pSS. To this end, PB samples were drawn from 16 patients with active pSS and 16 healthy blood donors, and the phosphorylation of STAT-1, -3, -4, -5 and -6 proteins was studied in T cells, B cells and monocytes using multi-colour flow cytometry. In addition, mRNA expression of STAT molecules in PB mononuclear cells (PBMC) was studied with quantitative reverse transcriptase–polymerase chain reaction (RT–PCR). Basal phosphorylation of STAT-5 was found to be significantly higher in PB T cells, B cells and monocytes in patients with pSS than in healthy controls. The expression of STAT-5 mRNA was not increased in PBMC. pSTAT-5 levels in B cells and monocytes showed a significant correlation with serum immunoglobulin (Ig)G levels and anti-SSB antibody titres. Constitutive STAT-5 activation in monocytes and CD4+ T cells was associated with purpura. There were no major differences in the activation of other STATs between pSS patients and healthy controls. In conclusion, STAT-5 is activated constitutively in PB leucocytes in patients with pSS, and basal STAT-5 phosphorylation seems to associate with hypergammaglobulinaemia, anti-SSB antibody production and purpura. PMID:25736842

  7. STAT-5 is activated constitutively in T cells, B cells and monocytes from patients with primary Sjögren's syndrome.

    PubMed

    Pertovaara, M; Silvennoinen, O; Isomäki, P

    2015-07-01

    The expression and phosphorylation of signal transducer and activator of transcription-1 (STAT-1) have been shown to be markedly increased in the salivary gland epithelial cells of patients with primary Sjögren's syndrome (pSS). The present aim was to investigate the activation status of different STAT proteins in peripheral blood (PB) lymphocytes and monocytes, and their correlations with clinical parameters in patients with pSS. To this end, PB samples were drawn from 16 patients with active pSS and 16 healthy blood donors, and the phosphorylation of STAT-1, -3, -4, -5 and -6 proteins was studied in T cells, B cells and monocytes using multi-colour flow cytometry. In addition, mRNA expression of STAT molecules in PB mononuclear cells (PBMC) was studied with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Basal phosphorylation of STAT-5 was found to be significantly higher in PB T cells, B cells and monocytes in patients with pSS than in healthy controls. The expression of STAT-5 mRNA was not increased in PBMC. pSTAT-5 levels in B cells and monocytes showed a significant correlation with serum immunoglobulin (Ig)G levels and anti-SSB antibody titres. Constitutive STAT-5 activation in monocytes and CD4(+) T cells was associated with purpura. There were no major differences in the activation of other STATs between pSS patients and healthy controls. In conclusion, STAT-5 is activated constitutively in PB leucocytes in patients with pSS, and basal STAT-5 phosphorylation seems to associate with hypergammaglobulinaemia, anti-SSB antibody production and purpura. PMID:25736842

  8. Antiviral activity of aloe-emodin against influenza A virus via galectin-3 up-regulation.

    PubMed

    Li, Shih-Wen; Yang, Tsuey-Ching; Lai, Chien-Chen; Huang, Su-Hua; Liao, Jun-Ming; Wan, Lei; Lin, Ying-Ju; Lin, Cheng-Wen

    2014-09-01

    Novel influenza A H7N9 virus, which emerged in 2013, and highly pathogenic H5N1 virus, identified since 2003, pose challenges to public health and necessitate quest for new anti-influenza compounds. Anthraquinone derivatives like aloe-emodin, emodin and chrysophanol, reportedly exhibit antiviral activity. This study probes their inhibitory mechanism and effect against influenza A virus. Of three anthraquinone derivatives, aloe-emodin, with a lower cytotoxicity showed concentration-dependently reducing virus-induced cytopathic effect and inhibiting replication of influenza A in MDCK cells. 50% inhibitory concentration value of aloe-emodin on virus yield was less than 0.05 μg/ml. Proteomics and Western blot of MDCK cells indicated aloe-emodin up-regulating galectin-3, and thioredoxin as well as down-regulating nucleoside diphosphate kinase A. Western blot and quantitative PCR confirmed aloe-emodin up-regulating galectin-3 expression; recombinant galectin-3 augmented expression of antiviral genes IFN-β, IFN-γ, PKR and 2'5',-OAS in infected cells, agreeing with expression pattern of those treated with aloe-emodin. Galectin-3 also inhibited influenza A virus replication. Proteomic analysis of treated cells indicated galectin-3 up-regulation as one anti-influenza A virus action by aloe-emodin. Since galectin-3 exhibited cytokine-like regulatory actions via JAK/STAT pathways, aloe-emodin also restored NS1-inhibited STAT1-mediated antiviral responses in transfected cells: e.g., STAT1 phosphorylation of interferon (IFN) stimulation response element (ISRE)-driven promoter, RNA-dependent protein kinase (PKR) and 2'5',-oligoadenylate synthetase (2'5',-OAS) expression. Treatment with aloe-emodin could control influenza infection in humans. PMID:24877694

  9. Janus kinase signaling activation mediates peritoneal inflammation and injury in vitro and in vivo in response to dialysate.

    PubMed

    Dai, Tiane; Wang, Ying; Nayak, Aditi; Nast, Cynthia C; Quang, Lan; LaPage, Janine; Andalibi, Ali; Adler, Sharon G

    2014-12-01

    Peritoneal membrane pathology limits long-term peritoneal dialysis (PD). Here, we tested whether JAK/STAT signaling is implicated and if its attenuation might be salutary. In cultured mesothelial cells, PD fluid activated, and the pan-JAK inhibitor P6 reduced, phospho-STAT1 and phospho-STAT3, periostin secretion, and cleaved caspase-3. Ex vivo, JAK was phosphorylated in PD effluent cells from long-term but not new PD patients. MCP-1 and periostin were increased in PD effluent in long term compared with new patients. In rats, twice daily, PD fluid infusion induced phospho-JAK, mesothelial cell hyperplasia, inflammation, fibrosis, and hypervascularity after 10 days of exposure to PD fluid. Concomitant instillation of a JAK1/2 inhibitor virtually completely attenuated these changes. Thus, our studies directly implicate JAK/STAT signaling in the mediation of peritoneal membrane pathology as a consequence of PD. PMID:25007168

  10. Surfactant Protein A Prevents IFN-γ/IFN-γ Receptor Interaction and Attenuates Classical Activation of Human Alveolar Macrophages.

    PubMed

    Minutti, Carlos M; García-Fojeda, Belén; Sáenz, Alejandra; de Las Casas-Engel, Mateo; Guillamat-Prats, Raquel; de Lorenzo, Alba; Serrano-Mollar, Anna; Corbí, Ángel L; Casals, Cristina

    2016-07-15

    Lung surfactant protein A (SP-A) plays an important function in modulating inflammation in the lung. However, the exact role of SP-A and the mechanism by which SP-A affects IFN-γ-induced activation of alveolar macrophages (aMϕs) remains unknown. To address these questions, we studied the effect of human SP-A on rat and human aMϕs stimulated with IFN-γ, LPS, and combinations thereof and measured the induction of proinflammatory mediators as well as SP-A's ability to bind to IFN-γ or IFN-γR1. We found that SP-A inhibited (IFN-γ + LPS)-induced TNF-α, iNOS, and CXCL10 production by rat aMϕs. When rat macrophages were stimulated with LPS and IFN-γ separately, SP-A inhibited both LPS-induced signaling and IFN-γ-elicited STAT1 phosphorylation. SP-A also decreased TNF-α and CXCL10 secretion by ex vivo-cultured human aMϕs and M-CSF-derived macrophages stimulated by either LPS or IFN-γ or both. Hence, SP-A inhibited upregulation of IFN-γ-inducible genes (CXCL10, RARRES3, and ETV7) as well as STAT1 phosphorylation in human M-CSF-derived macrophages. In addition, we found that SP-A bound to human IFN-γ (KD = 11 ± 0.5 nM) in a Ca(2+)-dependent manner and prevented IFN-γ interaction with IFN-γR1 on human aMϕs. We conclude that SP-A inhibition of (IFN-γ + LPS) stimulation is due to SP-A attenuation of both inflammatory agents and that the binding of SP-A to IFN-γ abrogates IFN-γ effects on human macrophages, suppressing their classical activation and subsequent inflammatory response. PMID:27271568

  11. The poly-γ-d-glutamic acid capsule surrogate of the Bacillus anthracis capsule induces nitric oxide production via the platelet activating factor receptor signaling pathway.

    PubMed

    Lee, Hae-Ri; Jeon, Jun Ho; Park, Ok-Kyu; Chun, Jeong-Hoon; Park, Jungchan; Rhie, Gi-Eun

    2015-12-01

    The poly-γ-d-glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis, confers protection of the bacillus from phagocytosis and allows its unimpeded growth in the host. PGA capsules released from B. anthracis are associated with lethal toxin in the blood of experimentally infected animals and enhance the cytotoxic effect of lethal toxin on macrophages. In addition, PGA capsule itself activates macrophages and dendritic cells to produce proinflammatory cytokine such as IL-1β, indicating multiple roles of PGA capsule in anthrax pathogenesis. Here we report that PGA capsule of Bacillus licheniformis, a surrogate of B. anthracis capsule, induces production of nitric oxide (NO) in RAW264.7 cells and bone marrow-derived macrophages. NO production was induced by PGA in a dose-dependent manner and was markedly reduced by inhibitors of inducible NO synthase (iNOS), suggesting iNOS-dependent production of NO. Induction of NO production by PGA was not observed in macrophages from TLR2-deficient mice and was also substantially inhibited in RAW264.7 cells by pretreatment of TLR2 blocking antibody. Subsequently, the downstream signaling events such as ERK, JNK and p38 of MAPK pathways as well as NF-κB activation were required for PGA-induced NO production. In addition, the induced NO production was significantly suppressed by treatment with antagonists of platelet activating factor receptor (PAFR) or PAFR siRNA, and mediated through PAFR/Jak2/STAT-1 signaling pathway. These findings suggest that PGA capsule induces NO production in macrophages by triggering both TLR2 and PAFR signaling pathways which lead to activation of NF-kB and STAT-1, respectively. PMID:26350415

  12. A novel STAT-like factor mediates lipopolysaccharide, interleukin 1 (IL-1), and IL-6 signaling and recognizes a gamma interferon activation site-like element in the IL1B gene.

    PubMed Central

    Tsukada, J; Waterman, W R; Koyama, Y; Webb, A C; Auron, P E

    1996-01-01

    Binding of many cytokines to their cognate receptors immediately activates Jak tyrosine kinases and their substrates, STAT (signal transducers and activators of transcription) DNA-binding proteins. The DNA binding targets of STATs are sequence elements related to the archetypal gamma interferon activation site, GAS. However, association of interleukin 1 (IL-1) with Jak-STAT signaling has remained unresolved. We now report an element termed LILRE (lipopolysaccharide [LPS] and IL-1-responsive element) in the human prointerleukin 1beta gene (IL1B) which can be immediately induced by either lipopolysaccharide (LPS) or IL-1 protein to bind a tyrosine-phosphorylated protein. This LPS- and IL-1-induced factor (LIL factor) is recognized by an antibody raised against the N terminus of Stat1, but not by those specific for either the C terminus of Stat1 or any other GAS-binding STAT. Phosphotyrosine (P-Tyr) specifically inhibits formation of the LIL factor-DNA complex, suggesting the importance of P-Tyr for the DNA-binding activity, as has been found for all STAT dimers. Analysis of DNA-binding specificity demonstrates that the LIL factor possesses a novel GAS-like binding activity that contrasts with those of other STATs in a requirement for a G residue at position 8 (TTCCTGAGA). Further investigation has revealed that IL-6, but neither IL-4 nor gamma interferon, activates the LIL factor. Thus, the existence of such a STAT-like factor (LIL-Stat) relates the LPS and IL-1 signaling pathway to other cytokine receptor signaling pathways via the activation of STATs. Moreover, the unique DNA-binding specificity and antigenicity of this factor suggest that LPS, IL-1, and IL-6 may use a common signaling pathway. PMID:8628285

  13. Triggering through NOD-2 Differentiates Bone Marrow Precursors to Dendritic Cells with Potent Bactericidal activity

    PubMed Central

    Khan, Nargis; Aqdas, Mohammad; Vidyarthi, Aurobind; Negi, Shikha; Pahari, Susanta; Agnihotri, Tapan; Agrewala, Javed N.

    2016-01-01

    Dendritic cells (DCs) play a crucial role in bridging innate and adaptive immunity by activating naïve T cells. The role of pattern recognition receptors like Toll-Like Receptors and Nod-Like Receptors expressed on DCs is well-defined in the recognition of the pathogens. However, nothing is precisely studied regarding the impact of NOD-2 signaling during the differentiation of DCs. Consequently, we explored the role of NOD-2 signaling in the differentiation of DCs and therefore their capability to activate innate and adaptive immunity. Intriguingly, we observed that NOD-2 stimulated DCs (nDCs) acquired highly activated and matured phenotype and exhibited substantially greater bactericidal activity by robust production of nitric oxide. The mechanism involved in improving the functionality of nDCs was dependent on IFN-αβ signaling, leading to the activation of STAT pathways. Furthermore, we also observed that STAT-1 and STAT-4 dependent maturation and activation of DCs was under the feedback mechanism of SOCS-1 and SOCS-3 proteins. nDCs acquired enhanced potential to activate chiefly Th1 and Th17 immunity. Taken together, these results suggest that nDCs can be exploited as an immunotherapeutic agent in bolstering host immunity and imparting protection against the pathogens. PMID:27265209

  14. Bone marrow mesenchymal stromal cells with CD47 high expression via the signal transducer and activators of transcription signaling pathway preventing myocardial fibrosis

    PubMed Central

    Deng, Wei; Chen, Qing-Wei; Li, Xing-Sheng; Yuan, Zhong-Ming; Li, Gui-Qiong; Ke, Da-Zhi; Wang, Li; Wu, Zhi-Qing; Luo, Shi-Lan

    2015-01-01

    This study was initiated to investigate the efficacy of myocardial fibrosis intervention via signal transducer and activators of transcription (STAT) signaling using bone marrow (BM) mesenchymal stromal cells (MSC) in which being over-expressed with the aid of bispecific antibody (BiAb) and ultrasound-mediated microbubbles (MB). BiAb was prepared and combined with isolated MSC with CD47 overexpression from male mice and trans-fused into female mice with isoproterenol-induced myocardial fibrosis via the tail vein, followed by MB. This study included five groups. Five weeks after treatment, expression levels of the sex-determining region of Y-chromosome (SRY), matrix metalloproteinases (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and vascular endothelial growth factor (VEGF) in myocardium were detected by fluorescent quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of signal transducer and activators of transcription (STAT) 1 and STAT 3 was detected by Western blot. Results: The highest homing number of MSC was in the CD47 + MSC + BiAb + MB group, second highest in the CD47 + MSC + BiAb group, and lowest in MSC alone. Compared with the Control group, CD47 + MSC + BiAb + MB, CD47 + MSC + BiAb, CD47 + MSC and MSC groups had decreased levels of MMP-9, TIMP-1, STAT 1 and collagen deposition, and increased levels of STAT 3. Up regulated STAT 3 and down regulated TIMP-1 were significantly different in CD47 + MSC + BiAb + MB compared with CD47 + MSC or CD47 + MSC + BiAb. Conclusion: CD47 can enhance the homing rate and repairing efficacy of MSC. MSC can improve MMP-TIMP expression in injured myocardium and interfere with myocardial fibrosis after homing, a mechanism that may be related to the STAT-mediated signaling pathway. PMID:26617765

  15. Different STAT transcription complexes drive early and delayed responses to type I Interferons

    PubMed Central

    Plumlee, Courtney R.; Perry, Stuart; Gu, Ai Di; Lee, Carolyn; Shresta, Sujan; Decker, Thomas; Schindler, Christian

    2015-01-01

    Interferons, which transduce pivotal signals through signal transducer and activator of transcription (Stat)1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Whereas the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-α/β unexpectedly suppresses L. pneumophila growth in both Stat1 and Stat2 deficient macrophages. New studies demonstrating that the robust response to IFN-α/β is lost in Stat1-Stat2 double knockout macrophages, suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response towards L. pneumophila. Since the ability of IFN-α/β to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-α/β in the absence of Stat1. These studies reveal that IFN-α/β is able to drive the formation of a Stat2 and IRF9 complex that drives the expression of a subset of IFN stimulated genes (ISGs), but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1 dependent responses. PMID:26019270

  16. Activating STAT6 mutations in follicular lymphoma

    PubMed Central

    Yildiz, Mehmet; Li, Hongxiu; Bernard, Denzil; Amin, Nisar A.; Ouillette, Peter; Jones, Siân; Saiya-Cork, Kamlai; Parkin, Brian; Jacobi, Kathryn; Shedden, Kerby; Wang, Shaomeng; Chang, Alfred E.; Kaminski, Mark S.

    2015-01-01

    Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world. FL cell-intrinsic and cell-extrinsic factors influence FL biology and clinical outcome. To further our understanding of the genetic basis of FL, we performed whole-exome sequencing of 23 highly purified FL cases and 1 transformed FL case and expanded findings to a combined total of 114 FLs. We report recurrent mutations in the transcription factor STAT6 in 11% of FLs and identified the STAT6 amino acid residue 419 as a novel STAT6 mutation hotspot (p.419D/G, p.419D/A, and p.419D/H). FL-associated STAT6 mutations were activating, as evidenced by increased transactivation in HEK293T cell–based transfection/luciferase reporter assays, heightened interleukin-4 (IL-4) –induced activation of target genes in stable STAT6 transfected lymphoma cell lines, and elevated baseline expression levels of STAT6 target genes in primary FL B cells harboring mutant STAT6. Mechanistically, FL-associated STAT6 mutations facilitated nuclear residency of STAT6, independent of IL-4–induced STAT6-Y641 phosphorylation. Structural modeling of STAT6 based on the structure of the STAT1-DNA complex revealed that most FL-associated STAT6 mutants locate to the STAT6-DNA interface, potentially facilitating heightened interactions. The genetic and functional data combined strengthen the recognition of the IL-4/JAK/STAT6 axis as a driver of FL pathogenesis. PMID:25428220

  17. A novel small molecule inhibits STAT3 phosphorylation and DNA binding activity and exhibits potent growth suppressive activity in human cancer cells

    PubMed Central

    2010-01-01

    Background Targeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an attractive therapeutic approach for most types of human cancers with constitutively activated STAT3. A novel small molecular STAT3 inhibitor, FLLL32 was specifically designed from dietary agent, curcumin to inhibit constitutive STAT3 signaling in multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells. Results FLLL32 was found to be a potent inhibitor of STAT3 phosphorylation, STAT3 DNA binding activity, and the expression of STAT3 downstream target genes in vitro, leading to the inhibition of cell proliferation as well as the induction of Caspase-3 and PARP cleavages in human multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cell lines. However, FLLL32 exhibited little inhibition on some tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein and lipid kinases using a kinase profile assay. FLLL32 was also more potent than four previously reported JAK2 and STAT3 inhibitors as well as curcumin to inhibit cell viability in these cancer cells. Furthermore, FLLL32 selectively inhibited the induction of STAT3 phosphorylation by Interleukin-6 but not STAT1 phosphorylation by IFN-γ. Conclusion Our findings indicate that FLLL32 exhibits potent inhibitory activity to STAT3 and has potential for targeting multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells expressing constitutive STAT3 signaling. PMID:20712901

  18. Molecular Mechanism of Macrophage Activation by Red Ginseng Acidic Polysaccharide from Korean Red Ginseng

    PubMed Central

    Byeon, Se Eun; Lee, Jaehwi; Kim, Ji Hye; Yang, Woo Seok; Kwak, Yi-Seong; Kim, Sun Young; Choung, Eui Su; Rhee, Man Hee; Cho, Jae Youl

    2012-01-01

    Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng, displays immunostimulatory and antitumor activities. Even though numerous studies have been reported, the mechanism as to how RGAP is able to stimulate the immune response is not clear. In this study, we aimed to explore the mechanism of molecular activation of RGAP in macrophages. RGAP treatment strongly induced NO production in RAW264.7 cells without altering morphological changes, although the activity was not strong compared to LPS-induced dendritic-like morphology in RAW264.7 cells. RGAP-induced NO production was accompanied with enhanced mRNA levels of iNOS and increases in nuclear transcription factors such as NF-κB, AP-1, STAT-1, ATF-2, and CREB. According to pharmacological evaluation with specific enzyme inhibitors, Western blot analysis of intracellular signaling proteins and inhibitory pattern using blocking antibodies, ERK, and JNK were found to be the most important signaling enzymes compared to LPS signaling cascade. Further, TLR2 seems to be a target surface receptor of RGAP. Lastly, macrophages isolated from RGS2 knockout mice or wortmannin exposure strongly upregulated RGAP-treated NO production. Therefore, our results suggest that RGAP can activate macrophage function through activation of transcription factors such as NF-κB and AP-1 and their upstream signaling enzymes such as ERK and JNK. PMID:22474399

  19. Simultaneous Pathway Activity Inference and Gene Expression Analysis Using RNA Sequencing.

    PubMed

    O'Connell, Daniel J; Kolde, Raivo; Sooknah, Matthew; Graham, Daniel B; Sundberg, Thomas B; Latorre, Isabel J; Mikkelsen, Tarjei S; Xavier, Ramnik J

    2016-05-25

    Reporter gene assays are a venerable tool for studying signaling pathways, but they lack the throughput and complexity necessary to contribute to a systems-level understanding of endogenous signaling networks. We present a parallel reporter assay, transcription factor activity sequencing (TF-seq), built on synthetic DNA enhancer elements, which enables parallel measurements in primary cells of the transcriptome and transcription factor activity from more than 40 signaling pathways. Using TF-seq in Myd88(-/-) macrophages, we captured dynamic pathway activity changes underpinning the global transcriptional changes of the innate immune response. We also applied TF-seq to investigate small molecule mechanisms of action and find a role for NF-κB activation and coordination of the STAT1 response in the macrophage reaction to the anti-inflammatory natural product halofuginone. Simultaneous TF-seq and global gene expression profiling represent an integrative approach for gaining mechanistic insight into pathway activity and transcriptional changes that result from genetic and small molecule perturbations. PMID:27211859

  20. Mechanism of Plasminogen Activator Inhibitor-1 regulation by Oncostatin M and Interleukin-1 in human astrocytes

    PubMed Central

    Kasza, Aneta; Kiss, Daniel L.; Gopalan, Sunita; Xu, Weili; Rydel, Russell E.; Koj, Aleksander; Kordula, Tomasz

    2015-01-01

    Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that downregulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor kB (IkB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. We conclude that OSM and IL-1 regulate the PAI-1 gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the PAI-1 gene. PMID:12390531

  1. Modular analysis of bioinformatics demonstrates a critical role for NF-κB in macrophage activation.

    PubMed

    Zhang, Yingmei; Wang, Yingmei; Lu, Ming; Qiao, Xin; Sun, Bei; Zhang, Weihui; Xue, Dongbo

    2014-08-01

    To achieve the goal of identifying the gene groups that regulated macrophage activation, a total of 925 differentially expressed genes of activated macrophages were found at the intersection of the three series (GSE5099-1, GSE5099-2, and GSE18686) from the Gene Expression Omnibus (GEO) database, and a sub-network was constructed based on the protein-protein interaction (PPI) network. Four communities (K = 3) were identified from the sub-network using the CFinder software. Community 1 was considered as the gene group of interest base on the heat map. GO-BP and KEGG enrichment analysis with the DAVID software showed that the functions of the 14 genes in community 1 were mainly related to the NF-κB pathway. A network was constructed using the Cytoscape software. The diagram showed that STAT1, NFKBIA, NFKAIB, JUN, and RELA were the key genes in the regulation of macrophage activation. Among these genes, RELA (NF-κB P65) was an important member of the NF-κB family, while NFKBIA (IκBα) and NFKAIB (IκBβ) were the inhibitory factors of NF-κB. Small molecules capable of regulating these five genes were identified via the CMap software, and a network diagram was generated using the Cytoscape software to provide a reference for the development of new drugs that regulate macrophage activation. PMID:24577727

  2. Parainfluenza Virus 3 Blocks Antiviral Mediators Downstream of the Interferon Lambda Receptor by Modulating Stat1 Phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paramyxoviruses are known to inhibit type I interferon (IFN) production, however there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, the IFN-'R1/IL-10R2, which is primarily expressed by epithelial cells. ...

  3. Parainfluenza virus 3 blocks antiviral mediators downstream of the interferon lambda receptor by modulating stat1 phosphorylation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Paramyxoviruses are known to inhibit type I interferon (IFN) production, however there is a lack of information regarding the type III IFN response during infection. Type III IFNs signal through a unique heterodimeric receptor, the IFN-'R1/IL-10R2, which is primarily expressed by epithelial cells. ...

  4. High hydrostatic pressure processing of murine norovirus 1-contaminated oysters inhibits oral infection in STAT-1 -/- deficient female mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously demonstrated that high pressure processing (HPP) is effective in preventing in vitro replication of murine norovirus strain 1 (MNV-1), a human norovirus surrogate, in a monocyte cell line following extraction from MNV-1-contaminated oysters. In the present study, the efficacy of ...

  5. Anti-Tumor and Immune Enhancing Activities of Rice Bran Gramisterol on Acute Myelogenous Leukemia

    PubMed Central

    Somintara, Somsuda; Leardkamolkarn, Vijittra; Suttiarporn, Panawan; Mahatheeranont, Sugunya

    2016-01-01

    Background Acute myelogenous leukemia (AML) is a cancer of the blood that most commonly affects human adults. The specific cause of AML is unclear, but it induces abnormality of white blood cells that grow rapidly and accumulate in bone marrow interfering with the production and functions of the normal blood cells. AML patients face poor prognosis and low quality of life during chemotherapy or transplantation of hematopoietic stem cells due to the progressive impairment of their immune system. The goal of this study is to find natural products that have the potential to delay growth or eliminate the abnormal leukemic cells but cause less harmful effect to the body’s immune system. Methods and Findings The unsaponified fraction of Riceberry rice bran (RBDS) and the main pure compound, gramisterol, were studied for cytotoxicity and biological activities in WEHI-3 cells and in the leukemic mouse model induced by transplantation of WEHI-3 cells intraperitoneally. In the in vitro assay, RBDS and gramisterol exerted sub-G1 phase cell cycle arrest with a potent induction of apoptosis. Both of them effectively decreased cell cycle controlling proteins (cyclin D1 and cyclin E), suppressed cellular DNA synthesis and mitotic division, and reduced anti-apoptosis Bcl-2 protein, but increased apoptotic proteins (p53 and Bax) and activated caspase-3 enzyme in the intrinsic cell death stimulation pathway. In leukemic mice, daily feeding of RBDS significantly increased the amount of immune function-related cells including CD3+, CD19+, and CD11b+, and elevated the serum levels of IFN-γ, TNF-α, IL-2, and IL-12β cytokines, but suppressed IL-10 level. At the tumor sites, CD11b+ cells were polarized and became active phagocytotic cells. Treatment of mice normal immune cells with gramisterol alone or a combination of gramisterol with cytokines released from RBDS-treated leukemic mice splenocytes culture synergistically increased pSTAT1 transcriptional factor that up-regulated the

  6. Increased Early RNA Replication by Chimeric West Nile Virus W956IC Leads to IPS-1-Mediated Activation of NF-κB and Insufficient Virus-Mediated Counteraction of the Resulting Canonical Type I Interferon Signaling

    PubMed Central

    Scherbik, S. V.; Pulit-Penaloza, J. A.; Basu, M.; Courtney, S. C.

    2013-01-01

    Although infections with “natural” West Nile virus (WNV) and the chimeric W956IC WNV infectious clone virus produce comparable peak virus yields in type I interferon (IFN) response-deficient BHK cells, W956IC infection produces higher levels of “unprotected” viral RNA at early times after infection. Analysis of infections with these two viruses in IFN-competent cells showed that W956IC activated NF-κB, induced higher levels of IFN-β, and produced lower virus yields than WNV strain Eg101. IPS-1 was required for both increased induction of IFN-β and decreased yields of W956IC. In Eg101-infected cells, phospho-STAT1/STAT2 nuclear translocation was blocked at all times analyzed, while some phospho-STAT1/STAT2 nuclear translocation was still detected at 8 h after infection in W956IC-infected mouse embryonic fibroblasts (MEFs), and early viral protein levels were lower in these cells. A set of additional chimeras was made by replacing various W956IC gene regions with the Eg101 equivalents. As reported previously, for three of these chimeras, the low early RNA phenotype of Eg101 was restored in BHK cells. Analysis of infections with two of these chimeric viruses in MEFs detected lower early viral RNA levels, higher early viral protein levels, lower early IFN-β levels, and higher virus yields similar to those seen after Eg101 infection. The data suggest that replicase protein interactions directly or indirectly regulate genome switching between replication and translation at early times in favor of translation to minimize NF-κB activation and IFN induction by decreasing the amount of unprotected viral RNA, to produce sufficient viral protein to block canonical type I IFN signaling, and to efficiently remodel cell membranes for exponential genome amplification. PMID:23678179

  7. Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3.

    PubMed

    Haapaniemi, Emma M; Kaustio, Meri; Rajala, Hanna L M; van Adrichem, Arjan J; Kainulainen, Leena; Glumoff, Virpi; Doffinger, Rainer; Kuusanmäki, Heikki; Heiskanen-Kosma, Tarja; Trotta, Luca; Chiang, Samuel; Kulmala, Petri; Eldfors, Samuli; Katainen, Riku; Siitonen, Sanna; Karjalainen-Lindsberg, Marja-Liisa; Kovanen, Panu E; Otonkoski, Timo; Porkka, Kimmo; Heiskanen, Kaarina; Hänninen, Arno; Bryceson, Yenan T; Uusitalo-Seppälä, Raija; Saarela, Janna; Seppänen, Mikko; Mustjoki, Satu; Kere, Juha

    2015-01-22

    The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity. PMID:25349174

  8. Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3

    PubMed Central

    Haapaniemi, Emma M.; Kaustio, Meri; Rajala, Hanna L. M.; van Adrichem, Arjan J.; Kainulainen, Leena; Glumoff, Virpi; Doffinger, Rainer; Kuusanmäki, Heikki; Heiskanen-Kosma, Tarja; Trotta, Luca; Chiang, Samuel; Kulmala, Petri; Eldfors, Samuli; Katainen, Riku; Siitonen, Sanna; Karjalainen-Lindsberg, Marja-Liisa; Kovanen, Panu E.; Otonkoski, Timo; Porkka, Kimmo; Heiskanen, Kaarina; Hänninen, Arno; Bryceson, Yenan T.; Uusitalo-Seppälä, Raija; Saarela, Janna; Seppänen, Mikko; Kere, Juha

    2015-01-01

    The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked–like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4−CD8−) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity. PMID:25349174

  9. TYK2 Kinase Activity Is Required for Functional Type I Interferon Responses In Vivo

    PubMed Central

    Prchal-Murphy, Michaela; Semper, Christian; Lassnig, Caroline; Wallner, Barbara; Gausterer, Christian; Teppner-Klymiuk, Ingeborg; Kobolak, Julianna; Müller, Simone; Kolbe, Thomas; Karaghiosoff, Marina; Dinnyés, Andras; Rülicke, Thomas; Leitner, Nicole R.; Strobl, Birgit; Müller, Mathias

    2012-01-01

    Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors. PMID:22723949

  10. 2,2',4,6,6'-pentachlorobiphenyl (PCB 104) induces apoptosis of human microvascular endothelial cells through the caspase-dependent activation of CREB.

    PubMed

    Lee, Yong Woo; Park, Hyen Joo; Son, Kwang Won; Hennig, Bernhard; Robertson, Larry W; Toborek, Michal

    2003-05-15

    It has been proposed that endothelial integrity can play an active regulatory role in the extravasation of tumor cells during cancer metastasis. Since polychlorinated biphenyls (PCBs) have been shown to cause endothelial cell activation or injury and to lead to various diseases that involve dysfunction of the vascular endothelium, the present study was designed to determine the cellular and molecular signaling mechanisms of PCB-induced apoptosis in human microvascular endothelial cells (HMEC-1). A significant and marked decrease in cell viability was observed in HMEC-1 treated with 2,2',4,6,6'-pentachlorobiphenyl (PCB 104) in a time- and dose-dependent manner. Exposure of HMEC-1 to PCB 104 also dramatically induced internucleosomal DNA fragmentation. However, the caspase inhibitor zVAD-fmk significantly reversed the PCB 104-induced DNA fragmentation in HMEC-1, suggesting that endothelial cell death induced by PCB 104 exposure is, at least in part, due to caspase-dependent apoptotic pathways. To elucidate the molecular signaling mechanisms of PCB 104-induced apoptotic cell death in human microvascular endothelial cells, the present study focused on the effects of acute exposure of PCB 104 on the activation of several transcription factors, such as cAMP responsive element-binding protein (CREB), activator protein-1 (AP-1), nuclear factor-kappaB (NF-kappaB), and signal transducers and activators of transcription (STAT1), which have been known to play a pivotal role in the molecular signaling cascades for the induction of apoptosis. A series of electrophoretic mobility shift assay showed that PCB 104 specifically increased only CREB DNA-binding activity in a dose-dependent manner. AP-1, NF-kappaB, and STAT1, however, were not activated. In addition, zVAD-fmk significantly and dose-dependently blocked the CREB activation enhanced by PCB 104 exposure. These results suggest that PCB-induced death of human microvascular endothelial cells is mediated, at least in part, via

  11. Identifying Mechanisms by Which Escherichia coli O157:H7 Subverts Interferon-γ Mediated Signal Transducer and Activator of Transcription-1 Activation

    PubMed Central

    Ho, Nathan K.; Crandall, Ian; Sherman, Philip M.

    2012-01-01

    Enterohemorrhagic Escherichia coli serotype O157:H7 is a food borne enteric bacterial pathogen that causes significant morbidity and mortality in both developing and industrialized nations. E. coli O157:H7 infection of host epithelial cells inhibits the interferon gamma pro-inflammatory signaling pathway, which is important for host defense against microbial pathogens, through the inhibition of Stat-1 tyrosine phosphorylation. The aim of this study was to determine which bacterial factors are involved in the inhibition of Stat-1 tyrosine phosphorylation. Human epithelial cells were challenged with either live bacteria or bacterial-derived culture supernatants, stimulated with interferon-gamma, and epithelial cell protein extracts were then analyzed by immunoblotting. The results show that Stat-1 tyrosine phosphorylation was inhibited by E. coli O157:H7 secreted proteins. Using sequential anion exchange and size exclusion chromatography, YodA was identified, but not confirmed to mediate subversion of the Stat-1 signaling pathway using isogenic mutants. We conclude that E. coli O157:H7 subverts Stat-1 tyrosine phosphorylation in response to interferon-gamma through a still as yet unidentified secreted bacterial protein. PMID:22253910

  12. Identifying mechanisms by which Escherichia coli O157:H7 subverts interferon-γ mediated signal transducer and activator of transcription-1 activation.

    PubMed

    Ho, Nathan K; Crandall, Ian; Sherman, Philip M

    2012-01-01

    Enterohemorrhagic Escherichia coli serotype O157:H7 is a food borne enteric bacterial pathogen that causes significant morbidity and mortality in both developing and industrialized nations. E. coli O157:H7 infection of host epithelial cells inhibits the interferon gamma pro-inflammatory signaling pathway, which is important for host defense against microbial pathogens, through the inhibition of Stat-1 tyrosine phosphorylation. The aim of this study was to determine which bacterial factors are involved in the inhibition of Stat-1 tyrosine phosphorylation. Human epithelial cells were challenged with either live bacteria or bacterial-derived culture supernatants, stimulated with interferon-gamma, and epithelial cell protein extracts were then analyzed by immunoblotting. The results show that Stat-1 tyrosine phosphorylation was inhibited by E. coli O157:H7 secreted proteins. Using sequential anion exchange and size exclusion chromatography, YodA was identified, but not confirmed to mediate subversion of the Stat-1 signaling pathway using isogenic mutants. We conclude that E. coli O157:H7 subverts Stat-1 tyrosine phosphorylation in response to interferon-gamma through a still as yet unidentified secreted bacterial protein. PMID:22253910

  13. Both Cyclophilin Inhibitors and Direct-Acting Antivirals Prevent PKR Activation in HCV-Infected Cells

    PubMed Central

    Bobardt, Michael; Chatterji, Udayan; Lim, Precious; Gawlik, Katarzyna; Gallay, Philippe

    2014-01-01

    We and others demonstrated that the contact between NS5A and the host factor CypA is critical for HCV replication. CypI, by disrupting NS5A-CypA complexes, block HCV replication both in vitro and in patients. Since NS5A also binds to PKR, a central component of the IFN response, we investigated the possibility of a relationship between CypA, NS5A and PKR in the IFN response to HCV. HCV-infected cells treated with CypI, DAAs or IFN were analyzed for the expression and activation of various components of the innate response. We found that CypI (cyclosporine A, alisporivir, NIM811 and sanglifehrins), drastically prevented the activation/phosphorylation, but not the expression of IFN-induced PKR in HCV-infected cells. CypI had no effect on the expression or phosphorylation of other components of the innate response such as eiF2, NF-kB, IRF3, IRF9, STAT1 and STAT2, suggesting a specific effect on PKR. No significant activation of IFN-induced PKR was observed in the absence of HCV. Importantly, we found that several classes of DAAs such as NS3/4A protease, NS5B polymerase and NS5A inhibitors also prevented PKR activation. Furthermore, we found that PKR activation by the dsRNA mimic poly I:C cannot be prevented by CypI or DAAs. Our findings suggest that CypI do not have a unique effect on PKR activation, but rather the suppression of HCV replication by any anti-HCV inhibitor, abrogates PKR activation induced by IFN. Moreover, they suggest that the accumulation of dsRNA intermediates allows HCV to exploit the activation of PKR to counteract the IFN response. PMID:24799968

  14. Suppression of USP18 Potentiates the Anti-HBV Activity of Interferon Alpha in HepG2.2.15 Cells via JAK/STAT Signaling

    PubMed Central

    Li, Lin; Lei, Qing-song; Zhang, Shu-Jun; Kong, Ling-na; Qin, Bo

    2016-01-01

    Ubiquitin-specific protease 18 (USP18, also known as UBP43) has both interferon stimulated gene 15 (ISG15) dependent and ISG15-independent functions. By silencing the expression of USP18 in HepG2.2.15 cells, we studied the effect of USP18 on the anti-HBV activity of IFN-α and demonstrated that knockdown of USP18 significantly Inhibited the HBV expression and increased the expression of ISGs. Levels of hepatitis B virus surface antigen (HBsAg), hepatitis B virus e antigen (HBeAg), HBV DNA and intracellular hepatitis B virus core antigen (HBcAg) were dramatically decreased with or without treatment of indicated dose of IFN-α. Suppression of USP18 activated the JAK/STAT signaling pathway as shown by the increased and prolonged expression of phosphorylated signal transducer and activator of transcription 1 (p-STAT1) in combination with enhanced expression of several interferon stimulated genes (ISGs). Our results indicated that USP18 modulates the anti-HBV activity of IFN-α via activation of the JAK/STAT signaling pathway in Hepg2.2.15 cells. PMID:27227879

  15. Interferon-Gamma Enhances TLR3 Expression and Anti-Viral Activity in Keratinocytes.

    PubMed

    Kajita, Ai; Morizane, Shin; Takiguchi, Tetsuya; Yamamoto, Takenobu; Yamada, Masao; Iwatsuki, Keiji

    2015-08-01

    Toll-like receptors (TLRs) recognize specific microbial products in the innate immune response. TLR3, a double-stranded RNA sensor, is thought to have an important role in viral infections, but the regulation of TLR3 expression and its function in keratinocytes are not fully understood. Here we show the Th1 cytokine IFN-γ increased the TLR3 expression via STAT1 in cultured normal human epidermal keratinocytes (NHEKs). Co-stimulation with IFN-γ and the TLR3 ligand poly (I:C) synergistically increased the expression of IFN-β, IL-6, IL-8, and human β-defensin-2 in NHEKs compared with poly (I:C) or IFN-γ alone. These synergistic inductions were significantly inhibited by an endosomal acidification inhibitor, chloroquine, and by TLR3 siRNA. Co-stimulation with IFN-γ and poly (I:C) also significantly enhanced the anti-viral activity against herpes simplex virus type-1 in NHEKs compared with poly (I:C) or IFN-γ alone. In addition to the in vitro findings, an immunohistochemical analysis revealed IFN-γ-positive cells surrounding herpetic vesicles. These findings indicate that IFN-γ might contribute to the innate immune response to cutaneous viral infections by enhancing TLR3 expression and function in keratinocytes. PMID:25822580

  16. Mitochondrial translocation of signal transducer and activator of transcription 5 (STAT5) in leukemic T cells and cytokine-stimulated cells

    SciTech Connect

    Chueh, Fu-Yu; Leong, King-Fu; Yu, Chao-Lan

    2010-11-26

    Research highlights: {yields} STAT5 interacts with a mitochondrial protein PDC-E2 in a leukemic T cell line LSTRA. {yields} Tyrosine-phosphorylated STAT5, but not STAT3, is present in LSTRA mitochondria. {yields} Cytokines induce mitochondrial translocation of STAT5, but not STAT1 or STAT3. {yields} Cytokine-induced mitochondrial translocation of tyrosine-phosphorylated STAT5 is transient. {yields} Mitochondrial STAT5 binds to a putative STAT5 site in the mitochondrial DNA in vitro. -- Abstract: Signal transducers and activators of transcription (STATs) were first identified as key signaling molecules in response to cytokines. Constitutive STAT activation also has been widely implicated in oncogenesis. We analyzed STAT5-associated proteins in a leukemic T cell line LSTRA, which exhibits constitutive tyrosine phosphorylation and activation of STAT5. A cellular protein was found to specifically interact with STAT5 in LSTRA cells by co-immunoprecipitation. Sequencing analysis and subsequent immunoblotting confirmed the identity of this STAT5-associated protein as the E2 component of mitochondrial pyruvate dehydrogenase complex (PDC-E2). Consistent with this interaction, both subcellular fractionation and immunofluorescence microscopy revealed mitochondrial localization of STAT5 in LSTRA cells. Mitochondrial localization of tyrosine-phosphorylated STAT5 also occurred in cytokine-stimulated cells. A time course experiment further demonstrated the transient kinetics of STAT5 mitochondrial translocation after cytokine stimulation. In contrast, cytokine-induced STAT1 and STAT3 activation did not result in their translocation into mitochondria. Furthermore, we showed that mitochondrial STAT5 bound to the D-loop regulatory region of mitochondrial DNA in vitro. It suggests a potential role of STAT5 in regulating the mitochondrial genome. Proliferative metabolism toward aerobic glycolysis is well known in cancer cells as the Warburg effect and is also observed in cytokine

  17. Characterization of a Novel Human-Specific STING Agonist that Elicits Antiviral Activity Against Emerging Alphaviruses

    PubMed Central

    Sali, Tina M.; Pryke, Kara M.; Abraham, Jinu; Liu, Andrew; Archer, Iris; Broeckel, Rebecca; Staverosky, Julia A.; Smith, Jessica L.; Al-Shammari, Ahmed; Amsler, Lisi; Sheridan, Kayla; Nilsen, Aaron; Streblow, Daniel N.; DeFilippis, Victor R.

    2015-01-01

    Pharmacologic stimulation of innate immune processes represents an attractive strategy to achieve multiple therapeutic outcomes including inhibition of virus replication, boosting antitumor immunity, and enhancing vaccine immunogenicity. In light of this we sought to identify small molecules capable of activating the type I interferon (IFN) response by way of the transcription factor IFN regulatory factor 3 (IRF3). A high throughput in vitro screen yielded 4-(2-chloro-6-fluorobenzyl)-N-(furan-2-ylmethyl)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (referred to herein as G10), which was found to trigger IRF3/IFN-associated transcription in human fibroblasts. Further examination of the cellular response to this molecule revealed expression of multiple IRF3-dependent antiviral effector genes as well as type I and III IFN subtypes. This led to the establishment of a cellular state that prevented replication of emerging Alphavirus species including Chikungunya virus, Venezuelan Equine Encephalitis virus, and Sindbis virus. To define cellular proteins essential to elicitation of the antiviral activity by the compound we employed a reverse genetics approach that utilized genome editing via CRISPR/Cas9 technology. This allowed the identification of IRF3, the IRF3-activating adaptor molecule STING, and the IFN-associated transcription factor STAT1 as required for observed gene induction and antiviral effects. Biochemical analysis indicates that G10 does not bind to STING directly, however. Thus the compound may represent the first synthetic small molecule characterized as an indirect activator of human STING-dependent phenotypes. In vivo stimulation of STING-dependent activity by an unrelated small molecule in a mouse model of Chikungunya virus infection blocked viremia demonstrating that pharmacologic activation of this signaling pathway may represent a feasible strategy for combating emerging Alphaviruses. PMID:26646986

  18. The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by Type I interferon

    PubMed Central

    Rajsbaum, Ricardo; Macleod, Jesica M. Levingston; Pisanelli, Giuseppe; Pham, Alissa; Ayllon, Juan; Miorin, Lisa; Martinez, Carles; tenOever, Benjamin R; García-Sastre, Adolfo

    2014-01-01

    Summary To successfully establish infection Flaviviruses have to overcome the antiviral state induced by type I interferon (IFN-I). The nonstructural NS5 proteins of several flaviviruses antagonize IFN-I signaling. Here we show that yellow fever virus (YFV) inhibits IFN-I signaling through a unique mechanism that involves binding of YFV NS5 to the IFN-activated transcription factor STAT2 only in cells that have been stimulated with IFN-I. This NS5-STAT2 interaction requires IFN-I-induced tyrosine phosphorylation of STAT1 and the K63-linked polyubiquitination at a lysine in the N-terminal region of YFV NS5. We identified TRIM23 as the E3 ligase that interacts with and polyubiquitinates YFV NS5 to promote its binding to STAT2 and trigger IFN-I signaling inhibition. Our results demonstrate the importance of YFV NS5 in overcoming the antiviral action of IFN-I and offer a unique example of a viral protein that is activated by the same host pathway that it inhibits. PMID:25211074

  19. Microarray profiling of L1-overexpressing endothelial cells reveals STAT3 activation via IL-6/IL-6Rα axis

    PubMed Central

    Magrini, Elena; Cavallaro, Ugo; Bianchi, Fabrizio

    2015-01-01

    We recently identified a novel role for the L1 transmembrane glycoprotein (also known as L1CAM or CD171) in the regulation of tumor angiogenesis and vessels stabilization. L1 overexpression in cultured endothelial cells of the lung (luECs) exerted a pleiotropic effect in that it regulated proliferation, migration, tubulogenesis, vascular permeability, and endothelial-to-mesenchymal transition (EndMT). In addition, we provided strong evidence that antibody-mediated targeting of L1 may be an effective strategy for vessel normalization with the potential to increase efficacy of chemotherapeutic agents. High-throughput microarray expression profile revealed that L1 modulates the expression of hundreds of genes mainly involved in cell cycle regulation, DNA replication, cellular assembly, migration, development and organization. By using a ‘pathway-oriented’ analysis strategy we were able to identify a network of 105 genes modulated by L1 through the predicted activation of five transcription factors: STAT1, STAT2, STAT3, IRF7, and ATF4. Indeed, L1 overexpression resulted in the strong induction of STAT3 phosphorylation which was abolished by antibody-mediated neutralization of IL-6Rα. These results indicated that L1 promoted STAT3 activation via the IL-6/IL-6Rα axis. PMID:26484199

  20. Cytomegalovirus generates long-lived antigen-specific NK cells with diminished bystander activation to heterologous infection

    PubMed Central

    Min-Oo, Gundula

    2014-01-01

    Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)–induced memory Ly49H+ NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes, a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H+ or Ly49H− NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge. PMID:25422494

  1. Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome

    PubMed Central

    He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

    2016-01-01

    The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

  2. Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome.

    PubMed

    He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

    2016-01-26

    The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

  3. Interleukin-21 enhances NK cell activation in response to antibody-coated targets.

    PubMed

    Roda, Julie M; Parihar, Robin; Lehman, Amy; Mani, Aruna; Tridandapani, Susheela; Carson, William E

    2006-07-01

    NK cells express an activating FcR (FcgammaRIIIa) that mediates Ab-dependent cellular cytotoxicity and the production of immune modulatory cytokines in response to Ab-coated targets. IL-21 has antitumor activity in murine models that depends in part on its ability to promote NK cell cytotoxicity and IFN-gamma secretion. We hypothesized that the NK cell response to FcR stimulation would be enhanced by the administration of IL-21. Human NK cells cultured with IL-21 and immobilized IgG or human breast cancer cells coated with a therapeutic mAb (trastuzumab) secreted large amounts of IFN-gamma. Increased secretion of TNF-alpha and the chemokines IL-8, MIP-1alpha, and RANTES was also observed under these conditions. NK cell IFN-gamma production was dependent on distinct signals mediated by the IL-21R and the FcR and was abrogated in STAT1-deficient NK cells. Supernatants derived from NK cells that had been stimulated with IL-21 and mAb-coated breast cancer cells were able to drive the migration of naive and activated T cells in an in vitro chemotaxis assay. IL-21 also enhanced NK cell lytic activity against Ab-coated tumor cells. Coadministration of IL-21 and Ab-coated tumor cells to immunocompetent mice led to synergistic production of IFN-gamma by NK cells. Furthermore, the administration of IL-21 augmented the effects of an anti-HER2/neu mAb in a murine tumor model, an effect that required IFN-gamma. These findings demonstrate that IL-21 significantly enhances the NK cell response to Ab-coated targets and suggest that IL-21 would be an effective adjuvant to administer in combination with therapeutic mAbs. PMID:16785506

  4. STAT2 Is Required for TLR-Induced Murine Dendritic Cell Activation and Cross-Presentation.

    PubMed

    Xu, Jun; Lee, Michael H; Chakhtoura, Marita; Green, Benjamin L; Kotredes, Kevin P; Chain, Robert W; Sriram, Uma; Gamero, Ana M; Gallucci, Stefania

    2016-07-01

    TLR-stimulated cross-presentation by conventional dendritic cells (cDCs) is important in host defense and antitumor immunity. We recently reported that cDCs lacking the type I IFN signaling molecule STAT2 are impaired in cross-presenting tumor Ags to CD8(+) T cells. To investigate how STAT2 affects cross-presentation, we determined its requirements for dendritic cell activation. In this study, we report that STAT2 is essential for the activation of murine female cDCs upon TLR3, -4, -7, and -9 stimulation. In response to various TLR ligands, Stat2(-/-) cDCs displayed reduced expression of costimulatory molecules and type I IFN-stimulated genes. The cDC responses to exogenous IFN-α that we evaluated required STAT2 activation, indicating that the canonical STAT1-STAT2 heterodimers are the primary signaling transducers of type I IFNs in cDCs. Interestingly, LPS-induced production of IL-12 was STAT2 and type I IFN receptor (IFNAR) dependent, whereas LPS-induced production of TNF-α and IL-6 was STAT2 and IFNAR independent, suggesting a specific role of the IFNAR-STAT2 axis in the stimulation of proinflammatory cytokines by LPS in cDCs. In contrast, R848- and CpG-induced cytokine production was less influenced by the IFNAR-STAT2 axis. Short kinetics and IFNAR blockade studies showed that STAT2 main function is to transduce signals triggered by autocrine type I IFNs. Importantly, Stat2(-/-) cDCs were deficient in cross-presenting to CD8(+) T cells in vitro upon IFN-α, CpG, and LPS stimulation, and also in cross-priming and licensing cytotoxic T cell killers in vivo. We conclude that STAT2 plays a critical role in TLR-induced dendritic cell activation and cross-presentation, and thus is vital in host defense. PMID:27233962

  5. Low Molecular Weight Antagonists of Plasminogen Activator Inhibitor-1: Therapeutic Potential in Cardiovascular Disease.

    PubMed

    Simone, Tessa M; Higgins, Paul J

    2012-08-01

    Plasminogen activator inhibitor-1 (PAI-1; SERPINE1) is the major physiologic regulator of the plasmin-based pericellular proteolytic cascade, a modulator of vascular smooth muscle cell (VSMC) migration and a causative factor in cardiovascular disease and restenosis, particularly in the context of increased vessel transforming growth factor- β1 (TGF-β1) levels. PAI-1 limits conversion of plasminogen to plasmin (and, thereby, fibrin degradation) by inhibiting its protease targets urokinase and tissue-type plasminogen activators (uPA, tPA). PAI-1 also has signaling functions and binds to the low density lipoprotein receptor-related protein 1 (LRP1) to regulate LRP1-dependent cell motility that, in turn, contributes to neointima formation. PAI-1/uPA/uPA receptor/LRPI/integrin complexes are endocytosed with subsequent uPAR/LRP1/integrin redistribution to the leading edge, initiating an "adhesion-detachment-readhesion" cycle to promote cell migration. PAI-1 also interacts with LRP1 in a uPA/uPAR-independent manner triggering Jak/Stat1 pathway activation to stimulate cell motility. PAI-1 itself is a substrate for extracellular proteases and exists in a "cleaved" form which, while unable to interact with uPA and tPA, retains LRP1-binding and migratory activity. These findings suggest that there are multiple mechanisms through which inhibition of PAI-1 may promote cardiovascular health. Several studies have focused on the design, synthesis and preclinical assessment of PAI-1 antagonists including monoclonal antibodies, peptides and low molecular weight (LMW) antagonists. This review discusses the translational impact of LMW PAI-1 antagonists on cardiovascular disease addressing PAI-1-initiated signaling, PAI-1 structure, the design and characteristics of PAI-1-targeting drugs, results of in vitro and in vivo studies, and their clinical implications. PMID:23936868

  6. Ciliary neurotrophic factor and stress stimuli activate the Jak-STAT pathway in retinal neurons and glia.

    PubMed

    Peterson, W M; Wang, Q; Tzekova, R; Wiegand, S J

    2000-06-01

    Ciliary neurotrophic factor (CNTF) is pleiotrophic for central, peripheral, and sensory neurons. In the mature retina, CNTF treatment enhances survival of retinal ganglion and photoreceptor cells exposed to otherwise lethal perturbation. To understand its mechanism of action in vivo, the adult rat retina was used as a model to investigate CNTF-mediated activation of Janus kinase/signal transducer and activator of transcription (Jak-STAT) and ras-mitogen activated protein kinase (ras-MAPK). Intravitreal injection of Axokine, an analog of CNTF, phosphorylates STAT3 and MAPK and produces delayed upregulation of total STAT3 and STAT1 protein in rat retina. Activated STAT3 is predominantly localized in nuclei of retinal Müller (glial) cells, ganglion cells, and astrocytes, but not in photoreceptors. Although CNTF alpha-receptor (CNTFRalpha) mRNA and protein are localized predominantly if not exclusively in retinal neurons, coincident CNTF-mediated STAT3 signaling was observed in both glia and neurons. CNTF-induced activation of Jak-STAT signaling prompted us to investigate STAT3 phosphorylation after a variety of stress-mediated, conditioning stimuli. We show that STAT3 is activated in the retina after exposure to subtoxic bright light, mechanical trauma, and systemic administration of the alpha(2)-adrenergic agonist xylazine, all of which have been shown previously to condition photoreceptors to resist light-induced degeneration. These results demonstrate that CNTF directly stimulates Jak-STAT and ras-MAPK cascades in vivo and strongly suggest that STAT3 signaling is an underlying component of neural responsiveness to stress stimuli. The observation that CNTF activates STAT3 in ganglion cells, but not in photoreceptors, suggests that Jak-STAT signaling influences neuronal survival via both direct and indirect modes of action. PMID:10818143

  7. Pomalidomide Shows Significant Therapeutic Activity against CNS Lymphoma with a Major Impact on the Tumor Microenvironment in Murine Models

    PubMed Central

    Li, Zhimin; Qiu, Yushi; Personett, David; Huang, Peng; Edenfield, Brandy; Katz, Jason; Babusis, Darius; Tang, Yang; Shirely, Michael A.; Moghaddam, Mehran F.; Copland, John A.; Tun, Han W.

    2013-01-01

    Primary CNS lymphoma carries a poor prognosis. Novel therapeutic agents are urgently needed. Pomalidomide (POM) is a novel immunomodulatory drug with anti-lymphoma activity. CNS pharmacokinetic analysis was performed in rats to assess the CNS penetration of POM. Preclinical evaluation of POM was performed in two murine models to assess its therapeutic activity against CNS lymphoma. The impact of POM on the CNS lymphoma immune microenvironment was evaluated by immunohistochemistry and immunofluorescence. In vitro cell culture experiments were carried out to further investigate the impact of POM on the biology of macrophages. POM crosses the blood brain barrier with CNS penetration of ~ 39%. Preclinical evaluations showed that it had significant therapeutic activity against CNS lymphoma with significant reduction in tumor growth rate and prolongation of survival, that it had a major impact on the tumor microenvironment with an increase in macrophages and natural killer cells, and that it decreased M2-polarized tumor-associated macrophages and increased M1-polarized macrophages when macrophages were evaluated based on polarization status. In vitro studies using various macrophage models showed that POM converted the polarization status of IL4-stimulated macrophages from M2 to M1, that M2 to M1 conversion by POM in the polarization status of lymphoma-associated macrophages is dependent on the presence of NK cells, that POM induced M2 to M1 conversion in the polarization of macrophages by inactivating STAT6 signaling and activating STAT1 signaling, and that POM functionally increased the phagocytic activity of macrophages. Based on our findings, POM is a promising therapeutic agent for CNS lymphoma with excellent CNS penetration, significant preclinical therapeutic activity, and a major impact on the tumor microenvironment. It can induce significant biological changes in tumor-associated macrophages, which likely play a major role in its therapeutic activity against CNS

  8. CD81 controls immunity to Listeria infection through rac-dependent inhibition of proinflammatory mediator release and activation of cytotoxic T cells.

    PubMed

    Martínez del Hoyo, Gloria; Ramírez-Huesca, Marta; Levy, Shoshana; Boucheix, Claude; Rubinstein, Eric; Minguito de la Escalera, María; González-Cintado, Leticia; Ardavín, Carlos; Veiga, Esteban; Yáñez-Mó, María; Sánchez-Madrid, Francisco

    2015-06-15

    Despite recent evidence on the involvement of CD81 in pathogen binding and Ag presentation by dendritic cells (DCs), the molecular mechanism of how CD81 regulates immunity during infection remains to be elucidated. To investigate the role of CD81 in the regulation of defense mechanisms against microbial infections, we have used the Listeria monocytogenes infection model to explore the impact of CD81 deficiency in the innate and adaptive immune response against this pathogenic bacteria. We show that CD81(-/-) mice are less susceptible than wild-type mice to systemic Listeria infection, which correlates with increased numbers of inflammatory monocytes and DCs in CD81(-/-) spleens, the main subsets controlling early bacterial burden. Additionally, our data reveal that CD81 inhibits Rac/STAT-1 activation, leading to a negative regulation of the production of TNF-α and NO by inflammatory DCs and the activation of cytotoxic T cells by splenic CD8α(+) DCs. In conclusion, this study demonstrates that CD81-Rac interaction exerts an important regulatory role on the innate and adaptive immunity against bacterial infection and suggests a role for CD81 in the development of novel therapeutic targets during infectious diseases. PMID:25972472

  9. Control of pathogenic effector T-cell activities in situ by PD-L1 expression on respiratory inflammatory dendritic cells during respiratory syncytial virus infection

    PubMed Central

    Yao, S; Jiang, L; Moser, EK; Jewett, LB; Wright, J; Du, J; Zhou, B; Davis, SD; Krupp, NL; Braciale, TJ; Sun, J

    2015-01-01

    Respiratory syncytial virus (RSV) infection is a leading cause of severe lower respiratory tract illness in young infants, the elderly and immunocompromised individuals. We demonstrate here that the co-inhibitory molecule programmed cell death 1 (PD-1) is selectively upregulated on T cells within the respiratory tract during both murine and human RSV infection. Importantly, the interaction of PD-1 with its ligand PD-L1 is vital to restrict the pro-inflammatory activities of lung effector T cells in situ, thereby inhibiting the development of excessive pulmonary inflammation and injury during RSV infection. We further identify that PD-L1 expression on lung inflammatory dendritic cells is critical to suppress inflammatory T-cell activities, and an interferon–STAT1–IRF1 axis is responsible for increased PD-L1 expression on lung inflammatory dendritic cells. Our findings suggest a potentially critical role of PD-L1 and PD-1 interactions in the lung for controlling host inflammatory responses and disease progression in clinical RSV infection. PMID:25465101

  10. Endothelial Cell Proteomic Response to Rickettsia conorii Infection Reveals Activation of the Janus Kinase (JAK)-Signal Transducer and Activator of Transcription (STAT)-Inferferon Stimulated Gene (ISG)15 Pathway and Reprogramming Plasma Membrane Integrin/Cadherin Signaling.

    PubMed

    Zhao, Yingxin; Valbuena, Gustavo; Walker, David H; Gazi, Michal; Hidalgo, Marylin; DeSousa, Rita; Oteo, Jose Antonio; Goez, Yenny; Brasier, Allan R

    2016-01-01

    Rickettsia conorii is the etiologic agent of Mediterranean spotted fever, a re-emerging infectious disease with significant mortality. This Gram-negative, obligately intracellular pathogen is transmitted via tick bites, resulting in disseminated vascular endothelial cell infection with vascular leakage. In the infected human, Rickettsia conorii infects endothelial cells, stimulating expression of cytokines and pro-coagulant factors. However, the integrated proteomic response of human endothelial cells to R. conorii infection is not known. In this study, we performed quantitative proteomic profiling of primary human umbilical vein endothelial cells (HUVECs) with established R conorii infection versus those stimulated with endotoxin (LPS) alone. We observed differential expression of 55 proteins in HUVEC whole cell lysates. Of these, we observed induction of signal transducer and activator of transcription (STAT)1, MX dynamin-like GTPase (MX1), and ISG15 ubiquitin-like modifier, indicating activation of the JAK-STAT signaling pathway occurs in R. conorii-infected HUVECs. The down-regulated proteins included those involved in the pyrimidine and arginine biosynthetic pathways. A highly specific biotinylated cross-linking enrichment protocol was performed to identify dysregulation of 11 integral plasma membrane proteins that included up-regulated expression of a sodium/potassium transporter and down-regulation of α-actin 1. Analysis of Golgi and soluble Golgi fractions identified up-regulated proteins involved in platelet-endothelial adhesion, phospholipase activity, and IFN activity. Thirty four rickettsial proteins were identified with high confidence in the Golgi, plasma membrane, or secreted protein fractions. The host proteins associated with rickettsial infections indicate activation of interferon-STAT signaling pathways; the disruption of cellular adhesion and alteration of antigen presentation pathways in response to rickettsial infections are distinct from

  11. Preclinical Characterization of Signal Transducer and Activator of Transcription 3 Small Molecule Inhibitors for Primary and Metastatic Brain Cancer Therapy

    PubMed Central

    Assi, Hikmat H.; Paran, Chris; VanderVeen, Nathan; Savakus, Jonathan; Doherty, Robert; Petruzzella, Emanuele; Hoeschele, James D.; Appelman, Henry; Raptis, Leda; Mikkelsen, Tom; Lowenstein, Pedro R.

    2014-01-01

    Signal transducer and activator of transcription 3 (STAT3) has been implicated as a hub for multiple oncogenic pathways. The constitutive activation of STAT3 is present in several cancers, including gliomas (GBMs), and is associated with poor therapeutic responses. Phosphorylation of STAT3 triggers its dimerization and nuclear transport, where it promotes the transcription of genes that stimulate tumor growth. In light of this role, inhibitors of the STAT3 pathway are attractive therapeutic targets for cancer. To this end, we evaluated the STAT3-inhibitory activities of three compounds (CPA-7 [trichloronitritodiammineplatinum(IV)], WP1066 [(S,E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide, C17H14BrN3O], and ML116 [4-benzyl-1-{thieno[2,3-d]pyrimidin-4-yl}piperidine, C18H19N3S]) in cultured rodent and human glioma cells, including GBM cancer stem cells. Our results demonstrate a potent induction of growth arrest in GBM cells after drug treatment with a concomitant induction of cell death. Although these compounds were effective at inhibiting STAT3 phosphorylation, they also displayed variable dose-dependent inhibition of STAT1, STAT5, and nuclear factor κ light-chain enhancer of activated B cells. The therapeutic efficacy of these compounds was further evaluated in peripheral and intracranial mouse tumor models. Whereas CPA-7 elicited regression of peripheral tumors, both melanoma and GBM, its efficacy was not evident when the tumors were implanted within the brain. Our data suggest poor permeability of this compound to tumors located within the central nervous system. WP1066 and ML116 exhibited poor in vivo efficacy. In summary, CPA-7 constitutes a powerful anticancer agent in models of peripheral solid cancers. Our data strongly support further development of CPA-7–derived compounds with increased permeability to enhance their efficacy in primary and metastatic brain tumors. PMID:24696041

  12. Trypanosoma cruzi Evades the Protective Role of Interferon-Gamma-Signaling in Parasite-Infected Cells

    PubMed Central

    Stahl, Philipp; Ruppert, Volker; Schwarz, Ralph T.; Meyer, Thomas

    2014-01-01

    The protozoan parasite Trypanosoma cruzi is responsible for the zoonotic Chagas disease, a chronic and systemic infection in humans and warm-blooded animals typically leading to progressive dilated cardiomyopathy and gastrointestinal manifestations. In the present study, we report that the transcription factor STAT1 (signal transducer and activator of transcription 1) reduces the susceptibility of human cells to infection with T. cruzi. Our in vitro data demonstrate that interferon -γ (IFNγ) pre-treatment causes T. cruzi-infected cells to enter an anti-parasitic state through the activation of the transcription factor STAT1. Whereas stimulation of STAT1-expressing cells with IFNγ significantly impaired intracellular replication of parasites, no protective effect of IFNγ was observed in STAT1-deficient U3A cells. The gene encoding indoleamine 2, 3-dioxygenase (ido) was identified as a STAT1-regulated target gene engaged in parasite clearance. Exposure of cells to T. cruzi trypomastigotes in the absence of IFNγ resulted in both sustained tyrosine and serine phosphorylation of STAT1 and its increased DNA binding. Furthermore, we found that in response to T. cruzi the total amount of intracellular STAT1 increased in an infectious dose-dependent manner, both at the mRNA and protein level. While STAT1 activation is a potent strategy of the host in the fight against the invading pathogen, amastigotes replicating intracellularly antagonize this pathway by specifically promoting the dephosphorylation of STAT1 serine 727, thereby partially circumventing its protective effects. These findings point to the crucial role of the IFNγ/STAT1 signal pathway in the evolutionary combat between T. cruzi parasites and their host. PMID:25340519

  13. Curcumin suppresses Janus kinase-STAT inflammatory signaling through activation of Src homology 2 domain-containing tyrosine phosphatase 2 in brain microglia.

    PubMed

    Kim, Hee Young; Park, Eun Jung; Joe, Eun-Hye; Jou, Ilo

    2003-12-01

    Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that the inhibitory action of curcumin on Janus kinase (JAK)-STAT signaling can contribute to its anti-inflammatory activity in the brain. In both rat primary microglia and murine BV2 microglial cells, curcumin effectively suppressed the ganglioside-, LPS-, or IFN-gamma-stimulated induction of cyclooxygenase-2 and inducible NO synthase, important enzymes that mediate inflammatory processes. These anti-inflammatory effects appear to be due, at least in part, to the suppression of the JAK-STAT inflammatory signaling cascade. Curcumin markedly inhibited the phosphorylation of STAT1 and 3 as well as JAK1 and 2 in microglia activated with gangliosides, LPS, or IFN-gamma. Curcumin consistently suppressed not only NF binding to IFN-gamma-activated sequence/IFN-stimulated regulatory element, but also the expression of inflammation-associated genes, including ICAM-1 and monocyte chemoattractant protein 1, whose promoters contain STAT-binding elements. We further show that activation of Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-2, a negative regulator of JAK activity, is likely to be one of the mechanisms underlying the curcumin-mediated inhibition of JAK-STAT signaling. Treatment of microglial cells with curcumin led to an increase in phosphorylation and association with JAK1/2 of SHP-2, which inhibit the initiation of JAK-STAT inflammatory signaling in activated microglia. Taken together, these data suggest curcumin suppresses JAK-STAT signaling via activation of SHP-2, thus attenuating inflammatory response of brain microglial cells. PMID:14634121

  14. Protein-DNA array-based identification of transcription factor activities differentially regulated in skeletal muscle of normal and dystrophin-deficient mdx mice.

    PubMed

    Dogra, Charu; Srivastava, Daya Shankar; Kumar, Ashok

    2008-05-01

    Inactivation of dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and mdx mice. However, the underpinning mechanisms, which govern the pathogenesis of dystrophin-deficient skeletal muscle, remain poorly understood. We have previously reported activation of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-kappaB), and phosphatidyl-inositol 3-kinase/Akt (PI3K/Akt) signaling pathways in diaphragm muscle of mdx mice. In this study, using a protein-DNA array-based approach, we have investigated the activation of 345 transcription factors in diaphragm muscle of 6-week old normal and dystrophin-deficient mdx mice. Our data demonstrate increased activation of a number nuclear transcription factors including AP1, HFH-3, PPARalpha, c.myb BP, ETF, Fra-1/JUN, kBF-A, N-rasBP, lactoferrin BP, Myb(2), EBP40_45, EKLF(1), p53(2), TFEB, Myc-Max; c-Rel; E2, ISRE; NF-kB; Stat1 p84/p91, Antioxidant RE, EVI-1, Stat3, AP3, p53, Stat4, AP4, HFH-1, FAST-1, Pax-5, and Beta-RE in the diaphragm muscle of mdx mice compared to corresponding normal mice. The level of activation for p53 was highest among all the transcription factors studied. Furthermore, higher activation of p53 in diaphragm muscle of mdx mice was associated with its increased phosphorylation and nuclear translocation. Collectively, our data suggest that the primary deficiency of dystrophin leads to the aberrant activation of nuclear transcription factors which might further contribute to muscle pathogenesis in mdx mice. PMID:18278580

  15. Active-R filter

    DOEpatents

    Soderstrand, Michael A.

    1976-01-01

    An operational amplifier-type active filter in which the only capacitor in the circuit is the compensating capacitance of the operational amplifiers, the various feedback and coupling elements being essentially solely resistive.

  16. Signal transducer and activator of transcription 3 (STAT3) and survivin induction by varicella-zoster virus promote replication and skin pathogenesis.

    PubMed

    Sen, Nandini; Che, Xibing; Rajamani, Jaya; Zerboni, Leigh; Sung, Phillip; Ptacek, Jason; Arvin, Ann M

    2012-01-10

    Varicella-zoster virus (VZV) is a human α-herpesvirus that causes varicella (chickenpox) during primary infection and zoster (shingles) upon reactivation. Like other viruses, VZV must subvert the intrinsic antiviral defenses of differentiated human cells to produce progeny virions. Accordingly, VZV inhibits the activation of the cellular transcription factors IFN regulatory factor 3 (IRF3) and signal transducers and activators of transcription 1 (STAT1), thereby downregulating antiviral factors, including IFNs. Conversely, in this study, we found that VZV triggers STAT3 phosphorylation in cells infected in vitro and in human skin xenografts in SCID mice in vivo and that STAT3 activation induces the anti-apoptotic protein survivin. Small-molecule inhibitors of STAT3 phosphorylation and survivin restrict VZV replication in vitro, and VZV infection of skin xenografts in vivo is markedly impaired by the administration of the phospho-STAT3 inhibitor S3I-201. STAT3 and survivin are required for malignant transformation caused by γ-herpesviruses, such as Kaposi's sarcoma virus. We show that STAT3 activation is also critical for VZV, a nononcogenic herpesvirus, via a survivin-dependent mechanism. Furthermore, STAT3 activation is critical for the life cycle of the virus because VZV skin infection is necessary for viral transmission and persistence in the human population. Therefore, we conclude that takeover of this major cell-signaling pathway is necessary, independent of cell transformation, for herpesvirus pathogenesis and that STAT3 activation and up-regulation of survivin is a common mechanism important for the pathogenesis of lytic as well as tumorigenic herpesviruses. PMID:22190485

  17. The transcriptional PPARβ/δ network in human macrophages defines a unique agonist-induced activation state

    PubMed Central

    Adhikary, Till; Wortmann, Annika; Schumann, Tim; Finkernagel, Florian; Lieber, Sonja; Roth, Katrin; Toth, Philipp M.; Diederich, Wibke E.; Nist, Andrea; Stiewe, Thorsten; Kleinesudeik, Lara; Reinartz, Silke; Müller-Brüsselbach, Sabine; Müller, Rolf

    2015-01-01

    Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a lipid ligand-inducible transcription factor with established metabolic functions, whereas its anti-inflammatory function is poorly understood. To address this issue, we determined the global PPARβ/δ-regulated signaling network in human monocyte-derived macrophages. Besides cell type-independent, canonical target genes with metabolic and immune regulatory functions we identified a large number of inflammation-associated NFκB and STAT1 target genes that are repressed by agonists. Accordingly, PPARβ/δ agonists inhibited the expression of multiple pro-inflammatory mediators and induced an anti-inflammatory, IL-4-like morphological phenotype. Surprisingly, bioinformatic analyses also identified immune stimulatory effects. Consistent with this prediction, PPARβ/δ agonists enhanced macrophage survival under hypoxic stress and stimulated CD8+ T cell activation, concomitantly with the repression of immune suppressive target genes and their encoded products CD274 (PD-1 ligand), CD32B (inhibitory Fcγ receptor IIB) and indoleamine 2,3-dioxygenase 1 (IDO-1), as well as a diminished release of the immune suppressive IDO-1 metabolite kynurenine. Comparison with published data revealed a significant overlap of the PPARβ/δ transcriptome with coexpression modules characteristic of both anti-inflammatory and pro-inflammatory cytokines. Our findings indicate that PPARβ/δ agonists induce a unique macrophage activation state with strong anti-inflammatory but also specific immune stimulatory components, pointing to a context-dependent function of PPARβ/δ in immune regulation. PMID:25934804

  18. Systemic Activation of TLR3-Dependent TRIF Signaling Confers Host Defense against Gram-Negative Bacteria in the Intestine

    PubMed Central

    Ruiz, Jose; Kanagavelu, Saravana; Flores, Claudia; Romero, Laura; Riveron, Reldy; Shih, David Q.; Fukata, Masayuki

    2016-01-01

    Recognition of Gram-negative bacteria by toll-like receptor (TLR)4 induces MyD88 and TRIF mediated responses. We have shown that TRIF-dependent responses play an important role in intestinal defense against Gram-negative enteropathogens. In the current study, we examined underlying mechanisms of how systemic TRIF activation enhances intestinal immune defense against Gram-negative bacteria. First we confirmed that the protective effect of poly I:C against enteric infection of mice with Yersinia enterocolitica was dependent on TLR3-mediated TRIF signaling by using TLR3-deficient mice. This protection was unique in TRIF-dependent TLR signaling because systemic stimulation of mice with agonists for TLR2 (Pam3CSK4) or TLR5 (flagellin) did not reduce mortality on Y. enterocolitica infection. Systemic administration of poly I:C mobilized CD11c+, F4/80+, and Gr−1hi cells from lamina propria and activated NK cells in the mesenteric lymph nodes (MLN) within 24 h. This innate immune cell rearrangement was type I IFN dependent and mediated through upregulation of TLR4 followed by CCR7 expression in these innate immune cells found in the intestinal mucosa. Poly I:C induced IFN-γ expression by NK cells in the MLN, which was mediated through type I IFNs and IL-12p40 from antigen presenting cells and consequent activation of STAT1 and STAT4 in NK cells. This formation of innate immunity significantly contributed to the elimination of bacteria in the MLN. Our results demonstrated an innate immune network in the intestine that can be established by systemic stimulation of TRIF, which provides a strong host defense against Gram-negative pathogens. The mechanism underlying TRIF-mediated protective immunity may be useful to develop novel therapies for enteric bacterial infection. PMID:26793623

  19. Vorinostat Modulates the Imbalance of T Cell Subsets, Suppresses Macrophage Activity, and Ameliorates Experimental Autoimmune Uveoretinitis.

    PubMed

    Fang, Sijie; Meng, Xiangda; Zhang, Zhuhong; Wang, Yang; Liu, Yuanyuan; You, Caiyun; Yan, Hua

    2016-03-01

    The purpose of the study was to investigate the anti-inflammatory efficiency of vorinostat, a histone deacetylase inhibitor, in experimental autoimmune uveitis (EAU). EAU was induced in female C57BL/6J mice immunized with interphotoreceptor retinoid-binding protein peptide. Vorinostat or the control treatment, phosphate-buffered saline, was administrated orally from 3 days before immunization until euthanasia at day 21 after immunization. The clinical and histopathological scores of mice were graded, and the integrity of the blood-retinal barrier was examined by Evans blue staining. T helper cell subsets were measured by flow cytometry, and the macrophage functions were evaluated with immunohistochemistry staining and immunofluorescence assays. The mRNA levels of tight junction proteins were measured by qRT-PCR. The expression levels of intraocular cytokines and transcription factors were examined by western blotting. Vorinostat relieved both clinical and histopathological manifestations of EAU in our mouse model, and the BRB integrity was maintained in vorinostat-treated mice, which had less vasculature leakage and higher mRNA and protein expressions of tight junction proteins than controls. Moreover, vorinostat repressed Th1 and Th17 cells and increased Th0 and Treg cells. Additionally, the INF-γ and IL-17A expression levels were significantly decreased, while the IL-10 level was increased by vorinostat treatment. Furthermore, due to the reduced TNF-α level, the macrophage activity was considerably inhibited in EAU mice. Finally, transcription factors, including STAT1, STAT3, and p65, were greatly suppressed by vorinostat treatment. Our data suggest that vorinostat might be a potential anti-inflammatory agent in the management of uveitis and other autoimmune inflammatory diseases. PMID:26798022

  20. Prep1 (pKnox1) regulates mouse embryonic HSC cycling and self-renewal affecting the Stat1-Sca1 IFN-dependent pathway.

    PubMed

    Modica, Livia; Iotti, Giorgio; D'Avola, Annalisa; Blasi, Francesco

    2014-01-01

    A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs. PMID:25233378

  1. Prep1 (pKnox1) Regulates Mouse Embryonic HSC Cycling and Self-Renewal Affecting the Stat1-Sca1 IFN-Dependent Pathway

    PubMed Central

    Modica, Livia; Iotti, Giorgio; D’Avola, Annalisa; Blasi, Francesco

    2014-01-01

    A hypomorphic Prep1 mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to de novo generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs. PMID:25233378

  2. Physical activity

    MedlinePlus

    ... this page: //medlineplus.gov/ency/article/001941.htm Physical activity To use the sharing features on this page, please enable JavaScript. Physical activity -- which includes an active lifestyle and routine exercise -- ...

  3. Expression of microRNA-34a in Alzheimer's disease brain targets genes linked to synaptic plasticity, energy metabolism, and resting state network activity.

    PubMed

    Sarkar, S; Jun, S; Rellick, S; Quintana, D D; Cavendish, J Z; Simpkins, J W

    2016-09-01

    Polygenetic risk factors and reduced expression of many genes in late-onset Alzheimer's disease (AD) impedes identification of a target(s) for disease-modifying therapies. We identified a single microRNA, miR-34a that is over expressed in specific brain regions of AD patients as well as in the 3xTg-AD mouse model. Specifically, increased miR-34a expression in the temporal cortex region compared to age matched healthy control correlates with severity of AD pathology. miR-34a over expression in patient's tissue and forced expression in primary neuronal culture correlates with concurrent repression of its target genes involved in synaptic plasticity, oxidative phosphorylation and glycolysis. The repression of oxidative phosphorylation and glycolysis related proteins correlates with reduced ATP production and glycolytic capacity, respectively. We also found that miR-34a overexpressed neurons secrete miR-34a containing exosomes that are taken up by neighboring neurons. Furthermore, miR-34a targets dozens of genes whose expressions are known to be correlated with synchronous activity in resting state functional networks. Our analysis of human genomic sequences from the tentative promoter of miR-34a gene shows the presence of NFκB, STAT1, c-Fos, CREB and p53 response elements. Together, our results raise the possibilities that pathophysiology-induced activation of specific transcription factor may lead to increased expression of miR-34a gene and miR-34a mediated concurrent repression of its target genes in neural networks may result in dysfunction of synaptic plasticity, energy metabolism, and resting state network activity. Thus, our results provide insights into polygenetic AD mechanisms and disclose miR-34a as a potential therapeutic target for AD. PMID:27235866

  4. In vitro and in vivo downregulation of C3 by lipoteichoic acid isolated from Lactobacillus plantarum K8 suppressed cytokine-mediated complement system activation.

    PubMed

    Jeon, Boram; Kim, Hye Rim; Kim, Hangeun; Chung, Dae Kyun

    2016-07-01

    Complement component 3 (C3) is one of the proteins associated with complement cascades. C3 plays an essential role in three different pathways-the alternative, classical and lectin pathways. It is well known that cytokines activate complement system and increase complement component C3 production. In the current study, we found that lipoteichoic acid isolated from Lactobacillus plantarum K8 (pLTA) inhibited tumor necrosis factor-alpha (TNF-α) or interferon-gamma (IFN-γ)-mediated C3 mRNA and protein expression in HaCaT cells. pLTA inhibited C3 expression through the inhibition of the phosphorylation of p65 and p38 in the TNF-α-treated cells, while the inhibition of STAT1/2 and JAK2 phosphorylation by pLTA contributed to the reduction of C3 in IFN-γ-treated cells. When mice were pre-injected with pLTA followed by re-injection of TNF-α, serum C3 level was decreased as compared to TNF-α-injected only. Further studies revealed that membrane attack complex (MAC) increased by TNF-α injection was lessened in pLTA-pre-injected mice. A bactericidal assay using mouse sera showed that MAC activity in pLTA-pre-injected mice was lower than in TNF-α only-injected mice. These results suggest that pLTA can suppress inflammatory cytokine-mediated complement activation through the inhibition of C3 synthesis. pLTA application has the potential to alleviate complement-mediated diseases caused by excessive inflammation. PMID:27231239

  5. Activation detector

    DOEpatents

    Bell, Zane William [Oak Ridge, TN; Boatner, Lynn Allen [Oak Ridge, TN

    2009-12-08

    A method of detecting an activator, the method including impinging with an activator a receptor material lacking a photoluminescent material and generating a by-product of a radioactive decay due to the activator impinging the reeptor material. The method further including, generating light from the by-product via the Cherenkov effect and identifying a characteristic of the activator based on the light.

  6. Cobalt Protoporphyrin Upregulates Cyclooxygenase-2 Expression Through a Heme Oxygenase-Independent Mechanism.

    PubMed

    Lin, Hsiao-Yun; Tsai, Chon-Haw; Lin, Chingju; Yeh, Wei-Lan; Tsai, Cheng-Fang; Chang, Pei-Chun; Wu, Ling-Hsuan; Lu, Dah-Yuu

    2016-09-01

    Cobalt protoporphyrin (CoPP) is a potent HO-1 inducer and generally known to be an antioxidant in various cell types. Little is known about the CoPP-induced cyclooxygenase-2 (COX-2) expression and its downstream signaling in microglial cells. In current study, CoPP caused concentration- and time-dependent increases in COX-2 expression in microglial cells. Furthermore, activation of apoptosis signal-regulating kinase (ASK) 1/MAP kinase involved in CoPP-induced COX-2 expression in microglia. CoPP also induced P2X7 receptor activation, and treatment of P2X7 inhibitors effectively reduced CoPP-induced COX-2 expression. Protein inhibitor of activated STAT (PIAS) 1 is reported to be involved in modulating anti-inflammatory response through negative regulation of transcription factors. Interestingly, treatment with CoPP markedly induced PIAS1 degradation which is regulated by PI3K, Akt, and glycogen synthase kinase 3α/β (GSK3α/β) signaling pathways. These results suggest that CoPP induces COX-2 expression through activating P2X7 receptors and ASK1/MAP kinases as well as PIAS1 degradation signaling pathways. Our study provides a new insight into the regulatory effect of CoPP on neuroinflammation in microglial cells. PMID:26255181

  7. Active turbulence in active nematics

    NASA Astrophysics Data System (ADS)

    Thampi, S. P.; Yeomans, J. M.

    2016-07-01

    Dense, active systems show active turbulence, a state characterised by flow fields that are chaotic, with continually changing velocity jets and swirls. Here we review our current understanding of active turbulence. The development is primarily based on the theory and simulations of active liquid crystals, but with accompanying summaries of related literature.

  8. Unabated Adenovirus Replication following Activation of the cGAS/STING-Dependent Antiviral Response in Human Cells

    PubMed Central

    Lam, Eric

    2014-01-01

    ABSTRACT The cGAS/STING DNA sensing complex has recently been established as a predominant pathogen recognition receptor (PRR) for DNA-directed type I interferon (IFN) innate immune activation. Using replication-defective adenovirus vectors and replication-competent wild-type adenovirus, we have modeled the influence of the cGAS/STING cascade in permissive human cell lines (A549, HeLa, ARPE19, and THP1). Wild-type adenovirus induced efficient early activation of the cGAS/STING cascade in a cell-specific manner. In all responsive cell lines, cGAS/STING short hairpin RNA (shRNA) knockdown resulted in a loss of TBK1 and interferon response factor 3 (IRF3) activation, a lack of beta interferon transcript induction, loss of interferon-dependent STAT1 activation, and diminished induction of interferon-stimulated genes (ISGs). Adenoviruses that infect through the coxsackievirus-adenovirus receptor (CAR) (Ad2 and Ad5) and the CD46 (Ad35) and desmoglein-2 (Ad7) viral receptors all induce the cGAS/STING/TBK1/IRF3 cascade. The magnitude of the IRF3/IFN/ISG antiviral response was strongly influenced by serotype, with Ad35>Ad7>Ad2. For each serotype, no enhancement of viral DNA replication or virus production occurred in cGAS or STING shRNA-targeted cell line pools. We found no replication advantage in permissive cell lines that do not trigger the cGAS/STING cascade following infection. The cGAS/STING/TBK1/IRF3 cascade was not a direct target of viral antihost strategies, and we found no evidence that Ad stimulation of the cGAS/STING DNA response had an impact on viral replication efficiency. IMPORTANCE This study shows for the first time that the cGAS DNA sensor directs a dominant IRF3/IFN/ISG antiviral response to adenovirus in human cell lines. Activation of cGAS occurs with viruses that infect through different high-affinity receptors (CAR, CD46, and desmoglein-2), and the magnitude of the cGAS/STING DNA response cascade is influenced by serotype-specific functions

  9. Early Changes in Interferon Signaling Define Natural Killer Cell Response and Refractoriness To Interferon-based Therapy of Hepatitis C

    PubMed Central

    Edlich, Birgit; Ahlenstiel, Golo; Azpiroz, Aintzane Zabaleta; Stoltzfus, Jonathan; Noureddin, Mazen; Serti, Elisavet; Feld, Jordan J.; Liang, T. Jake; Rotman, Yaron; Rehermann, Barbara

    2012-01-01

    Natural killer (NK) cells exhibit a polarized phenotype with increased cytotoxicity and decreased IFN- γ production in chronic hepatitis C virus (HCV) infection. Here we asked whether this is due to type I interferon (IFN)-induced expression and phosphorylation levels of signal transducer and activator of transcription (STAT) molecules in NK cells and whether it affects the response and refractoriness of NK cells to IFN-α-based therapy of hepatitis C. STAT1 levels in NK cells were significantly higher in patients with chronic HCV infection than in uninfected controls. STAT1 levels and induction of phosphorylated STAT1 (pSTAT1) increased further during IFN-α-based therapy with preferential STAT1 over STAT4 phosphorylation. Induction of pSTAT1 correlated with increased NK cytotoxicity (TRAIL expression and degranulation) and decreased IFN-γ production. NK cells from patients with a greater than 2 log10 first phase HCV RNA decline to IFN-α-based therapy (>99% IFN effectiveness) displayed strong pSTAT1 induction in vivo and were refractory to further stimulation in vitro. In contrast, NK cells from patients with a less than 2 log10 first phase HCV RNA decline exhibited lower pSTAT1 induction in vivo (p=0.024) but retained greater IFN-α responsiveness in vitro (p=0.024). NK cells of all patients became refractory to in vivo and in vitro stimulation by IFN-α during the second phase virological response. Conclusion These data show that IFN-α-induced modulation of STAT1/4 phosphorylation underlies the polarization of NK cells towards increased cytotoxicity and decreased IFN-γ production in HCV infection, and that NK cell responsiveness and refractoriness correlate to the antiviral effectiveness of IFN-α-based therapy. PMID:21898483

  10. Physical activity

    MedlinePlus

    ... time they spend watching TV and using a computer and other electronic devices. All of these activities ... U.S. Department of Health and Human Services. Physical Activity Guidelines for Americans: Recommendation ... Page last updated: ...

  11. Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

    PubMed Central

    Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R.

    2014-01-01

    Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and

  12. Transfected Poly(I:C) Activates Different dsRNA Receptors, Leading to Apoptosis or Immunoadjuvant Response in Androgen-independent Prostate Cancer Cells*

    PubMed Central

    Palchetti, Sara; Starace, Donatella; De Cesaris, Paola; Filippini, Antonio; Ziparo, Elio; Riccioli, Anna

    2015-01-01

    Despite the effectiveness of surgery or radiation therapy for the treatment of early-stage prostate cancer (PCa), there is currently no effective strategy for late-stage disease. New therapeutic targets are emerging; in particular, dsRNA receptors Toll-like receptor 3 (TLR3) and cytosolic helicases expressed by cancer cells, once activated, exert a pro-apoptotic effect in different tumors. We previously demonstrated that the synthetic analog of dsRNA poly(I:C) induces apoptosis in the androgen-dependent PCa cell line LNCaP in a TLR3-dependent fashion, whereas only a weak apoptotic effect is observed in the more aggressive and androgen-independent PCa cells PC3 and DU145. In this paper, we characterize the receptors and the signaling pathways involved in the remarkable apoptosis induced by poly(I:C) transfected by Lipofectamine (in-poly(I:C)) compared with the 12-fold higher free poly(I:C) concentration in PC3 and DU145 cells. By using genetic inhibition of different poly(I:C) receptors, we demonstrate the crucial role of TLR3 and Src in in-poly(I:C)-induced apoptosis. Therefore, we show that the increased in-poly(I:C) apoptotic efficacy is due to a higher binding of endosomal TLR3. On the other hand, we show that in-poly(I:C) binding to cytosolic receptors MDA5 and RIG-I triggers IRF3-mediated signaling, leading uniquely to the up-regulation of IFN-β, which likely in turn induces increased TLR3, MDA5, and RIG-I proteins. In summary, in-poly(I:C) activates two distinct antitumor pathways in PC3 and DU145 cells: one mediated by the TLR3/Src/STAT1 axis, leading to apoptosis, and the other one mediated by MDA5/RIG-I/IRF3, leading to immunoadjuvant IFN-β expression. PMID:25568326

  13. Activity Scale.

    ERIC Educational Resources Information Center

    Kerpelman, Larry C.; Weiner, Michael J.

    This twenty-four item scale assesses students' actual and desired political-social activism in terms of physical participation, communication activities, and information-gathering activities. About ten minutes are required to complete the instrument. The scale is divided into two subscales. The first twelve items (ACT-A) question respondents on…

  14. Active ratchets

    NASA Astrophysics Data System (ADS)

    Angelani, L.; Costanzo, A.; Di Leonardo, R.

    2011-12-01

    We analyze self-propelling organisms, or active particles, in a periodic asymmetric potential. Unlike standard ratchet effect for Brownian particles requiring external forcing, in the case of active particles asymmetric potential alone produces a net drift speed (active ratchet effect). By using theoretical models and numerical simulations we demonstrate the emergence of the rectification process in the presence of an asymmetric piecewise periodic potential. The broken spatial symmetry (external potential) and time symmetry (active particles) are sufficient ingredients to sustain unidirectional transport. Our findings open the way to new mechanisms to move in directional manner motile organisms by using external periodic static fields.

  15. EGF-receptor phosphorylation and downstream signaling are activated by benzo[a]pyrene 3,6-quinone and benzo[a]pyrene 1,6-quinone in human mammary epithelial cells

    SciTech Connect

    Rodriguez-Fragoso, Lourdes; Melendez, Karla; Hudson, Laurie G.; Lauer, Fredine T.; Burchiel, Scott W.

    2009-03-15

    Benzo[a]pyrene (BaP) is activated by xenobiotic-metabolizing enzymes to highly mutagenic and carcinogenic metabolites. Previous studies in this laboratory have shown that benzo[a]pyrene quinones (BPQs), 1,6-BPQ and 3,6-BPQ, are able to induce epidermal growth factor receptor (EGFR) cell signaling through the production of reactive oxygen species. Recently, we have reported that BPQs have the potential to induce the expression of genes involved in numerous pathways associated with cell proliferation and survival in human mammary epithelial cells. In the present study we demonstrated that BPQs not only induced EGFR tyrosine autophosphorylation, but also induced EGFR-dependent tyrosine phosphorylation of phospholipase C-{gamma}1 and several signal transducers and activators of transcription (STATs). The effects of BPQs were evaluated in a model of EGF withdrawal in MCF10-A cells. We found that BPQs (1 {mu}M), induced EGFR tyrosine phosphorylation at positions Y845, Y992, Y1068, and Y1086. PLC-{gamma}1 phosphorylation correlated with the phosphorylation of tyrosine-Y992, a proposed docking site for PLC-{gamma}1 on the EGFR. Additionally, we found that BPQs induced the activation of STAT-1, STAT-3, STAT-5a and STAT-5b. STAT5 was shown to translocate to the nucleus following 3,6-BPQ and 1,6-BPQ exposures. Although the patterns of phosphorylation at EGFR, PLC-{gamma}1 and STATs were quite similar to those induced by EGF, an important difference between BPQ-mediated signaling of the EGFR was observed. Signaling produced by EGF ligand produced a rapid disappearance of EGFR from the cell surface, whereas BPQ signaling maintained EGFR receptors on the cell membrane. Thus, the results of these studies show that 1,6-BPQ and 3,6-BPQ can produce early events as evidenced by EGFR expression, and a prolonged transactivation of EGFR leading to downstream cell signaling pathways.

  16. Faculty Activism

    ERIC Educational Resources Information Center

    Academe, 2005

    2005-01-01

    Blending scholarship and activism, whether domestic or international, takes some real work. Two scholar-activists reflect on why and how activism can be more than academic labor in this feature of the "Academe" journal. This feature includes the following brief reflections on political work, both local and global that demonstrates how on campus…

  17. Indoor Activities

    MedlinePlus

    ... so you can do some lifting while you watch TV. Walk around the house when you talk on the phone. Make an extra trip up and down the stairs when you do the laundry. Download the Tip Sheet Indoor Activities (PDF, 739.53 KB) You Might Also Like Sun Safety Have Fun. Be Active with Your Dog! ...

  18. Catalyst activator

    DOEpatents

    McAdon, Mark H.; Nickias, Peter N.; Marks, Tobin J.; Schwartz, David J.

    2001-01-01

    A catalyst activator particularly adapted for use in the activation of metal complexes of metals of Group 3-10 for polymerization of ethylenically unsaturated polymerizable monomers, especially olefins, comprising two Group 13 metal or metalloid atoms and a ligand structure including at least one bridging group connecting ligands on the two Group 13 metal or metalloid atoms.

  19. Outdoor Activities.

    ERIC Educational Resources Information Center

    Minneapolis Independent School District 275, Minn.

    Twenty-four activities suitable for outdoor use by elementary school children are outlined. Activities designed to make children aware of their environment include soil painting, burr collecting, insect and pond water collecting, studies of insect galls and field mice, succession studies, and a model of natural selection using dyed toothpicks. A…

  20. Astronomy Activities.

    ERIC Educational Resources Information Center

    Greenstone, Sid

    This document consists of activities and references for teaching astronomy. The activities (which include objectives, list of materials needed, and procedures) focus on: observing the Big Dipper and locating the North Star; examining the Big Dipper's stars; making and using an astrolabe; examining retograde motion of Mars; measuring the Sun's…

  1. Activated Charcoal

    MedlinePlus

    ... ACTIVATED CHARCOAL are as follows:Trapping chemicals to stop some types of poisoning when used as a ... Charbon Végétal, Charbon Végétal Activé, Charcoal, Gas Black, Lamp Black, Medicinal Charcoal, Noir de Gaz, Noir de ...

  2. Role for herpes simplex virus 1 ICP27 in the inhibition of type I interferon signaling

    SciTech Connect

    Johnson, Karen E.; Song, Byeongwoon; Knipe, David M.

    2008-05-10

    Host cells respond to viral infection by many mechanisms, including the production of type I interferons which act in a paracrine and autocrine manner to induce the expression of antiviral interferon-stimulated genes (ISGs). Viruses have evolved means to inhibit interferon signaling to avoid induction of the innate immune response. Herpes simplex virus 1 (HSV-1) has several mechanisms to inhibit type I interferon production, the activities of ISGs, and the interferon signaling pathway itself. We report that the inhibition of the Jak/STAT pathway by HSV-1 requires viral gene expression and that viral immediate-early protein ICP27 plays a role in downregulating STAT-1 phosphorylation and in preventing the accumulation of STAT-1 in the nucleus. We also show that expression of ICP27 by transfection causes an inhibition of IFN-induced STAT-1 nuclear accumulation. Therefore, ICP27 is necessary and sufficient for at least some of the effects of HSV infection on STAT-1.

  3. Activated Charcoal

    MedlinePlus

    ... is used to treat poisonings, reduce intestinal gas (flatulence), lower cholesterol levels, prevent hangover, and treat bile ... lower cholesterol levels in the blood. Decreasing gas (flatulence). Some studies show that activated charcoal is effective ...

  4. Activation analysis

    SciTech Connect

    Alfassi, Z.B. . Dept. of Nuclear Engineering)

    1990-01-01

    This volume contains 16 chapters on the application of activation analysis in the fields of life sciences, biological materials, coal and its effluents, environmental samples, archaeology, material science, and forensics. Each chapter is processed separately for the data base.

  5. Get Active

    MedlinePlus

    ... section Health Conditions 4 of 10 sections Take Action! Take Action: How Active Are You? First, think about your ... section Learn More 5 of 10 sections Take Action: Get Started I’m just getting started. Start ...

  6. PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

    PubMed Central

    Schildbach, Stefan; Blumert, Conny; Horn, Friedemann; von Bergen, Martin; Labudde, Dirk

    2016-01-01

    The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6. PMID:26966684

  7. Integrin activation

    PubMed Central

    Ginsberg, Mark H.

    2014-01-01

    Integrin-mediated cell adhesion is important for development, immune responses, hemostasis and wound healing. Integrins also function as signal transducing receptors that can control intracellular pathways that regulate cell survival, proliferation, and cell fate. Conversely, cells can modulate the affinity of integrins for their ligands a process operationally defined as integrin activation. Analysis of activation of integrins has now provided a detailed molecular understanding of this unique form of “inside-out” signal transduction and revealed new paradigms of how transmembrane domains (TMD) can transmit long range allosteric changes in transmembrane proteins. Here, we will review how talin and mediates integrin activation and how the integrin TMD can transmit these inside out signals. [BMB Reports 2014; 47(12): 655-659] PMID:25388208

  8. Active Cytokinins

    PubMed Central

    Mornet, René; Theiler, Jane B.; Leonard, Nelson J.; Schmitz, Ruth Y.; Moore, F. Hardy; Skoog, Folke

    1979-01-01

    Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N6-benzyladenines, -N6-(Δ2-isopentenyl)adenines, and -zeatins, and N6-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10−5 micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10−6 micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10−5 micromolar, representing a sensitivity rarely attained. All of the azido compounds have been submitted to photolysis in aqueous ethanol, and the photoproducts have been detected and identified by low and high resolution mass spectrometry. They are rationalized as products of abstraction and insertion reactions of the intermediate nitrenes. The potential of the major released products as cytokinins was also assessed by bioassay. 2-Azido-N6-(Δ2-isopentenyl)adenine competed with [14C]kinetin for the cytokinin-binding protein isolated from wheat germ. When the azido compound was photolysed in the presence of this protein, its attachment effectively blocked the binding of [14C]kinetin. PMID:16661017

  9. Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

    SciTech Connect

    Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro

    2011-05-25

    Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression of C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-{gamma} signaling pathway via inhibition of phosphorylated STAT1 dimerization.

  10. Get Active

    MedlinePlus

    ... Lifting small weights – you can even use bottled water or cans of food as weights Watch these videos for muscle strengthening exercises to do at home or at the gym. If you do muscle-strengthening activities with weights, check out the do’s and don’ ...

  11. Learning Activities.

    ERIC Educational Resources Information Center

    Tipton, Tom, Ed.

    1983-01-01

    Presents a flow chart for naming inorganic compounds. Although it is not necessary for students to memorize rules, preliminary skills needed before using the chart are outlined. Also presents an activity in which the mass of an imaginary atom is determined using lead shot, Petri dishes, and a platform balance. (JN)

  12. Activated Sludge.

    ERIC Educational Resources Information Center

    Saunders, F. Michael

    1978-01-01

    Presents the 1978 literature review of wastewater treatment. This review covers: (1) activated sludge process; (2) process control; (3) oxygen uptake and transfer; (4) phosphorus removal; (5) nitrification; (6) industrial wastewater; and (7) aerobic digestion. A list of 136 references is also presented. (HM)

  13. Activity report

    SciTech Connect

    Yu, S W

    2008-08-11

    This report is aimed to show the author's activities to support the LDRD. The title is 'Investigation of the Double-C Behavior in the Pu-Ga Time-Temperature-Transformation Diagram' The sections are: (1) Sample Holder Test; (2) Calculation of x-ray diffraction patterns; (3) Literature search and preparing publications; (4) Tasks Required for APS Experiments; and (5) Communications.

  14. Laboratory Activities

    SciTech Connect

    Brown, Christopher F.; Serne, R. Jeffrey

    2008-01-17

    This chapter summarizes the laboratory activities performed by PNNL’s Vadose Zone Characterization Project in support of the Tank Farm Vadose Zone Program, led by CH2M HILL Hanford Group, Inc. The results of these studies are contained in numerous reports (Lindenmeier et al. 2002; Serne et al. 2002a, 2002b, 2002c, 2002d, 2002e; Lindenmeier et al. 2003; Serne et al. 2004a, 2004b; Brown et al. 2005, 2006a, 2007; Serne et al. 2007) and have generated much of the data reported in Chapter 22 (Geochemistry-Contaminant Movement), Appendix G (Geochemistry-Contaminant Movement), and Cantrell et al. (2007, SST WMA Geochemistry Data Package – in preparation). Sediment samples and characterization results from PNNL’s Vadose Zone Characterization Project are also shared with other science and technology (S&T) research projects, such as those summarized in Chapter 12 (Associated Science Activities).

  15. Hepatoprotective Activity.

    PubMed

    2016-01-01

    The liver performs a vital role in metabolism, secretion, storage, and detoxification of endogenous and exogenous substances. Oxidative stress and free radicals enhance the severity of hepatic damage, which can be overcome by the antioxidant mechanism. Plant extracts can be the best source of such antioxidants and mediate hepatoprotective activity. In this chapter, high-dose paracetamol-induced hepatotoxicity in rat model is discussed with explanations of biochemical and histopathological studies. PMID:26939279

  16. Analgesic Activity.

    PubMed

    2016-01-01

    Analgesics are agents which selectively relieve pain by acting in the CNS and peripheral pain mediators without changing consciousness. Analgesics may be narcotic or non-narcotic. The study of pain in animals raises ethical, philosophical, and technical problems. Both peripheral and central pain models are included to make the test more evident for the analgesic property of the plant. This chapter highlights methods such as hot plate and formalin and acetic acid-induced pain models to check the analgesic activity of medicinal plants. PMID:26939272

  17. Active Sonar

    NASA Astrophysics Data System (ADS)

    Sullivan, Edmund J.

    An active sonar system is one in which pulses of acoustic energy are launched into the water for the purpose of producing echoes. By examining the echoes of transmitted pulses, it affords the capability of both detecting the presence of and estimating the range, and in certain cases, the bearing, of an underwater target. In its most common arrangement, the transmitter (or projector) and the receiver are colocated. This is known as the monostatic configuration and is depicted in Figure 1. When this is not so, it is known as a bistatic or multistatic configuration.

  18. Resveratrol Suppresses Cytokine Production Linked to FcεRI-MAPK Activation in IgE-Antigen Complex-Exposed Basophilic Mast Cells and Mice.

    PubMed

    Han, Seon-Young; Choi, Yean-Jung; Kang, Min-Kyung; Park, Jung Han Yoon; Kang, Young-Hee

    2015-01-01

    A complicated interplay between resident mast cells and other recruited inflammatory cells contributes to the development and progression of allergic inflammation entailing the promotion of T helper 2 (Th2) cytokine responses. The current study examined whether resveratrol suppressed the production of inflammatory Th2 cytokines in cultured rat basophilic leukemia RBL-2H3 cells. Cells pre-treated with resveratrol nontoxic at 1–25 μM were sensitized with anti-dinitrophenyl (anti-DNP), and subsequently stimulated by dinitrophenyl-human serum albumin (DNP–HSA) antigen. Resveratrol dose-dependently diminished the secretion of interleukin (IL)-3, IL-4, IL-13 as well as tumor necrosis factor (TNF)-α by the antigen stimulation from sensitized cells. It was found that resveratrol mitigated the phosphorylation of p38 MAPK, ERK, and JNK elevated in mast cells exposed to Fc epsilon receptor I (FcεRI)-mediated immunoglobulin E (IgE)-antigen complex. The FcεRI aggregation was highly enhanced on the surface of mast cells following the HSA stimulation, which was retarded by treatment with 1–25 μM resveratrol. The IgE-receptor engagement rapidly induced tyrosine phosphorylation of c-Src-related focal adhesion protein paxillin involved in the cytoskeleton rearrangement. The FcεRI-mediated rapid activation of c-Src and paxillin was attenuated in a dose-dependent manner. In addition, the paxillin activation entailed p38 MAPK and ERK-responsive signaling, but the JNK activation was less involved. Consistently, oral administration of resveratrol reduced the tissue level of phosphorylated paxillin in the dorsal skin of DNP–HSA-challenged mice. The other tyrosine kinase Tyk2-STAT1 signaling was activated in the dorsal epidermis of antigen-exposed mice, which was associated with allergic inflammation. These results showed that resveratrol inhibited Th2 cytokines- and paxillin-linked allergic responses dependent upon MAPK signaling. Therefore, resveratrol may possess the

  19. Active packaging with antifungal activities.

    PubMed

    Nguyen Van Long, N; Joly, Catherine; Dantigny, Philippe

    2016-03-01

    There have been many reviews concerned with antimicrobial food packaging, and with the use of antifungal compounds, but none provided an exhaustive picture of the applications of active packaging to control fungal spoilage. Very recently, many studies have been done in these fields, therefore it is timely to review this topic. This article examines the effects of essential oils, preservatives, natural products, chemical fungicides, nanoparticles coated to different films, and chitosan in vitro on the growth of moulds, but also in vivo on the mould free shelf-life of bread, cheese, and fresh fruits and vegetables. A short section is also dedicated to yeasts. All the applications are described from a microbiological point of view, and these were sorted depending on the name of the species. Methods and results obtained are discussed. Essential oils and preservatives were ranked by increased efficacy on mould growth. For all the tested molecules, Penicillium species were shown more sensitive than Aspergillus species. However, comparison between the results was difficult because it appeared that the efficiency of active packaging depended greatly on the environmental factors of food such as water activity, pH, temperature, NaCl concentration, the nature, the size, and the mode of application of the films, in addition to the fact that the amount of released antifungal compounds was not constant with time. PMID:26803804

  20. Active tectonics

    SciTech Connect

    Not Available

    1986-01-01

    This study is part of a series of Studies in Geophysics that have been undertaken for the Geophysics Research Forum by the Geophysics Study Committee. One purpose of each study is to provide assessments from the scientific community to aid policymakers in decisions on societal problems that involve geophysics. An important part of such assessments is an evaluation of the adequacy of current geophysical knowledge and the appropriateness of current research programs as a source of information required for those decisions. The study addresses our current scientific understanding of active tectonics --- particularly the patterns and rates of ongoing tectonic processes. Many of these processes cannot be described reasonably using the limited instrumental or historical records; however, most can be described adequately for practical purposes using the geologic record of the past 500,000 years. A program of fundamental research focusing especially on Quaternary tectonic geology and geomorphology, paleoseismology, neotectonics, and geodesy is recommended to better understand ongoing, active tectonic processes. This volume contains 16 papers. Individual papers are indexed separately on the Energy Database.

  1. The C-terminus of interferon gamma receptor beta chain (IFNgammaR2) has antiapoptotic activity as a Bax inhibitor.

    PubMed

    Gomez, Jose A; Sun, Weiyon; Gama, Vivian; Hajkova, Dagmar; Yoshida, Tomoyuki; Wu, Zhengrong; Miyagi, Masaru; Pink, John J; Jackson, Mark W; Danielpour, David; Matsuyama, Shigemi

    2009-09-01

    Bax is a pro-apoptotic protein that mediates intrinsic cell-death signaling. Using a yeast-based functional screening approach, we identified interferon gamma receptor beta chain (IFNgammaR2) as a new Bax suppressor. IFNgammaR2 is a component of the IFNgamma receptor complex along with the IFNgammaR alpha chain (IFNgammaR1). Upon IFNgamma binding, a conformational change in the receptor complex occurs that activates the Jak2/STAT1 signaling cascade. We found that the C-terminal region (amino acids 296-337) of IFNgammaR2 (IFNgammaR2(296-337)) contains a novel Bax inhibitory domain. This portion does not contain the Jak2-binding domain; therefore, the antiapoptotic function of IFNgammaR2 is independent of JAK/STAT signaling. IFNgammaR2(296-337) rescued human cells from apoptosis induced by overexpression of Bax but not Bak. Overexpression of IFNgammaR2 (wild type and IFNgammaR2(296-337)) rescued cells from etoposide and staurosporine, which are known to induce Bax-mediated cell death. Interestingly, IFNgammaR2 inhibited apoptosis induced by the BH3-only protein Bim-EL, suggesting that IFNgammaR2 inhibits Bax activation through a BH3-only protein. Bax and IFNgammaR2 were co-immunoprecipitated from cell lysates prepared from HEK293 and DAMI cells. Furthermore, direct binding of purified recombinant proteins of Bax and IFNgammaR2 was also confirmed. Addition of recombinant Bcl-2 protein to cell lysates significantly reduced the interaction of IFNgammaR2 and Bax, suggesting that Bcl-2 and IFNgammaR2 bind a similar domain of Bax. We found that the C-terminal fragment (cytoplasmic domain) of IFNgammaR2 is expressed in human cancer cell lines of megakaryocytic cancer (DAMI), breast cancer (MDA-MD-468), and prostate cancer (PC3 cells). The presence of the C-terminal fragment of IFNgammaR2 may confer on cancer cells resistance to apoptotic stresses. Our discovery of the anti-Bax activity of the cytoplasmic domain of IFNgammaR2 may shed new light on the mechanism of how cell

  2. DAVIC activities

    NASA Astrophysics Data System (ADS)

    Fujiwara, Hiroshi

    1995-12-01

    DAVIC (Digital Audio Visual Council) is the defacto standardization organization established in Mar. 1994, based on international consensus for digital audio visual services. After completion of MPEG2 standardization, the broadcasting industry, the communication industry, the computer industry, and consumer electronics industry have started development of concrete services and products. Especially the interactive digital audio visual services, such as Video On Demand (VOD) or Near Video On Demand (NVOD), have become hot topics all over the world. Such interactive digital audio visual services are combined technologies of multi-media coding, digital transmission and computer networking. Therefore more than 150 organizations from all industry sectors have participated in DAVIC and are contributing from their own industrial contexts. DAVIC's basic policy is to use the available technologies specified by the other standards bodies as much as possible. So DAVIC's standardization activities have close relationship with ISO IEC/JTC1/SC29, ITU-T SG 9, ATM-Forum, IETF, IMA, DVB, etc. DAVIC is trying to specify Applications, Reference Models, Security, Usage Information Control, and the interfaces and protocols among the Content Provider, the Server, the core network, the access network, and the Set Top Unit. DAVIC's first goal is to specify DAVIC1.0 based on CFP1 (Call for Proposal) and CFP2 by Dec. 1995, and the next direction is under preparation for further progress based on CFP3 and CFP4.

  3. Activities update

    NASA Astrophysics Data System (ADS)

    Smith, Gerald A.

    1994-07-01

    The present report is an update on activities for the second year of funding. Research leading to a detailed characterization of antiproton annihilation in nuclei has resulted in a published analysis of fast deuteron production from carbon and uranium targets. This follows previously reported work and publications by our group on gamma-ray, neutral and charged pion, proton, and neutron production. The deuteron measurements are important to our SHIVA Star antiproton- catalyzed microfission experiment at the Phillips Laboratory, Kirtland AFB, in that they help constrain theoretical models of light nuclei production and subsequent energy deposition in the target. Work continues at SHIVA Star on working fluid formation and target compression for the microfission experiment. Excellent progress has been made, both theoretically and experimentally, on these important aspects of the experiment. The Penn State group, working in collaboration with Los Alamos National Laboratory physicists, trapped and held up to 721,000 antiprotons per beam injection pulse from the LEAR accelerator during July, 1993. This was a crucial step to the ultimate goal of transferring large numbers of antiprotons to the Phillips Laboratory for the antiproton-catalyzed microfission experiment.

  4. Active Segmentation

    PubMed Central

    Mishra, Ajay; Aloimonos, Yiannis

    2009-01-01

    The human visual system observes and understands a scene/image by making a series of fixations. Every fixation point lies inside a particular region of arbitrary shape and size in the scene which can either be an object or just a part of it. We define as a basic segmentation problem the task of segmenting that region containing the fixation point. Segmenting the region containing the fixation is equivalent to finding the enclosing contour- a connected set of boundary edge fragments in the edge map of the scene - around the fixation. This enclosing contour should be a depth boundary. We present here a novel algorithm that finds this bounding contour and achieves the segmentation of one object, given the fixation. The proposed segmentation framework combines monocular cues (color/intensity/texture) with stereo and/or motion, in a cue independent manner. The semantic robots of the immediate future will be able to use this algorithm to automatically find objects in any environment. The capability of automatically segmenting objects in their visual field can bring the visual processing to the next level. Our approach is different from current approaches. While existing work attempts to segment the whole scene at once into many areas, we segment only one image region, specifically the one containing the fixation point. Experiments with real imagery collected by our active robot and from the known databases 1 demonstrate the promise of the approach. PMID:20686671

  5. IASS Activity

    NASA Astrophysics Data System (ADS)

    Hojaev, Alisher S.; Ibragimova, Elvira M.

    2015-08-01

    It’s well known, astronomy in Uzbekistan has ancient roots and traditions (e.g., Mirzo Ulugh Beg, Abū al-Rayhān al-Bīrūnī, Abū ‘Abdallāh al-Khwārizmī) and astronomical heritage carefully preserved. Nowadays uzbek astronomers play a key role in scientific research but also in OAD and Decadal Plan activity in the Central Asia region. International Aerospace School (IASS) is an amazing and wonderful event held annually about 30 years. IASS is unique project in the region, and at the beginning we spent the Summer and Winter Schools. At present in the summer camp we gather about 50 teenage and undergraduate students over the country and abroad (France, Malaysia, Turkey, Azerbaijan, Pakistan, Russia, etc.). They are selected on the basis of tests of astronomy and space issues. During two weeks of IASS camp the invited scientists, cosmonauts and astronauts as well as other specialists give lectures and engage in practical exercises with IASS students in astronomy, including daily observations of the Sun and night sky observations with meniscus telescope, space research and exploration, aerospace modelling, preparation and presentation of original projects. This is important that IASS gives not theoretical grounds only but also practically train the students and the hands-on training is the major aims of IASS. Lectures and practice in the field of astronomy carried out with the direct involvement and generous assistance of Uranoscope Association (Paris, France). The current 26-th IASS is planned to held in July 2015.

  6. Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation

    SciTech Connect

    Johnson, Karen E.; Knipe, David M.

    2010-01-05

    Host cells respond to viral infection by the production of type I interferons (IFNs), which induce the expression of antiviral genes. Herpes simplex virus I (HSV-1) encodes many mechanisms that inhibit the type I IFN response, including the ICP27-dependent inhibition of type I IFN signaling. Here we show inhibition of Stat-1 nuclear accumulation in cells that express ICP27. ICP27 expression also induces the secretion of a small, heat-stable type I IFN antagonizing protein that inhibits Stat-1 nuclear accumulation. We show that the inhibition of IFN-induced Stat-1 phosphorylation occurs at or upstream of Jak-1 phosphorylation. Finally, we show that ISG15 expression is induced after IFNalpha treatment in mock-infected cells, but not cells infected with WT HSV-1 or ICP27{sup -} HSV-1. These data suggest that HSV-1 has evolved multiple mechanisms to inhibit IFN signaling not only in infected cells, but also in neighboring cells, thereby allowing for increased viral replication and spread.

  7. Hepatitis C virus core protein enhances hepatocellular carcinoma cells to be susceptible to oncolytic vesicular stomatitis virus through down-regulation of HDAC4.

    PubMed

    Moon, Jeong; Kaowinn, Sirichat; Cho, Il-Rae; Min, Do Sik; Myung, Heejoon; Oh, Sangtaek; Kaewpiboon, Chutima; Kraemer, Olive H; Chung, Young-Hwa

    2016-06-01

    Since hepatitis C virus (HCV) core protein is known to possess potential oncogenic activity, we explored whether oncolytic vesicular stomatitis virus (VSV) could efficiently induce cytolysis in hepatocellular carcinoma cells stably expressing HCV core protein (Hep3B-Core). We found that Hep3B-Core cells were more susceptible to VSV as compared to control (Hep3B-Vec) cells owing to core-mediated inactivation of STAT1 and STAT2 proteins. Core expression induced lower phosphorylation levels of type I IFN signaling proteins such as Tyk2 and Jak1, and a reduced response to exogenous IFN-α, which resulted in susceptibility to VSV. Furthermore, as STAT1 acetylation by switching phosphorylation regulated its activity, the role of STAT1 acetylation in susceptibility of Hep3B-Core cells to VSV was investigated. Treatment with trichostatin A, an inhibitor of histone deacetylase (HDAC), increased STAT1 acetylation but blocked IFN-α-induced phosphorylation of STAT1, leading to increase of susceptibility to VSV. Interestingly, the core protein decreased HDCA4 transcript levels, leading to down-regulation of HDAC4 protein. However, ectopic expression of HDAC4 conversely enforced phosphorylation of STAT1 and hindered VSV replication, indicating that core-mediated reduction of HDAC4 provides a suitable intracellular circumstance for VSV replication. Collectively, we suggest that VSV treatment will be a useful therapeutic strategy for HCV-infected hepatocellular carcinoma cells because HCV core protein suppresses the anti-viral threshold by down-regulation of the STAT1-HDAC4 signaling axis. PMID:27150631

  8. Global changes in STAT target selection and transcription regulation upon interferon treatments

    PubMed Central

    Hartman, Stephen E.; Bertone, Paul; Nath, Anjali K.; Royce, Thomas E.; Gerstein, Mark; Weissman, Sherman; Snyder, Michael

    2005-01-01

    The STAT (signal transducer and activator of transcription) proteins play a crucial role in the regulation of gene expression, but their targets and the manner in which they select them remain largely unknown. Using chromatin immunoprecipitation and DNA microarray analysis (ChIP-chip), we have identified the regions of human chromosome 22 bound by STAT1 and STAT2 in interferon-treated cells. Analysis of the genomic loci proximal to these binding sites introduced new candidate STAT1 and STAT2 target genes, several of which are affiliated with proliferation and apoptosis. The genes on chromosome 22 that exhibited interferon-induced up- or down-regulated expression were determined and correlated with the STAT-binding site information, revealing the potential regulatory effects of STAT1 and STAT2 on their target genes. Importantly, the comparison of STAT1-binding sites upon interferon (IFN)-γ and IFN-α treatments revealed dramatic changes in binding locations between the two treatments. The IFN-α induction revealed nonconserved STAT1 occupancy at IFN-γ-induced sites, as well as novel sites of STAT1 binding not evident in IFN-γ-treated cells. Many of these correlated with binding by STAT2, but others were STAT2 independent, suggesting that multiple mechanisms direct STAT1 binding to its targets under different activation conditions. Overall, our results reveal a wealth of new information regarding IFN/STAT-binding targets and also fundamental insights into mechanisms of regulation of gene expression in different cell states. PMID:16319195

  9. Activation Energy

    NASA Technical Reports Server (NTRS)

    Gadeken, Owen

    2002-01-01

    Teaming is so common in today's project management environment that most of us assume it comes naturally. We further assume that when presented with meaningful and challenging work, project teams will naturally engage in productive activity to complete their tasks. This assumption is expressed in the simple (but false) equation: Team + Work = Teamwork. Although this equation appears simple and straightforward, it is far from true for most project organizations whose reality is a complex web of institutional norms based on individual achievement and rewards. This is illustrated by the very first successful team experience from my early Air Force career. As a young lieutenant, I was sent to Squadron Officer School, which was the first in the series of Air Force professional military education courses I was required to complete during my career. We were immediately formed into teams of twelve officers. Much of the course featured competition between these teams. As the most junior member of my team, I quickly observed the tremendous pressure to show individual leadership capability. At one point early in the course, almost everyone in our group was vying to become the team leader. This conflict was so intense that it caused us to fail miserably in our first outdoor team building exercise. We spent so much time fighting over leadership that we were unable to complete any of the events on the outdoor obstacle course. This complete lack of success was so disheartening to me that I gave our team little hope for future success. What followed was a very intense period of bickering, conflict, and even shouting matches as our dysfunctional team tried to cope with our early failures and find some way to succeed. British physician and researcher Wilfred Bion (Experiences in Groups, 1961) discovered that there are powerful psychological forces inherent in all groups that divert from accomplishing their primary tasks. To overcome these restraining forces and use the potential

  10. Physical Activity (Exercise)

    MedlinePlus

    ... fitness. Your fitness routine should include aerobic and strength-training activities, and may also include stretching activities. Aerobic ... Examples include walking, jogging, bicycling, swimming, and tennis. Strength-training activities These activities increase the strength and endurance ...

  11. Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation

    PubMed Central

    Dicay, Michael S.; Hirota, Christina L.; Ronaghan, Natalie J.; Peplowski, Michael A.; Zaheer, Raza S.; Carati, Colin A.; MacNaughton, Wallace K.

    2015-01-01

    Inflammatory bowel diseases are associated with dysregulated electrolyte and water transport and resultant diarrhea. Aquaporins are transmembrane proteins that function as water channels in intestinal epithelial cells. We investigated the effect of the inflammatory cytokine, interferon-γ, which is a major player in inflammatory bowel diseases, on aquaporin-1 expression in a mouse colonic epithelial cell line, CMT93. CMT93 monolayers were exposed to 10 ng/mL interferon-γ and aquaporin-1 mRNA and protein expressions were measured by real-time PCR and western blot, respectively. In other experiments, CMT93 cells were pretreated with inhibitors or were transfected with siRNA to block the effects of Janus kinases, STATs 1 and 3, or interferon regulatory factor 2, prior to treatment with interferon-γ. Interferon-γ decreased aquaporin-1 expression in mouse intestinal epithelial cells in a manner that did not depend on the classical STAT1/JAK2/IRF-1 pathway, but rather, on an alternate Janus kinase (likely JAK1) as well as on STAT3. The pro-inflammatory cytokine, interferon-γ may contribute to diarrhea associated with intestinal inflammation in part through regulation of the epithelial aquaporin-1 water channel via a non-classical JAK/STAT receptor signalling pathway. PMID:25793528

  12. [Adapting physical activities for an active retirement].

    PubMed

    Renaudie, François

    2016-01-01

    The benefits of doing adapted physical exercise for elderly people have been proven. For more than thirty years, the French Federation for an Active Retirement has been striving to help people age well by proposing multiple activities to remain in good health after the age of 50. Doctors, activity leaders and federal instructors are attentive to each individual's capacities. PMID:27449307

  13. Antimycotics suppress the Malassezia extract-induced production of CXC chemokine ligand 10 in human keratinocytes.

    PubMed

    Hau, Carren S; Kanda, Naoko; Makimura, Koichi; Watanabe, Shinichi

    2014-02-01

    Malassezia, a lipophilic yeast, exacerbates atopic dermatitis. Malassezia products can penetrate the disintegrated stratum corneum and encounter subcorneal keratinocytes in the skin of atopic dermatitis patients. Type 1 helper T (Th1) cells infiltrate chronic lesions with atopic dermatitis, and antimycotic agents improve its symptoms. We aimed to identify Malassezia-induced chemokines in keratinocytes and examine whether antimycotics suppressed this induction. Normal human keratinocytes were incubated with a Malassezia restricta extract and antimycotics. Chemokine expression was analyzed by enzyme-linked immunosorbent assays and real-time polymerase chain reaction. Signal transducer and activator of transcription (STAT)1 activity was examined by luciferase assays. The tyrosine-phosphorylation of STAT1 was analyzed by western blotting. The M. restricta extract increased the mRNA and protein expression of Th1-attracting CXC chemokine ligand (CXCL)10 and STAT1 activity and phosphorylation in keratinocytes, which was suppressed by a Janus kinase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine suppressed M. restricta extract-induced CXCL10 mRNA and protein expression and STAT1 activity and phosphorylation. These effects were similarly induced by 15-deoxy-Δ-(12,14) -prostaglandin J2 (15d-PGJ2 ), a prostaglandin D2 metabolite. Antimycotics increased the release of 15d-PGJ2 from keratinocytes. The antimycotic-induced suppression of CXCL10 production and STAT1 activity was counteracted by a lipocalin-type prostaglandin D synthase inhibitor. The antimycotics itraconazole, ketoconazole, luliconazole, terbinafine, butenafine and amorolfine may suppress the M. restricta-induced production of CXCL10 by inhibiting STAT1 through an increase in 15d-PGJ2 production in keratinocytes. These antimycotics may block the Th1-mediated inflammation triggered by Malassezia in the chronic phase of atopic dermatitis. PMID

  14. Learning as Activity.

    ERIC Educational Resources Information Center

    Jonassen, David H.

    2002-01-01

    Integrates contemporary theories of learning into a theory of learning as activity. Explains ecological psychology, changes in understanding of learning, activity systems and activity theory (including the integration of consciousness and activity), and activity structure; and discusses learning as a cognitive and social process. (LRW)

  15. Coagulant Activity of Leukocytes. TISSUE FACTOR ACTIVITY

    PubMed Central

    Niemetz, J.

    1972-01-01

    Peritoneal leukocytes harvested from rabbits which have received two spaced doses of endotoxin have significantly greater (10-fold) coagulant activity than leukocytes from control rabbits. The coagulant activity accelerates the clotting of normal plasma and activates factor X in the presence of factor VII and calcium and is therefore regarded as tissue factor. A total of 40-80 mg tissue factor activity was obtained from the peritoneal cavity of single endotoxin-treated rabbits. In leukocyte subcellular fractions, separated by centrifugation, the specific tissue factor activity sedimented mainly at 14,500 g and above. The procoagulant activity was destroyed after heating for 10 min at 65°C but was preserved at lower temperatures. Polymyxin B, when given with the first dose of endotoxin, reduced both the number of peritoneal leukocytes and their tissue factor activity by two-thirds. When given immediately before the second dose of endotoxin, polymyxin B had no inhibitory effect. PMID:4333021

  16. Facts about Physical Activity

    MedlinePlus

    ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs Facts about Physical Activity ... Physical Activity Overweight & Obesity Healthy Weight Breastfeeding Micronutrient Malnutrition State and Local Programs File Formats Help: How ...

  17. Physical Activity Assessment

    Cancer.gov

    Current evidence convincingly indicates that physical activity reduces the risk of colon and breast cancer. Physical activity may also reduce risk of prostate cancer. Scientists are also evaluating potential relationships between physical activity and other cancers.

  18. A20 Regulates Atherogenic Interferon (IFN)-γ Signaling in Vascular Cells by Modulating Basal IFNβ Levels*

    PubMed Central

    Moll, Herwig P.; Lee, Andy; Minussi, Darlan C.; da Silva, Cleide G.; Csizmadia, Eva; Bhasin, Manoj; Ferran, Christiane

    2014-01-01

    IFNγ signaling in endothelial (EC) and smooth muscle cells (SMC) is a key culprit of pathologic vascular remodeling. The impact of NF-κB inhibitory protein A20 on IFNγ signaling in vascular cells remains unknown. In gain- and loss-of-function studies, A20 inversely regulated expression of IFNγ-induced atherogenic genes in human EC and SMC by modulating STAT1 transcription. In vivo, inadequate A20 expression in A20 heterozygote mice aggravated intimal hyperplasia following partial carotid artery ligation. This outcome uniquely associated with increased levels of Stat1 and super-induction of Ifnγ-dependent genes. Transcriptome analysis of the aortic media from A20 heterozygote versus wild-type mice revealed increased basal Ifnβ signaling as the likely cause for higher Stat1 transcription. We confirmed higher basal IFNβ levels in A20-silenced human SMC and showed that neutralization or knockdown of IFNβ abrogates heightened STAT1 levels in these cells. Upstream of IFNβ, A20-silenced EC and SMC demonstrated higher levels of phosphorylated/activated TANK-binding kinase-1 (TBK1), a regulator of IFNβ transcription. This suggested that A20 knockdown increased STAT1 transcription by enhancing TBK1 activation and subsequently basal IFNβ levels. Altogether, these results uncover A20 as a key physiologic regulator of atherogenic IFNγ/STAT1 signaling. This novel function of A20 added to its ability to inhibit nuclear factor-κB (NF-κB) activation solidifies its promise as an ideal therapeutic candidate for treatment and prevention of vascular diseases. In light of recently discovered A20/TNFAIP3 (TNFα-induced protein 3) single nucleotide polymorphisms that impart lower A20 expression or function, these results also qualify A20 as a reliable clinical biomarker for vascular risk assessment. PMID:25217635

  19. Chromium(VI) stimulates Fyn to initiate innate immune gene induction in human airway epithelial cells

    PubMed Central

    Nemec, Antonia A.; Zubritsky, Lindsey M.; Barchowsky, Aaron

    2009-01-01

    Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes and we hypothesized this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation. PMID:19994902

  20. Molecular mechanisms of mucocutaneous immunity against Candida and Staphylococci

    PubMed Central

    Maródi, László; Cypowyj, Sophie; Tóth, Beáta; Chernyshova, Liudmyla; Puel, Anne; Casanova, Jean-Laurent

    2013-01-01

    Signal transducer and activator of transcription (STAT) proteins are key components of the innate and adaptive immune responses to pathogenic microorganisms. Recent research on primary immunodeficiency disorders and the identification of patients carrying germline mutations in STAT1, STAT3, and STAT5B have highlighted the role of human STATs in host defense against various viruses, bacteria, and fungi. Mutations in STAT1 and STAT3 may disrupt various cytokine pathways that control mucocutaneous immunity against Candida species, especially Candida albicans, and Staphylococci, especially Staphylococcus aureus. Here, we consider inborn errors of immunity arising from mutations in either STAT1 or STAT3 that affect mucocutaneous immunity to Candida and Staphylococci. PMID:23040277

  1. Active commuting to school

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Declines in physical activity levels have coincided with increasing rates of obesity in children. This is problematic because physical activity has been shown to attenuate weight gain in children. Active commuting to school is one way of increasing children's physical activity. However, given the hi...

  2. Civil Law: 12 Activities.

    ERIC Educational Resources Information Center

    Dresbach, Debra

    These learning activities on civil law are intended to supplement the secondary level Scholastic materials "Living Law." Case studies, simulations, and role-play activities are included. Information provided for each activity includes a brief overview, background information, teacher instructions and a description of each activity. Activities…

  3. Increasing Youth Physical Activity with Activity Calendars

    ERIC Educational Resources Information Center

    Eckler, Seth

    2016-01-01

    Physical educators often struggle with ways to get their students to be active beyond the school day. One strategy to accomplish this is the use of physical activity calendars (PACs). The purpose of this article is to support the use of PACs and give practical advice for creating effective PACs.

  4. Persistent active longitudes in sunspot activity

    NASA Astrophysics Data System (ADS)

    Berdyugina, S.; Usoskin, I.

    It has been recently shown that spot activity of cool stars including solar analogues, is grouped in two clearly distinguished active longitudes which are persistent within at least one starspot cycle. Solar data including positional information of individual sunspots / groups extends back for about 130 years covering 12 solar cycles. Here we present the results of our research of longitudinal distribution of sunspot activity using an analysis similar to that applied to the stars. First, we synthesized, from the actual sunspot data, the sun's light curve as if it was defined only by spots. Then solar images were calculated from this light curve, giving a natural smoothing of the spot pattern. For each Carrington rotation, longitudinal position of these smoothed spot regions was calculated. The analysis reveals the following main features: - Sunspot activity is grouped in two active longitudes (with the differential rotation taken into account) 180o apart from each other which are persistent through the entire studied period of 12 cycles, similarly to stars. - The longitude migration is determined by changing the mean latitude of sunspot activity (the Maunder butterfly) and differential rotation. - The two longitudes periodically alternate the dominant activity with about 3.7 year period implying for the existence of the Sflip-flopT phenomenon known in - starspot activity.

  5. Signal transduction mechanism of a peptide mimetic of interferon-gamma.

    PubMed

    Subramaniam, Prem S; Flowers, Lawrence O; Haider, S Mohammed I; Johnson, Howard M

    2004-05-11

    The C-terminus of interferon-gamma (IFNgamma) contains a nuclear localization sequence (NLS) required for the activation and nuclear translocation of the transcription factor STAT1alpha and induction of IFNgamma-activated genes. On the basis of this and other studies, we developed a peptide mimetic of IFNgamma that possesses the IFNgamma functions of antiviral activity and upregulation of MHC class II molecules. The mimetic also shares with IFNgamma the ability to induce the activation and nuclear translocation of STAT1alpha and the IFNgamma receptor (IFNGR)-1 subunit. The mimetic, IFNgamma(95-132), is a peptide that consists of the C-terminal residues 95-132 of murine IFNgamma and contains a required alpha-helical domain and the NLS of IFNgamma. In this study, we determined the mechanism of the intracellular action of the mimetic at the level of signal transduction. We show that the mimetic mediates the nuclear transport of IFNGR-1 through its interaction with IFNGR-1 cytoplasmic region 253-287 via both the helical region and the NLS of IFNgamma(95-132). Alanine substitutions of the NLS of the mimetic showed that the NLS was required for nuclear translocation and that the nuclear transport properties of the mimetic correlated with its ability to bind IFNGR-1. These data also show that the NLS of IFNgamma(95-132) can interact simultaneously with IFNGR-1 and the nuclear import machinery. We found that in in vitro nuclear transport assays tyrosine-phosphorylated STAT1alpha failed to undergo nuclear translocation in the presence of nuclear import factors, but was transported to nucleus in the presence of IFNgamma(95-132) and JAK2-phosphorylated IFNGR-1, to which STAT1alpha binds, as a complex of IFNgamma(95-132)/IFNGR-1/STAT1alpha. Thus, the mimetic, which possesses IFNgamma function, is directly involved as a chaperone in the nuclear transport of STAT1alpha and shares this mechanism of action with that previously described for IFNgamma. The mimetic, like IFNgamma, is

  6. Exacerbated inflammatory arthritis in response to hyperactive gp130 signalling is independent of IL-17A

    PubMed Central

    Jones, G W; Greenhill, C J; Williams, J O; Nowell, M A; Williams, A S; Jenkins, B J; Jones, S A

    2013-01-01

    Objective Interleukin (IL)-17A producing CD4 T-cells (TH-17 cells) are implicated in rheumatoid arthritis (RA). IL-6/STAT3 signalling drives TH-17 cell differentiation, and hyperactive gp130/STAT3 signalling in the gp130F/F mouse promotes exacerbated pathology. Conversely, STAT1-activating cytokines (eg, IL-27, IFN-γ) inhibit TH-17 commitment. Here, we evaluate the impact of STAT1 ablation on TH-17 cells during experimental arthritis and relate this to IL-17A-associated pathology. Methods Antigen-induced arthritis (AIA) was established in wild type (WT), gp130F/F mice displaying hyperactive gp130-mediated STAT signalling and the compound mutants gp130F/F:Stat1−/− and gp130F/F:Il17a−/− mice. Joint pathology and associated peripheral TH-17 responses were compared. Results Augmented gp130/STAT3 signalling enhanced TH-17 commitment in vitro and exacerbated joint pathology. Ablation of STAT1 in gp130F/F mice (gp130F/F:Stat1−/−) promoted the hyperexpansion of TH-17 cells in vitro and in vivo during AIA. Despite this heightened peripheral TH-17 cell response, disease severity and the number of joint-infiltrating T-cells were comparable with that of WT mice. Thus, gp130-mediated STAT1 activity within the inflamed synovium controls T-cell trafficking and retention. To determine the contribution of IL-17A, we generated gp130F/F:IL-17a−/− mice. Here, loss of IL-17A had no impact on arthritis severity. Conclusions Exacerbated gp130/STAT-driven disease in AIA is associated with an increase in joint infiltrating T-cells but synovial pathology is IL-17A independent. PMID:23894061

  7. Interleukin 2 signaling involves the phosphorylation of Stat proteins.

    PubMed

    Frank, D A; Robertson, M J; Bonni, A; Ritz, J; Greenberg, M E

    1995-08-15

    One of the most important cytokines involved in immune response regulation is interleukin 2 (IL-2), a potent activator of the proliferation and function of T lymphocytes and natural killer cells. The mechanisms by which the effects of IL-2 are propagated within cells are not understood. While the binding of IL-2 to its receptor was recently shown to lead to the activation of two kinases, Jak-1 and Jak-3, subsequent steps in the signaling pathway to the nucleus that lead to the activation of specific genes had not been characterized. Since many cytokines that activate Jak kinases also lead to the tyrosine phosphorylation and activation of members of the Stat family of transcription factors, the ability of IL-2 to trigger Stat phosphorylation was examined. Exposure of activated human T lymphocytes or of a natural killer cell line (NKL) to IL-2 leads to the phosphorylation of Stat1 alpha, Stat1 beta, and Stat3, as well as of two Stat-related proteins, p94 and p95. p94 and p95 share homology with Stat1 at the phosphorylation site and in the Src homology 2 (SH2) domain, but otherwise are immunologically distinct from Stat1. These Stat proteins were found to translocate to the nucleus and to bind to a specific DNA sequence. These findings suggest a mechanism by which IL-2 binding to its receptor may activate specific genes involved in immune cell function. PMID:7544001

  8. Antifeedant activity of quassinoids.

    PubMed

    Leskinen, V; Polonsky, J; Bhatnagar, S

    1984-10-01

    The antifeedant activity of 13 quassinoids of different structural types has been studied against the Mexican bean beetle (Epilachna varivestis Mulsant) 4th instar larvae and the southern armyworm (Spodoptera eridania Crawer) 5th instar larvae. All quassinoids tested displayed significant activity against the Mexican bean beetle and, thus, do not reveal a simple structure-activity relationship. Five quassinoids were active against the southern armyworm. Interestingly, four of these-bruceantin (I), glaucarubinone (VI), isobruceine A (VIII), and simalikalactone D (XI)-possess the required structural features for antineoplastic activity. The noncytotoxic quassin (X) is an exception; it is active against both pests. PMID:24318349

  9. CATALASE ACTIVITY IN LEPTOSPIRA

    PubMed Central

    Rao, P. J.; Larson, A. D.; Cox, C. D.

    1964-01-01

    Rao, P. J. (University of Illinois, Urbana), A. D. Larson, and C. D. Cox. Catalase activity in Leptospira. J. Bacteriol. 88:1045–1048. 1964.—A number of serotypes of Leptospira were found to possess catalase activity, although considerable variation in activity existed among various serotypes. Catalase activity of L. pomona was reduced by inhibitors commonly employed for arresting catalase activity in other biological systems. Catalase activity was increased three to five times by growing cultures under conditions of oxygen availability; however, aeration had no beneficial effect on total viable cell crop. The relationship of oxygen to metabolism and future studies on virulence of the leptospirae is discussed. PMID:14219017

  10. Active Fire Mapping Program

    MedlinePlus

    ... Incidents (Home) New Large Incidents Fire Detection Maps MODIS Satellite Imagery VIIRS Satellite Imagery Fire Detection GIS ... Data Web Services Latest Detected Fire Activity Other MODIS Products Frequently Asked Questions About Active Fire Maps ...

  11. Preschoolers’ Physical Activity Behaviours

    PubMed Central

    Irwin, Jennifer D.; He, Meizi; Bouck, L. Michelle Sangster; Tucker, Patricia; Pollett, Graham L.

    2016-01-01

    Objectives To understand parents’ perspectives of their preschoolers’ physical activity behaviours. Methods A maximum variation sample of 71 parents explored their preschoolers’ physical activity behaviours through 10 semi-structured focus group discussions. Results Parents perceived Canada’s Physical Activity Guidelines for Children as inadequate; that their preschoolers get and need more than 30–90 minutes of activity daily; and that physical activity habits must be established during the preschool years. Nine barriers against and facilitators toward adequate physical activity were proposed: child’s age, weather, daycare, siblings, finances, time, society and safety, parents’ impact, and child’s activity preferences. Discussion The need for education and interventions that address current barriers are essential for establishing physical activity as a lifestyle behaviour during early childhood and, consequently, helping to prevent both childhood and adulthood obesity. PMID:16625802

  12. Balance Food and Activity

    MedlinePlus

    ... For Health Professionals Tools and Resources Promotional Materials Programming Materials Weight Management Nutrition Physical Activity Reduce Screen ... Training For Health Professionals Tools & Resources Promotional ... Programming Materials Weight Management Nutrition Physical Activity Reduce Screen ...

  13. Active magnetic regenerator

    DOEpatents

    Barclay, John A.; Steyert, William A.

    1982-01-01

    The disclosure is directed to an active magnetic regenerator apparatus and method. Brayton, Stirling, Ericsson, and Carnot cycles and the like may be utilized in an active magnetic regenerator to provide efficient refrigeration over relatively large temperature ranges.

  14. Exercise and Physical Activity

    MedlinePlus

    Alzheimer ’s Caregiving Tips Exercise and Physical Activity Being active and getting exercise helps people with Alzheimer’s disease feel better. Exercise helps keep their muscles, joints, and heart in ...

  15. Antimicrobial activity of isopteropodine.

    PubMed

    García, Rubén; Cayunao, Cesia; Bocic, Ronny; Backhouse, Nadine; Delporte, Carle; Zaldivar, Mercedes; Erazo, Silvia

    2005-01-01

    Bioassay-directed fractionation for the determination of antimicrobial activity of Uncaria tomentosa, has led to the isolation of isopteropodine (0.3%), a known Uncaria pentacyclic oxindol alkaloid that exhibited antibacterial activity against Gram positive bacteria. PMID:16042336

  16. Population Education. Awareness Activities.

    ERIC Educational Resources Information Center

    Brouse, Deborah E.

    1990-01-01

    Described are awareness activities that deal with human population growth, resources, and the environment. Activities include simulations, mathematical exercises, and discussions of the topic. Specific examples of what individuals can do to help are listed. (KR)

  17. Major operations and activities

    SciTech Connect

    Black, D.G.

    1995-06-01

    This section of the 1994 Hanford Site Environmental Report summarizes the major operations and activities on the site. These operations and activities include site management, waste management, environmental restoration and corrective actions, and research and technology development.

  18. Family Activities for Fitness

    ERIC Educational Resources Information Center

    Grosse, Susan J.

    2009-01-01

    This article discusses how families can increase family togetherness and improve physical fitness. The author provides easy ways to implement family friendly activities for improving and maintaining physical health. These activities include: walking, backyard games, and fitness challenges.

  19. Active at Any Size

    MedlinePlus

    ... beginners. Daily life activities Lifestyle activities, such as gardening or washing the car, are great ways to ... bags. Doing chores like lawn mowing, raking leaves, gardening, and housework also count. What questions should I ...

  20. WASTE ACTIVATED SLUDGE PROCESSING

    EPA Science Inventory

    A study was made at pilot scale of a variety of processes for dewatering and stabilization of waste activated sludge from a pure oxygen activated sludge system. Processes evaluated included gravity thickening, dissolved air flotation thickening, basket centrifugation, scroll cent...

  1. Green Schools Activity Booklet.

    ERIC Educational Resources Information Center

    Sacramento Tree Foundation, CA.

    This collection of interdisciplinary hands-on activities covers a variety of topics related to trees and conservation. Twenty-four activities integrate the subjects of social studies, fine arts, science, language arts, math, geography, and music. Although activity instructions are not consistent they usually contain details on objectives and…

  2. Technology Systems. Laboratory Activities.

    ERIC Educational Resources Information Center

    Brame, Ray; And Others

    This guide contains 43 modules of laboratory activities for technology education courses. Each module includes an instructor's resource sheet and the student laboratory activity. Instructor's resource sheets include some or all of the following elements: module number, course title, activity topic, estimated time, essential elements, objectives,…

  3. Climate Change: An Activity.

    ERIC Educational Resources Information Center

    Lewis, Garry

    1995-01-01

    Presents a segment of the Geoscience Education booklet, Climate Change, that contains information and activities that enable students to gain a better appreciation of the possible effects human activity has on the Earth's climate. Describes the Terrace Temperatures activity that leads students through an investigation using foraminifera data to…

  4. Bonus Activity Book.

    ERIC Educational Resources Information Center

    Learning, 1992

    1992-01-01

    Provides on-task activities to fill in unexpected extra moments in elementary classes. The activities require little preparation and take 5-15 minutes to complete. There are activities for math, language arts, social science, science, critical thinking, and computer. An outer space board game is also included. (SM)

  5. Activity Sheets. Draft Copy.

    ERIC Educational Resources Information Center

    Duke Power Company, Educational Services Dept., Charlotte, NC.

    This document consists of energy vocabulary activities, three games, worksheets, laboratory activities/exercises, and an introductory classroom exercise designed to introduce energy concepts to students. Vocabulary activities focus on coal and energy consumption. The three games (with instructions) focus on various aspects of energy and energy…

  6. Hepatitis and activity

    PubMed Central

    Krikler, Dennis M.

    1971-01-01

    The effects of physical activity during an attack of infectious hepatitis are discussed. There is no evidence that activity during convalescence produces any ill-effects. On the other hand, strenuous physical activity in the acute stage may be dangerous, possibly because hepatic blood-flow is reduced. PMID:5560143

  7. Measurement of Physical Activity.

    ERIC Educational Resources Information Center

    Dishman, Rod K.; Washburn, Richard A.; Schoeller, Dale A.

    2001-01-01

    Valid assessment of physical activity must be unobtrusive, practical to administer, and specific about physical activity type, frequency, duration, and intensity. Assessment methods can be categorized according to whether they provide direct or indirect (e.g., self-report) observation of physical activity, body motion, physiological response…

  8. Activity Theory and Ontology

    ERIC Educational Resources Information Center

    Peim, Nick

    2009-01-01

    This paper seeks to re-examine Yrio Engestrom's activity theory as a technology of knowledge designed to enable positive transformations of specific practices. The paper focuses on a key paper where Engestrom defines the nature and present state of activity theory. Beginning with a brief account of the relations between activity theory and…

  9. Technology Learning Activities I.

    ERIC Educational Resources Information Center

    International Technology Education Association, Reston, VA.

    This guide contains 30 technology learning activities. Activities may contain all or some of the following: an introduction, objectives, materials and equipment, challenges, limitations, notes and investigations, resources and references used, and evaluation ideas. Activity titles are: (1) Occupations in Construction Technology; (2) Designing a…

  10. Woodsy Owl Activity Guide.

    ERIC Educational Resources Information Center

    Forest Service (USDA), Washington, DC.

    This guide offers teachers and after-school group leaders 12 fun and engaging activities. Activities feature lessons on trees, water, wind, the earth, food, and waste. The activities are designed to help children aged 5-8 become more aware of the natural environment and fundamental conservation principles. Titles of children's books are embedded…

  11. FLES Games and Activities.

    ERIC Educational Resources Information Center

    Irujo, Suzanne, Ed.

    A number of activities for teaching foreign language in the elementary school (FLES) are presented. The activities were developed by participants in a FLES teacher training workshop, Project INTERACT, in the Boston area. The first section contains games, thematic units, and other activities specifically related to French language instruction,…

  12. FL Activities & Festivals.

    ERIC Educational Resources Information Center

    American Council on the Teaching of Foreign Languages, Hastings-on-Hudson, NY.

    A collection of student, class, and school foreign language activities suggests a variety of projects and describes three specific school efforts. The suggested activities include: (1) individual student efforts such as writing to pen-pals; (2) group activities such as a foreign language auction or sing-along; (3) group projects for the school…

  13. Highlights of 1981 activities

    NASA Technical Reports Server (NTRS)

    1981-01-01

    The highlights of NASA's 1981 activities are presented, including the results of the two flights of the space shuttle Columbia and the Voyager 2 encounter with Saturn. Accomplishments in the areas of space transportation operations; space science; aeronautical, energy, and space research and development; as well as space tracking, international activities, and 1981 launch activities are discussed.

  14. Anticancer activity of ferrocenylthiosemicarbazones.

    PubMed

    Sandra, Cortez-Maya; Elena, Klimova; Marcos, Flores-Alamo; Elena, Martínez-Klimova; Arturo, Ramírez-Ramírez; Teresa, Ramírez Apan; Marcos, Martínez-García

    2014-03-01

    Aliphatic and aromatic ferrocenylthiosemicarbazones were synthesized. The characterization of the new ferrocenylthiosemicarbazones was done by IR, (1)H-NMR and (13)C-NMR spectroscopy, elemental analysis and X-ray diffraction studies. The biological activity of the obtained compounds was assessed in terms of anticancer activity. Their activity against U251 (human glyoblastoma), PC-3 (human prostatic adenocarcinoma), K562 (human chronic myelogenous leukemia), HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma) and SKLU-1 (human lung adenocarcinoma) cell lines was studied and compared with cisplatin. All tested compounds showed good activity and the aryl-chloro substituted ferrocenylthiosemicarbazones showed the best anticancer activity. PMID:24144199

  15. Transcriptional activators in yeast

    PubMed Central

    2006-01-01

    Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested ∼6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix–loop–helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2). PMID:16464826

  16. Vestibular activation of sympathetic nerve activity

    NASA Technical Reports Server (NTRS)

    Ray, C. A.; Carter, J. R.

    2003-01-01

    AIM: The vestibulosympathetic reflex refers to sympathetic nerve activation by the vestibular system. Animal studies indicate that the vestibular system assists in blood pressure regulation during orthostasis. Although human studies clearly demonstrate activation of muscle sympathetic nerve activity (MSNA) during engagement of the otolith organs, the role of the vestibulosympathetic reflex in maintaining blood pressure during orthostasis is not well-established. Examination of the vestibulosympathetic reflex with other cardiovascular reflexes indicates that it is a powerful and independent reflex. Ageing, which is associated with an increased risk for orthostatic hypotension, attenuates the vestibulosympathetic reflex. The attenuated reflex is associated with a reduction in arterial pressure. CONCLUSION: These findings suggest that the vestibulosympathetic reflex assists in blood pressure regulation in humans, but future studies examining this reflex in other orthostatically intolerant populations are necessary to address this hypothesis.

  17. Activated carbon from biomass

    NASA Astrophysics Data System (ADS)

    Manocha, S.; Manocha, L. M.; Joshi, Parth; Patel, Bhavesh; Dangi, Gaurav; Verma, Narendra

    2013-06-01

    Activated carbon are unique and versatile adsorbents having extended surface area, micro porous structure, universal adsorption effect, high adsorption capacity and high degree of surface reactivity. Activated carbons are synthesized from variety of materials. Most commonly used on a commercial scale are cellulosic based precursors such as peat, coal, lignite wood and coconut shell. Variation occurs in precursors in terms of structure and carbon content. Coir having very low bulk density and porous structure is found to be one of the valuable raw materials for the production of highly porous activated carbon and other important factor is its high carbon content. Exploration of good low cost and non conventional adsorbent may contribute to the sustainability of the environment and offer promising benefits for the commercial purpose in future. Carbonization of biomass was carried out in a horizontal muffle furnace. Both carbonization and activation were performed in inert nitrogen atmosphere in one step to enhance the surface area and to develop interconnecting porosity. The types of biomass as well as the activation conditions determine the properties and the yield of activated carbon. Activated carbon produced from biomass is cost effective as it is easily available as a waste biomass. Activated carbon produced by combination of chemical and physical activation has higher surface area of 2442 m2/gm compared to that produced by physical activation (1365 m2/gm).

  18. Function of miR-146a in controlling Treg cell-mediated regulation of Th1 responses

    PubMed Central

    Lu, Li-Fan; Boldin, Mark P.; Chaudhry, Ashutosh; Lin, Ling-Li; Taganov, Konstantin D.; Hanada, Toshikatsu; Yoshimura, Akihiko; Baltimore, David; Rudensky, Alexander Y.

    2010-01-01

    Summary Foxp3+ regulatory T (Treg) cells maintain immune homeostasis by limiting different types of inflammatory responses. Here, we report that miR-146a, one of the miRNAs prevalently expressed in Treg cells, is critical for their suppressor function. The deficiency of miR-146a in Treg cells resulted in a breakdown of immunological tolerance manifested in a fatal IFNγ-dependent immune-mediated lesions in a variety of organs. This was likely due to augmented expression and activation of signal transducer and activator transcription 1 (Stat1), a direct target of miR146a. Likewise, heightened Stat1 activation in Treg cells subjected to a selective ablation of SOCS1, a key negative regulator of Stat1 phosphorylation downstream of IFNγ receptor, was associated with analogous Th1-mediated pathology. Our results suggest that specific aspects of Treg suppressor function are controlled by a single miRNA and that an optimal range of Stat1 activation is important for Treg-mediated control of Th1 responses and associated autoimmunity. PMID:20850013

  19. Marine Biology Activities. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  20. Patterns in Active Nematics

    NASA Astrophysics Data System (ADS)

    Yeomans, Julia M.

    Active systems, from bacterial suspensions to cellular monolayers, are continuously driven out of equilibrium by local injection of energy from their constituent elements and exhibit turbulent-like, chaotic patterns. We describe how active systems can be stabilised by tuning a physical feature of the system, friction. We demonstrate how the crossover between wet active systems, whose behaviour is dominated by hydrodynamics, and dry active matter where any flow is screened, can be achieved by using friction as a control parameter and demonstrate vortex ordering at the wet-dry crossover. We show that the self organisation of vortices into lattices is accompanied by the spatial ordering of topological defects leading to active crystal-like structures. The emergence of vortex lattices which leads to the positional ordering of topological defects may be a useful step towards the design and control of active materials.

  1. Thermally Activated Driver

    NASA Technical Reports Server (NTRS)

    Kinard, William H.; Murray, Robert C.; Walsh, Robert F.

    1987-01-01

    Space-qualified, precise, large-force, thermally activated driver (TAD) developed for use in space on astro-physics experiment to measure abundance of rare actinide-group elements in cosmic rays. Actinide cosmic rays detected using thermally activated driver as heart of event-thermometer (ET) system. Thermal expansion and contraction of silicone oil activates driver. Potential applications in fluid-control systems where precise valve controls are needed.

  2. Activity in distant comets

    NASA Technical Reports Server (NTRS)

    Luu, Jane X.

    1992-01-01

    Activity in distant comets remains a mystery in the sense that we still have no complete theory to explain the various types of activity exhibited by different comets at large distances. This paper explores the factors that should play a role in determining activity in a distant comet, especially in the cases of comet P/Tempel 2, comet Schwassmann-Wachmann 1, and 2060 Chiron.

  3. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  4. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  5. Physical Activity and Cancer

    MedlinePlus

    ... of scientists, ranging from experts in basic biological science to those with expertise in community behavioral interventions to increase physical activity. This combination of scientists and expertise will ...

  6. NASA metrication activities

    NASA Technical Reports Server (NTRS)

    Vlannes, P. N.

    1978-01-01

    NASA's organization and policy for metrification, history from 1964, NASA participation in Federal agency activities, interaction with nongovernmental metrication organizations, and the proposed metrication assessment study are reviewed.

  7. Active material based active sealing technology: Part 1. Active seal requirements vs. active material actuator properties

    NASA Astrophysics Data System (ADS)

    Henry, Christopher P.; Carter, William; Herrera, Guillermo A.; McKnight, Geoffrey P.; Browne, Alan L.; Johnson, Nancy L.; Bazzi, Imad F.

    2010-04-01

    Current seals used for vehicle closures/swing panels are essentially flexible, frequently hollow structures whose designs are constrained by numerous requirements, many of them competing, including door closing effort (both air bind and seal compression), sound isolation, prevention of water leaks, and accommodation of variations in vehicle build. This paper documents the first portion of a collaborative research study/exploration of the feasibility of and approaches for using active materials with shape and stiffness changing attributes to produce active seal technologies, seals with improved performance. An important design advantage of an active material approach compared to previous active seal technologies is the distribution of active material regions throughout the seal length, which would enable continued active function even with localized failure. Included as a major focus of this study was the assessment of polymeric active materials because of their potential ease of integration into the current seal manufacturing process. In Part 1 of this study, which is documented in this paper, potential materials were evaluated in terms of their cost, activation mechanisms, and mechanical and actuation properties. Based on these properties, simple designs were proposed and utilized to help determine which materials are best suited for active seals. Shape memory alloys (SMA) and electroactive polymers (EAP) were judged to be the most promising.

  8. Active Flow Control Activities at NASA Langley

    NASA Technical Reports Server (NTRS)

    Anders, Scott G.; Sellers, William L., III; Washburn, Anthony E.

    2004-01-01

    NASA Langley continues to aggressively investigate the potential advantages of active flow control over more traditional aerodynamic techniques. This paper provides an update to a previous paper and describes both the progress in the various research areas and the significant changes in the NASA research programs. The goals of the topics presented are focused on advancing the state of knowledge and understanding of controllable fundamental mechanisms in fluids as well as to address engineering challenges. An organizational view of current research activities at NASA Langley in active flow control as supported by several projects is presented. On-center research as well as NASA Langley funded contracts and grants are discussed at a relatively high level. The products of this research are to be demonstrated either in bench-top experiments, wind-tunnel investigations, or in flight as part of the fundamental NASA R&D program and then transferred to more applied research programs within NASA, DOD, and U.S. industry.

  9. Activating Event Knowledge

    ERIC Educational Resources Information Center

    Hare, Mary; Jones, Michael; Thomson, Caroline; Kelly, Sarah; McRae, Ken

    2009-01-01

    An increasing number of results in sentence and discourse processing demonstrate that comprehension relies on rich pragmatic knowledge about real-world events, and that incoming words incrementally activate such knowledge. If so, then even outside of any larger context, nouns should activate knowledge of the generalized events that they denote or…

  10. Endonuclease activity in lipocalins.

    PubMed Central

    Yusifov, T N; Abduragimov, A R; Gasymov, O K; Glasgow, B J

    2000-01-01

    Several lipocalins contain conserved amino acid sequences similar to the phosphodiester bond cleavage domain of sugar non-specific magnesium-dependent nucleases of the Serratia marcescens type. His-89 and Glu-127 of the S. marcescens endonuclease are believed to have a role in the active catalytic site by the attack of a water molecule at the phosphorus atom of the bridging phosphate. Tear lipocalin contains both amino acids in analogous regions, and is active as a nuclease. Two forms of beta-lactoglobulin contain only Glu-134 (analogous to Glu-127 of the Serratia nuclease) yet retain nuclease activity equal to or greater than that of tear lipocalin. However, retinol-binding protein lacks both of these motifs and shows no detectable activity. DNA-nicking activity is decreased by 80% in the mutant of tear lipocalin that replaces Glu-128 but is unchanged by mutations of His-84. The endonuclease activity of tear lipocalin is dependent on the bivalent cations Mg(2+) or Mn(2+) but is decreased at high concentrations of NaCl. These findings indicate that some lipocalins have non-specific endonuclease activity similar in characteristics to the Mg(2+)-dependent nucleases and related to the conserved sequence LEDFXR (where 'X' denotes 'any other residue'), in which the glutamic residue seems to be important for activity. PMID:10769187

  11. Endonuclease activity in lipocalins.

    PubMed

    Yusifov, T N; Abduragimov, A R; Gasymov, O K; Glasgow, B J

    2000-05-01

    Several lipocalins contain conserved amino acid sequences similar to the phosphodiester bond cleavage domain of sugar non-specific magnesium-dependent nucleases of the Serratia marcescens type. His-89 and Glu-127 of the S. marcescens endonuclease are believed to have a role in the active catalytic site by the attack of a water molecule at the phosphorus atom of the bridging phosphate. Tear lipocalin contains both amino acids in analogous regions, and is active as a nuclease. Two forms of beta-lactoglobulin contain only Glu-134 (analogous to Glu-127 of the Serratia nuclease) yet retain nuclease activity equal to or greater than that of tear lipocalin. However, retinol-binding protein lacks both of these motifs and shows no detectable activity. DNA-nicking activity is decreased by 80% in the mutant of tear lipocalin that replaces Glu-128 but is unchanged by mutations of His-84. The endonuclease activity of tear lipocalin is dependent on the bivalent cations Mg(2+) or Mn(2+) but is decreased at high concentrations of NaCl. These findings indicate that some lipocalins have non-specific endonuclease activity similar in characteristics to the Mg(2+)-dependent nucleases and related to the conserved sequence LEDFXR (where 'X' denotes 'any other residue'), in which the glutamic residue seems to be important for activity. PMID:10769187

  12. Activity Book: Ocean Ecology.

    ERIC Educational Resources Information Center

    Learning, 1992

    1992-01-01

    Presents a collection of activities to help elementary students study ocean ecology. The activities have students investigate ocean inhabitants, analyze animal adaptations, examine how temperature and saltiness affect ocean creatures, and learn about safeguarding the sea. Student pages offer reproducible learning sheets. (SM)

  13. Activity Book. Celebrating Diversity.

    ERIC Educational Resources Information Center

    Angell, Pat; And Others

    1993-01-01

    This activity book presents elementary level multicultural books designed to foster self-esteem, understanding, and critical reading skills. It includes a resource list and general discussion boosters for analyzing characters and setting, class activities related to the books, and a student page on character study. (SM)

  14. Active Students in Webinars

    ERIC Educational Resources Information Center

    Kolås, Line; Nordseth, Hugo; Yri, Jørgen Sørlie

    2015-01-01

    To ensure student activity in webinars we have defined 10 learning tasks focusing on production and communication e.g. collaborative writing, discussion and polling, and investigated how the technology supports the learning activities. The three project partners in the VisPed-project use different video-conferencing systems, and we analyzed how it…

  15. PM ACTIVITY PATTERN RESEARCH

    EPA Science Inventory

    Human activity/uptake rate data are necessary to estimate potential human exposure and intake dose to environmental pollutants and to refine human exposure models. Personal exposure monitoring studies have demonstrated the critical role that activities play in explaining and pre...

  16. Activation of fly ash

    DOEpatents

    Corbin, David R.; Velenyi, Louis J.; Pepera, Marc A.; Dolhyj, Serge R.

    1986-01-01

    Fly ash is activated by heating a screened magnetic fraction of the ash in a steam atmosphere and then reducing, oxidizing and again reducing the hydrothermally treated fraction. The activated fly ash can be used as a carbon monoxide disproportionating catalyst useful in the production of hydrogen and methane.

  17. Laboratory Activities in Israel

    ERIC Educational Resources Information Center

    Mamlok-Naaman, Rachel; Barnea, Nitza

    2012-01-01

    Laboratory activities have long had a distinctive and central role in the science curriculum, and science educators have suggested that many benefits accrue from engaging students in science laboratory activities. Many research studies have been conducted to investigate the educational effectiveness of laboratory work in science education in…

  18. Active Healthy Summer

    ERIC Educational Resources Information Center

    Elliott, Eloise

    2005-01-01

    Summer break is almost here for most elementary teachers and students. Warmer weather and additional free time to make choices create more opportunities to be physically active, whether home alone or out with friends and family. This article describes ways by which physical education specialists can encourage students' physical activity by…

  19. Ecological Structure Activity Relationships

    EPA Science Inventory

    Ecological Structure Activity Relationships, v1.00a, February 2009
    ECOSAR (Ecological Structure Activity Relationships) is a personal computer software program that is used to estimate the toxicity of chemicals used in industry and discharged into water. The program predicts...

  20. The Activity of Trypsin

    ERIC Educational Resources Information Center

    Russo, Salvatore F.; Holzman, Tom

    1977-01-01

    Describes an experiment that illustrates the following points concerning the experimental determination of trypsin activity: (1) there is a difference in basing enzyme concentration on weight, absorbance, or active sites; and (2) the method of expressing enzyme concentration determines the value of specific, molecular, and catalytic center…

  1. Obesity, Physical Activity - Children.

    ERIC Educational Resources Information Center

    Gilliam, Thomas B.

    Childhood obesity starts at a very early age, and preventive measures taken early enough may retard the development of fat cells. It appears that physical activity plays an important role in reducing obesity. The activity program must start early, in preschool days. It is felt that screening children for obesity when they first enter school and…

  2. Warm-Up Activities.

    ERIC Educational Resources Information Center

    Mingguang, Yang

    1999-01-01

    Discusses how warm-up activities can help to make the English-as-a-foreign-language classroom a lively and interesting place. Warm-up activities are games carried out at the beginning of each class to motivate students to make good use of class time. (Author/VWL)

  3. Directory of Development Activities.

    ERIC Educational Resources Information Center

    Control Data Corp., Minneapolis, Minn.

    Assembled in a loose leaf notebook, this collection of independent on-the-job activities is designed to facilitate employee development and intended to help improve an organization's performance appraisal system. The on-the-job development activities described derive from job descriptions, performance appraisal forms, and discussions with job…

  4. Active and Healthy Schools

    ERIC Educational Resources Information Center

    Ball, Stephen; Kovarik, Jessica; Leidy, Heather

    2015-01-01

    The Active and Healthy School Program (AHS) can be used to alter the culture and environment of a school to help children make healthier choices. The purpose of this study was to determine the effectiveness of AHS to increase physical activity while decreasing total screen time, increase healthy food choices, and improve knowledge about physical…

  5. Chemical Activities. Teacher Edition.

    ERIC Educational Resources Information Center

    Borgford, Christie L.; Summerlin, Lee R.

    This sourcebook for chemical activities is designed to be used as a student laboratory book for both junior and senior high school students. The student's role as a knowledgeable consumer and informed citizen is stressed. Each activity includes a list of needed materials, procedures, reactions, questions, and notes for the teacher which include…

  6. Rainy Day Activities.

    ERIC Educational Resources Information Center

    Texas Child Care, 1997

    1997-01-01

    Experienced caregivers plan ahead for rainy days. This article describes specific rainy day activities for young children, such as books and crafts to learn about rain (rain in a jar, making a rainbow), simple cooking activities (taffy pull, cinnamon candy tea), and games (mummy wrap, hunt the thimble, rain lotto). (EV)

  7. Reflections on Activity Theory

    ERIC Educational Resources Information Center

    Bakhurst, David

    2009-01-01

    It is sometimes suggested that activity theory represents the most important legacy of Soviet philosophy and psychology. But what exactly "is" activity theory? The canonical account in the West is given by Engestrom, who identifies three stages in the theory's development: from Vygotsky's insights, through Leontiev's articulation of the…

  8. Emotionally Intense Science Activities

    ERIC Educational Resources Information Center

    King, Donna; Ritchie, Stephen; Sandhu, Maryam; Henderson, Senka

    2015-01-01

    Science activities that evoke positive emotional responses make a difference to students' emotional experience of science. In this study, we explored 8th Grade students' discrete emotions expressed during science activities in a unit on Energy. Multiple data sources including classroom videos, interviews and emotion diaries completed at the end of…

  9. Activities: More Calculator Capers.

    ERIC Educational Resources Information Center

    Schmalz, Rosemary

    1983-01-01

    Provided is an activity designed to give grades 7-12 students opportunities to discover numerical patterns and to derive general conclusions from observing data. The activity focuses attention on patterns in products such as 33x34, 333x334, and 3333x3334. Three worksheets and answers are included. (JN)

  10. Vegetable Soup Activities.

    ERIC Educational Resources Information Center

    Shepard, Mary; Shepard, Ray

    Vegetable Soup is a new children's television series whose purpose is to counter the negative and destructive effects of racial isolation. This manual gives detailed instructions for discussion of activities that are presented during the television series such as: crafts, games, recipes, language activities, and children's questions. A list of…

  11. ZOOMsci Activity Guide.

    ERIC Educational Resources Information Center

    Wade, Meredith

    This activity guide is based on the Public Broadcasting System's (PBS) program "ZOOM." It is designed for educators with activities that are categorized into three themes: (1) Things That Go, which includes "Air" which explores air pressure, "Rubber Bands" which discovers the potential energy of rubber bands, "Baking Soda and Vinegar" which…

  12. Science World Activities Book.

    ERIC Educational Resources Information Center

    Wisconsin Academy of Sciences, Arts and Letters, Madison.

    This document consists of three sections. Section I contains 19 activities developed by master teachers for the Science World '84 summer science program. These activities focus on studies involving airplane controls, trash bag kites, computers, meteorology, compass orienteering, soils, aquatic ecosystems, bogs, and others. Objectives, materials…

  13. HUMAN EXPOSURE ACTIVITY PATTERNS

    EPA Science Inventory

    Human activity/uptake rate data are necessary to estimate potential human exposure and intake dose to environmental pollutants and to refine human exposure models. Personal exposure monitoring studies have demonstrated the critical role that activities play in explaining and pre...

  14. Coordinating Shared Activities

    NASA Technical Reports Server (NTRS)

    Clement, Bradley

    2004-01-01

    Shared Activity Coordination (ShAC) is a computer program for planning and scheduling the activities of an autonomous team of interacting spacecraft and exploratory robots. ShAC could also be adapted to such terrestrial uses as helping multiple factory managers work toward competing goals while sharing such common resources as floor space, raw materials, and transports. ShAC iteratively invokes the Continuous Activity Scheduling Planning Execution and Replanning (CASPER) program to replan and propagate changes to other planning programs in an effort to resolve conflicts. A domain-expert specifies which activities and parameters thereof are shared and reports the expected conditions and effects of these activities on the environment. By specifying these conditions and effects differently for each planning program, the domain-expert subprogram defines roles that each spacecraft plays in a coordinated activity. The domain-expert subprogram also specifies which planning program has scheduling control over each shared activity. ShAC enables sharing of information, consensus over the scheduling of collaborative activities, and distributed conflict resolution. As the other planning programs incorporate new goals and alter their schedules in the changing environment, ShAC continually coordinates to respond to unexpected events.

  15. Elementary Environmental Activities.

    ERIC Educational Resources Information Center

    Larson, Robert J.

    This guide presents suggestions for field trips, out-of-doors activities, material for centers, and individualized activities in the teaching of elementary school science and particularly environmental education at the elementary level. The guide includes a section on preparation and procedures for conducting field trips, including sample…

  16. Ten Minute Writing Activities.

    ERIC Educational Resources Information Center

    Markus, Sharyn

    Designed with junior high school students in mind, the activities in this booklet are offered as ways to stimulate interest in writing using as little as ten minutes of class time. The activities are arranged in six sections: (1) developing observation skills and paying attention to details; (2) word play, descriptive words, and word collections…

  17. Activation of fly ash

    DOEpatents

    Corbin, D.R.; Velenyi, L.J.; Pepera, M.A.; Dolhyj, S.R.

    1986-08-19

    Fly ash is activated by heating a screened magnetic fraction of the ash in a steam atmosphere and then reducing, oxidizing and again reducing the hydrothermally treated fraction. The activated fly ash can be used as a carbon monoxide disproportionating catalyst useful in the production of hydrogen and methane.

  18. Peak Longevity Physical Activity

    Cancer.gov

    People who engage in three to five times the recommended minimum level of leisure-time physical activity derive the greatest benefit in terms of mortality reduction when compared with people who do not engage in leisure-time physical activity.

  19. Student Activities. Managing Liability.

    ERIC Educational Resources Information Center

    Bennett, Barbara; And Others

    This monograph suggests ways that college or university administrations can undertake a systematic and careful review of the risks posed by students' activities. Its purpose is to provide guidance in integrating the risk management process into a school's existing approaches to managing student organizations and activities. It is noted that no…

  20. Curriculum Activities on Aging.

    ERIC Educational Resources Information Center

    Schmall, Vicki L.; Benge, Nancy

    This paper contains learning activities on aging for use with elementary, high school, and university students in health, family relationships, social studies, and art courses. The activities are intended to help youth develop a more realistic understanding of the aging process and to become aware of both the problems and benefits associated with…

  1. Aging and Semantic Activation.

    ERIC Educational Resources Information Center

    Howard, Darlene V.

    Three studies tested the theory that long term memory consists of a semantically organized network of concept nodes interconnected by leveled associations or relations, and that when a stimulus is processed, the corresponding concept node is assumed to be temporarily activated and this activation spreads to nearby semantically related nodes. In…

  2. [Field Learning Activities].

    ERIC Educational Resources Information Center

    Nolde Forest Environmental Education Center, Reading, PA.

    Seventy field activities, pertinent to outdoor, environmental studies, are described in this compilation. Designed for elementary and junior high school students, the activities cover many discipline areas--science, social studies, language arts, health, history, mathematics, and art--and many are multidisciplinary in use. Topics range from soil…

  3. Active galactic nuclei

    PubMed Central

    Fabian, Andrew C.

    1999-01-01

    Active galactic nuclei are the most powerful, long-lived objects in the Universe. Recent data confirm the theoretical idea that the power source is accretion into a massive black hole. The common occurrence of obscuration and outflows probably means that the contribution of active galactic nuclei to the power density of the Universe has been generally underestimated. PMID:10220363

  4. Activity in F stars

    NASA Technical Reports Server (NTRS)

    Wolff, Sidney C.; Boesgaard, Ann Merchant; Simon, Theodore

    1986-01-01

    Measurements of He I 5876 A and IUE measurements of chromospheric and transition region lines in a large sample of F-type stars are presented. The data show that activity is detectable in nearly all early F-type stars and differs in several of its characteristics from that typically seen in cooler stars with slow rotation and fully developed convective zones. The onset of activity occurs near B-V = 0.28, which corresponds approximately to spectral type F0 and T(eff) = 7300 K. There is no correlation between the level of activity and the abundances of lithium and beryllium in F stars hotter than T(eff) = 6600 K. All but one of the stars in the 6600-7300 K temperature interval are active. The levels of activity in these stars are independent of Rossby number.

  5. Determinants of Physical Activity in Active and Low-Active, Sixth Grade African-American Youth.

    ERIC Educational Resources Information Center

    Trost, Stewart G.; Pate, Russell R.; Ward, Dianne S.; Saunders, Ruth; Riner, William

    1999-01-01

    Compared determinants of physical activity in active and low-active African-American sixth graders, surveying students and making objective assessments of physical activity over seven days. Results indicated that physical activity self-efficacy, beliefs about physical activity outcomes, involvement in community-based physical activity, perception…

  6. Active optical zoom system

    DOEpatents

    Wick, David V.

    2005-12-20

    An active optical zoom system changes the magnification (or effective focal length) of an optical imaging system by utilizing two or more active optics in a conventional optical system. The system can create relatively large changes in system magnification with very small changes in the focal lengths of individual active elements by leveraging the optical power of the conventional optical elements (e.g., passive lenses and mirrors) surrounding the active optics. The active optics serve primarily as variable focal-length lenses or mirrors, although adding other aberrations enables increased utility. The active optics can either be LC SLMs, used in a transmissive optical zoom system, or DMs, used in a reflective optical zoom system. By appropriately designing the optical system, the variable focal-length lenses or mirrors can provide the flexibility necessary to change the overall system focal length (i.e., effective focal length), and therefore magnification, that is normally accomplished with mechanical motion in conventional zoom lenses. The active optics can provide additional flexibility by allowing magnification to occur anywhere within the FOV of the system, not just on-axis as in a conventional system.

  7. Physical activity and cancer.

    PubMed

    Shephard, R J

    1990-12-01

    Evidence that physical activity may protect against various forms of cancer is examined in relation to occupational demands, leisure activities and participation in sport while at university. The variety of forms of neoplasm and equally varied physical activity histories militate against finding any simple relationship between the risk of malignancy and the individual's physical activity history. Nevertheless, five of seven major occupational studies suggest that a physically active occupation offers some protection against colon cancer, and an application of Bradford Hill's criteria generally supports the causal nature of the relationship between physical inactivity and an increased risk of intestinal neoplasia. However, existing reports are by no means conclusive; there thus remains a need for well-designed epidemiological studies of this issue. Data from one laboratory also suggest that in women a history of active leisure is associated with a reduced prevalence of breast and reproductive system cancers. Physical activity potentially encourages a healthy lifestyle, and it could have more direct effects on certain forms of carcinogenesis (for instance, by a speeding of gastro-intestinal transit, or a moderation of sex hormone levels). However, there are also potential negative effects from some types of exercise, particularly an excessive exposure to ultra-violet light in certain water sports. Since moderate exercise elevates mood and helps to conserve lean tissue, it may finally be a helpful component of treatment after a neoplasm has been diagnosed. PMID:2286478

  8. Active touch sensing

    PubMed Central

    Prescott, Tony J.; Diamond, Mathew E.; Wing, Alan M.

    2011-01-01

    Active sensing systems are purposive and information-seeking sensory systems. Active sensing usually entails sensor movement, but more fundamentally, it involves control of the sensor apparatus, in whatever manner best suits the task, so as to maximize information gain. In animals, active sensing is perhaps most evident in the modality of touch. In this theme issue, we look at active touch across a broad range of species from insects, terrestrial and marine mammals, through to humans. In addition to analysing natural touch, we also consider how engineering is beginning to exploit physical analogues of these biological systems so as to endow robots with rich tactile sensing capabilities. The different contributions show not only the varieties of active touch—antennae, whiskers and fingertips—but also their commonalities. They explore how active touch sensing has evolved in different animal lineages, how it serves to provide rapid and reliable cues for controlling ongoing behaviour, and even how it can disintegrate when our brains begin to fail. They demonstrate that research on active touch offers a means both to understand this essential and primary sensory modality, and to investigate how animals, including man, combine movement with sensing so as to make sense of, and act effectively in, the world. PMID:21969680

  9. Measuring Physical Activity Environments

    PubMed Central

    Sallis, James F.

    2010-01-01

    Physical activity is usually done in specific types of places, referred to as physical activity environments. These often include parks, trails, fitness centers, schools, and streets. In recent years, scientific interest has increased notably in measuring physical activity environments. The present paper provides an historical overview of the contributions of the health, planning, and leisure studies fields to the development of contemporary measures. The emphasis is on attributes of the built environment that can be affected by policies to contribute to the promotion of physical activity. Researchers from health fields assessed a wide variety of built environment variables expected to be related to recreational physical activity. Settings of interest were schools, workplaces, and recreation facilities, and most early measures used direct observation methods with demonstrated inter-observer reliability. Investigators from the city planning field evaluated aspects of community design expected to be related to people’s ability to walk from homes to destinations. GIS was used to assess walkability defined by the 3Ds of residential density, land-use diversity, and pedestrian-oriented designs. Evaluating measures for reliability or validity was rarely done in the planning-related fields. Researchers in the leisure studies and recreation fields studied mainly people’s use of leisure time rather than physical characteristics of parks and other recreation facilities. Although few measures of physical activity environments were developed, measures of aesthetic qualities are available. Each of these fields made unique contributions to the contemporary methods used to assess physical activity environments. PMID:19285214

  10. Emotionally Intense Science Activities

    NASA Astrophysics Data System (ADS)

    King, Donna; Ritchie, Stephen; Sandhu, Maryam; Henderson, Senka

    2015-08-01

    Science activities that evoke positive emotional responses make a difference to students' emotional experience of science. In this study, we explored 8th Grade students' discrete emotions expressed during science activities in a unit on Energy. Multiple data sources including classroom videos, interviews and emotion diaries completed at the end of each lesson were analysed to identify individual student's emotions. Results from two representative students are presented as case studies. Using a theoretical perspective drawn from theories of emotions founded in sociology, two assertions emerged. First, during the demonstration activity, students experienced the emotions of wonder and surprise; second, during a laboratory activity, students experienced the intense positive emotions of happiness/joy. Characteristics of these activities that contributed to students' positive experiences are highlighted. The study found that choosing activities that evoked strong positive emotional experiences, focused students' attention on the phenomenon they were learning, and the activities were recalled positively. Furthermore, such positive experiences may contribute to students' interest and engagement in science and longer term memorability. Finally, implications for science teachers and pre-service teacher education are suggested.

  11. THE ACTIVE CENTAURS

    SciTech Connect

    Jewitt, David

    2009-05-15

    The Centaurs are recent escapees from the Kuiper Belt that are destined either to meet fiery oblivion in the hot inner regions of the solar system or to be ejected to the interstellar medium by gravitational scattering from the giant planets. Dynamically evolved Centaurs, when captured by Jupiter and close enough to the Sun for near-surface water ice to sublimate, are conventionally labeled as 'short-period' (specifically, Jupiter-family) comets. Remarkably, some Centaurs show comet-like activity even when far beyond the orbit of Jupiter, suggesting mass loss driven by a process other than the sublimation of water ice. We observed a sample of 23 Centaurs and found nine to be active, with mass-loss rates measured from several kg s{sup -1} to several tonnes s{sup -1}. Considered as a group, we find that the 'active Centaurs' in our sample have perihelia smaller than the inactive Centaurs (median 5.9 AU versus 8.7 AU), and smaller than the median perihelion distance computed for all known Centaurs (12.4 AU). This suggests that their activity is thermally driven. We consider several possibilities for the origin of the mass loss from the active Centaurs. Most are too cold for activity at the observed levels to originate via the sublimation of crystalline water ice. Solid carbon monoxide and carbon dioxide have the opposite problem: they are so volatile that they should drive activity in Centaurs at much larger distances than observed. We consider the possibility that activity in the Centaurs is triggered by the conversion of amorphous ice into the crystalline form accompanied by the release of trapped gases, including carbon monoxide. By imposing the condition that crystallization should occur when the crystallization time is shorter than the orbital period we find a qualitative match to the perihelion distribution of the active Centaurs and conclude that the data are consistent with the hypothesis that the Centaurs contain amorphous ice.

  12. Physics of solar activity

    NASA Technical Reports Server (NTRS)

    Sturrock, Peter A.

    1993-01-01

    The aim of the research activity was to increase our understanding of solar activity through data analysis, theoretical analysis, and computer modeling. Because the research subjects were diverse and many researchers were supported by this grant, a select few key areas of research are described in detail. Areas of research include: (1) energy storage and force-free magnetic field; (2) energy release and particle acceleration; (3) radiation by nonthermal electrons; (4) coronal loops; (5) flare classification; (6) longitude distributions of flares; (7) periodicities detected in the solar activity; (8) coronal heating and related problems; and (9) plasma processes.

  13. Extravehicular activity technology discipline

    NASA Technical Reports Server (NTRS)

    Webbon, Bruce W.

    1990-01-01

    Viewgraphs on extravehicular activity technology discipline for Space Station Freedom are presented. Topics covered include: extravehicular mobility unit; airlock and EMU support equipment; tools, mobility aids, and workstations; and telerobotic work aids interfaces.

  14. Microglial Activation & Chronic Neurodegeneration

    PubMed Central

    Lull, Melinda E.; Block, Michelle L.

    2010-01-01

    Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurodegenerative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including TNFα, NO, IL1-β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (ex. LPS or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss over time. While the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson’s Disease. Here, we review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype. PMID:20880500

  15. Activities: Pick's Rule

    ERIC Educational Resources Information Center

    Hirsch, Christian R.

    1974-01-01

    A series of activities are presented on worksheets marked off with lattice points (like a geoboard). Students are led to discover and apply Pick's Theorem for finding the area of a polygon whose vertices are lattice points. (JP)

  16. Finding Activities You Enjoy

    MedlinePlus

    ... choose physical activities that match your interests! Love music? Take dancing lessons. Sign up for an aerobics ... a Safe Environment Feel Down? Get Up—Emotional Benefits of Exercise STAY INFORMED Follow us on Twitter ...

  17. Exercise and activity - children

    MedlinePlus

    ... other organized sports (such as soccer, swimming, and dancing) Younger children cannot stick with the same activity ... in a water sprinkler, or splashing in puddles. Dance to music. Skate, ice-skate, skate-board, or ...

  18. French space activities

    NASA Technical Reports Server (NTRS)

    Blanc, R.

    1982-01-01

    The four main points of research and development of space programs by France are explained. The National Center of Space Studies is discussed, listing the missions of the Center and describing the activities of the staff.

  19. Island Watershed Activity.

    ERIC Educational Resources Information Center

    Benson, Rod

    2003-01-01

    Describes a 90-minute "Island Watershed" activity to help earth science students understand the concept of the water cycle. Introduces a surface waters unit appropriate for students in grades 7-10. Includes watershed project guidelines. (Author/KHR)

  20. Algorithm-development activities

    NASA Technical Reports Server (NTRS)

    Carder, Kendall L.

    1994-01-01

    The task of algorithm-development activities at USF continues. The algorithm for determining chlorophyll alpha concentration, (Chl alpha) and gelbstoff absorption coefficient for SeaWiFS and MODIS-N radiance data is our current priority.

  1. A Big Gulp Activity.

    ERIC Educational Resources Information Center

    Kelly, Bruce

    1997-01-01

    Explains how to implement an activity in which students measure the volume of their oral cavities. Enables students to develop skills in estimation, measurement, connections, statistics, applying concepts and procedures, and communication. (DDR)

  2. Active terahertz metamaterials

    SciTech Connect

    Chen, Hou-tong

    2009-01-01

    We demonstrate planar terahertz metamaterial devices enabling actively controllable transmission amplitude, phase, or frequency at room temperature via carrier depletion or photoexcitation in the semiconductor substrate or in semiconductor materials incorporated into the metamaterial structure.

  3. PRESSURE ACTIVATED SEALANT TECHNOLOGY

    SciTech Connect

    Michael A. Romano

    2004-04-01

    The objective of this project is to develop new, efficient, cost effective methods of internally sealing natural gas pipeline leaks through the application of differential pressure activated sealants. In researching the current state of the art for gas pipeline sealing technologies we concluded that if the project was successful, it appeared that pressure activated sealant technology would provide a cost effective alternative to existing pipeline repair technology. From our analysis of current field data for a 13 year period from 1985 to 1997 we were able to identify 205 leaks that were candidates for pressure activated sealant technology, affirming that pressure activated sealant technology is a viable option to traditional external leak repairs. The data collected included types of defects, areas of defects, pipe sizes and materials, incident and operating pressures, ability of pipeline to be pigged and corrosion states. This data, and subsequent analysis, was utilized as a basis for constructing applicable sealant test modeling.

  4. Intercreativity: Mapping Online Activism

    NASA Astrophysics Data System (ADS)

    Meikle, Graham

    How do activists use the Internet? This article maps a wide range of activist practice and research by applying and developing Tim Berners-Lee's concept of ‘intercreativity' (1999). It identifies four dimensions of Net activism: intercreative texts, tactics, strategies and networks. It develops these through examples of manifestations of Net activism around one cluster of issues: support campaigns for refugees and asylum seekers.

  5. Tinnitus activities treatment.

    PubMed

    Tyler, Richard S; Gogel, Stephanie A; Gehringer, Anne K

    2007-01-01

    Tinnitus Activities Treatment includes counseling of the whole person, and considers individual differences and needs. We consider four areas: thoughts and emotions, hearing and communication, sleep, and concentration. We typically use Partial Masking Sound Therapy, with a noise or music set to the lowest level that provides relief. A picture-based approach facilitates engagement of the patient, and provides thorough and structured counseling. We engage the patient by including homework and activities to demonstrate understanding and facilitate progress. PMID:17956807

  6. Low activation ferritic alloys

    DOEpatents

    Gelles, David S.; Ghoniem, Nasr M.; Powell, Roger W.

    1986-01-01

    Low activation ferritic alloys, specifically bainitic and martensitic stainless steels, are described for use in the production of structural components for nuclear fusion reactors. They are designed specifically to achieve low activation characteristics suitable for efficient waste disposal. The alloys essentially exclude molybdenum, nickel, nitrogen and niobium. Strength is achieved by substituting vanadium, tungsten, and/or tantalum in place of the usual molybdenum content in such alloys.

  7. RMS active damping augmentation

    NASA Technical Reports Server (NTRS)

    Gilbert, Michael G.; Scott, Michael A.; Demeo, Martha E.

    1992-01-01

    The topics are presented in viewgraph form and include: RMS active damping augmentation; potential space station assembly benefits to CSI; LaRC/JSC bridge program; control law design process; draper RMS simulator; MIMO acceleration control laws improve damping; potential load reduction benefit; DRS modified to model distributed accelerations; accelerometer location; Space Shuttle aft cockpit simulator; simulated shuttle video displays; SES test goals and objectives; and SES modifications to support RMS active damping augmentation.

  8. RAVEN Quality Assurance Activities

    SciTech Connect

    Cogliati, Joshua Joseph

    2015-09-01

    This report discusses the quality assurance activities needed to raise the Quality Level of Risk Analysis in a Virtual Environment (RAVEN) from Quality Level 3 to Quality Level 2. This report also describes the general RAVEN quality assurance activities. For improving the quality, reviews of code changes have been instituted, more parts of testing have been automated, and improved packaging has been created. For upgrading the quality level, requirements have been created and the workflow has been improved.

  9. Low activation ferritic alloys

    DOEpatents

    Gelles, D.S.; Ghoniem, N.M.; Powell, R.W.

    1985-02-07

    Low activation ferritic alloys, specifically bainitic and martensitic stainless steels, are described for use in the production of structural components for nuclear fusion reactors. They are designed specifically to achieve low activation characteristics suitable for efficient waste disposal. The alloys essentially exclude molybdenum, nickel, nitrogen and niobium. Strength is achieved by substituting vanadium, tungsten, and/or tantalum in place of the usual molybdenum content in such alloys.

  10. Double-stranded RNA-dependent protein kinase is required for bone calcification in MC3T3-E1 cells in vitro.

    PubMed

    Yoshida, Kaya; Okamura, Hirohiko; Amorim, Bruna Rabelo; Ozaki, Akiko; Tanaka, Hiroaki; Morimoto, Hiroyuki; Haneji, Tatsuji

    2005-11-15

    In this study, we demonstrated that double-stranded RNA-dependent protein kinase (PKR) is required for the calcification of osteoblasts via the signal transducers and activators of transcription 1alpha (STAT1alpha) signaling in vitro. A dominant-negative mutant PKR cDNA, in which the amino acid lysine at 296 was replaced with arginine and which does not have catalytic activity, was transfected into mouse osteoblastic MC3T3-E1 cells; thereby, we established cells that stably expressed the PKR mutant gene (PKR-K/R). Phosphorylation of PKR was not stimulated by polyinosic-polycytidylic acid in the mutant cells. The PKR-K/R mutant cells exhibited up-regulated cell growth and had low alkaline phosphatase (ALP) activity. The PKR-K/R mutant cells were not able to form bone nodules in vitro. In the PKR-K/R mutant cells, runt-related gene 2 (Runx2)-mediated transcription decreased compared with the levels in the control cells. The expression of STAT1alpha protein increased and the protein was translocated to the nucleus in the PKR-K/R mutant cells. When the expression of STAT1alpha protein in PKR mutant cells was suppressed using RNAi, the activity of Runx2-mediated transcription recovered to the control level. Our results indicate that PKR is a stimulator of Runx2 transcription and is a negative modulator of STAT1alpha expression. Our findings also suggest that PKR plays important roles in the differentiation and calcification of osteoblasts by modulating STAT1alpha and/or Runx2 expression. PMID:16216244

  11. Rheology of Active Gels

    NASA Astrophysics Data System (ADS)

    Chen, Daniel

    2015-03-01

    Active networks drive a diverse range of critical processes ranging from motility to division in living cells, yet a full picture of their rheological capabilities in non-cellular contexts is still emerging, e.g., How does the rheological response of a network capable of remodeling under internally-generated stresses differ from that of a passive biopolymer network? In order to address this and other basic questions, we have engineered an active gel composed of microtubules, bidirectional kinesin motors, and molecular depletant that self-organizes into a highly dynamic network of active bundles. The network continually remodels itself under ATP-tunable cycles of extension, buckling, fracturing, and self-healing. Using confocal rheometry we have simultaneously characterized the network's linear and non-linear rheological responses to shear deformation along with its dynamic morphology. We find several surprising and unique material properties for these active gels; most notably, rheological cloaking, the ability of the active gel to drive large-scale fluid mixing over several orders of flow magnitude while maintaining an invariant, solid-like rheological profile and spontaneous flow under confinement, the ability to exert micro-Newton forces to drive persistent directed motion of the rheometer tool. Taken together, these results and others to be discussed highlight the rich stress-structure-dynamics relationships in this class of biologically-derived active gels.

  12. ABB: active bandwidth broker

    NASA Astrophysics Data System (ADS)

    Wong, Kason; Law, Eddie

    2001-07-01

    In this paper, we shall discuss a novel design on the policy-based management for the Internet. This design deploys the concept of active networking. As opposed to the traditional network design, active network empowers network node with the ability to manipulate data and program code in packets, and configure the network properties according to the needs of different applications. The policy-based management can control network routers in order to realize end-to-end Quality of Service (QoS), such as differentiated and integrated services, across the Internet. For the moment, the Internet Engineering Task Force (IETF) has defined the framework of the policy-based management. It employs a simple client/server model that uses Common Open Policy Service (COPS) protocol to facilitate policy management and control. Our design of Active Bandwidth Broker (ABB) belongs to an active application. Our goals are to distribute centralized workload of the policy-based management over multiple active nodes in the active networks, introduce mobility of the bandwidth brokers, and allows load sharing to the policy-based management. This results a network-wide intelligent, highly available, and consistent QoS control that allows performance protection for voice, video and Internet business application while reducing costs for growing networks.

  13. Phytase activity in lichens.

    PubMed

    Higgins, Niall F; Crittenden, Peter D

    2015-10-01

    Phytase activity was investigated in 13 lichen species using a novel assay method. The work tested the hypothesis that phytase is a component of the suite of surface-bound lichen enzymes that hydrolyse simple organic forms of phosphorus (P) and nitrogen (N) deposited onto the thallus surface. Hydrolysis of inositol hexaphosphate (InsP6 , the substrate for phytase) and appearance of lower-order inositol phosphates (InsP5 -InsP1 ), the hydrolysis products, were measured by ion chromatography. Phytase activity in Evernia prunastri was compared among locations with contrasting rates of N deposition. Phytase activity was readily measurable in epiphytic lichens (e.g. 11.3 μmol InsP6 hydrolysed g(-1)  h(-1) in Bryoria fuscescens) but low in two terricolous species tested (Cladonia portentosa and Peltigera membranacea). Phytase and phosphomonoesterase activities were positively correlated amongst species. In E. prunastri both enzyme activities were promoted by N enrichment and phytase activity was readily released into thallus washings. InsP6 was not detected in tree canopy throughfall but was present in pollen leachate. Capacity to hydrolyse InsP6 appears widespread amongst lichens potentially promoting P capture from atmospheric deposits and plant leachates, and P cycling in forest canopies. The enzyme assay used here might find wider application in studies on plant root-fungal-soil systems. PMID:25963718

  14. Youth Physical Activity Resources Use and Activity Measured by Accelerometry

    PubMed Central

    Maslow, Andréa L.; Colabianchi, Natalie

    2014-01-01

    Objectives To examine whether utilization of physical activity resources (eg, parks) was associated with daily physical activity measured by accelerometry. Methods 111 adolescents completed a travel diary with concurrent accelerometry. The main exposure was self-reported utilization of a physical activity resource (none/1+ resources). The main outcomes were total minutes spent in daily 1) moderate-vigorous physical activity and 2) vigorous physical activity. Results Utilizing a physical activity resource was significantly associated with total minutes in moderate-vigorous physical activity. African-Americans and males had significantly greater moderate-vigorous physical activity. Conclusions Results from this study support the development and use of physical activity resources. PMID:21204684

  15. Abrogation of fibroblast activation protein enzymatic activity attenuates tumor growth.

    PubMed

    Cheng, Jonathan D; Valianou, Matthildi; Canutescu, Adrian A; Jaffe, Eileen K; Lee, Hyung-Ok; Wang, Hao; Lai, Jack H; Bachovchin, William W; Weiner, Louis M

    2005-03-01

    Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting

  16. Photon-activation therapy

    SciTech Connect

    Fairchild, R.G.; Bond, V.P.

    1982-01-01

    Photon Activation Therapy (PAT) is a technique in which radiation dose to tumor is enhanced via introduction of stable /sup 127/I in the form of iodinated deoxyuridine (IdUrd). Stimulation of cytotoxic effects from IdUrd is accomplished by activation with external (or implanted) radiation sources. Thus, accumulations of this nucleoside in actively competing cellpools do not preclude therapy in so far as such tissues can be excluded from the radiation field. Calculations show that 5% replacement of thymidine (Tyd) in tumor DNA should enhance the biological effectiveness of a given photon radiotherapy dose by a factor of approx. 3. Proportionally higher gains would result from higher replacements of Tyd and IdUrd. In addition, biological response is enhanced by chemical sensitization with IdUrd. The data indicate that damage from photon activation as well as chemical sensitization does not repair. Thus, at low dose rates, a further increase in therapeutic gain should accrue as normal tissues are allowed to repair and regenerate. A samarium-145 source has been developed for PAT, with activating x-ray energies of from 38 to 45 keV. Favorable clinical results can be expected through the use of IdUrd and protracted irradiations with low energy x-rays. In particular, PAT may provide unique advantages at selected sites such as brain, or head and neck tumors. (ERB)

  17. [Inflammasome: activation mechanisms].

    PubMed

    Suárez, Raibel; Buelvas, Neudo

    2015-03-01

    Inflammation is a rapid biologic response of the immune system in vascular tissues, directed to eliminate stimuli capable of causing damage and begin the process of repair. The macromolecular complexes known as "inflammasomes" are formed by a receptor, either NOD (NLR) or ALR, the receptor absent in melanoma 2 (AIM2). In addition, the inflammasome is formed by the speck-like protein associated to apoptosis (ASC) and procaspase-1, that may be activated by variations in the ionic and intracellular and extracellular ATP concentrations; and the loss of stabilization of the fagolisosomme by internalization of insoluble crystals and redox mechanisms. As a result, there is activation of the molecular platform and the processing of inflammatory prointerleukins to their active forms. There are two modalities of activation of the inflammasome: canonical and non-canonical, both capable of generating effector responses. Recent data associate NLRP 3, IL-1β and IL-18 in the pathogenesis of a variety of diseases, including atherosclerosis, type II diabetes, hyperhomocysteinemia, gout, malaria and hypertension. The inflammasome cascade is emerging as a new chemotherapeutic target in these diseases. In this review we shall discuss the mechanisms of activation and regulation of the inflammasome that stimulate, modulate and resolve inflammation. PMID:25920188

  18. Mechanochemically Active Soft Robots.

    PubMed

    Gossweiler, Gregory R; Brown, Cameron L; Hewage, Gihan B; Sapiro-Gheiler, Eitan; Trautman, William J; Welshofer, Garrett W; Craig, Stephen L

    2015-10-14

    The functions of soft robotics are intimately tied to their form-channels and voids defined by an elastomeric superstructure that reversibly stores and releases mechanical energy to change shape, grip objects, and achieve complex motions. Here, we demonstrate that covalent polymer mechanochemistry provides a viable mechanism to convert the same mechanical potential energy used for actuation in soft robots into a mechanochromic, covalent chemical response. A bis-alkene functionalized spiropyran (SP) mechanophore is cured into a molded poly(dimethylsiloxane) (PDMS) soft robot walker and gripper. The stresses and strains necessary for SP activation are compatible with soft robot function. The color change associated with actuation suggests opportunities for not only new color changing or camouflaging strategies, but also the possibility for simultaneous activation of latent chemistry (e.g., release of small molecules, change in mechanical properties, activation of catalysts, etc.) in soft robots. In addition, mechanochromic stress mapping in a functional robotic device might provide a useful design and optimization tool, revealing spatial and temporal force evolution within the robot in a way that might be coupled to autonomous feedback loops that allow the robot to regulate its own activity. The demonstration motivates the simultaneous development of new combinations of mechanophores, materials, and soft, active devices for enhanced functionality. PMID:26390078

  19. Active imaging at DARPA

    NASA Astrophysics Data System (ADS)

    Ricklin, J. C.; Tomlinson, P. G.

    2005-08-01

    Active systems, because they provide their own illumination, are capable of operating 24 hours a day and are not dependent upon the angle of the sun. Unlike passive systems, they can provide three-dimensional imaging. DARPA is currently developing systems, technologies, and signal processing to pioneer new or improve existing capabilities that employ active imaging capabilities. These involve both radar and ladar, ranging from a few MHz for foliage penetration to near-visible IR to achieve ultra-high resolution at long range. These capabilities would improve Battlefield Awareness (BA) and provide persistent Intelligence, Surveillance, and Reconnaissance (ISR) to perform target detection, recognition, and identification. This paper discusses two different approaches to active optical imaging. One is a coherent approach that uses synthetic aperture techniques with infrared laser radar, and another approach uses only the intensity of the speckle pattern in the aperture plane. Both are capable of produc