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Sample records for activity negatively regulates

  1. DEC1 negatively regulates AMPK activity via LKB1

    PubMed Central

    Sato, Fuyuki; Muragaki, Yasuteru; Zhang, Yanping

    2016-01-01

    Basic helix-loop-helix (bHLH) transcription factor DEC1 (bHLHE40/Stra13/Sharp2) is one of the clock genes that show a circadian rhythm in various tissues. AMP-activated protein kinase (AMPK) activity plays important roles in the metabolic process and in cell death induced by glucose depletion. Recent reports have shown that AMPK activity exhibited a circadian rhythm. However, little is known regarding the regulatory mechanisms involved in the circadian rhythm of AMPK activity. The aim of this study is to investigate whether there is a direct correlation between DEC1 expression and AMPK activity. DEC1 protein and AMPK activity showed a circadian rhythm in the mouse liver with different peak levels. Knocking down DEC1 expression increased AMPK activity, whereas overexpression of DEC1 decreased it. Overexpressing the DEC1 basic mutants had little effect on the AMPK activity. DEC1 bound to the E-box of the LKB1 promoter, decreased LKB1 activity and total protein levels. There was an inverse relationship between DEC1 expression and AMPK activity. Our results suggest that DEC1 negatively regulates AMPK activity via LKB1. PMID:26498531

  2. RGS10 Negatively Regulates Platelet Activation and Thrombogenesis

    PubMed Central

    Druey, Kirk M.; Tansey, Malú G.; Khasawneh, Fadi T.

    2016-01-01

    Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10-/- mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states. PMID:27829061

  3. FANCI is a negative regulator of Akt activation.

    PubMed

    Zhang, Xiaoshan; Lu, Xiaoyan; Akhter, Shamima; Georgescu, Maria-Magdalena; Legerski, Randy J

    2016-01-01

    Akt is a critical mediator of the oncogenic PI3K pathway, and its activation is regulated by kinases and phosphatases acting in opposition. We report here the existence of a novel protein complex that is composed minimally of Akt, PHLPP1, PHLPP2, FANCI, FANCD2, USP1 and UAF1. Our studies show that depletion of FANCI, but not FANCD2 or USP1, results in increased phosphorylation and activation of Akt. This activation is due to a reduction in the interaction between PHLPP1 and Akt in the absence of FANCI. In response to DNA damage or growth factor treatment, the interactions between Akt, PHLPP1 and FANCI are reduced consistent with the known phosphorylation of Akt in response to these stimuli. Furthermore, depletion of FANCI results in reduced apoptosis after DNA damage in accord with its role as a negative regular of Akt. Our findings describe an unexpected function for FANCI in the regulation of Akt and define a previously unrecognized intersection between the PI3K-Akt and FA pathways.

  4. Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity

    PubMed Central

    Ormerod, Kiel G.

    2017-01-01

    Abstract Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. Here, we describe an analysis of how Ca2+ signals within Drosophila astrocyte-like glia regulate excitability in the nervous system. We find that Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, we find that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. We observe that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. Our data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability. PMID:28303263

  5. MERTK as negative regulator of human T cell activation

    PubMed Central

    Cabezón, Raquel; Carrera-Silva, E. Antonio; Flórez-Grau, Georgina; Errasti, Andrea E.; Calderón-Gómez, Elisabeth; Lozano, Juan José; España, Carolina; Ricart, Elena; Panés, Julián; Rothlin, Carla Vanina; Benítez-Ribas, Daniel

    2015-01-01

    The aim of this study was to test the hypothesis whether MERTK, which is up-regulated in human DCs treated with immunosuppressive agents, is directly involved in modulating T cell activation. MERTK is a member of the TAM family and contributes to regulating innate immune response to ACs by inhibiting DC activation in animal models. However, whether MERTK interacts directly with T cells has not been addressed. Here, we show that MERTK is highly expressed on dex-induced human tol-DCs and participates in their tolerogenic effect. Neutralization of MERTK in allogenic MLR, as well as autologous DC–T cell cultures, leads to increased T cell proliferation and IFN-γ production. Additionally, we identify a previously unrecognized noncell-autonomous regulatory function of MERTK expressed on DCs. Mer-Fc protein, used to mimic MERTK on DCs, suppresses naïve and antigen-specific memory T cell activation. This mechanism is mediated by the neutralization of the MERTK ligand PROS1. We find that MERTK and PROS1 are expressed in human T cells upon TCR activation and drive an autocrine proproliferative mechanism. Collectively, these results suggest that MERTK on DCs controls T cell activation and expansion through the competition for PROS1 interaction with MERTK in the T cells. In conclusion, this report identified MERTK as a potent suppressor of T cell response. PMID:25624460

  6. Osterix represses adipogenesis by negatively regulating PPARγ transcriptional activity.

    PubMed

    Han, Younho; Kim, Chae Yul; Cheong, Heesun; Lee, Kwang Youl

    2016-10-18

    Osterix is a novel bone-related transcription factor involved in osteoblast differentiation, and bone maturation. Because a reciprocal relationship exists between adipocyte and osteoblast differentiation of bone marrow derived mesenchymal stem cells, we hypothesized that Osterix might have a role in adipogenesis. Ablation of Osterix enhanced adipogenesis in 3T3-L1 cells, whereas overexpression suppressed this process and inhibited the expression of adipogenic markers including CCAAT/enhancer-binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ). Further studies indicated that Osterix significantly decreased PPARγ-induced transcriptional activity. Using co-immunoprecipitation and GST-pull down analysis, we found that Osterix directly interacts with PPARγ. The ligand-binding domain (LBD) of PPARγ was responsible for this interaction, which was followed by repression of PPARγ-induced transcriptional activity, even in the presence of rosiglitazone. Taken together, we identified the Osterix has an important regulatory role on PPARγ activity, which contributed to the mechanism of adipogenesis.

  7. Chaperone-mediated autophagy regulates T cell responses through targeted degradation of negative regulators of T cell activation.

    PubMed

    Valdor, Rut; Mocholi, Enric; Botbol, Yair; Guerrero-Ros, Ignacio; Chandra, Dinesh; Koga, Hiroshi; Gravekamp, Claudia; Cuervo, Ana Maria; Macian, Fernando

    2014-11-01

    Chaperone-mediated autophagy (CMA) targets soluble proteins for lysosomal degradation. Here we found that CMA was activated in T cells in response to engagement of the T cell antigen receptor (TCR), which induced expression of the CMA-related lysosomal receptor LAMP-2A. In activated T cells, CMA targeted the ubiquitin ligase Itch and the calcineurin inhibitor RCAN1 for degradation to maintain activation-induced responses. Consequently, deletion of the gene encoding LAMP-2A in T cells caused deficient in vivo responses to immunization or infection with Listeria monocytogenes. Impaired CMA activity also occurred in T cells with age, which negatively affected their function. Restoration of LAMP-2A in T cells from old mice resulted in enhancement of activation-induced responses. Our findings define a role for CMA in regulating T cell activation through the targeted degradation of negative regulators of T cell activation.

  8. Drosophila protein kinase N (Pkn) is a negative regulator of actin-myosin activity during oogenesis.

    PubMed

    Ferreira, Tânia; Prudêncio, Pedro; Martinho, Rui Gonçalo

    2014-10-15

    Nurse cell dumping is an actin-myosin based process, where 15 nurse cells of a given egg chamber contract and transfer their cytoplasmic content through the ring canals into the growing oocyte. We isolated two mutant alleles of protein kinase N (pkn) and showed that Pkn negatively-regulates activation of the actin-myosin cytoskeleton during the onset of dumping. Using live-cell imaging analysis we observed that nurse cell dumping rates sharply increase during the onset of fast dumping. Such rate increase was severely impaired in pkn mutant nurse cells due to excessive nurse cell actin-myosin activity and/or loss of tissue integrity. Our work demonstrates that the transition between slow and fast dumping is a discrete event, with at least a five to six-fold dumping rate increase. We show that Pkn negatively regulates nurse cell actin-myosin activity. This is likely to be important for directional cytoplasmic flow. We propose Pkn provides a negative feedback loop to help avoid excessive contractility after local activation of Rho GTPase.

  9. All-trans retinoic acid negatively regulates cytotoxic activities of nature killer cell line 92

    SciTech Connect

    Li Ang . E-mail: liang3829@sina.com.cn; He Meilan; Wang Hui; Qiao Bin; Chen Ping; Gu Hua; Zhang Mengjie; He Shengxiang

    2007-01-05

    NK cells are key components of innate immune systems and their activities are regulated by cytokines and hormones. All-trans retinoic acid (ATRA), as a metabolite of vitamin A and an immunomodulatory hormone, plays an important role in regulating immune responses. In the present study, we investigated the effect of ATRA on human NK cell line NK92. We found that ATRA dose-dependently suppressed cytotoxic activities of NK92 cells without affecting their proliferation. To explore the mechanisms underlying the ATRA influence on NK92 cells, we examined the production of cytokines (TNF-{alpha}, IFN-{gamma}), gene expression of cytotoxic-associated molecules (perforin, granzyme B, nature killer receptors (NCRs), and NKG2D), and the activation of NF-{kappa}B pathways related with immune response. Our results demonstrated that ATRA suppressed NF-{kappa}B activity and prevented I{kappa}B{alpha} degradation in a dose-dependent way, inhibited IFN-{gamma} production and gene expression of granzyme B and NKp46. Our findings suggest that ATRA is a negative regulator of NK92 cell activation and may act as a potential regulator of anti-inflammatory functions in vivo.

  10. Positive and negative regulation of T-cell activation through kinases and phosphatases.

    PubMed Central

    Mustelin, Tomas; Taskén, Kjetil

    2003-01-01

    The sequence of events in T-cell antigen receptor (TCR) signalling leading to T-cell activation involves regulation of a number of protein tyrosine kinases (PTKs) and the phosphorylation status of many of their substrates. Proximal signalling pathways involve PTKs of the Src, Syk, Csk and Tec families, adapter proteins and effector enzymes in a highly organized tyrosine-phosphorylation cascade. In intact cells, tyrosine phosphorylation is rapidly reversible and generally of a very low stoichiometry even under induced conditions due to the fact that the enzymes removing phosphate from tyrosine-phosphorylated substrates, the protein tyrosine phosphatases (PTPases), have a capacity that is several orders of magnitude higher than that of the PTKs. It follows that a relatively minor change in the PTK/PTPase balance can have a major impact on net tyrosine phosphorylation and thereby on activation and proliferation of T-cells. This review focuses on the involvement of PTKs and PTPases in positive and negative regulation of T-cell activation, the emerging theme of reciprocal regulation of each type of enzyme by the other, as well as regulation of phosphotyrosine turnover by Ser/Thr phosphorylation and regulation of localization of signal components. PMID:12485116

  11. CD40 Negatively Regulates ATP-TLR4-Activated Inflammasome in Microglia.

    PubMed

    Gaikwad, Sagar; Patel, Divyesh; Agrawal-Rajput, Reena

    2017-03-01

    During acute brain injury and/or sterile inflammation, release of danger-associated molecular patterns (DAMPs) activates pattern recognition receptors (PRRs). Microglial toll-like receptor (TLR)-4 activated by DAMPs potentiates neuroinflammation through inflammasome-induced IL-1β and pathogenic Th17 polarization which critically influences brain injury. TLR4 activation accompanies increased CD40, a cognate costimulatory molecule, involved in microglia-mediated immune responses in the brain. During brain injury, excessive release of extracellular ATP (DAMPs) is involved in promoting the damage. However, the regulatory role of CD40 in microglia during ATP-TLR4-mediated inflammasome activation has never been explored. We report that CD40, in the absence of ATP, synergizes TLR4-induced proinflammatory cytokines but not IL-1β, suggesting that the response is independent of inflammasome. The presence of ATP during TLR4 activation leads to NLRP3 inflammasome activation and caspase-1-mediated IL-1β secretion which was inhibited during CD40 activation, accompanied with inhibition of ERK1/2 and reactive oxygen species (ROS), and elevation in p38 MAPK phosphorylation. Experiments using selective inhibitors prove indispensability of ERK 1/2 and ROS for inflammasome activation. The ATP-TLR4-primed macrophages polarize the immune response toward pathogenic Th17 cells, whereas CD40 activation mediates Th1 response. Exogenous supplementation of IFN-γ (a Th1 cytokine and CD40 inducer) results in decreased IL-1β, suggesting possible feedback loop mechanism of inflammasome inhibition, whereby IFN-γ-mediated increase in CD40 expression and activation suppress neurotoxic inflammasome activation required for Th17 response. Collectively, the findings indicate that CD40 is a novel negative regulator of ATP-TLR4-mediated inflammasome activation in microglia, thus providing a checkpoint to regulate excessive inflammasome activation and Th17 response during DAMP-mediated brain injury.

  12. Effects of negative air ions on activity of neural substrates involved in autonomic regulation in rats

    NASA Astrophysics Data System (ADS)

    Suzuki, Satoko; Yanagita, Shinya; Amemiya, Seiichiro; Kato, Yumi; Kubota, Natsuko; Ryushi, Tomoo; Kita, Ichiro

    2008-07-01

    The neural mechanism by which negative air ions (NAI) mediate the regulation of autonomic nervous system activity is still unknown. We examined the effects of NAI on physiological responses, such as blood pressure (BP), heart rate (HR), and heart rate variability (HRV) as well as neuronal activity, in the paraventricular nucleus of the hypothalamus (PVN), locus coeruleus (LC), nucleus ambiguus (NA), and nucleus of the solitary tract (NTS) with c-Fos immunohistochemistry in anesthetized, spontaneously breathing rats. In addition, we performed cervical vagotomy to reveal the afferent pathway involved in mediating the effects of NAI on autonomic regulation. NAI significantly decreased BP and HR, and increased HF power of the HRV spectrum. Significant decreases in c-Fos positive nuclei in the PVN and LC, and enhancement of c-Fos expression in the NA and NTS were induced by NAI. After vagotomy, these physiological and neuronal responses to NAI were not observed. These findings suggest that NAI can modulate autonomic regulation through inhibition of neuronal activity in PVN and LC as well as activation of NA neurons, and that these effects of NAI might be mediated via the vagus nerves.

  13. Tetratricopeptide Repeat Domain 9A Negatively Regulates Estrogen Receptor Alpha Activity

    PubMed Central

    Shrestha, Smeeta; Sun, Yang; Lufkin, Thomas; Kraus, Petra; Or, Yuzuan; Garcia, Yenni A.; Guy, Naihsuan; Ramos, Paola; Cox, Marc B.; Tay, Fiona; Lin, Valerie CL

    2015-01-01

    Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51. PMID:25798063

  14. Tetratricopeptide repeat domain 9A negatively regulates estrogen receptor alpha activity.

    PubMed

    Shrestha, Smeeta; Sun, Yang; Lufkin, Thomas; Kraus, Petra; Or, Yuzuan; Garcia, Yenni A; Guy, Naihsuan; Ramos, Paola; Cox, Marc B; Tay, Fiona; Lin, Valerie C L

    2015-01-01

    Tetratricopeptide repeat domain 9A (TTC9A) is a target gene of estrogen and progesterone. It is over-expressed in breast cancer. However, little is known about the physiological function of TTC9A. The objectives of this study were to establish a Ttc9a knockout mouse model and to study the consequence of Ttc9a gene inactivation. The Ttc9a targeting vector was generated by replacing the Ttc9a exon 1 with a neomycin cassette. The mice homozygous for Ttc9a exon 1 deletion appear to grow normally and are fertile. However, further characterization of the female mice revealed that Ttc9a deficiency is associated with greater body weight, bigger thymus and better mammary development in post-pubertal mice. Furthermore, Ttc9a deficient mammary gland was more responsive to estrogen treatment with greater mammary ductal lengthening, ductal branching and estrogen target gene induction. Since Ttc9a is induced by estrogen in estrogen target tissues, these results suggest that Ttc9a is a negative regulator of estrogen function through a negative feedback mechanism. This is supported by in vitro evidence that TTC9A over-expression attenuated ERα activity in MCF-7 cells. Although TTC9A does not bind to ERα or its chaperone protein Hsp90 directly, TTC9A strongly interacts with FKBP38 and FKBP51, both of which interact with ERα and Hsp90 and modulate ERα activity. It is plausible therefore that TTC9A negatively regulates ERα activity through interacting with co-chaperone proteins such as FKBP38 and FKBP51.

  15. Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

    PubMed Central

    Kim, Won Sam; Kim, Mi Jeong; Kim, Dong Oh; Byun, Jae-Eun; Huy, Hangsak; Song, Hae Young; Park, Young-Jun; Kim, Tae-Don; Yoon, Suk Ran; Choi, Eun-Ji; Jung, Haiyoung; Choi, Inpyo

    2017-01-01

    Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction. PMID:28383049

  16. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  17. JOSD1 Negatively Regulates Type-I Interferon Antiviral Activity by Deubiquitinating and Stabilizing SOCS1.

    PubMed

    Wang, Xiaofang; Zhang, Liting; Zhang, Yunli; Zhao, Peng; Qian, Liping; Yuan, Yukang; Liu, Jin; Cheng, Qiao; Xu, Wenqian; Zuo, Yibo; Guo, Tingting; Yu, Zhengyuan; Zheng, Hui

    2017-03-29

    The Josephin domain-containing (JOSD) protein 1 (JOSD1) is recognized as one member of deubiquitinases (DUBs) due to its catalytic "Josephin" domain. However, the in vivo deubiquitinating activity of JOSD1 remains unidentified, and the biological functions of JOSD1 are largely unknown. In this study, we report that JOSD1 plays an important role in regulating type-I interferon (IFN-I)-mediated antiviral activity. JOSD1 physically interacts with SOCS1, which is an essential negative regulator of many cytokines signaling, and enhances SOCS1 stability by deubiquitinating K48-linked polyubiquitination of SOCS1. Furthermore, JOSD1 inhibits IFN-I-induced signaling pathway and antiviral response. Interestingly, during the early stage of viral infections, the levels of JOSD1 and SOCS1 undergo downregulation, which may facilitate activation of IFN-I signaling and efficient antiviral activity. Thus, our finding identified the first deubiquitinating substrate of JOSD1 and a novel biological function of JOSD1 and may provide a potential target for IFNs-based antiviral therapy.

  18. Phosphorylation acts positively and negatively to regulate MRTF-A subcellular localisation and activity

    PubMed Central

    Panayiotou, Richard; Miralles, Francesc; Pawlowski, Rafal; Diring, Jessica; Flynn, Helen R; Skehel, Mark; Treisman, Richard

    2016-01-01

    The myocardin-related transcription factors (MRTF-A and MRTF-B) regulate cytoskeletal genes through their partner transcription factor SRF. The MRTFs bind G-actin, and signal-regulated changes in cellular G-actin concentration control their nuclear accumulation. The MRTFs also undergo Rho- and ERK-dependent phosphorylation, but the function of MRTF phosphorylation, and the elements and signals involved in MRTF-A nuclear export are largely unexplored. We show that Rho-dependent MRTF-A phosphorylation reflects relief from an inhibitory function of nuclear actin. We map multiple sites of serum-induced phosphorylation, most of which are S/T-P motifs and show that S/T-P phosphorylation is required for transcriptional activation. ERK-mediated S98 phosphorylation inhibits assembly of G-actin complexes on the MRTF-A regulatory RPEL domain, promoting nuclear import. In contrast, S33 phosphorylation potentiates the activity of an autonomous Crm1-dependent N-terminal NES, which cooperates with five other NES elements to exclude MRTF-A from the nucleus. Phosphorylation thus plays positive and negative roles in the regulation of MRTF-A. DOI: http://dx.doi.org/10.7554/eLife.15460.001 PMID:27304076

  19. PCDH10 Interacts With hTERT and Negatively Regulates Telomerase Activity

    PubMed Central

    Zhou, Li-Na; Hua, Xing; Deng, Wu-Quan; Wu, Qi-Nan; Mei, Hao; Chen, Bing

    2015-01-01

    Abstract Telomerase catalyzes telomeric DNA synthesis, an essential process to maintain the length of telomere for continuous cell proliferation and genomic stability. Telomerase is activated in gametes, stem cells, and most tumor cells, and its activity is tightly controlled by a catalytic human telomerase reverse transcriptase (hTERT) subunit and a collection of associated proteins. In the present work, normal human testis tissue was used for the first time to identify proteins involved in the telomerase regulation under normal physiological conditions. Immunoprecipitation was performed using total protein lysates from the normal testis tissue and the proteins of interest were identified by microfluidic high-performance liquid chromatography and tandem mass spectrometry (HPLC-Chip-MS/MS). The regulatory role of PCDH10 in telomerase activity was confirmed by a telomeric repeat amplification protocol (TRAP) assay, and the biological functions of it were characterized by in vitro proliferation, migration, and invasion assays. A new in vivo hTERT interacting protein, protocadherin 10 (PCDH10), was identified. Overexpression of PCDH10 in pancreatic cancer cells impaired telomere elongation by inhibiting telomerase activity while having no obvious effect on hTERT expression at mRNA and protein levels. As a result of this critical function in telomerase regulation, PCDH10 was found to inhibit cell proliferation, migration, and invasion, suggesting a tumor suppressive role of this protein. Our data suggested that PCDH10 played a critical role in cancer cell growth, by negatively regulating telomerase activity, implicating a potential value in future therapeutic development against cancer. PMID:26683936

  20. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  1. Slamf8 is a negative regulator of Nox2 activity in macrophages

    PubMed Central

    Wang, Guoxing; Abadía-Molina, Ana C; Berger, Scott B; Romero, Xavier; O'Keeffe, Michael; Rojas-Barros, Domingo I.; Aleman, Marta; Liao, Gongxian; Maganto-García, Elena; Fresno, Manuel; Wang, Ninghai; Detre, Cynthia; Terhorst, Cox

    2012-01-01

    Slamf8 (CD353) is a cell surface receptor that is expressed upon activation of macrophages by interferon-gamma or bacteria. Here we report that a very high Nox2 activity enzyme was found in Slamf8−/− macrophages in response to E.coli or S.aureus, but also to phorbol myristate acetate. The elevated Nox2 activity in Slamf8−/− macrophages was also demonstrated in E.coli or S.aureus phagosomes by using a pH indicator system, and was further confirmed by a reduction of the enzyme activity after transfection of the receptor into Slamf8-deficient primary macrophages or RAW 264.7 cells. Upon exposure to bacteria and/or phorbol myristate acetate, PKC activity in Slamf8−/− macrophages is increased. This results in an enhanced phosphorylation of p40phox, one key component of the Nox2 enzyme complex, which in turn leads to greater Nox2 activity. Taken together, the data show that upon response to inflammation-associated stimuli the inducible receptor Slamf8 negatively regulates inflammatory responses. PMID:22593622

  2. TRIF Is a Critical Negative Regulator of TLR Agonist Mediated Activation of Dendritic Cells In Vivo

    PubMed Central

    Appledorn, Daniel M.; Aylsworth, Charles F.; Godbehere, Sarah; Liu, Chyong-Jy Joyce; Quiroga, Dionisia; Amalfitano, Andrea

    2011-01-01

    Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNγ production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy. PMID:21760953

  3. Drosophila Mi-2 negatively regulates dDREF by inhibiting its DNA-binding activity.

    PubMed

    Hirose, Fumiko; Ohshima, Nobuko; Kwon, Eun-Jeong; Yoshida, Hideki; Yamaguchi, Masamitsu

    2002-07-01

    Drosophila melanogaster DNA replication-related element (DRE) factor (dDREF) is a transcriptional regulatory factor required for the expression of genes carrying the 5'-TATCGATA DRE. dDREF has been reported to bind to a sequence in the chromatin boundary element, and thus, dDREF may play a part in regulating insulator activity. To generate further insights into dDREF function, we carried out a Saccharomyces cerevisiae two-hybrid screening with DREF polypeptide as bait and identified Mi-2 as a DREF-interacting protein. Biochemical analyses revealed that the C-terminal region of Drosophila Mi-2 (dMi-2) specifically binds to the DNA-binding domain of dDREF. Electrophoretic mobility shift assays showed that dMi-2 thereby inhibits the DNA-binding activity of dDREF. Ectopic expression of dDREF and dMi-2 in eye imaginal discs resulted in severe and mild rough-eye phenotypes, respectively, whereas flies simultaneously expressing both proteins exhibited almost-normal eye phenotypes. Half-dose reduction of the dMi-2 gene enhanced the DREF-induced rough-eye phenotype. Immunostaining of polytene chromosomes of salivary glands showed that dDREF and dMi-2 bind in mutually exclusive ways. These lines of evidence define a novel function of dMi-2 in the negative regulation of dDREF by its DNA-binding activity. Finally, we postulated that dDREF and dMi-2 may demonstrate reciprocal regulation of their functions.

  4. Smart conjugated polymer nanocarrier for healthy weight loss by negative feedback regulation of lipase activity

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Lei; Zhu, Sha; Zhang, Lei; Feng, Pei-Jian; Yao, Xi-Kuang; Qian, Cheng-Gen; Zhang, Can; Jiang, Xi-Qun; Shen, Qun-Dong

    2016-02-01

    Healthy weight loss represents a real challenge when obesity is increasing in prevalence. Herein, we report a conjugated polymer nanocarrier for smart deactivation of lipase and thus balancing calorie intake. After oral administration, the nanocarrier is sensitive to lipase in the digestive tract and releases orlistat, which deactivates the enzyme and inhibits fat digestion. It also creates negative feedback to control the release of itself. The nanocarrier smartly regulates activity of the lipase cyclically varied between high and low levels. In spite of high fat diet intervention, obese mice receiving a single dose of the nanocarrier lose weight over eight days, whereas a control group continues the tendency to gain weight. Daily intragastric administration of the nanocarrier leads to lower weight of livers or fat pads, smaller adipocyte size, and lower total cholesterol level than that of the control group. Near-infrared fluorescence of the nanocarrier reveals its biodistribution.Healthy weight loss represents a real challenge when obesity is increasing in prevalence. Herein, we report a conjugated polymer nanocarrier for smart deactivation of lipase and thus balancing calorie intake. After oral administration, the nanocarrier is sensitive to lipase in the digestive tract and releases orlistat, which deactivates the enzyme and inhibits fat digestion. It also creates negative feedback to control the release of itself. The nanocarrier smartly regulates activity of the lipase cyclically varied between high and low levels. In spite of high fat diet intervention, obese mice receiving a single dose of the nanocarrier lose weight over eight days, whereas a control group continues the tendency to gain weight. Daily intragastric administration of the nanocarrier leads to lower weight of livers or fat pads, smaller adipocyte size, and lower total cholesterol level than that of the control group. Near-infrared fluorescence of the nanocarrier reveals its biodistribution

  5. TIPE1 induces apoptosis by negatively regulating Rac1 activation in hepatocellular carcinoma cells.

    PubMed

    Zhang, Z; Liang, X; Gao, L; Ma, H; Liu, X; Pan, Y; Yan, W; Shan, H; Wang, Z; Chen, Y H; Ma, C

    2015-05-14

    TIPE1 (tumor necrosis factor-α-induced protein 8-like 1 or TNFAIP8L1) is a newly identified member of the TIPE (TNFAIP8) family, which play roles in regulating cell death. However, the biologic functions of TIPE1 in physiologic and pathologic conditions are largely unknown. Here, we report the roles of TIPE1 in hepatocellular carcinoma (HCC). Evaluated by immunohistochemical staining, HCC tissues showed significantly downregulated TIPE1 expression compared with adjacent non-tumor tissues, which positively correlated with tumor pathologic grades and patient survival. Using a homograft tumor model in Balb/c mice, we discovered that TIPE1 significantly diminished the growth and tumor weight of murine liver cancer homografts. Consistently, TIPE1 inhibited both cell growth and colony formation ability of cultured HCC cell lines, which was further identified to be due to TIPE1-inducing apoptosis in a caspase-independent, necrostatin-1 (Nec-1)-insensitive manner. Furthermore, mechanistic investigations revealed that TIPE1 interacted with Rac1, and inhibited the activation of Rac1 and its downstream p65 and c-Jun N-terminal kinase pathway. Moreover, overexpression of constitutively active Rac1 partially rescued the apoptosis induced by TIPE1, and Rac1 knockdown significantly restored the deregulated cell growth induced by TIPE1 small interfering RNA. Our findings revealed that TIPE1 induced apoptosis in HCC cells by negatively regulating Rac1 pathway, and loss of TIPE1 might be a new prognostic indicator for HCC patients.

  6. PCTK3/CDK18 regulates cell migration and adhesion by negatively modulating FAK activity

    PubMed Central

    Matsuda, Shinya; Kawamoto, Kohei; Miyamoto, Kenji; Tsuji, Akihiko; Yuasa, Keizo

    2017-01-01

    PCTAIRE kinase 3 (PCTK3) is a member of the cyclin dependent kinase family, but its physiological function remains unknown. We previously reported that PCTK3-knockdown HEK293T cells showed actin accumulation at the leading edge, suggesting that PCTK3 is involved in the regulation of actin reorganization. In this study, we investigated the physiological function and downstream signal transduction molecules of PCTK3. PCTK3 knockdown in HEK293T cells increased cell motility and RhoA/Rho-associated kinase activity as compared with control cells. We also found that phosphorylation at residue Tyr-397 in focal adhesion kinase (FAK) was increased in PCTK3-knockdown cells. FAK phosphorylation at Tyr-397 was increased in response to fibronectin stimulation, whereas its phosphorylation was suppressed by PCTK3. In addition, excessive expression of PCTK3 led to the formation of filopodia during the early stages of cell adhesion in HeLa cells. These results indicate that PCTK3 controls actin cytoskeleton dynamics by negatively regulating the FAK/Rho signaling pathway. PMID:28361970

  7. Phosphodiesterase 4B negatively regulates endotoxin-activated interleukin-1 receptor antagonist responses in macrophages

    PubMed Central

    Yang, Jing-Xing; Hsieh, Kou-Chou; Chen, Yi-Ling; Lee, Chien-Kuo; Conti, Marco; Chuang, Tsung-Hsien; Wu, Chin-Pyng; Jin, S.-L. Catherine

    2017-01-01

    Activation of TLR4 by lipopolysaccharide (LPS) induces both pro-inflammatory and anti-inflammatory cytokine production in macrophages. Type 4 phosphodiesterases (PDE4) are key cAMP-hydrolyzing enzymes, and PDE4 inhibitors are considered as immunosuppressors to various inflammatory responses. We demonstrate here that PDE4 inhibitors enhance the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) secretion in LPS-activated mouse peritoneal macrophages, and this response was regulated at the transcriptional level rather than an increased IL-1Ra mRNA stability. Studies with PDE4-deficient macrophages revealed that the IL-1Ra upregulation elicited by LPS alone is PKA-independent, whereas the rolipram-enhanced response was mediated by inhibition of only PDE4B, one of the three PDE4 isoforms expressed in macrophages, and it requires PKA but not Epac activity. However, both pathways activate CREB to induce IL-1Ra expression. PDE4B ablation also promoted STAT3 phosphorylation (Tyr705) to LPS stimulation, but this STAT3 activation is not entirely responsible for the IL-1Ra upregulation in PDE4B-deficient macrophages. In a model of LPS-induced sepsis, only PDE4B-deficient mice displayed an increased circulating IL-1Ra, suggesting a protective role of PDE4B inactivation in vivo. These findings demonstrate that PDE4B negatively modulates anti-inflammatory cytokine expression in innate immune cells, and selectively targeting PDE4B should retain the therapeutic benefits of nonselective PDE4 inhibitors. PMID:28383060

  8. Negative regulation of glial engulfment activity by Draper terminates glial responses to axon injury

    PubMed Central

    Logan, Mary A.; Hackett, Rachel; Doherty, Johnna; Sheehan, Amy; Speese, Sean D.; Freeman, Marc R.

    2012-01-01

    Neuronal injury elicits potent cellular responses from glia, but molecular pathways modulating glial activation, phagocytic function, and termination of reactive responses remain poorly defined. Here we show that positive or negative regulation of glial reponses to axon injury are molecularly encoded by unique isoforms of the Drosophila engulfment receptor Draper. Draper-I promotes engulfment of axonal debris through an immunoreceptor tyrosine-based activation motif (ITAM). In contrast, Draper-II, an alternative splice variant, potently inhibits glial engulfment function. Draper-II suppresses Draper-I signaling through a novel immunoreceptor tyrosine-based inhibitory motif (ITIM)-like domain and the tyrosine phosphatase Corkscrew (Csw). Intriguingly, loss of Draper-II/Csw signaling prolongs expression of glial engulfment genes after axotomy and reduces the ability of glia to respond to secondary axotomy. Our work highlights a novel role for Draper-II in inhibiting glial responses to neurodegeneration, and indicates a balance of opposing Draper-I/-II signaling events is essential to maintain glial sensitivity to brain injury. PMID:22426252

  9. Drosophila distal-less negatively regulates dDREF by inhibiting its DNA binding activity.

    PubMed

    Hayashi, Yuko; Kato, Masaki; Seto, Hirokazu; Yamaguchi, Masamitsu

    2006-07-01

    The Drosophila DNA replication-related element binding factor (dDREF) is required for expression of many proliferation-related genes carrying the DRE sequence, 5'-TATCGATA. Over-expression of dDREF in the eye imaginal disc induces ectopic DNA synthesis, apoptosis and inhibition of photoreceptor cell specification, and results in rough eye phenotype in adults. In the present study, half dose reduction of the Distal-less (Dll) gene enhanced the dDREF-induced rough eye phenotype, suggesting that Dll negatively regulates dDREF activity in eye imaginal disc cells. Biochemical analyses revealed the N-terminal (30aa to 124aa) and C-terminal (190aa to 327aa) regions of Dll to interact with the DNA binding domain (16aa to 125aa) of dDREF, although it is not clear yet whether the interaction is direct or indirect. Electrophoretic mobility shift assays showed that Dll thereby inhibits DNA binding. The repression of this dDREF-function by a homeodomain protein like Dll may contribute to the differentiation-coupled repression of cell proliferation during development.

  10. The Y’s that bind: negative regulators of Src family kinase activity in platelets

    PubMed Central

    NEWMAN, D. K.

    2015-01-01

    Summary Members of the Src family of protein tyrosine kinases play important roles in platelet adhesion, activation, and aggregation. The purpose of this review is to summarize current knowledge regarding how Src family kinase activity is regulated in general, to describe what is known about mechanisms underlying SFK activation in platelets, and to discuss platelet proteins that contribute to SFK inactivation, particularly those that use phosphotyrosine-containing sequences to recruit phosphatases and kinases to sites of SFK activity. PMID:19630799

  11. A20 is a negative regulator of BCL10- and CARMA3-mediated activation of NF-kappaB.

    PubMed

    Stilo, Romania; Varricchio, Ettore; Liguoro, Domenico; Leonardi, Antonio; Vito, Pasquale

    2008-04-15

    The molecular complex containing CARMA proteins, BCL10 and TRAF6 has been identified recently as a key component in the signal transduction pathways that regulate activation of the nuclear factor kappaB (NF-kappaB) transcription factor. Here, we report that the inducible protein A20 negatively regulates these signaling cascades by means of its deubiquitylation activity. We show that A20 perturbs assembly of the complex containing CARMA3, BCL10 and IKKgamma/NEMO, thereby suppressing activation of NF-kappaB. Together, our results further define the molecular mechanisms that control activation of NF-kappaB and reveal a function for A20 in the regulation of CARMA and BCL10 activity in lymphoid and non-lymphoid cells.

  12. Negative regulators of cell proliferation

    NASA Technical Reports Server (NTRS)

    Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    Cell proliferation is governed by the influence of both mitogens and inhibitors. Although cell contact has long been thought to play a fundamental role in cell cycling regulation, and negative regulators have long been suspected to exist, their isolation and purification has been complicated by a variety of technical difficulties. Nevertheless, over recent years an ever-expanding list of putative negative regulators have emerged. In many cases, their biological inhibitory activities are consistent with density-dependent growth inhibition. Most likely their interactions with mitogenic agents, at an intracellular level, are responsible for either mitotic arrest or continued cell cycling. A review of naturally occurring cell growth inhibitors is presented with an emphasis on those factors shown to be residents of the cell surface membrane. Particular attention is focused on a cell surface sialoglycopeptide, isolated from intact bovine cerebral cortex cells, which has been shown to inhibit the proliferation of an unusually wide range of target cells. The glycopeptide arrest cells obtained from diverse species, both fibroblasts and epithelial cells, and a broad variety of transformed cells. Signal transduction events and a limited spectrum of cells that are refractory to the sialoglycopeptide have provided insight into the molecular events mediated by this cell surface inhibitor.

  13. miR-181b negatively regulates activation-induced cytidine deaminase in B cells.

    PubMed

    de Yébenes, Virginia G; Belver, Laura; Pisano, David G; González, Susana; Villasante, Aranzazu; Croce, Carlo; He, Lin; Ramiro, Almudena R

    2008-09-29

    Activated B cells reshape their primary antibody repertoire after antigen encounter by two molecular mechanisms: somatic hypermutation (SHM) and class switch recombination (CSR). SHM and CSR are initiated by activation-induced cytidine deaminase (AID) through the deamination of cytosine residues on the immunoglobulin loci, which leads to the generation of DNA mutations or double-strand break intermediates. As a bystander effect, endogenous AID levels can also promote the generation of chromosome translocations, suggesting that the fine tuning of AID expression may be critical to restrict B cell lymphomagenesis. To determine whether microRNAs (miRNAs) play a role in the regulation of AID expression, we performed a functional screening of an miRNA library and identified miRNAs that regulate CSR. One such miRNA, miR-181b, impairs CSR when expressed in activated B cells, and results in the down-regulation of AID mRNA and protein levels. We found that the AID 3' untranslated region contains multiple putative binding sequences for miR-181b and that these sequences can be directly targeted by miR-181b. Overall, our results provide evidence for a new regulatory mechanism that restricts AID activity and can therefore be relevant to prevent B cell malignant transformation.

  14. EBV LMP-1 negatively regulates expression and pro-apoptotic activity of Par-4 in nasopharyngeal carcinoma cells.

    PubMed

    Lee, Jeng-Woei; Liu, Po-Fan; Hsu, Lee-Ping; Chen, Peir-Rong; Chang, Chung-Hsing; Shih, Wen-Ling

    2009-07-08

    Latent membrane protein-1 (LMP-1) of the Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC), and in this study we sought to determine whether the pro-apoptotic activity of prostate apoptosis response-4 (Par-4) is modulated by LMP-1 in NPC cells. We found that LMP-1 diminished the pro-apoptotic activity of Par-4 and negatively regulated Par-4 protein by de novo synthesis; moreover, although LMP-1 accelerated a Par-4 activator, PKA, we demonstrated that LMP-1 also activated the PI3K/Akt pathway and increased Bcl-2 expression to suppress the activity of Par-4. Consequently, our results revealed a novel negative action of LMP-1 on the pro-apoptosis protein Par-4 by the coordination of multiple signaling pathways.

  15. An aza-anthrapyrazole negatively regulates Th1 activity and suppresses experimental autoimmune encephalomyelitis.

    PubMed

    Clark, Matthew P; Leaman, Douglas W; Hazelhurst, Lori A; Hwang, Eun S; Quinn, Anthony

    2016-02-01

    Previously we showed that BBR3378, a novel analog of the anticancer drug mitoxantrone, had the ability to ameliorate ascending paralysis in MOG35-55-induced experimental autoimmune encephalomyelitis (EAE), a murine model of human multiple sclerosis, without the drug-induced cardiotoxicity or lymphopenia associated with mitoxantrone therapy. Chemotherapeutic drugs like mitoxantrone, a topoisomerase inhibitor, are thought to provide protection in inflammatory autoimmune diseases like EAE by inducing apoptosis in rapidly proliferating autoreactive lymphocytes. Here, we show that while BR3378 blocked cell division, T cells were still able to respond to antigenic stimulation and upregulate surface molecules indicative of activation. However, in contrast to mitoxantrone, BBR3378 inhibited the production of the proinflammatory cytokine IFN-γ both in recently activated T cell blasts and established Th1 effectors, while sparing the activities of IL-13-producing Th2 cells. IFN-γ is known to be regulated by the transcription factor T-bet. In addition to IFN-γ, in vitro and in vivo exposure to BBR3378 suppressed the expression of other T-bet regulated proteins, including CXCR3 and IL-2Rβ. Microarray analysis revealed BBR3378-induced suppression of additional T-bet regulated genes, suggesting that the drug might disrupt global Th1 programming. Importantly, BBR3378 antagonized ongoing Th1 autoimmune responses in vivo, modulated clinical disease and CNS inflammation in acute and relapsing forms of EAE. Therefore, BBR3378 may be a unique inhibitor of T-bet regulated genes and may have potential as a therapeutic intervention in human autoimmune disease.

  16. Liver X Receptor (LXR) activation negatively regulates visfatin expression in macrophages

    SciTech Connect

    Mayi, Therese Hervee; Rigamonti, Elena; Pattou, Francois; Staels, Bart; Chinetti-Gbaguidi, Giulia

    2011-01-07

    Research highlights: {yields} Synthetic LXR ligands decreased visfatin expression in human macrophages. {yields} LXR activation leads to a modest and transient decrease of NAD{sup +} concentration. {yields} LXR activation decreased PPAR{gamma}-induced visfatin in human macrophages. -- Abstract: Adipose tissue macrophages (ATM) are the major source of visfatin, a visceral fat adipokine upregulated during obesity. Also known to play a role in B cell differentiation (pre-B cell colony-enhancing factor (PBEF)) and NAD biosynthesis (nicotinamide phosphoribosyl transferase (NAMPT)), visfatin has been suggested to play a role in inflammation. Liver X Receptor (LXR) and Peroxisome Proliferator-Activated Receptor (PPAR){gamma} are nuclear receptors expressed in macrophages controlling the inflammatory response. Recently, we reported visfatin as a PPAR{gamma} target gene in human macrophages. In this study, we examined whether LXR regulates macrophage visfatin expression. Synthetic LXR ligands decreased visfatin gene expression in a LXR-dependent manner in human and murine macrophages. The decrease of visfatin mRNA was paralleled by a decrease of protein secretion. Consequently, a modest and transient decrease of NAD{sup +} concentration was observed. Interestingly, LXR activation decreased the PPAR{gamma}-induced visfatin gene and protein secretion in human macrophages. Our results identify visfatin as a gene oppositely regulated by the LXR and PPAR{gamma} pathways in human macrophages.

  17. MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity

    PubMed Central

    Moretti, Irene; Ciciliot, Stefano; Dyar, Kenneth A.; Abraham, Reimar; Murgia, Marta; Agatea, Lisa; Akimoto, Takayuki; Bicciato, Silvio; Forcato, Mattia; Pierre, Philippe; Uhlenhaut, N. Henriette; Rigby, Peter W. J.; Carvajal, Jaime J.; Blaauw, Bert; Calabria, Elisa; Schiaffino, Stefano

    2016-01-01

    The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia. PMID:27484840

  18. TAM receptors affect adult brain neurogenesis by negative regulation of microglial cell activation.

    PubMed

    Ji, Rui; Tian, Shifu; Lu, Helen J; Lu, Qingjun; Zheng, Yan; Wang, Xiaomin; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

    2013-12-15

    TAM tyrosine kinases play multiple functional roles, including regulation of the target genes important in homeostatic regulation of cytokine receptors or TLR-mediated signal transduction pathways. In this study, we show that TAM receptors affect adult hippocampal neurogenesis and loss of TAM receptors impairs hippocampal neurogenesis, largely attributed to exaggerated inflammatory responses by microglia characterized by increased MAPK and NF-κB activation and elevated production of proinflammatory cytokines that are detrimental to neuron stem cell proliferation and neuronal differentiation. Injection of LPS causes even more severe inhibition of BrdU incorporation in the Tyro3(-/-)Axl(-/-)Mertk(-/-) triple-knockout (TKO) brains, consistent with the LPS-elicited enhanced expression of proinflammatory mediators, for example, IL-1β, IL-6, TNF-α, and inducible NO synthase, and this effect is antagonized by coinjection of the anti-inflammatory drug indomethacin in wild-type but not TKO brains. Conditioned medium from TKO microglia cultures inhibits neuron stem cell proliferation and neuronal differentiation. IL-6 knockout in Axl(-/-)Mertk(-/-) double-knockout mice overcomes the inflammatory inhibition of neurogenesis, suggesting that IL-6 is a major downstream neurotoxic mediator under homeostatic regulation by TAM receptors in microglia. Additionally, autonomous trophic function of the TAM receptors on the proliferating neuronal progenitors may also promote progenitor differentiation into immature neurons.

  19. PLZF is a negative regulator of retinoic acid receptor transcriptional activity.

    PubMed

    Martin, Perrine J; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2003-09-06

    BACKGROUND: Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. RESULTS: We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. CONCLUSION: Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled.

  20. TRAF family member-associated NF-κB activator (TANK) induced by RANKL negatively regulates osteoclasts survival and function.

    PubMed

    Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

    2012-01-01

    Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.

  1. E3 Ubiquitin Ligase RLIM Negatively Regulates c-Myc Transcriptional Activity and Restrains Cell Proliferation

    PubMed Central

    Wang, Lan; Cai, Hao; Zhu, Jingjing; Yu, Long

    2016-01-01

    RNF12/RLIM is a RING domain-containing E3 ubiquitin ligase whose function has only begun to be elucidated recently. Although RLIM was reported to play important roles in some biological processes such as imprinted X-chromosome inactivation and regulation of TGF-β pathway etc., other functions of RLIM are largely unknown. Here, we identified RLIM as a novel E3 ubiquitin ligase for c-Myc, one of the most frequently deregulated oncoproteins in human cancers. RLIM associates with c-Myc in vivo and in vitro independently of the E3 ligase activity of RLIM. Moreover, RLIM promotes the polyubiquitination of c-Myc protein independently of Ser62 and Thr58 phosphorylation of c-Myc. However, RLIM-mediated ubiquitination does not affect c-Myc stability. Instead, RLIM inhibits the transcriptional activity of c-Myc through which RLIM restrains cell proliferation. Our results suggest that RLIM may function as a tumor suppressor by controlling the activity of c-Myc oncoprotein. PMID:27684546

  2. Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

    PubMed Central

    Pradère, Jean-Philippe; Hernandez, Céline; Koppe, Christiane; Friedman, Richard A; Luedde, Tom; Schwabe, Robert F.

    2016-01-01

    Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of kB (IκB) kinase (IKK)–dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. Here, we investigated the contribution of the phosphorylation of the NF-κ B subunit p65 at the IKK phosphorylation site serine 536 (Ser536) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser536), we observed increased expression of NF-κ B–dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κ B signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser534-phosphorylated wild-type protein. Together, our results suggest that Ser534 phosphorylation of p65 in mice (and, by extension, Ser536 phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κ B signaling to prevent deleterious inflammation. PMID:27555662

  3. PLZF is a negative regulator of retinoic acid receptor transcriptional activity

    PubMed Central

    Martin, Perrine J; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2003-01-01

    Background Retinoic acid receptors (RARs) are ligand-regulated transcription factors controlling cellular proliferation and differentiation. Receptor-interacting proteins such as corepressors and coactivators play a crucial role in specifying the overall transcriptional activity of the receptor in response to ligand treatment. Little is known however on how receptor activity is controlled by intermediary factors which interact with RARs in a ligand-independent manner. Results We have identified the promyelocytic leukemia zinc finger protein (PLZF), a transcriptional corepressor, to be a RAR-interacting protein using the yeast two-hybrid assay. We confirmed this interaction by GST-pull down assays and show that the PLZF N-terminal zinc finger domain is necessary and sufficient for PLZF to bind RAR. The RAR ligand binding domain displayed the highest affinity for PLZF, but corepressor and coactivator binding interfaces did not contribute to PLZF recruitment. The interaction was ligand-independent and correlated to a decreased transcriptional activity of the RXR-RAR heterodimer upon overexpression of PLZF. A similar transcriptional interference could be observed with the estrogen receptor alpha and the glucocorticoid receptor. We further show that PLZF is likely to act by preventing RXR-RAR heterodimerization, both in-vitro and in intact cells. Conclusion Thus RAR and PLZF interact physically and functionally. Intriguingly, these two transcription factors play a determining role in hematopoiesis and regionalization of the hindbrain and may, upon chromosomal translocation, form fusion proteins. Our observations therefore define a novel mechanism by which RARs activity may be controlled. PMID:14521715

  4. PPARγ negatively regulates T cell activation to prevent follicular helper T cells and germinal center formation.

    PubMed

    Park, Hong-Jai; Kim, Do-Hyun; Choi, Jin-Young; Kim, Won-Ju; Kim, Ji Yun; Senejani, Alireza G; Hwang, Soo Seok; Kim, Lark Kyun; Tobiasova, Zuzana; Lee, Gap Ryol; Craft, Joseph; Bothwell, Alfred L M; Choi, Je-Min

    2014-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a transcription factor that regulates lipid and glucose metabolism. Although studies of PPARγ ligands have demonstrated its regulatory functions in inflammation and adaptive immunity, its intrinsic role in T cells and autoimmunity has yet to be fully elucidated. Here we used CD4-PPARγKO mice to investigate PPARγ-deficient T cells, which were hyper-reactive to produce higher levels of cytokines and exhibited greater proliferation than wild type T cells with increased ERK and AKT phosphorylation. Diminished expression of IκBα, Sirt1, and Foxo1, which are inhibitors of NF-κB, was observed in PPARγ-deficient T cells that were prone to produce all the signature cytokines under Th1, Th2, Th17, and Th9 skewing condition. Interestingly, 1-year-old CD4-PPARγKO mice spontaneously developed moderate autoimmune phenotype by increased activated T cells, follicular helper T cells (TFH cells) and germinal center B cells with glomerular inflammation and enhanced autoantibody production. Sheep red blood cell immunization more induced TFH cells and germinal centers in CD4-PPARγKO mice and the T cells showed increased of Bcl-6 and IL-21 expression suggesting its regulatory role in germinal center reaction. Collectively, these results suggest that PPARγ has a regulatory role for TFH cells and germinal center reaction to prevent autoimmunity.

  5. Siglec-E Negatively Regulates the Activation of TLR4 by Controlling Its Endocytosis.

    PubMed

    Wu, Yin; Ren, Dongren; Chen, Guo-Yun

    2016-10-15

    TLR4 signaling is critical for providing effective immune protection, but it must be tightly controlled to avoid inflammation-induced pathology. Previously, we reported extensive and direct interactions between TLR and Siglec families of pattern recognition receptors. In this study, we examined the biological significance of this interaction during infection. We show that Siglec-E is required for Escherichia coli-induced endocytosis of TLR4. Siglec-E-deficient dendritic cells infected with E. coli fail to internalize TLR4. This leads to sustained TLR4 on the cell surface and activation of NF-κB and MAPK p38, resulting in high levels of TNF-α and IL-6 compared with wild-type dendritic cells. In contrast to the signaling events occurring at the plasma membrane, as a result of the inability to internalize TLR4, Siglec-E-deficient dendritic cells were also defective for TRIF-mediated IFN-β production in response to E. coli infection. Furthermore, we found that accumulation of ubiquitinated TLR4 and binding of E3 ubiquitin ligase Triad3A to TLR4 was increased significantly in bone marrow-derived dendritic cells from wild-type mice, but not from Siglec-E-deficient mice, after E. coli infection. This represents a newly discovered mechanism that regulates the signaling of TLR4 during E. coli infection.

  6. The catecholamines up (Catsup) protein of Drosophila melanogaster functions as a negative regulator of tyrosine hydroxylase activity.

    PubMed Central

    Stathakis, D G; Burton, D Y; McIvor, W E; Krishnakumar, S; Wright, T R; O'Donnell, J M

    1999-01-01

    We report the genetic, phenotypic, and biochemical analyses of Catecholamines up (Catsup), a gene that encodes a negative regulator of tyrosine hydroxylase (TH) activity. Mutations within this locus are semidominant lethals of variable penetrance that result in three broad, overlapping effective lethal phases (ELPs), indicating that the Catsup gene product is essential throughout development. Mutants from each ELP exhibit either cuticle defects or catecholamine-related abnormalities, such as melanotic salivary glands or pseudotumors. Additionally, Catsup mutants have significantly elevated TH activity that may arise from a post-translational modification of the enzyme. The hyperactivation of TH in Catsup mutants results in abnormally high levels of catecholamines, which can account for the lethality, visible phenotypes, and female sterility observed in these mutants. We propose that Catsup is a component of a novel system that downregulates TH activity, making Catsup the fourth locus found within the Dopa decarboxylase (Ddc) gene cluster that functions in catecholamine metabolism. PMID:10471719

  7. The protein activator of protein kinase R, PACT/RAX, negatively regulates protein kinase R during mouse anterior pituitary development.

    PubMed

    Dickerman, Benjamin K; White, Christine L; Kessler, Patricia M; Sadler, Anthony J; Williams, Bryan R G; Sen, Ganes C

    2015-12-01

    The murine double-stranded RNA-binding protein termed protein kinase R (PKR)-associated protein X (RAX) and the human homolog, protein activator of PKR (PACT), were originally characterized as activators of PKR. Mice deficient in RAX show reproductive and developmental defects, including reduced body size, craniofacial defects and anterior pituitary hypoplasia. As these defects are not observed in PKR-deficient mice, the phenotype has been attributed to PKR-independent activities of RAX. Here we further investigated the involvement of PKR in the physiological function of RAX, by generating rax(-/-) mice deficient in PKR, or carrying a kinase-inactive mutant of PKR (K271R) or an unphosphorylatable mutant of the PKR substrate eukaryotic translation initiation factor 2 α subunit (eIF2α) (S51A). Ablating PKR expression rescued the developmental and reproductive deficiencies in rax(-/-) mice. Generating rax(-/-) mice with a kinase-inactive mutant of PKR resulted in similar rescue, confirming that the rax(-/-) defects are PKR dependent; specifically that the kinase activity of PKR was required for these defects. Moreover, generating rax(-/-) mice that were heterozygous for an unphosphorylatable mutant eIF2α provides partial rescue of the rax(-/-) defect, consistent with mutation of one copy of the Eif2s1 gene. These observations were further investigated in vitro by reducing RAX expression in anterior pituitary cells, resulting in increased PKR activity and induction of the PKR-regulated cyclin-dependent kinase inhibitor p21(WAF1/CIP1). These results demonstrate that PKR kinase activity is required for onset of the rax(-/-) phenotype, implying an unexpected function for RAX as a negative regulator of PKR in the context of postnatal anterior pituitary tissue, and identify a critical role for the regulation of PKR activity for normal development.

  8. Structure-based mutational analysis of ICAT residues mediating negative regulation of β-catenin co-transcriptional activity

    PubMed Central

    Domingues, Mélanie J.; Martinez-Sanz, Juan; Papon, Laura; Larue, Lionel; Mouawad, Liliane

    2017-01-01

    ICAT (Inhibitor of β-CAtenin and TCF) is a small acidic protein that negatively regulates β-catenin co-transcriptional activity by competing with TCF/LEF factors in their binding to β-catenin superhelical core. In melanoma cells, ICAT competes with LEF1 to negatively regulate the M-MITF and NEDD9 target genes. The structure of ICAT consists of two domains: the 3-helix bundle N-terminal domain binds to β-catenin Armadillo (Arm) repeats 10–12 and the C-terminal tail binds to Arm repeats 5–9. To elucidate the structural mechanisms governing ICAT/β-catenin interactions in melanoma cells, three ICAT residues Y15, K19 and V22 in the N-terminal domain, contacting hydrophobic β-catenin residue F660, were mutated and interaction was assessed by immunoprecipitation. Despite the moderate hydrophobicity of the contact, its removal completely abolished the interaction. In the ICAT C-terminal tail consensus sequence, neutralization of the electrostatic interactions between residues D66, E75 and β-catenin residues K435, K312, coupled to deletion of the hydrophobic contact between F71 and β-catenin R386, markedly reduced, but failed to abolish the ICAT-mediated negative regulation of M-MITF and NEDD9 promoters. We conclude that in melanoma cells, anchoring of ICAT N-terminal domain to β-catenin through the hook made by residue F660, trapped in the pincers formed by ICAT residues Y15 and V22, is crucial for stabilizing the ICAT/β-catenin complex. This is a prerequisite for binding of the consensus peptide to Arm repeats 5–9 and competition with LEF1. Differences between ICAT and LEF1 in their affinity for β-catenin may rely on the absence in ICAT of hydrophilic residues between D66 and F71. PMID:28273108

  9. MKP-1 negatively regulates LPS-mediated IL-1β production through p38 activation and HIF-1α expression.

    PubMed

    Talwar, Harvinder; Bauerfeld, Christian; Bouhamdan, Mohamad; Farshi, Pershang; Liu, Yusen; Samavati, Lobelia

    2017-02-24

    Interleukin 1 beta (IL-1β) is a pro-inflammatory cytokine that plays a major role in inflammatory diseases as well as cancer. The inflammatory response after Toll-like receptor (TLR) 4 activation is tightly regulated through phosphorylation of MAP kinases, including p38 and JNK pathways. The activation of MAP kinases is negatively regulated by MAPK phosphatases (MKPs). MKP-1 preferentially dephosphorylates p38 and JNK. IL-1β is regulated through the activation of MAPK, including p38 as well as several transcription factors. The oxygen-sensitive transcription factor HIF-1α is a known transcription factor for several inflammatory cytokines including IL-1β and IL-6. Here, we report that MKP-1 regulates HIF-1α expression in response to LPS. MKP-1 deficient bone marrow derived macrophages (BMDMs) exhibited increased reactive oxygen species (ROS) production and higher HIF-1α expression. In contrast, the expression of all three isoforms of prolyl hydroxylases (PHDs), which are important in destabilizing HIF-1α through hydroxylation, were significantly decreased in MKP-1 deficient BMDMs. LPS challenge of MKP-1 deficient BMDMs led to a substantial increase in IL-1β production. An inhibitor of HIF-1α significantly decreased LPS mediated IL-1β production both at the transcript and protein levels. Similarly, inhibition of p38 MAP kinase reduced LPS mediated pro-IL-1β and HIF-1α protein levels as well as ROS production in MKP-1 deficient BMDMs. These findings demonstrate a regulatory function for MKP-1 in modulating IL-1β expression through p38 activation, ROS production and HIF-1α expression.

  10. The proapoptotic and antimitogenic protein p66SHC acts as a negative regulator of lymphocyte activation and autoimmunity.

    PubMed

    Finetti, Francesca; Pellegrini, Michela; Ulivieri, Cristina; Savino, Maria Teresa; Paccagnini, Eugenio; Ginanneschi, Chiara; Lanfrancone, Luisa; Pelicci, Pier Giuseppe; Baldari, Cosima T

    2008-05-15

    The ShcA locus encodes 3 protein isoforms that differ in tissue specificity, subcellular localization, and function. Among these, p66Shc inhibits TCR coupling to the Ras/MAPK pathway and primes T cells to undergo apoptotic death. We have investigated the outcome of p66Shc deficiency on lymphocyte development and homeostasis. We show that p66Shc(-/-) mice develop an age-related lupus-like autoimmune disease characterized by spontaneous peripheral T- and B-cell activation and proliferation, autoantibody production, and immune complex deposition in kidney and skin, resulting in autoimmune glomerulonephritis and alopecia. p66Shc(-/-) lymphocytes display enhanced proliferation in response to antigen receptor engagement in vitro and more robust immune responses both to vaccination and to allergen sensitization in vivo. The data identify p66Shc as a negative regulator of lymphocyte activation and show that loss of this protein results in breaking of immunologic tolerance and development of systemic autoimmunity.

  11. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    PubMed

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

  12. The GTPase-activating protein GIT2 protects against colitis by negatively regulating Toll-like receptor signaling

    PubMed Central

    Wei, Juncheng; Wei, Chao; Wang, Min; Qiu, Xiao; Li, Yang; Yuan, Yanzhi; Jin, Chaozhi; Leng, Ling; Wang, Jian; Yang, Xiaoming; He, Fuchu

    2014-01-01

    G protein-coupled receptor kinase-interactor 2 (GIT2) regulates thymocyte positive selection, neutrophil-direction sensing, and cell motility during immune responses by regulating the activity of the small GTPases ADP ribosylation factors (Arfs) and Ras-related C3 botulinum toxin substrate 1 (Rac1). Here, we show that Git2-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis, Escherichia coli, or endotoxin-shock challenge, and a dramatic increase in proinflammatory cytokines was observed in Git2 knockout mice and macrophages. GIT2 is a previously unidentified negative regulator of Toll-like receptor (TLR)-induced NF-κB signaling. The ubiquitination of TNF receptor associated factor 6 (TRAF6) is critical for the activation of NF-κB. GIT2 terminates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis to inhibit the ubiquitination of TRAF6. Finally, we show that the susceptibility of Git2-deficient mice to DSS-induced colitis depends on TLR signaling. Thus, we show that GIT2 is an essential terminator of TLR signaling and that loss of GIT2 leads to uncontrolled inflammation and severe organ damage. PMID:24879442

  13. A cytoplasmic negative regulator isoform of ATF7 impairs ATF7 and ATF2 phosphorylation and transcriptional activity.

    PubMed

    Diring, Jessica; Camuzeaux, Barbara; Donzeau, Mariel; Vigneron, Marc; Rosa-Calatrava, Manuel; Kedinger, Claude; Chatton, Bruno

    2011-01-01

    Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor) are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK), phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors.

  14. A Cytoplasmic Negative Regulator Isoform of ATF7 Impairs ATF7 and ATF2 Phosphorylation and Transcriptional Activity

    PubMed Central

    Diring, Jessica; Camuzeaux, Barbara; Donzeau, Mariel; Vigneron, Marc; Rosa-Calatrava, Manuel; Kedinger, Claude; Chatton, Bruno

    2011-01-01

    Alternative splicing and post-translational modifications are processes that give rise to the complexity of the proteome. The nuclear ATF7 and ATF2 (activating transcription factor) are structurally homologous leucine zipper transcription factors encoded by distinct genes. Stress and growth factors activate ATF2 and ATF7 mainly via sequential phosphorylation of two conserved threonine residues in their activation domain. Distinct protein kinases, among which mitogen-activated protein kinases (MAPK), phosphorylate ATF2 and ATF7 first on Thr71/Thr53 and next on Thr69/Thr51 residues respectively, resulting in transcriptional activation. Here, we identify and characterize a cytoplasmic alternatively spliced isoform of ATF7. This variant, named ATF7-4, inhibits both ATF2 and ATF7 transcriptional activities by impairing the first phosphorylation event on Thr71/Thr53 residues. ATF7-4 indeed sequesters the Thr53-phosphorylating kinase in the cytoplasm. Upon stimulus-induced phosphorylation, ATF7-4 is poly-ubiquitinated and degraded, enabling the release of the kinase and ATF7/ATF2 activation. Our data therefore conclusively establish that ATF7-4 is an important cytoplasmic negative regulator of ATF7 and ATF2 transcription factors. PMID:21858082

  15. MCPIP1 endoribonuclease activity negatively regulates interleukin-17-mediated signaling and inflammation

    PubMed Central

    Garg, Abhishek V.; Amatya, Nilesh; Chen, Kong; Cruz, J. Agustin; Grover, Prerna; Whibley, Natasha; Conti, Heather R.; Mir, Gerard Hernandez; Sirakova, Tatiana; Childs, Erin C.; Smithgall, Thomas E.; Biswas, Partha S.; Kolls, Jay K.; McGeachy, Mandy J.; Kolattukudy, Pappachan E.; Gaffen, Sarah L.

    2015-01-01

    SUMMARY Interleukin-17 (IL-17) induces pathology in autoimmunity and infections; therefore constraint of this pathway is an essential component of its regulation. We demonstrate that the signaling intermediate MCPIP1 (also termed Regnase-1, encoded by Zc3h12a) is a feedback inhibitor of IL-17 receptor signal transduction. MCPIP1 knockdown enhanced IL-17-mediated signaling, requiring MCPIP1’s endoribonuclease but not deubiquitinase domain. MCPIP1 haploinsufficient mice showed enhanced resistance to disseminated Candida albicans infection, which was reversed in an Il17ra−/− background. Conversely, IL-17-dependent pathology in Zc3h12a+/− mice was exacerbated in both EAE and pulmonary inflammation. MCPIP1 degraded Il6 mRNA directly, but only modestly downregulated the IL-6 promoter. However, MCPIP1 strongly inhibited the Lcn2 promoter by regulating the mRNA stability of Nfkbiz, encoding the IκBζ transcription factor. Unexpectedly, MCPIP1 degraded Il17ra and Il17rc mRNA, independently of the 3’ UTR. The cumulative impact of MCPIP1 on IL-6, IκBζ and possibly IL-17R subunits results in a biologically relevant inhibition of IL-17 signaling. PMID:26320658

  16. PGC-1-related coactivator (PRC) negatively regulates endothelial adhesion of monocytes via inhibition of NF κB activity

    SciTech Connect

    Chengye, Zhan; Daixing, Zhou Qiang, Zhong; Shusheng, Li

    2013-09-13

    Highlights: •First time to display that LPS downregulate the expression of PRC. •First time to show that PRC inhibits the induction of VCAM-1 and E-selectin. •First time to show that PRC inhibit monocytes attachment to endothelial cells. •First time to display that PRC inhibits transcriptional activity of NF-κB. •PRC protects the respiration rate and suppresses the glycolysis rate against LPS. -- Abstract: PGC-1-related coactivator (PRC) is a growth-regulated transcriptional cofactor known to activate many of the nuclear genes specifying mitochondrial respiratory function. Endothelial dysfunction is a prominent feature found in many inflammatory diseases. Adhesion molecules, such as VCAM-1, mediate the attachment of monocytes to endothelial cells, thereby playing an important role in endothelial inflammation. The effects of PRC in regards to endothelial inflammation remain unknown. In this study, our findings show that PRC can be inhibited by the inflammatory cytokine LPS in cultured human umbilical vein endothelial cells (HUVECs). In the presence of LPS, the expression of endothelial cell adhesion molecular, such as VCAM1 and E-selectin, is found to be increased. These effects can be negated by overexpression of PRC. Importantly, monocyte adhesion to endothelial cells caused by LPS is significantly attenuated by PRC. In addition, overexpression of PRC protects mitochondrial metabolic function and suppresses the rate of glycolysis against LPS. It is also found that overexpression of PRC decreases the transcriptional activity of NF-κB. These findings suggest that PRC is a negative regulator of endothelial inflammation.

  17. Exercise Activates p53 and Negatively Regulates IGF-1 Pathway in Epidermis within a Skin Cancer Model

    PubMed Central

    Yu, Miao; King, Brenee; Ewert, Emily; Su, Xiaoyu; Mardiyati, Nur; Zhao, Zhihui; Wang, Weiqun

    2016-01-01

    Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study aims to investigate the underlying mechanisms by which exercise may down-regulate the IGF-1 pathway via p53 and p53-related regulators in the skin epidermis. Female SENCAR mice were pair-fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min, 60 min/day and 5 days/week. Animals were topically treated with TPA 2 hours before sacrifice and the target proteins in the epidermis were analyzed by both immunohistochemistry and Western blot. Under TPA or vehicle treatment, MDM2 expression was significantly reduced in exercised mice when compared with sedentary control. Meanwhile, p53 was significantly elevated. In addition, p53-transcriptioned proteins, i.e., p21, IGFBP-3, and PTEN, increased in response to exercise. There was a synergy effect between exercise and TPA on the decreased MDM2 and increased p53, but not p53-transcripted proteins. Taken together, exercise appeared to activate p53, resulting in enhanced expression of p21, IGFBP-3, and PTEN that might induce a negative regulation of IGF-1 pathway and thus contribute to the observed cancer prevention by exercise in this skin cancer model. PMID:27509024

  18. Exercise Activates p53 and Negatively Regulates IGF-1 Pathway in Epidermis within a Skin Cancer Model.

    PubMed

    Yu, Miao; King, Brenee; Ewert, Emily; Su, Xiaoyu; Mardiyati, Nur; Zhao, Zhihui; Wang, Weiqun

    2016-01-01

    Exercise has been previously reported to lower cancer risk through reducing circulating IGF-1 and IGF-1-dependent signaling in a mouse skin cancer model. This study aims to investigate the underlying mechanisms by which exercise may down-regulate the IGF-1 pathway via p53 and p53-related regulators in the skin epidermis. Female SENCAR mice were pair-fed an AIN-93 diet with or without 10-week treadmill exercise at 20 m/min, 60 min/day and 5 days/week. Animals were topically treated with TPA 2 hours before sacrifice and the target proteins in the epidermis were analyzed by both immunohistochemistry and Western blot. Under TPA or vehicle treatment, MDM2 expression was significantly reduced in exercised mice when compared with sedentary control. Meanwhile, p53 was significantly elevated. In addition, p53-transcriptioned proteins, i.e., p21, IGFBP-3, and PTEN, increased in response to exercise. There was a synergy effect between exercise and TPA on the decreased MDM2 and increased p53, but not p53-transcripted proteins. Taken together, exercise appeared to activate p53, resulting in enhanced expression of p21, IGFBP-3, and PTEN that might induce a negative regulation of IGF-1 pathway and thus contribute to the observed cancer prevention by exercise in this skin cancer model.

  19. Stress-activated protein kinases are negatively regulated by cell density.

    PubMed Central

    Lallemand, D; Ham, J; Garbay, S; Bakiri, L; Traincard, F; Jeannequin, O; Pfarr, C M; Yaniv, M

    1998-01-01

    Stimulation by UV irradiation, TNFalpha, as well as PDGF or EGF activates the JNK/SAPK signalling pathway in mouse fibroblasts. This results in the phosphorylation of the N-terminal domain of c-Jun, increasing its transactivation potency. Using an antibody that specifically recognizes c-Jun phosphorylated at Ser63, we show that culture confluency drastically inhibited c-Jun N-terminal phosphorylation due to the inhibition of the JNK/SAPK pathway. Transfection experiments demonstrate that the inhibition occurs at the same level as, or upstream of, the small G-proteins cdc42 and Rac1. In contrast, the classical MAPK pathway was insensitive to confluency. The inhibition of JNK/SAPK activation depended on the integrity of the actin microfilament network. These results were confirmed and extended in monolayer wounding experiments. After PDGF, EGF or UV stimulation, c-Jun was predominantly phosphorylated in cells bordering the wound, which are the cells that move to occupy the wounded area. Thus, modulation of the stress-dependent signal cascade by confluency will restrict c-Jun N-terminal phosphorylation in response to mitogenic or chemotactic agents to cells that border a wounded area. PMID:9755162

  20. The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

    PubMed

    Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

    2009-02-01

    The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

  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. p21-activated kinase1 (Pak1) is a negative regulator of NADPH-oxidase 2 in ventricular myocytes

    PubMed Central

    DeSantiago, Jaime; Bare, Dan J; Xiao, Lei; Ke, Yunbo; Solaro, R. John; Banach, Kathrin

    2014-01-01

    Ischemic conditions reduce the activity of the p21-activated kinase (Pak1) resulting in increased arrhythmic activity. Triggered arrhythmic activity during ischemia is based on changes in cellular ionic balance and the cells Ca2+ handling properties. In the current study we used isolated mouse ventricular myocytes (VMs) deficient for the expression of Pak1 (Pak1-/-) to determine the mechanism by which Pak1 influences the generation of arrhythmic activity during simulated ischemia. The Ca2+ transient amplitude and kinetics did not significantly change in wild type (WT) and Pak1-/- VMs during 15 min of simulated ischemia. However, Pak1-/- VMs exhibited an exaggerated increase in [Ca2+]i, which resulted in spontaneous Ca2+ release events and waves. The Ca2+ overload in Pak1-/- VMs could be suppressed with a reverse mode blocker (KB-R7943) of the sodium calcium exchanger (NCX), a cytoplasmic scavenger of reactive oxygen species (ROS; TEMPOL) or a RAC1 inhibitor (NSC23766). Measurements of the cytoplasmic ROS levels revealed that decreased Pak1 activity in Pak1-/- VMs or VMs treated with the Pak1 inhibitor (IPA3) enhanced cellular ROS production. The Pak1 dependent increase in ROS was attenuated in VMs deficient for NADPH oxidase 2 (NOX2; p47phox-/-) or in VMs where NOX2 was inhibited (gp91ds-tat). Voltage clamp recordings showed increased NCX activity in Pak1-/- VMs that depended on enhanced NOX2 induced ROS production. The exaggerated Ca2+ overload in Pak1-/- VMs could be mimicked by low concentrations of ouabain. Overall our data show that Pak1 is a critical negative regulator of NOX2 dependent ROS production and that a latent ROS dependent stimulation of NCX activity can predispose VMs to Ca2+ overload under conditions where no significant changes in excitation-contraction coupling are yet evident. PMID:24380729

  3. N-terminal domain of soluble epoxide hydrolase negatively regulates the VEGF-mediated activation of endothelial nitric oxide synthase

    PubMed Central

    Hou, Hsin-Han; Hammock, Bruce D.; Su, Kou-Hui; Morisseau, Christophe; Kou, Yu Ru; Imaoka, Susumu; Oguro, Ami; Shyue, Song-Kun; Zhao, Jin-Feng; Lee, Tzong-Shyuan

    2012-01-01

    Aims The mammalian soluble epoxide hydrolase (sEH) has both an epoxide hydrolase and a phosphatase domain. The role of sEH hydrolase activity in the metabolism of epoxyeicosatrienoic acids (EETs) and the activation of endothelial nitric oxide synthase (eNOS) in endothelial cells (ECs) has been well defined. However, far less is known about the role of sEH phosphatase activity in eNOS activation. In the present study, we investigated whether the phosphatase domain of sEH was involved in the eNOS activation in ECs. Methods and results The level of eNOS phosphorylation in aortas is higher in the sEH knockout (sEH−/−) mice than in wild-type mice. In ECs, pharmacological inhibition of sEH phosphatase or overexpressing sEH with an inactive phosphatase domain enhanced vascular endothelial growth factor (VEGF)-induced NO production and eNOS phosphorylation. In contrast, overexpressing the phosphatase domain of sEH prevented the VEGF-mediated NO production and eNOS phosphorylation at Ser617, Ser635, and Ser1179. Additionally, treatment with VEGF induced a c-Src kinase-dependent increase in transient tyrosine phosphorylation of sEH and the formation of a sEH–eNOS complex, which was abolished by treatment with a c-Src kinase inhibitor, PP1, or the c-Src dominant-negative mutant K298M. We also demonstrated that the phosphatase domain of sEH played a key role in VEGF-induced angiogenesis by detecting the tube formation in ECs and neovascularization in Matrigel plugs in mice. Conclusion In addition to epoxide hydrolase activity, phosphatase activity of sEH plays a pivotal role in the regulation of eNOS activity and NO-mediated EC functions. PMID:22072631

  4. Peroxiredoxin-6 Negatively Regulates Bactericidal Activity and NF-κB Activity by Interrupting TRAF6-ECSIT Complex

    PubMed Central

    Min, Yoon; Wi, Sae M.; Shin, Dongwoo; Chun, Eunyoung; Lee, Ki-Young

    2017-01-01

    A TRAF6-ECSIT complex is crucial for the generation of mitochondrial reactive oxygen species (mROS) and nuclear factor-kappa B (NF-κB) activation induced by Toll-like receptor 4 (TLR4). Peroxiredoxin-6 (Prdx6) as a member of the peroxiredoxin family of antioxidant enzymes is involved in antioxidant protection and cell signaling. Here, we report on a regulatory role of Prdx6 in mROS production and NF-κB activation by TLR4. Prdx6 was translocated into the mitochondria by TLR4 stimulation and Prdx6-knockdown (Prdx6KD) THP-1 cells had increased level of mitochondrial reactive oxygen species levels and were resistant to Salmonella typhimurium infection. Biochemical studies revealed Prdx6 interaction with the C-terminal TRAF-C domain of TRAF6, which drove translocation into the mitochondria. Interestingly, Prdx6 competitively interacted with ECSIT to TRAF6 through its C-terminal TRAF-C domain, leading to the interruption of TRAF6-ECSIT interaction. The inhibitory effect was critically implicated in the activation of NF-κB induced by TLR4. Overexpression of Prdx6 led to the inhibition of NF-κB induced by TLR4, whereas Prdx6KD THP-1 cells displayed enhanced production of pro-inflammatory cytokines including interleukin-6 and -1β, and the up-regulation of NF-κB-dependent genes induced by TLR4 stimulation. Taken together, the data demonstrate that Prdx6 interrupts the formation of TRAF6-ECSIT complex induced by TLR4 stimulation, leading to suppression of bactericidal activity because of inhibited mROS production in mitochondria and the inhibition of NF-κB activation in the cytoplasm. PMID:28393051

  5. Transcription of the Salmonella Invasion Gene Activator, hilA, Requires HilD Activation in the Absence of Negative Regulators

    PubMed Central

    Boddicker, Jennifer D.; Knosp, Boyd M.; Jones, Bradley D.

    2003-01-01

    Salmonella enterica serovar Typhimurium causes human gastroenteritis and a systemic typhoid-like infection in mice. Infection is initiated by entry of the bacteria into intestinal epithelial cells and is mediated by a type III secretion system that is encoded by genes in Salmonella pathogenicity island 1. The expression of invasion genes is tightly regulated by environmental conditions such as oxygen and osmolarity, as well as by many bacterial factors. The hilA gene encodes an OmpR/ToxR family transcriptional regulator that activates the expression of invasion genes in response to both environmental and genetic regulatory factors. HilD is an AraC/XylS regulator that has been postulated to act as a derepressor of hilA expression that promotes transcription by interfering with repressor binding at the hilA promoter. Our research group has identified four genes (hilE, hha, pag, and ams) that negatively affect hilA transcription. Since the postulated function of HilD at the hilA promoter is to counteract the effects of repressors, we examined this model by measuring hilA::Tn5lacZY expression in strains containing negative regulator mutations in the presence or absence of functional HilD. Single negative regulator mutations caused significant derepression of hilA expression, and two or more negative regulator mutations led to very high level expression of hilA. However, in all strains tested, the absence of hilD resulted in low-level expression of hilA, suggesting that HilD is required for activation of hilA expression, whether or not negative regulators are present. We also observed that deletion of the HilD binding sites in the chromosomal hilA promoter severely decreased hilA expression. In addition, we found that a single point mutation at leucine 289 in the C-terminal domain of the α subunit of RNA polymerase leads to very low levels of hilA::Tn5lacZY expression, suggesting that HilD activates transcription of hilA by contacting and recruiting RNA polymerase to

  6. A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability.

    PubMed

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Basco, Davide; Catalano, Francesco; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2014-02-01

    Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated.

  7. A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability

    PubMed Central

    De Bellis, Manuela; Pisani, Francesco; Mola, Maria Grazia; Basco, Davide; Catalano, Francesco; Nicchia, Grazia Paola; Svelto, Maria; Frigeri, Antonio

    2014-01-01

    Two major isoforms of aquaporin-4 (AQP4) have been described in human tissue. Here we report the identification and functional analysis of an alternatively spliced transcript of human AQP4, AQP4-Δ4, that lacks exon 4. In transfected cells AQP4-Δ4 is mainly retained in the endoplasmic reticulum and shows no water transport properties. When AQP4-Δ4 is transfected into cells stably expressing functional AQP4, the surface expression of the full-length protein is reduced. Furthermore, the water transport activity of the cotransfectants is diminished in comparison to transfectants expressing only AQP4. The observed down-regulation of both the expression and water channel activity of AQP4 is likely to originate from a dominant-negative effect caused by heterodimerization between AQP4 and AQP4-Δ4, which was detected in coimmunoprecipitation studies. In skeletal muscles, AQP4-Δ4 mRNA expression inversely correlates with the level of AQP4 protein and is physiologically associated with different types of skeletal muscles. The expression of AQP4-Δ4 may represent a new regulatory mechanism through which the cell-surface expression and therefore the activity of AQP4 can be physiologically modulated. PMID:24356448

  8. Signaling lymphocyte activation molecule-associated protein is a negative regulator of the CD8 T cell response in mice.

    PubMed

    Chen, Gang; Tai, Albert K; Lin, Miao; Chang, Francesca; Terhorst, Cox; Huber, Brigitte T

    2005-08-15

    The primary manifestation of X-linked lymphoproliferative syndrome, caused by a dysfunctional adapter protein, signaling lymphocyte activation molecule-associated protein (SAP), is an excessive T cell response upon EBV infection. Using the SAP-/- mouse as a model system for the human disease, we compared the response of CD8+ T cells from wild-type (wt) and mutant mice to various stimuli. First, we observed that CD8+ T cells from SAP-/- mice proliferate more vigorously than those from wt mice upon CD3/CD28 cross-linking in vitro. Second, we analyzed the consequence of SAP deficiency on CTL effector function and homeostasis. For this purpose, SAP-/- and wt mice were infected with the murine gamma-herpesvirus 68 (MHV-68). At 2 wk postinfection, the level of viral-specific CTL was much higher in mutant than in wt mice, measured both ex vivo and in vivo. In addition, we established that throughout 45 days of MHV-68 infection the frequency of virus-specific CD8+ T cells producing IFN-gamma was significantly higher in SAP-/- mice. Consequently, the level of latent infection by MHV-68 was considerably lower in SAP-/- mice, which indicates that SAP-/- CTL control this infection more efficiently than wt CTL. Finally, we found that the Vbeta4-specific CD8+ T cell expansion triggered by MHV-68 infection is also enhanced and prolonged in SAP-/- mice. Taken together, our data indicate that SAP functions as a negative regulator of CD8+ T cell activation.

  9. Interaction of Nck1 and PERK phosphorylated at Y⁵⁶¹ negatively modulates PERK activity and PERK regulation of pancreatic β-cell proinsulin content.

    PubMed

    Yamani, Lama; Latreille, Mathieu; Larose, Louise

    2014-03-01

    PERK, the PKR-like endoplasmic reticulum (ER) kinase, is an ER transmembrane serine/threonine protein kinase activated during ER stress. In this study, we provide evidence that the Src-homology domain-containing adaptor Nck1 negatively regulates PERK. We show that Nck directly binds to phosphorylated Y(561) in the PERK juxtamembrane domain through its SH2 domain. We demonstrate that mutation of Y(561) to a nonphosphorylatable residue (Y561F) promotes PERK activity, suggesting that PERK phosphorylation at Y(561) (pY(561)PERK) negatively regulates PERK. In agreement, we show that pY(561)PERK delays PERK activation and signaling during ER stress. Compatible with a role for PERK in pancreatic β-cells, we provide strong evidence that Nck1 contributes to PERK regulation of pancreatic β-cell proteostasis. In fact, we demonstrated that down-regulation of Nck1 in mouse insulinoma MIN6 cells results in faster dephosphorylation of pY(561)PERK, which correlates with enhanced PERK activation, increased insulin biosynthesis, and PERK-dependent increase in proinsulin content. Furthermore, we report that pancreatic islets in whole-body Nck1-knockout mice contain more insulin than control littermates. Together our data strongly suggest that Nck1 negatively regulates PERK by interacting with PERK and protecting PERK from being dephosphorylated at its inhibitory site pY(561) and in this way affects pancreatic β-cell proinsulin biogenesis.

  10. FRNK negatively regulates IL-4-mediated inflammation.

    PubMed

    Sharma, Ritu; Colarusso, Pina; Zhang, Hong; Stevens, Katarzyna M; Patel, Kamala D

    2015-02-15

    Focal adhesion kinase (FAK)-related nonkinase (PTK2 isoform 6 in humans, hereafter referred to as FRNK) is a cytoskeletal regulatory protein that has recently been shown to dampen lung fibrosis, yet its role in inflammation is unknown. Here, we show for the first time that expression of FRNK negatively regulates IL-4-mediated inflammation in a human model of eosinophil recruitment. Mechanistically, FRNK blocks eosinophil accumulation, firm adhesion and transmigration by preventing transcription and protein expression of VCAM-1 and CCL26. IL-4 activates STAT6 to induce VCAM-1 and CCL26 transcription. We now show that IL-4 also increases GATA6 to induce VCAM-1 expression. FRNK blocks IL-4-induced GATA6 transcription but has little effect on GATA6 protein expression and no effect on STAT6 activation. FRNK can block FAK or Pyk2 signaling and we, thus, downregulated these proteins using siRNA to determine whether signaling from either protein is involved in the regulation of VCAM-1 and CCL26. Knockdown of FAK, Pyk2 or both had no effect on VCAM-1 or CCL26 expression, which suggests that FRNK acts independently of FAK and Pyk2 signaling. Finally, we found that IL-4 induces the late expression of endogenous FRNK. In summary, FRNK represents a novel mechanism to negatively regulate IL-4-mediated inflammation.

  11. GADD45α induction by nickel negatively regulates JNKs/p38 activation via promoting PP2Cα expression.

    PubMed

    Yu, Yonghui; Li, Jingxia; Wan, Yu; Lu, Jianyi; Gao, Jimin; Huang, Chuanshu

    2013-01-01

    Growth arrest and DNA damage (GADD) 45α is a member of GADD inducible gene family, and is inducible in cell response to oxidative stress. GADD45α upregulation induces MKK4/JNK activation in some published experimental systems. However, we found here that the depletion of GADD45α (GADD45α-/-) in mouse embryonic fibroblasts (MEFs) resulted in an increase in the phosphorylation of MKK4/7, MKK3/6 and consequently specific up-regulated the activation of JNK/p38 and their downstream transcription factors, such as c-Jun and ATF2, in comparison to those in GADD45α+/+ MEFs cell following nickel exposure. This up-regulation of MKK-JNK/p38 pathway in GADD45α-/- cell could be rescued by the reconstitutional expression of HA-GADD45α in GADD45α-/- MEFs, GADD45α-/-(HA-GADD45α). Subsequent studies indicated that GADD45α deletion repressed expression of PP2Cα, the phosphotase of MKK3/6 and MKK4/7, whereas ectopic expression of HA-PP2Cα in GADD45α-/- cells attenuated activation of MKK3/6-p38 and MKK4/7-JNK pathways. Collectively, our results demonstrate a novel function and mechanism responsible for GADD45α regulation of MKK/MAPK pathway, further provides insight into understanding the big picture of GADD45α in the regulation of cellular responses to oxidative stress and environmental carcinogens.

  12. Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction.

    PubMed

    Patil, Ajay Vitthal; Hsieh, Tao-Shih

    2017-03-10

    Human RecQ-like helicase 4 (RECQL4) plays crucial roles in replication initiation and DNA repair; however, the contextual regulation of its unwinding activity is not fully described. Mutations in RECQL4 have been linked to three diseases including Rothmund-Thomson syndrome, which is characterized by osteoskeletal deformities, photosensitivity, and increased osteosarcoma susceptibility. Understanding regulation of RECQL4 helicase activity by interaction partners will allow deciphering its role as an enzyme and a signaling cofactor in different cellular contexts. We became interested in studying the interaction of RECQL4 with ribosomal protein S3 (RPS3) because previous studies have shown that RPS3 activity is sometimes associated with phenotypes mimicking those of mutated RECQL4. RPS3 is a small ribosomal protein that also has extraribosomal functions, including apurnic-apyrimidinic endonuclease-like activity suggested to be important during DNA repair. Here, we report a functional and physical interaction between RPS3 and RECQL4 and show that this interaction may be enhanced during cellular stress. We show that RPS3 inhibits ATPase, DNA binding, and helicase activities of RECQL4 through their direct interaction. Further domain analysis shows that N-terminal 1-320 amino acids of RECQL4 directly interact with the C-terminal 94-244 amino acids of RPS3 (C-RPS3). Biochemical analysis of C-RPS3 revealed that it comprises a standalone apurnic-apyrimidinic endonuclease-like domain. We used U2OS cells to show that oxidative stress and UV exposure could enhance the interaction between nuclear RPS3 and RECQL4. Regulation of RECQL4 biochemical activities by RPS3 along with nuclear interaction during UV and oxidative stress may serve to modulate active DNA repair.

  13. Cocaine users with comorbid Cluster B personality disorders show dysfunctional brain activation and connectivity in the emotional regulation networks during negative emotion maintenance and reappraisal.

    PubMed

    Albein-Urios, Natalia; Verdejo-Román, Juan; Soriano-Mas, Carles; Asensio, Samuel; Martínez-González, José Miguel; Verdejo-García, Antonio

    2013-12-01

    Cocaine dependence often co-occurs with Cluster B personality disorders. Since both disorders are characterized by emotion regulation deficits, we predicted that cocaine comorbid patients would exhibit dysfunctional patterns of brain activation and connectivity during reappraisal of negative emotions. We recruited 18 cocaine users with comorbid Cluster B personality disorders, 17 cocaine users without comorbidities and 21 controls to be scanned using functional magnetic resonance imaging (fMRI) during performance on a reappraisal task in which they had to maintain or suppress the emotions induced by negative affective stimuli. We followed region of interest (ROI) and whole-brain approaches to investigate brain activations and connectivity associated with negative emotion experience and reappraisal. Results showed that cocaine users with comorbid personality disorders had reduced activation of the subgenual anterior cingulate cortex during negative emotion maintenance and increased activation of the lateral orbitofrontal cortex and the amygdala during reappraisal. Amygdala activation correlated with impulsivity and antisocial beliefs in the comorbid group. Connectivity analyses showed that in the cocaine comorbid group the subgenual cingulate was less efficiently connected with the amygdala and the fusiform gyri and more efficiently connected with the anterior insula during maintenance, whereas during reappraisal the left orbitofrontal cortex was more efficiently connected with the amygdala and the right orbitofrontal cortex was less efficiently connected with the dorsal striatum. We conclude that cocaine users with comorbid Cluster B personality disorders have distinctive patterns of brain activation and connectivity during maintenance and reappraisal of negative emotions, which correlate with impulsivity and dysfunctional beliefs.

  14. Transforming growth factor β-activated kinase 1 negatively regulates interleukin-1α-induced stromal-derived factor-1 expression in vascular smooth muscle cells

    SciTech Connect

    Yang, Bin; Li, Wei; Zheng, Qichang; Qin, Tao; Wang, Kun; Li, Jinjin; Guo, Bing; Yu, Qihong; Wu, Yuzhe; Gao, Yang; Cheng, Xiang; Hu, Shaobo; Kumar, Stanley Naveen; Liu, Sanguang; Song, Zifang

    2015-07-17

    Stromal-derived Factor-1 (SDF-1) derived from vascular smooth muscle cells (VSMCs) contributes to vascular repair and remodeling in various vascular diseases. In this study, the mechanism underlying regulation of SDF-1 expression by interleukin-1α (IL-1α) was investigated in primary rat VSMCs. We found IL-1α promotes SDF-1 expression by up-regulating CCAAT-enhancer-binding protein β (C/EBPβ) in an IκB kinase β (IKKβ) signaling-dependent manner. Moreover, IL-1α-induced expression of C/EBPβ and SDF-1 was significantly potentiated by knockdown of transforming growth factor β-activated kinase 1 (TAK1), an upstream activator of IKKβ signaling. In addition, we also demonstrated that TAK1/p38 mitogen-activated protein kinase (p38 MAPK) signaling exerted negative effect on IL-1α-induced expression of C/EBPβ and SDF-1 through counteracting ROS-dependent up-regulation of nuclear factor erythroid 2-related factor 2 (NRF2). In conclusion, TAK1 acts as an important regulator of IL-1α-induced SDF-1 expression in VSMCs, and modulating activity of TAK1 may serve as a potential strategy for modulating vascular repair and remodeling. - Highlights: • IL-1α induces IKKβ signaling-dependent SDF-1 expression by up-regulating C/EBPβ. • Activation of TAK1 by IL-1α negatively regulates C/EBPβ-dependent SDF-1 expression. • IL-1α-induced TAK1/p38 MAPK signaling counteracts ROS-dependent SDF-1 expression. • TAK1 counteracts IL-1α-induced SDF-1 expression by attenuating NRF2 up-regulation.

  15. p62(dok), a negative regulator of Ras and mitogen-activated protein kinase (MAPK) activity, opposes leukemogenesis by p210(bcr-abl).

    PubMed

    Di Cristofano, A; Niki, M; Zhao, M; Karnell, F G; Clarkson, B; Pear, W S; Van Aelst, L; Pandolfi, P P

    2001-08-06

    p62(dok) has been identified as a substrate of many oncogenic tyrosine kinases such as the chronic myelogenous leukemia (CML) chimeric p210(bcr-abl) oncoprotein. It is also phosphorylated upon activation of many receptors and cytoplamic tyrosine kinases. However, the biological functions of p62(dok) in normal cell signaling as well as in p210(bcr-abl) leukemogenesis are as yet not fully understood. Here we show, in hemopoietic and nonhemopoietic cells derived from p62(dok)-(/)- mice, that the loss of p62(dok) results in increased cell proliferation upon growth factor treatment. Moreover, Ras and mitogen-activated protein kinase (MAPK) activation is markedly sustained in p62(dok)-(/)- cells after the removal of growth factor. However, p62(dok) inactivation does not affect DNA damage and growth factor deprivation-induced apoptosis. Furthermore, p62(dok) inactivation causes a significant shortening in the latency of the fatal myeloproliferative disease induced by retroviral-mediated transduction of p210(bcr-abl) in bone marrow cells. These data indicate that p62(dok) acts as a negative regulator of growth factor-induced cell proliferation, at least in part through downregulating Ras/MAPK signaling pathway, and that p62(dok) can oppose leukemogenesis by p210(bcr-abl).

  16. The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation.

    PubMed

    Kumar, Sachin; Xu, Juying; Kumar, Rupali Sani; Lakshmikanthan, Sribalaji; Kapur, Reuben; Kofron, Matthew; Chrzanowska-Wodnicka, Magdalena; Filippi, Marie-Dominique

    2014-08-25

    Neutrophils are the first line of cellular defense in response to infections and inflammatory injuries. However, neutrophil activation and accumulation into tissues trigger tissue damage due to release of a plethora of toxic oxidants and proteases, a cause of acute lung injury (ALI). Despite its clinical importance, the molecular regulation of neutrophil migration is poorly understood. The small GTPase Rap1b is generally viewed as a positive regulator of immune cell functions by controlling bidirectional integrin signaling. However, we found that Rap1b-deficient mice exhibited enhanced neutrophil recruitment to inflamed lungs and enhanced susceptibility to endotoxin shock. Unexpectedly, Rap1b deficiency promoted the transcellular route of diapedesis through endothelial cell. Increased transcellular migration of Rap1b-deficient neutrophils in vitro was selectively mediated by enhanced PI3K-Akt activation and invadopodia-like protrusions. Akt inhibition in vivo suppressed excessive Rap1b-deficient neutrophil migration and associated endotoxin shock. The inhibitory action of Rap1b on PI3K signaling may be mediated by activation of phosphatase SHP-1. Thus, this study reveals an unexpected role for Rap1b as a key suppressor of neutrophil migration and lung inflammation.

  17. The Nedd4-2/Ndfip1 axis is a negative regulator of IgE-mediated mast cell activation

    PubMed Central

    Yip, Kwok Ho; Kolesnikoff, Natasha; Hauschild, Nicholas; Biggs, Lisa; Lopez, Angel F.; Galli, Stephen J.; Kumar, Sharad; Grimbaldeston, Michele A.

    2016-01-01

    Cross-linkage of the high-affinity immunoglobulin E (IgE) receptor (FcɛRI) on mast cells by antigen ligation has a critical role in the pathology of IgE-dependent allergic disorders, such as anaphylaxis and asthma. Restraint of intracellular signal transduction pathways that promote release of mast cell-derived pro-inflammatory mediators is necessary to dampen activation and restore homoeostasis. Here we show that the ligase Nedd4-2 and the adaptor Ndfip1 (Nedd4 family interacting protein 1) limit the intensity and duration of IgE-FcɛRI-induced positive signal transduction by ubiquitinating phosphorylated Syk, a tyrosine kinase that is indispensable for downstream FcɛRI signalosome activity. Importantly, loss of Nedd4-2 or Ndfip1 in mast cells results in exacerbated and prolonged IgE-mediated cutaneous anaphylaxis in vivo. Our findings reveal an important negative regulatory function for Nedd4-2 and Ndfip1 in IgE-dependent mast cell activity. PMID:27786273

  18. Nitric oxide negatively regulates mammalian adult neurogenesis

    NASA Astrophysics Data System (ADS)

    Packer, Michael A.; Stasiv, Yuri; Benraiss, Abdellatif; Chmielnicki, Eva; Grinberg, Alexander; Westphal, Heiner; Goldman, Steven A.; Enikolopov, Grigori

    2003-08-01

    Neural progenitor cells are widespread throughout the adult central nervous system but only give rise to neurons in specific loci. Negative regulators of neurogenesis have therefore been postulated, but none have yet been identified as subserving a significant role in the adult brain. Here we report that nitric oxide (NO) acts as an important negative regulator of cell proliferation in the adult mammalian brain. We used two independent approaches to examine the function of NO in adult neurogenesis. In a pharmacological approach, we suppressed NO production in the rat brain by intraventricular infusion of an NO synthase inhibitor. In a genetic approach, we generated a null mutant neuronal NO synthase knockout mouse line by targeting the exon encoding active center of the enzyme. In both models, the number of new cells generated in neurogenic areas of the adult brain, the olfactory subependyma and the dentate gyrus, was strongly augmented, which indicates that division of neural stem cells in the adult brain is controlled by NO and suggests a strategy for enhancing neurogenesis in the adult central nervous system.

  19. An HSV-based library screen identifies PP1α as a negative TRPV1 regulator with analgesic activity in models of pain

    PubMed Central

    Reinhart, Bonnie; Goins, William F; Harel, Asaff; Chaudhry, Suchita; Goss, James R; Yoshimura, Naoki; de Groat, William C; Cohen, Justus B; Glorioso, Joseph C

    2016-01-01

    Transient receptor potential vanilloid 1 (TRPV1) is a pronociceptive cation channel involved in persistent inflammatory and neuropathic pain. Herpes simplex virus (HSV) vector expression of TRPV1 causes cell death in the presence of capsaicin, thereby completely blocking virus replication. Here we describe a selection system for negative regulators of TRPV1 based on rescue of virus replication. HSV-based coexpression of TRPV1 and a PC12 cell-derived cDNA library identified protein phosphatase 1α (PP1α) as a negative regulator of TRPV1, mimicking the activity of “poreless” (PL), a dominant-negative mutant of TRPV1. Vectors expressing PP1α or PL reduced thermal sensitivity following virus injection into rat footpads, but failed to reduce the nocifensive responses to menthol/icilin-activated cold pain or formalin, demonstrating that the activity identified in vitro is functional in vivo with a degree of specificity. This system should prove powerful for identifying other cellular factors that can inhibit ion channel activity. PMID:27382601

  20. Bruton's tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein.

    PubMed

    Yamadori, T; Baba, Y; Matsushita, M; Hashimoto, S; Kurosaki, M; Kurosaki, T; Kishimoto, T; Tsukada, S

    1999-05-25

    Bruton's tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase that is crucial for human and murine B cell development, and its deficiency causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency. In this report, we describe the function of the Btk-binding protein Sab (SH3-domain binding protein that preferentially associates with Btk), which we reported previously as a newly identified Src homology 3 domain-binding protein. Sab was shown to inhibit the auto- and transphosphorylation activity of Btk, which prompted us to propose that Sab functions as a transregulator of Btk. Forced overexpression of Sab in B cells led to the reduction of B cell antigen receptor-induced tyrosine phosphorylation of Btk and significantly reduced both early and late B cell antigen receptor-mediated events, including calcium mobilization, inositol 1, 4,5-trisphosphate production, and apoptotic cell death, where the involvement of Btk activity has been demonstrated previously. Together, these results indicate the negative regulatory role of Sab in the B cell cytoplasmic tyrosine kinase pathway.

  1. Bruton’s tyrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein

    PubMed Central

    Yamadori, Tomoki; Baba, Yoshihiro; Matsushita, Masato; Hashimoto, Shoji; Kurosaki, Mari; Kurosaki, Tomohiro; Kishimoto, Tadamitsu; Tsukada, Satoshi

    1999-01-01

    Bruton’s tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase that is crucial for human and murine B cell development, and its deficiency causes human X-linked agammaglobulinemia and murine X-linked immunodeficiency. In this report, we describe the function of the Btk-binding protein Sab (SH3-domain binding protein that preferentially associates with Btk), which we reported previously as a newly identified Src homology 3 domain-binding protein. Sab was shown to inhibit the auto- and transphosphorylation activity of Btk, which prompted us to propose that Sab functions as a transregulator of Btk. Forced overexpression of Sab in B cells led to the reduction of B cell antigen receptor-induced tyrosine phosphorylation of Btk and significantly reduced both early and late B cell antigen receptor-mediated events, including calcium mobilization, inositol 1,4,5-trisphosphate production, and apoptotic cell death, where the involvement of Btk activity has been demonstrated previously. Together, these results indicate the negative regulatory role of Sab in the B cell cytoplasmic tyrosine kinase pathway. PMID:10339589

  2. Zea mays Taxilin protein negatively regulates opaque-2 transcriptional activity by causing a change in its sub-cellular distribution.

    PubMed

    Zhang, Nan; Qiao, Zhenyi; Liang, Zheng; Mei, Bing; Xu, Zhengkai; Song, Rentao

    2012-01-01

    Zea mays (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as in vivo assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.

  3. LINGO-1 negatively regulates myelination by oligodendrocytes.

    PubMed

    Mi, Sha; Miller, Robert H; Lee, Xinhua; Scott, Martin L; Shulag-Morskaya, Svetlane; Shao, Zhaohui; Chang, Jufang; Thill, Greg; Levesque, Melissa; Zhang, Mingdi; Hession, Cathy; Sah, Dinah; Trapp, Bruce; He, Zhigang; Jung, Vincent; McCoy, John M; Pepinsky, R Blake

    2005-06-01

    The control of myelination by oligodendrocytes in the CNS is poorly understood. Here we show that LINGO-1 is an important negative regulator of this critical process. LINGO-1 is expressed in oligodendrocytes. Attenuation of its function by dominant-negative LINGO-1, LINGO-1 RNA-mediated interference (RNAi) or soluble human LINGO-1 (LINGO-1-Fc) leads to differentiation and increased myelination competence. Attenuation of LINGO-1 results in downregulation of RhoA activity, which has been implicated in oligodendrocyte differentiation. Conversely, overexpression of LINGO-1 leads to activation of RhoA and inhibition of oligodendrocyte differentiation and myelination. Treatment of oligodendrocyte and neuron cocultures with LINGO-1-Fc resulted in highly developed myelinated axons that have internodes and well-defined nodes of Ranvier. The contribution of LINGO-1 to myelination was verified in vivo through the analysis of LINGO-1 knockout mice. The ability to recapitulate CNS myelination in vitro using LINGO-1 antagonists and the in vivo effects seen in the LINGO-1 knockout indicate that LINGO-1 signaling may be critical for CNS myelination.

  4. ART3 regulates triple-negative breast cancer cell function via activation of Akt and ERK pathways

    PubMed Central

    Sun, Xin; Wang, Ning; Qu, Ying; Sun, Zhijun

    2016-01-01

    Triple-negative breast cancers (TNBCs) are defined by lack of expressions of estrogen, progesterone, and ERBB2 receptors. Because biology of TNBC is poorly understood, no targeted therapy has been developed for this breast cancer subtype and chemotherapy is its only systemic treatment modality. In this study, we firstly determined that the expression of human ecto-ADP-ribosyltransferase 3 (ART3) is significantly associated with the basal-like breast cancer subgroup, which is largely overlapped with TNBC, through analyzing published data sets. We also found that ART3 protein is significantly overexpressed in human TNBC tumors tissue and cell lines through using immunohistochemistry and immunoblotting. Overexpression of ART3 in MDA-MB-231 breast cancer cells increased cell proliferation, invasion, and survival in vitro and growth of xenograft tumors. Conversely, knockdown of ART3 in breast cancer cells inhibited cell proliferation and invasion. In addition, we showed that ART 3 overexpression activated AKT and ERK in vitro and in xenograft tumors. Together, our findings demonstrate that ART3 is a critical TNBC marker with functional significance. PMID:27374177

  5. The Src homology 2 domain-containing inositol 5-phosphatase negatively regulates Fcgamma receptor-mediated phagocytosis through immunoreceptor tyrosine-based activation motif-bearing phagocytic receptors.

    PubMed

    Nakamura, Koji; Malykhin, Alexander; Coggeshall, K Mark

    2002-11-01

    Molecular mechanisms by which the Src homology 2 domain-containing inositol 5-phosphatase (SHIP) negatively regulates phagocytosis in macrophages are unclear. We addressed the issue using bone marrow-derived macrophages from FcgammaR- or SHIP-deficient mice. Phagocytic activities of macrophages from FcgammaRII(b)(-/-) and SHIP(-/-) mice were enhanced to a similar extent, relative to those from wild type. However, calcium influx was only marginally affected in FcgammaRII(b)(-/-), but greatly enhanced in SHIP(-/-) macrophages. Furthermore, SHIP was phosphorylated on tyrosine residues upon FcgammaR aggregation even in macrophages from FcgammaRII(b)(-/-) mice or upon clustering of a chimeric receptor containing CD8 and the immunoreceptor tyrosine-based activation motif (ITAM)-bearing gamma-chain or human-restricted FcgammaRIIa. These findings indicate that, unlike B cells, SHIP is efficiently phosphorylated in the absence of an immunoreceptor tyrosine-based inhibition motif (ITIM)-bearing receptor. We further demonstrate that SHIP directly bound to phosphorylated peptides derived from FcgammaRIIa with a high affinity, comparable to that of FcgammaRII(b). Lastly, FcgammaRIIa-mediated phagocytosis was significantly enhanced in THP-1 cells overexpressing dominant-negative form of SHIP in the absence of FcgammaRII(b). These results indicate that SHIP negatively regulates FcgammaR-mediated phagocytosis through all ITAM-containing IgG receptors using a molecular mechanism distinct from that in B cells.

  6. The functions of serpin-3, a negative-regulator involved in prophenoloxidase activation and antimicrobial peptides expression of Chinese oak silkworm, Antheraea pernyi.

    PubMed

    Wang, Xialu; Wang, Kailin; He, Yuanyuan; Lu, Xinrui; Wen, Daihua; Wu, Chunfu; Zhang, Jinghai; Zhang, Rong

    2017-04-01

    Serpins are a superfamily of proteins engaged in various physiological processes in all kingdoms of life. To date, many striking results have demonstrated serpins are involved in the invertebrate immune system by regulating the proteolytic cascades. However, in most insect species, the immune functions of serpins in response against pathogen invasion remain obscure. In this study, we identified a full-length cDNA sequence of serpin, named serpin-3, from the Chinese oak silkworm Antheraea pernyi. Sequence alignments have indicated that Apserpin-3 might regulate the melanization reaction via inhibiting prophenoloxidases-activating protease(s) in plasma. Furthermore, it was detected to be primarily transcribed within the fat body, epidermis and hemocytes with significant induction following immune-challenge. Further studies have shown that the knockdown of serpin-3 up-regulated the prophenoloxidases cascade stimulated by pathogen in hemolymph, while the addition of recombinant serpin-3 along with the same elicitor led to the suppressed activation of prophenoloxidase. Besides, the injection of dsRNA of serpin-3 caused the elevated expression of antimicrobial peptides. Altogether, we arrived at a conclusion that serpin-3 might act as a negative-regulator in prophenoloxidases activation and inhibit the production of antimicrobial peptides in Antheraea pernyi larvae.

  7. Ubiquitin carboxyl terminal hydrolase L1 negatively regulates TNF{alpha}-mediated vascular smooth muscle cell proliferation via suppressing ERK activation

    SciTech Connect

    Ichikawa, Tomonaga; Li, Jinqing; Dong, Xiaoyu; Potts, Jay D.; Tang, Dong-Qi; Li, Dong-Sheng; Cui, Taixing

    2010-01-01

    Deubiquitinating enzymes (DUBs) appear to be critical regulators of a multitude of processes such as proliferation, apoptosis, differentiation, and inflammation. We have recently demonstrated that a DUB of ubiquitin carboxyl terminal hydrolase L1 (UCH-L1) inhibits vascular lesion formation via suppressing inflammatory responses in vasculature. However, the precise underlying mechanism remains to be defined. Herein, we report that a posttranscriptional up-regulation of UCH-L1 provides a negative feedback to tumor necrosis factor alpha (TNF{alpha})-mediated activation of extracellular signal-regulated kinases (ERK) and proliferation in vascular smooth muscle cells (VSMCs). In rat adult VSMCs, adenoviral over-expression of UCH-L1 inhibited TNF{alpha}-induced activation of ERK and DNA synthesis. In contrast, over-expression of UCH-L1 did not affect platelet derived growth factor (PDGF)-induced VSMC proliferation and activation of growth stimulating cascades including ERK. TNF{alpha} hardly altered UCH-L1 mRNA expression and stability; however, up-regulated UCH-L1 protein expression via increasing UCH-L1 translation. These results uncover a novel mechanism by which UCH-L1 suppresses vascular inflammation.

  8. Nuclear Factor E2-Related Factor-2 Negatively Regulates NLRP3 Inflammasome Activity by Inhibiting Reactive Oxygen Species-Induced NLRP3 Priming

    PubMed Central

    Liu, Xiuting; Zhang, Xin; Ding, Yang; Zhou, Wei; Tao, Lei; Lu, Ping; Wang, Yajing

    2017-01-01

    Abstract Aims: The NLRP3 inflammasome is a multiprotein complex that protects hosts against a variety of pathogens. However, the molecular mechanisms of modulating NLRP3 inflammasome activation, especially at the priming step, are still poorly understood. This study was designed to elucidate the negative regulation of nuclear factor E2-related factor-2 (Nrf2) on the activation of NLRP3 inflammasome. Results: We reported that Nrf2 activation inhibited NLRP3 expression, caspase-1 cleavage, and subsequent IL-1β generation. Compared with normal cells, Nrf2-deficient cells showed upregulated cleaved caspase-1, which were attributed to the increased transcription of NLRP3 caused by excess reactive oxygen species (ROS). Furthermore, priming of the NLRP3 inflammasome was sensitive to the exogenous ROS levels induced by H2O2 or rotenone. Combined with adenosine triphosphate, rotenone triggered higher activity of the NLRP3 inflammasome compared with lipopolysaccharide, suggesting that ROS promoted the priming step. In addition, Nrf2-induced NQO1 was involved in the inhibition of the NLRP3 inflammasome. In an in vivo alum-induced peritonitis mouse model, Nrf2 activation suppressed typical IL-1 signaling-dependent inflammation, whereas Nrf2−/− mice exhibited a significant increase in the recruitment of immune cell and the generation of IL-1β compared with wild-type mice. Innovation: We elucidated the effects and possible mechanisms of Nrf2 activation-induced NQO1 expression on NLRP3 inflammasome inactivation and established a novel regulatory role of the Nrf2 pathway in ROS-induced NLRP3 priming. Conclusions: We demonstrated Nrf2 negatively regulating NLRP3 inflammasome activity by inhibiting the priming step and suggested that Nrf2 could be a potential target for some uncontrolled inflammasome activation-associated diseases. Antioxid. Redox Signal. 26, 28–43. PMID:27308893

  9. Immunobiotic Lactobacillus jensenii elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the Toll-like receptor signaling pathway.

    PubMed

    Shimazu, Tomoyuki; Villena, Julio; Tohno, Masanori; Fujie, Hitomi; Hosoya, Shoichi; Shimosato, Takeshi; Aso, Hisashi; Suda, Yoshihito; Kawai, Yasushi; Saito, Tadao; Makino, Seiya; Ikegami, Shuji; Itoh, Hiroyuki; Kitazawa, Haruki

    2012-01-01

    The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

  10. FOREVER YOUNG FLOWER Negatively Regulates Ethylene Response DNA-Binding Factors by Activating an Ethylene-Responsive Factor to Control Arabidopsis Floral Organ Senescence and Abscission.

    PubMed

    Chen, Wei-Han; Li, Pei-Fang; Chen, Ming-Kun; Lee, Yung-I; Yang, Chang-Hsien

    2015-08-01

    In this study of Arabidopsis (Arabidopsis thaliana), we investigated the relationship between FOREVER YOUNG FLOWER (FYF) and Ethylene Response DNA-binding Factors (EDFs) and functionally analyzed a key FYF target, an Ethylene-Responsive Factor (ERF), that controls flower senescence/abscission. Ectopic expression of EDF1/2/3/4 caused promotion of flower senescence/abscission and the activation of the senescence-associated genes. The presence of a repressor domain in EDFs and the enhancement of the promotion of senescence/abscission in EDF1/2/3/4+SRDX (converting EDFs to strong repressors by fusion with the ERF-associated amphiphilic repression motif repression domain SRDX) transgenic plants suggested that EDFs act as repressors. The significant reduction of β-glucuronidase (GUS) expression by 35S:FYF in EDF1/2/3/4:GUS plants indicates that EDF1/2/3/4 functions downstream of FYF in regulating flower senescence/abscission. In this study, we also characterized an ERF gene, FOREVER YOUNG FLOWER UP-REGULATING FACTOR1 (FUF1), which is up-regulated by FYF during flower development. Ectopic expression of FUF1 caused similar delayed flower senescence/abscission as seen in 35S:FYF plants. This phenotype was correlated with deficient abscission zone formation, ethylene insensitivity, and down-regulation of EDF1/2/3/4 and abscission-associated genes in 35S:FUF1 flowers. In contrast, significant promotion of flower senescence/abscission and up-regulation of EDF1/2/3/4 were observed in 35S:FUF1+SRDX transgenic dominant-negative plants, in which FUF1 is converted to a potent repressor by fusion to an SRDX-suppressing motif. Thus, FUF1 acts as an activator in suppressing EDF1/2/3/4 function and senescence/abscission of the flowers. Our results reveal that FYF regulates flower senescence/abscission by negatively regulating EDF1/2/3/4, which is the downstream gene in the ethylene response, by activating FUF1 in Arabidopsis.

  11. Negative Immune Regulator TIPE2 Promotes M2 Macrophage Differentiation through the Activation of PI3K-AKT Signaling Pathway

    PubMed Central

    Geng, Wenwen; Chen, Youhai H.; Zhang, Cui

    2017-01-01

    Macrophages play important roles in the regulation of the innate and adaptive immune responses. Classically activated macrophages and alternatively activated macrophages are the two major forms of macrophages and have opposing functionalities. Tumor necrosis factor-α-induced protein 8–2 is expressed primarily by immune cells and negatively regulates type 1 innate and adaptive immune responses to maintain immune tolerance. While previous studies indicate that TIPE2 promotes M2 but inhibits M1 macrophage differentiation, the underlying molecular mechanism by which TIPE2 promotes M2 macrophage differentiation remains unclear. Our current study shows that TIPE2-deficient bone-marrow cells are defective in IL-4 induced M2 macrophage differentiation in vitro. Mechanistic studies revealed that TIPE2 promotes phosphoinositide metabolism and the activation of the down-stream AKT signaling pathway, which in turn leads to the expression of markers specific for M2 macrophages. In addition, our results showed that Tipe2-deficiency does not affect the activation of the JAK-STAT6 signaling pathway that also plays an important role during M2 macrophage differentiation. Taken together, these results indicate that TIPE2 promotes M2 macrophage differentiation through the activation of PI3K-AKT signaling pathway, and may play an important role during the resolution of inflammation, parasite control, as well as tissue repair. PMID:28122045

  12. Acute heat stress brings down milk secretion in dairy cows by up-regulating the activity of the milk-borne negative feedback regulatory system

    PubMed Central

    Silanikove, Nissim; Shapiro, Fira; Shinder, Dima

    2009-01-01

    Background The objective of this study was to determine if acute heat stress (HS) decreases milk secretion by activating the milk-borne negative feedback system, as an emergency physiological response to prevent a life-threatening situation. To induce HS, summer acclimatized dairy cows were exposed to full sun under mid-summer Mediterranean conditions, with and without conventional cooling procedures. Results Exposure to HS induced a rapid and acute (within 24 h) reduction in milk yield in proportion to the heat load. This decrease was moderated by cooler night-time ambient temperature. The reduction in milk yield was associated with corresponding responses in plasminogen activator/plasminogen-plasmin activities, and with increased activity (concentration) of the (1–28) N-terminal fragment peptide that is released by plasmin from β-casein (β-CN (1–28)). These metabolites constitute the regulatory negative feedback system. Previously, it has been shown that β-CN (1–28) down-regulated milk secretion by blocking potassium channels on the apical aspects of the mammary epithelial cells. Conclusion Here we demonstrate that the potassium channels in mammary tissue became more susceptible to β-CN (1–28) activity under HS. Thus, the present study highlighted two previously unreported features of this regulatory system: (i) that it modulates rapidly in response to stressor impact variations; and (ii) that the regulations of the mammary epithelial potassium channel sensitivity to the inhibitory effect of β-CN (1–28) is part of the regulatory system. PMID:19563620

  13. PUB1 Interacts with the Receptor Kinase DMI2 and Negatively Regulates Rhizobial and Arbuscular Mycorrhizal Symbioses through Its Ubiquitination Activity in Medicago truncatula1

    PubMed Central

    Camut, Sylvie; Camps, Céline; Rembliere, Céline; de Carvalho-Niebel, Fernanda; Timmers, Ton; Gasciolli, Virginie; Thompson, Richard; Lefebvre, Benoit; Cullimore, Julie; Hervé, Christine

    2016-01-01

    PUB1, an E3 ubiquitin ligase, which interacts with and is phosphorylated by the LYK3 symbiotic receptor kinase, negatively regulates rhizobial infection and nodulation during the nitrogen-fixing root nodule symbiosis in Medicago truncatula. In this study, we show that PUB1 also interacts with and is phosphorylated by DOES NOT MAKE INFECTIONS 2, the key symbiotic receptor kinase of the common symbiosis signaling pathway, required for both the rhizobial and the arbuscular mycorrhizal (AM) endosymbioses. We also show here that PUB1 expression is activated during successive stages of root colonization by Rhizophagus irregularis that is compatible with its interaction with DOES NOT MAKE INFECTIONS 2. Through characterization of a mutant, pub1-1, affected by the E3 ubiquitin ligase activity of PUB1, we have shown that the ubiquitination activity of PUB1 is required to negatively modulate successive stages of infection and development of rhizobial and AM symbioses. In conclusion, PUB1 represents, to our knowledge, a novel common component of symbiotic signaling integrating signal perception through interaction with and phosphorylation by two key symbiotic receptor kinases, and downstream signaling via its ubiquitination activity to fine-tune both rhizobial and AM root endosymbioses. PMID:26839127

  14. Suppressor of cytokine signaling 2 (SOCS2) negatively regulates the expression of antimicrobial peptides by affecting the Stat transcriptional activity in shrimp Marsupenaeus japonicus.

    PubMed

    Sun, Jie-Jie; Lan, Jiang-Feng; Xu, Ji-Dong; Niu, Guo-Juan; Wang, Jin-Xing

    2016-09-01

    The suppressor of cytokine signaling (SOCS) family is a kind of negative regulators in the Janus kinase/signal transducer and activator of transcription (Jak/Stat) pathway in mammals and Drosophila. In kuruma shrimp, Marsupenaeus japonicus, SOCS2 is identified and its expression can be stimulated by peptidoglycan and polycytidylic acid. However, if SOCS2 participates in regulating Jak/Stat pathway in shrimp still needs further study. In this study, SOCS2 with Src homology 2 domain and SOCS box was identified in kuruma shrimp, M. japonicus. SOCS2 existed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine, the expression of SOCS2 was upregulated significantly in the hemocytes and intestine of shrimp challenged with Vibrio anguillarum at 6 h. To analyze SOCS2 function in shrimp immunity, bacterial clearance and survival rate were analyzed after knockdown of SOCS2 in shrimp challenged with V. anguillarum. Results showed that bacterial clearance increased, and the survival rate improved significantly comparing with controls. The SOCS2 was expressed in Escherichia coli and the recombinant SOCS2 was injected into shrimp, and Stat phosphorylation and translocation were analyzed. The result showed that "overexpression" of SOCS2 declined Stat phosphorylation level and inhibited Stat translocation into the nucleus. After knockdown of SOCS2 in shrimp prior to V. anguillarum infection, the expression level of antimicrobial peptides, including anti-lipopolysaccharide factors C1, C2 and D1, and Crustin I was upregulated significantly, and the expression of the AMPs was declined after recombinant SOCS2 injection. The SOCS2 expression was also decreased in Stat-knockdown shrimp challenged by V. anguillarum at 6 and 12 h. Therefore, SOCS2 negatively regulates the AMP expression by inhibiting Stat phosphorylation and translocation into nucleus in shrimp, meanwhile, SOCS2 expression was also regulated by Jak/Stat pathway.

  15. Glycogen Synthase Kinase 3β Is a Negative Regulator of Growth Factor-induced Activation of the c-Jun N-terminal Kinase*

    PubMed Central

    Liu, Shuying; Yu, Shuangxing; Hasegawa, Yutaka; LaPushin, Ruth; Xu, Hong-Ji; Woodgett, James R.; Mills, Gordon B.; Fang, Xianjun

    2016-01-01

    The c-Jun N-terminal kinase (JNK)/stress activated protein kinase is preferentially activated by stress stimuli. Growth factors, particularly ligands for G protein-coupled receptors, usually induce only modest JNK activation, although they may trigger marked activation of the related extracellular signal-regulated kinase. In the present study, we demonstrated that homozygous disruption of glycogen synthase kinase 3β (GSK-3β) dramatically sensitized mouse embryonic fibroblasts (MEFs) to JNK activation induced by lysophosphatidic acid (LPA) and sphingosine-1-phosphate, two prototype ligands for G protein-coupled receptors. To a lesser degree, a lack of GSK-3β also potentiated JNK activation in response to epidermal growth factor. In contrast, the absence of GSK-3β decreased UV light-induced JNK activation. The increased JNK activation induced by LPA in GSK-3β null MEFs was insufficient to trigger apoptotic cell death or growth inhibition. Instead, the increased JNK activation observed in GSK-3β−/− MEFs was associated with an increased proliferative response to LPA, which was reduced by the inhibition of JNK. Ectopic expression of GSK-3β in GSK-3β-negative MEFs restrained LPA-triggered JNK phosphorylation and induced a concomitant decrease in the mitogenic response to LPA compatible with GSK-3β through the inhibition of JNK activation, thus limiting LPA-induced cell proliferation. Mutation analysis indicated that GSK-3β kinase activity was required for GSK-3β to optimally inhibit LPA-stimulated JNK activation. Thus GSK-3β serves as a physiological switch to specifically repress JNK activation in response to LPA, sphingosine-1-phosphate, or the epidermal growth factor. These results reveal a novel role for GSK-3β in signal transduction and cellular responses to growth factors. PMID:15466414

  16. Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion

    PubMed Central

    Brombacher, Eva; Baratto, Andrea; Dorel, Corinne; Landini, Paolo

    2006-01-01

    Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene. PMID:16513732

  17. Gene expression regulation by the Curli activator CsgD protein: modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion.

    PubMed

    Brombacher, Eva; Baratto, Andrea; Dorel, Corinne; Landini, Paolo

    2006-03-01

    Curli fibers, encoded by the csgBAC genes, promote biofilm formation in Escherichia coli and other enterobacteria. Curli production is dependent on the CsgD transcription activator, which also promotes cellulose biosynthesis. In this study, we investigated the effects of CsgD expression from a weak constitutive promoter in the biofilm formation-deficient PHL565 strain of E. coli. We found that despite its function as a transcription activator, the CsgD protein is localized in the cytoplasmic membrane. Constitutive CsgD expression promotes biofilm formation by PHL565 and activates transcription from the csgBAC promoter; however, csgBAC expression remains dependent on temperature and the growth medium. Constitutive expression of the CsgD protein results in altered transcription patterns for at least 24 novel genes, in addition to the previously identified CsgD-dependent genes. The cspA and fecR genes, encoding regulatory proteins responding to cold shock and to iron, respectively, and yoaD, encoding a putative negative regulator of cellulose biosynthesis, were found to be some of the novel CsgD-regulated genes. Consistent with the predicted functional role, increased expression of the yoaD gene negatively affects cell aggregation, while yoaD inactivation results in stimulation of cell aggregation and leads to increased cellulose production. Inactivation of fecR results in significant increases in both cell aggregation and biofilm formation, while the effects of cspA are not as strong in the conditions tested. Our results indicate that CsgD can modulate cellulose biosynthesis through activation of the yoaD gene. In addition, the positive effect of CsgD on biofilm formation might be enhanced by repression of the fecR gene.

  18. NK cell development requires Tsc1-dependent negative regulation of IL-15-triggered mTORC1 activation

    PubMed Central

    Yang, Meixiang; Chen, Shasha; Du, Juan; He, Junming; Wang, Yuande; Li, Zehua; Liu, Guangao; Peng, Wanwen; Zeng, Xiaokang; Li, Dan; Xu, Panglian; Guo, Wei; Chang, Zai; Wang, Song; Tian, Zhigang; Dong, Zhongjun

    2016-01-01

    Activation of metabolic signalling by IL-15 is required for natural killer (NK) cell development. Here we show that Tsc1, a repressor of mTOR, is dispensable for the terminal maturation, survival and function of NK cells but is critical to restrict exhaustive proliferation of immature NK cells and activation downstream of IL-15 during NK cell development. Tsc1 is expressed in immature NK cells and is upregulated by IL-15. Haematopoietic-specific deletion of Tsc1 causes a marked decrease in the number of NK cells and compromises rejection of ‘missing-self' haematopoietic tumours and allogeneic bone marrow. The residual Tsc1-null NK cells display activated, pro-apoptotic phenotype and elevated mTORC1 activity. Deletion of Raptor, a component of mTORC1, largely reverses these defects. Tsc1-deficient NK cells express increased levels of T-bet and downregulate Eomes and CD122, a subunit of IL-15 receptor. These results reveal a role for Tsc1-dependent inhibition of mTORC1 activation during immature NK cell development. PMID:27601261

  19. Interlocked positive and negative feedback network motifs regulate β-catenin activity in the adherens junction pathway

    PubMed Central

    Klinke, David J.; Horvath, Nicholas; Cuppett, Vanessa; Wu, Yueting; Deng, Wentao; Kanj, Rania

    2015-01-01

    The integrity of epithelial tissue architecture is maintained through adherens junctions that are created through extracellular homotypic protein–protein interactions between cadherin molecules. Cadherins also provide an intracellular scaffold for the formation of a multiprotein complex that contains signaling proteins, including β-catenin. Environmental factors and controlled tissue reorganization disrupt adherens junctions by cleaving the extracellular binding domain and initiating a series of transcriptional events that aim to restore tissue homeostasis. However, it remains unclear how alterations in cell adhesion coordinate transcriptional events, including those mediated by β-catenin in this pathway. Here were used quantitative single-cell and population-level in vitro assays to quantify the endogenous pathway dynamics after the proteolytic disruption of the adherens junctions. Using prior knowledge of isolated elements of the overall network, we interpreted these data using in silico model-based inference to identify the topology of the regulatory network. Collectively the data suggest that the regulatory network contains interlocked network motifs consisting of a positive feedback loop, which is used to restore the integrity of adherens junctions, and a negative feedback loop, which is used to limit β-catenin–induced gene expression. PMID:26224311

  20. Kisspeptin-10-induced signaling of GPR54 negatively regulates chemotactic responses mediated by CXCR4: a potential mechanism for the metastasis suppressor activity of kisspeptins.

    PubMed

    Navenot, Jean-Marc; Wang, Zixuan; Chopin, Michael; Fujii, Nobutaka; Peiper, Stephen C

    2005-11-15

    The product of the KiSS-1 gene is absent or expressed at low level in metastatic melanoma and breast cancer compared with their nonmetastatic counterparts. A polypeptide derived from the KiSS-1 product, designated kisspeptin-10 (Kp-10), activates a receptor coupled to Galphaq subunits (GPR54 or KiSS-1R). To study the mechanism by which Kp-10 antagonizes metastatic spread, the effect on CXCR4-mediated signaling, which has been shown to direct organ-specific migration of tumor cells, was determined. Kp-10 blocked chemotaxis of tumor cells expressing CXCR4 in response to low and high concentrations of SDF-1/CXCL12 and inhibited mobilization of calcium ions induced by this ligand. Pretreatment with Kp-10 did not induce down-modulation of cell surface CXCR4 expression, reduce affinity for SDF-1/CXCL12, or alter Galphai subunit activation stimulated by this ligand. Although Kp-10 stimulated prolonged phosphorylation of extracellular signal-regulated kinase 1/2, it inhibited the phosphorylation of Akt induced by SDF-1. The ability of Kp-10 to inhibit signaling and chemotaxis induced by SDF-1 indicates that activation of GPR54 signaling may negatively regulate the role of CXCR4 in programming tumor metastasis.

  1. Integrin alpha1beta1 controls reactive oxygen species synthesis by negatively regulating epidermal growth factor receptor-mediated Rac activation.

    PubMed

    Chen, Xiwu; Abair, Tristin D; Ibanez, Maria R; Su, Yan; Frey, Mark R; Dise, Rebecca S; Polk, D Brent; Singh, Amar B; Harris, Raymond C; Zent, Roy; Pozzi, Ambra

    2007-05-01

    Integrins control many cell functions, including generation of reactive oxygen species (ROS) and regulation of collagen synthesis. Mesangial cells, found in the glomerulus of the kidney, are able to produce large amounts of ROS via the NADPH oxidase. We previously demonstrated that integrin alpha1-null mice develop worse fibrosis than wild-type mice following glomerular injury and this is due, in part, to excessive ROS production by alpha1-null mesangial cells. In the present studies, we describe the mechanism whereby integrin alpha1-null mesangial cells produce excessive ROS. Integrin alpha1-null mesangial cells have constitutively increased basal levels of activated Rac1, which result in its increased translocation to the cell membrane, excessive ROS production, and consequent collagen IV deposition. Basal Rac1 activation is a direct consequence of ligand-independent increased epidermal growth factor receptor (EGFR) phosphorylation in alpha1-null mesangial cells. Thus, our study demonstrates that integrin alpha1beta1-EGFR cross talk is a key step in negatively regulating Rac1 activation, ROS production, and excessive collagen synthesis, which is a hallmark of diseases characterized by irreversible fibrosis.

  2. HECT E3 Ubiquitin Ligase Itch Functions as a Novel Negative Regulator of Gli-Similar 3 (Glis3) Transcriptional Activity

    PubMed Central

    ZeRuth, Gary T.; Williams, Jason G.; Cole, Yasemin C.; Jetten, Anton M.

    2015-01-01

    The transcription factor Gli-similar 3 (Glis3) plays a critical role in the generation of pancreatic ß cells and the regulation insulin gene transcription and has been implicated in the development of several pathologies, including type 1 and 2 diabetes and polycystic kidney disease. However, little is known about the proteins and posttranslational modifications that regulate or mediate Glis3 transcriptional activity. In this study, we identify by mass-spectrometry and yeast 2-hybrid analyses several proteins that interact with the N-terminal region of Glis3. These include the WW-domain-containing HECT E3 ubiquitin ligases, Itch, Smurf2, and Nedd4. The interaction between Glis3 and the HECT E3 ubiquitin ligases was verified by co-immunoprecipitation assays and mutation analysis. All three proteins interact through their WW-domains with a PPxY motif located in the Glis3 N-terminus. However, only Itch significantly contributed to Glis3 polyubiquitination and reduced Glis3 stability by enhancing its proteasomal degradation. Itch-mediated degradation of Glis3 required the PPxY motif-dependent interaction between Glis3 and the WW-domains of Itch as well as the presence of the Glis3 zinc finger domains. Transcription analyses demonstrated that Itch dramatically inhibited Glis3-mediated transactivation and endogenous Ins2 expression by increasing Glis3 protein turnover. Taken together, our study identifies Itch as a critical negative regulator of Glis3-mediated transcriptional activity. This regulation provides a novel mechanism to modulate Glis3-driven gene expression and suggests that it may play a role in a number of physiological processes controlled by Glis3, such as insulin transcription, as well as in Glis3-associated diseases. PMID:26147758

  3. HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling

    PubMed Central

    Chen, Duchu; Wang, Huiping; Aweya, Jude Juventus; Chen, Yanheng; Chen, Meihua; Wu, Xiaomeng; Chen, Xiaonan; Lu, Jing

    2016-01-01

    In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms. While prostratin strongly stimulates HMBA-induced HIV-1 transcription via improved P-TEFb activation, HMBA is capable of boosting NF-κB-dependent transcription initiation by suppressing prostratin-induced expression of the deubiquitinase A20, a negative feedback regulator in the NF-κB signaling pathway. In addition, HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-κB inhibitor IκBα, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-κB, a prerequisite for stimulation of transcription initiation. Thus, by blocking the negative feedback circuit, HMBA functions as a signaling enhancer of the NF-κB signaling pathway. PMID:27529070

  4. Expression of μ-protocadherin is negatively regulated by the activation of the β-catenin signaling pathway in normal and cancer colorectal enterocytes

    PubMed Central

    Montorsi, L; Parenti, S; Losi, L; Ferrarini, F; Gemelli, C; Rossi, A; Manco, G; Ferrari, S; Calabretta, B; Tagliafico, E; Zanocco-Marani, T; Grande, A

    2016-01-01

    Mu-protocadherin (MUCDHL) is an adhesion molecule predominantly expressed by colorectal epithelial cells which is markedly downregulated upon malignant transformation. Notably, treatment of colorectal cancer (CRC) cells with mesalazine lead to increased expression of MUCDHL, and is associated with sequestration of β-catenin on the plasma membrane and inhibition of its transcriptional activity. To better characterize the causal relationship between β-catenin and MUCDHL expression, we performed various experiments in which CRC cell lines and normal colonic organoids were subjected to culture conditions inhibiting (FH535 treatment, transcription factor 7-like 2 siRNA inactivation, Wnt withdrawal) or stimulating (LiCl treatment) β-catenin activity. We show here that expression of MUCDHL is negatively regulated by functional activation of the β-catenin signaling pathway. This finding was observed in cell culture systems representing conditions of physiological stimulation and upon constitutive activation of β-catenin in CRC. The ability of MUCDHL to sequester and inhibit β-catenin appears to provide a positive feedback enforcing the effect of β-catenin inhibitors rather than serving as the primary mechanism responsible for β-catenin inhibition. Moreover, MUCDHL might have a role as biomarker in the development of CRC chemoprevention drugs endowed with β-catenin inhibitory activity. PMID:27310872

  5. The serine and threonine residues in the Ig-alpha cytoplasmic tail negatively regulate immunoreceptor tyrosine-based activation motif-mediated signal transduction.

    PubMed

    Müller, R; Wienands, J; Reth, M

    2000-07-18

    The B cell antigen receptor (BCR) is a multiprotein complex consisting of the membrane-bound Ig molecule and the Ig-alpha/Ig-beta heterodimer. On BCR engagement, Ig-alpha and Ig-beta become phosphorylated not only on tyrosine residues of the immunoreceptor tyrosine-based activation motif but also on serine and threonine residues. We have mutated all serine and threonine residues in the Ig-alpha tail to alanine and valine, respectively. The mutated Ig-alpha sequence was expressed either as a single-chain Fv/Ig-alpha molecule or in the context of the complete BCR. In both cases, the mutated Ig-alpha showed a stronger tyrosine phosphorylation than the wild-type Ig-alpha and initiated increased signaling on stimulation. These findings suggest that serine/threonine kinases can negatively regulate signal transduction from the BCR.

  6. Rhizobial gibberellin negatively regulates host nodule number

    PubMed Central

    Tatsukami, Yohei; Ueda, Mitsuyoshi

    2016-01-01

    In legume–rhizobia symbiosis, the nodule number is controlled to ensure optimal growth of the host. In Lotus japonicus, the nodule number has been considered to be tightly regulated by host-derived phytohormones and glycopeptides. However, we have discovered a symbiont-derived phytohormonal regulation of nodule number in Mesorhizobium loti. In this study, we found that M. loti synthesized gibberellic acid (GA) under symbiosis. Hosts inoculated with a GA-synthesis-deficient M. loti mutant formed more nodules than those inoculated with the wild-type form at four weeks post inoculation, indicating that GA from already-incorporated rhizobia prevents new nodule formation. Interestingly, the genes for GA synthesis are only found in rhizobial species that inhabit determinate nodules. Our findings suggest that the already-incorporated rhizobia perform GA-associated negative regulation of nodule number to prevent delayed infection by other rhizobia. PMID:27307029

  7. Evolution of lipopolysaccharide (LPS) recognition and signaling: fish TLR4 does not recognize LPS and negatively regulates NF-kappaB activation.

    PubMed

    Sepulcre, María P; Alcaraz-Pérez, Francisca; López-Muñoz, Azucena; Roca, Francisco J; Meseguer, José; Cayuela, María L; Mulero, Victoriano

    2009-02-15

    It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-kappaB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.

  8. Grb2 negatively regulates epidermal growth factor-induced phospholipase C-gamma1 activity through the direct interaction with tyrosine-phosphorylated phospholipase C-gamma1.

    PubMed

    Choi, Jang Hyun; Hong, Won-Pyo; Yun, Sanguk; Kim, Hyeon Soo; Lee, Jong-Ryul; Park, Jong Bae; Bae, Yun Soo; Ryu, Sung Ho; Suh, Pann-Ghill

    2005-10-01

    Phospholipase C-gamma1 (PLC-gamma1) plays pivotal roles in cellular growth and proliferation. Upon the stimulation of growth factors and hormones, PLC-gamma1 is rapidly phosphorylated at three known sites; Tyr771, Tyr783 and Tyr1254 and its enzymatic activity is up-regulated. In this study, we demonstrate for the first time that Grb2, an adaptor protein, specifically interacts with tyrosine-phosphorylated PLC-gamma1 at Tyr783. The association of Grb2 with PLC-gamma1 was induced by the treatment with epidermal growth factor (EGF). Replacement of Tyr783 with Phe completely blocked EGF-induced interaction of PLC-gamma1 with Grb2, indicating that tyrosine phosphorylation of PLC-gamma1 at Tyr783 is essential for the interaction with Grb2. Interestingly, the depletion of Grb2 from HEK-293 cells by RNA interference significantly enhanced increased EGF-induced PLC-gamma1 enzymatic activity and mobilization of the intracellular Ca2+, while it did not affect EGF-induced tyrosine phosphorylation of PLC-gamma1. Furthermore, overexpression of Grb2 inhibited PLC-gamma1 enzymatic activity. Taken together, these results suggest Grb2, in addition to its key function in signaling through Ras, may have a negatively regulatory role on EGF-induced PLC-gamma1 activation.

  9. GATA4 negatively regulates bone sialoprotein expression in osteoblasts

    PubMed Central

    Song, Insun; Jeong, Byung-chul; Choi, Yong Jun; Chung, Yoon-Sok; Kim, Nacksung

    2016-01-01

    GATA4 has been reported to act as a negative regulator in osteoblast differentiation by inhibiting the Dlx5 transactivation of Runx2 via the attenuation of the binding ability of Dlx5 to the Runx2 promoter region. Here, we determine the role of GATA4 in the regulation of bone sialoprotein (Bsp) in osteoblasts. We observed that the overexpression of Runx2 or Sox9 induced the Bsp expression in osteoblastic cells. Silencing GATA4 further enhanced the Runx2- and Sox9-mediated Bsp promoter activity, whereas GATA4 overexpression down-regulated Bsp promoter activity mediated by Runx2 and Sox9. GATA4 also interacted with Runx2 and Sox9, by attenuating the binding ability of Runx2 and Sox9 to the Bsp promoter region. Our data suggest that GATA4 acts as a negative regulator of Bsp expression in osteoblasts. [BMB Reports 2016; 49(6): 343-348] PMID:26973342

  10. dUbc9 negatively regulates the Toll-NF-kappa B pathways in larval hematopoiesis and drosomycin activation in Drosophila.

    PubMed

    Chiu, Hsiling; Ring, Brian C; Sorrentino, Richard Paul; Kalamarz, Marta; Garza, Dan; Govind, Shubha

    2005-12-01

    Highly conserved during evolution, the enzyme Ubc9 activates the small ubiquitin-like modifier (SUMO) prior to its covalent ligation to target proteins. We have used mutations in the Drosophila Ubc9 (dUbc9) gene to understand Ubc9 functions in vivo. Loss-of-function mutations in dUbc9 cause strong mitotic defects in larval hematopoietic tissues, an increase in the number of hematopoietic precursors in the lymph gland and of mature blood cells in circulation, and an increase in the proportion of cyclin-B-positive cells. Some blood cells are polyploid and multinucleate, exhibiting signs of genomic instability. We also observe an overabundance of highly differentiated blood cells (lamellocytes), normally not found in healthy larvae. Lamellocytes in mutants are either free in circulation or recruited to form tumorous masses. Hematopoietic defects of dUbc9 mutants are strongly suppressed in the absence of the Rel/NF-kappaB-family transcription factors Dorsal and Dif or in the presence of a non-signaling allele of Cactus, the IkappaB protein in Drosophila. In the larval fat body, dUbc9 negatively regulates the expression of the antifungal peptide gene drosomycin, which is constitutively expressed in dUbc9 mutants in the absence of immune challenge. dUbc9-mediated drosomycin expression requires Dorsal and Dif. Together, our results support a role for dUbc9 in the negative regulation of the Drosophila NF-kappaB signaling pathways in larval hematopoiesis and humoral immunity.

  11. AICAR-induced activation of AMPK negatively regulates myotube hypertrophy through the HSP72-mediated pathway in C2C12 skeletal muscle cells.

    PubMed

    Egawa, Tatsuro; Ohno, Yoshitaka; Goto, Ayumi; Ikuta, Akihiro; Suzuki, Miho; Ohira, Tomotaka; Yokoyama, Shingo; Sugiura, Takao; Ohira, Yoshinobu; Yoshioka, Toshitada; Goto, Katsumasa

    2014-02-01

    5'-AMP-activated protein kinase (AMPK) plays an important role as a negative regulator of skeletal muscle mass. However, the precise mechanism of AMPK-mediated regulation of muscle mass is not fully clarified. Heat shock proteins (HSPs), stress-induced molecular chaperones, are related with skeletal muscle adaptation, but the association between AMPK and HSPs in skeletal muscle hypertrophy is unknown. Thus, we investigated whether AMPK regulates hypertrophy by mediating HSPs in C2C12 cells. The treatment with AICAR, a potent stimulator of AMPK, decreased 72-kDa HSP (HSP72) expression, whereas there were no changes in the expressions of 25-kDa HSP, 70-kDa heat shock cognate, and heat shock transcription factor 1 in myotubes. Protein content and diameter were less in the AICAR-treated myotubes in those without treatment. AICAR-induced suppression of myotube hypertrophy and HSP72 expression was attenuated in the siRNA-mediated AMPKα knockdown myotubes. AICAR increased microRNA (miR)-1, a modulator of HSP72, and the increase of miR-1 was not induced in AMPKα knockdown condition. Furthermore, siRNA-mediated HSP72 knockdown blocked AICAR-induced inhibition of myotube hypertrophy. AICAR upregulated the gene expression of muscle Ring-finger 1, and this alteration was suppressed in either AMPKα or HSP72 knockdown myotubes. The phosphorylation of p70 S6 kinase Thr(389) was downregulated by AICAR, whereas this was attenuated in AMPKα, but not in HSP72, knockdown myotubes. These results suggest that AMPK inhibits hypertrophy through, in part, an HSP72-associated mechanism via miR-1 and protein degradation pathways in skeletal muscle cells.

  12. The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma.

    PubMed

    Fionda, Cinzia; Abruzzese, Maria Pia; Zingoni, Alessandra; Cecere, Francesca; Vulpis, Elisabetta; Peruzzi, Giovanna; Soriani, Alessandra; Molfetta, Rosa; Paolini, Rossella; Ricciardi, Maria Rosaria; Petrucci, Maria Teresa; Santoni, Angela; Cippitelli, Marco

    2015-09-15

    Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells.Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells.

  13. The IMiDs targets IKZF-1/3 and IRF4 as novel negative regulators of NK cell-activating ligands expression in multiple myeloma

    PubMed Central

    Fionda, Cinzia; Abruzzese, Maria Pia; Zingoni, Alessandra; Cecere, Francesca; Vulpis, Elisabetta; Peruzzi, Giovanna; Soriani, Alessandra; Molfetta, Rosa; Paolini, Rossella; Ricciardi, Maria Rosaria; Petrucci, Maria Teresa

    2015-01-01

    Immunomodulatory drugs (IMiDs) have potent anti-tumor activities in multiple myeloma (MM) and are able to enhance the cytotoxic function of natural killer (NK) cells, important effectors of the immune response against MM. Here, we show that these drugs can enhance the expression of the NKG2D and DNAM-1 activating receptor ligands MICA and PVR/CD155 in human MM cell lines and primary malignant plasma cells. Depletion of cereblon (CRBN) by shRNA interference strongly impaired upregulation of these ligands and, more interestingly, IMiDs/CRBN-mediated downregulation of the transcription factors Ikaros (IKZF1), Aiolos (IKZF3) and IRF4 was critical for these regulatory mechanisms. Indeed, shRNA knockdown of IKZF1 or IKZF3 expression was both necessary and sufficient for the upregulation of MICA and PVR/CD155 expression, suggesting that these transcription factors can repress these genes; accordingly, the direct interaction and the negative role of IKZF1 and IKZF3 proteins on MICA and PVR/CD155 promoters were demonstrated. Finally, MICA expression was enhanced in IRF4-silenced cells, indicating a specific suppressive role of this transcription factor on MICA gene expression in MM cells. Taken together, these findings describe novel molecular pathways involved in the regulation of MICA and PVR/CD155 gene expression and identify the transcription factors IKZF-1/IKZF-3 and IRF4 as repressors of these genes in MM cells. PMID:26269456

  14. Inflammation, Prostate Cancer and Negative Regulation of Androgen Receptor Expression

    DTIC Science & Technology

    2009-05-01

    activity, 2) microRNA -mediated regulation of prostate cancer cell proliferation. My data establish that the human AR level is negatively regulated by... cancer , scanning of the cancer microRNA array shows that miR-454 is up regulated in androgen-independent C4-2 cells and overexpression of miR-454...TERMS Androgen receptor, prostate cancer , TNF-α, NF-κB, microRNA 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF

  15. Tyrosine kinase FYN negatively regulates NOX4 in cardiac remodeling

    PubMed Central

    Matsushima, Shouji; Kuroda, Junya; Zhai, Peiyong; Liu, Tong; Ikeda, Shohei; Nagarajan, Narayani; Yokota, Takashi; Kinugawa, Shintaro; Hsu, Chiao-Po; Li, Hong; Tsutsui, Hiroyuki

    2016-01-01

    NADPH oxidases (Noxes) produce ROS that regulate cell growth and death. NOX4 expression in cardiomyocytes (CMs) plays an important role in cardiac remodeling and injury, but the posttranslational mechanisms that modulate this enzyme are poorly understood. Here, we determined that FYN, a Src family tyrosine kinase, interacts with the C-terminal domain of NOX4. FYN and NOX4 colocalized in perinuclear mitochondria, ER, and nuclear fractions in CMs, and FYN expression negatively regulated NOX4-induced O2– production and apoptosis in CMs. Mechanistically, we found that direct phosphorylation of tyrosine 566 on NOX4 was critical for this FYN-mediated negative regulation. Transverse aortic constriction activated FYN in the left ventricle (LV), and FYN-deficient mice displayed exacerbated cardiac hypertrophy and dysfunction and increased ROS production and apoptosis. Deletion of Nox4 rescued the exaggerated LV remodeling in FYN-deficient mice. Furthermore, FYN expression was markedly decreased in failing human hearts, corroborating its role as a regulator of cardiac cell death and ROS production. In conclusion, FYN is activated by oxidative stress and serves as a negative feedback regulator of NOX4 in CMs during cardiac remodeling. PMID:27525436

  16. Transcription dynamics of inducible genes modulated by negative regulations.

    PubMed

    Li, Yanyan; Tang, Moxun; Yu, Jianshe

    2015-06-01

    Gene transcription is a stochastic process in single cells, in which genes transit randomly between active and inactive states. Transcription of many inducible genes is also tightly regulated: It is often stimulated by extracellular signals, activated through signal transduction pathways and later repressed by negative regulations. In this work, we study the nonlinear dynamics of the mean transcription level of inducible genes modulated by the interplay of the intrinsic transcriptional randomness and the repression by negative regulations. In our model, we integrate negative regulations into gene activation process, and make the conventional assumption on the production and degradation of transcripts. We show that, whether or not the basal transcription is temporarily terminated when cells are stimulated, the mean transcription level grows in the typical up and down pattern commonly observed in immune response genes. With the help of numerical simulations, we clarify the delicate impact of the system parameters on the transcription dynamics, and demonstrate how our model generates the distinct temporal gene-induction patterns in mouse fibroblasts discerned in recent experiments.

  17. CD23 can negatively regulate B-cell receptor signaling

    PubMed Central

    Liu, Chaohong; Richard, Katharina; Wiggins, Melvin; Zhu, Xiaoping; Conrad, Daniel H.; Song, Wenxia

    2016-01-01

    CD23 has been implicated as a negative regulator of IgE and IgG antibody responses. However, whether CD23 has any role in B-cell activation remains unclear. We examined the expression of CD23 in different subsets of peripheral B cells and the impact of CD23 expression on the early events of B-cell receptor (BCR) activation using CD23 knockout (KO) mice. We found that in addition to marginal zone B cells, mature follicular B cells significantly down regulate the surface expression level of CD23 after undergoing isotype switch and memory B-cell differentiation. Upon stimulation with membrane-associated antigen, CD23 KO causes significant increases in the area of B cells contacting the antigen-presenting membrane and the magnitude of BCR clustering. This enhanced cell spreading and BCR clustering is concurrent with increases in the levels of phosphorylation of tyrosine and Btk, as well as the levels of F-actin and phosphorylated Wiskott Aldrich syndrome protein, an actin nucleation promoting factor, in the contract zone of CD23 KO B cells. These results reveal a role of CD23 in the negative regulation of BCR signaling in the absence of IgE immune complex and suggest that CD23 down-regulates BCR signaling by influencing actin-mediated BCR clustering and B-cell morphological changes. PMID:27181049

  18. PDE11A negatively regulates lithium responsivity

    PubMed Central

    Pathak, G.; Agostino, M.J.; Bishara, K.; Capell, W.R.; Fisher, J.L.; Hegde, S.; Ibrahim, B.A.; Pilarzyk, Kaitlyn; Sabin, C.; Tuczkewycz, Taras; Wilson, Steven; Kelly, M.P.

    2016-01-01

    Lithium responsivity in patients with bipolar disorder has been genetically associated with Phosphodiesterase 11A (PDE11A), and lithium decreases PDE11A mRNA in IPSC-derived hippocampal neurons originating from lithium responsive patients. PDE11 is an enzyme uniquely enriched in the hippocampus that breaks down cAMP and cGMP. Here, we determined if decreasing PDE11A expression is sufficient to increase lithium responsivity in mice. In dorsal hippocampus (DHIPP) and ventral hippocampus (VHIPP), lithium-responsive C57BL/6J and 129S6/SvEvTac mice show decreased PDE11A4 protein expression relative to lithium-unresponsive BALB/cJ mice. In VHIPP, C57BL/6J mice also show differences in PDE11A4 compartmentalization relative to BALB/cJ mice. In contrast, neither PDE2A nor PDE10A expression differ among the strains. The compartment-specific differences in PDE11A4 protein expression are explained by a coding SNP at amino acid 499, which falls within the GAF-B homodimerization domain. Relative to the BALB/cJ 499T, the C57BL/6J 499A decreases PDE11A4 homodimerization, which removes PDE11A4 from the membrane. Consistent with the observation that lower PDE11A4 expression correlates with better lithium responsiveness, we found that Pde11a KO mice given 0.4% lithium chow for 3+ weeks exhibit greater lithium responsivity relative to WT littermates in tail suspension, an antidepressant predictive assay, and amphetamine hyperlocomotion, an anti-manic predictive assay. Reduced PDE11A4 expression may represent a lithium-sensitive pathophysiology, because both C57BL/6J and Pde11a KO mice show increased expression of the pro-inflammatory cytokine IL-6 relative to BALB/cJ and PDE11A WT mice, respectively. Our finding that PDE11A4 negatively regulates lithium responsivity in mice suggests that the PDE11A SNPs identified in patients may be functionally relevant. PMID:27646265

  19. Adrenocortical Activity and Emotion Regulation.

    ERIC Educational Resources Information Center

    Stansbury, Kathy; Gunnar, Megan R.

    1994-01-01

    This essay argues that the activity of the hypothalamic-pituitary-adrenocortical (HPA) system does not appear to be related to emotion regulation processes in children, although individual differences in emotion processes related to negative emotion temperaments appear to be associated with individual differences in HPA reactivity among normally…

  20. The p21-activated kinase PAK is negatively regulated by POPX1 and POPX2, a pair of serine/threonine phosphatases of the PP2C family.

    PubMed

    Koh, Cheng-Gee; Tan, E-Jean; Manser, Edward; Lim, Louis

    2002-02-19

    The Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division. The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases (reviewed by [1]). The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known. We now report two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that efficiently inactivate PAK. POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX. The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK. Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42(V12). The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family.

  1. Cultural differences in hedonic emotion regulation after a negative event.

    PubMed

    Miyamoto, Yuri; Ma, Xiaoming; Petermann, Amelia G

    2014-08-01

    Beliefs about emotions can influence how people regulate their emotions. The present research examined whether Eastern dialectical beliefs about negative emotions lead to cultural differences in how people regulate their emotions after experiencing a negative event. We hypothesized that, because of dialectical beliefs about negative emotions prevalent in Eastern culture, Easterners are less motivated than Westerners to engage in hedonic emotion regulation-up-regulation of positive emotions and down-regulation of negative emotions. By assessing online reactions to a recent negative event, Study 1 found that European Americans are more motivated to engage in hedonic emotion regulation. Furthermore, consistent with the reported motivation to regulate emotion hedonically, European Americans show a steeper decline in negative emotions 1 day later than do Asians. By examining retrospective memory of reactions to a past negative event, Study 2 further showed that cultural differences in hedonic emotion regulation are mediated by cultural differences in dialectical beliefs about motivational and cognitive utility of negative emotions, but not by personal deservingness or self-efficacy beliefs. These findings demonstrate the role of cultural beliefs in shaping emotion regulation and emotional experiences.

  2. Negative regulation of p120GAP GTPase promoting activity by p210bcr/abl: implication for RAS-dependent Philadelphia chromosome positive cell growth

    PubMed Central

    1994-01-01

    The p210bcr/abl tyrosine kinase appears to be responsible for initiating and maintaining the leukemic phenotype in chronic myelogenous leukemia (CML) patients. p21ras-p120GAP interactions play a central role in transducing mitogenic signals. Therefore, we investigated whether p21ras and p120GAP are regulated by p210bcr/abl, and whether this activation is functionally significant for CML cell proliferation. We report that transient expression of p210bcr/abl in fibroblast-like cells induces simultaneous activation of p21ras and inhibition of GTPase-promoting activity of p120GAP, and confirm these data showing that downregulation of p210bcr/abl expression in CML cells with bcr/abl antisense oligodeoxynucleotides induces both inhibition of p21ras activation and stimulation of GTPase-promoting activity of p120GAP. Tyrosine phosphorylation of two p120GAP-associated proteins, p190 and p62, which may affect p120GAP activity, also depends on p210bcr/abl tyrosine kinase expression. Direct dependence of these effects on the kinase activity is proven in experiments in which expression of c-MYB protein in fibroblast-like cells or downregulation of c-MYB expression resulting in analogous inhibition of CML cell proliferation does not result in the same changes. Use of specific antisense oligodeoxynucleotides to downregulate p21ras expression revealed a requirement for functional p21ras in the proliferation of Philadelphia chromosome-positive CML primary cells. Thus, the p210bcr/abl-dependent regulation of p120GAP activity is responsible, in part, for the maintenance of p21ras in the active GTP-bound form, a crucial requirement for CML cell proliferation. PMID:8195713

  3. Negative regulation of the inflammasome: keeping inflammation under control.

    PubMed

    Pedraza-Alva, Gustavo; Pérez-Martínez, Leonor; Valdez-Hernández, Laura; Meza-Sosa, Karla F; Ando-Kuri, Masami

    2015-05-01

    In addition to its roles in controlling infection and tissue repair, inflammation plays a critical role in diverse and distinct chronic diseases, such as cancer, metabolic syndrome, and neurodegenerative disorders, underscoring the harmful effect of an uncontrolled inflammatory response. Regardless of the nature of the stimulus, initiation of the inflammatory response is mediated by assembly of a multimolecular protein complex called the inflammasome, which is responsible for the production of inflammatory cytokines, such as interleukin-1β (IL-1β) and IL-18. The different stimuli and mechanisms that control inflammasome activation are fairly well understood, but the mechanisms underlying the control of undesired inflammasome activation and its inactivation remain largely unknown. Here, we review recent advances in our understanding of the molecular mechanisms that negatively regulate inflammasome activation to prevent unwanted activation in the resting state, as well as those involved in terminating the inflammatory response after a specific insult to maintain homeostasis.

  4. HUA ENHANCER1 is involved in posttranscriptional regulation of positive and negative regulators in Arabidopsis photomorphogenesis.

    PubMed

    Tsai, Huang-Lung; Li, Yi-Hang; Hsieh, Wen-Ping; Lin, Meng-Chun; Ahn, Ji Hoon; Wu, Shu-Hsing

    2014-07-01

    Light regulates growth and developmental processes in plants via global transcriptome adjustment, translational control, and multilayered posttranslational modification of proteins. The transcriptional activation and repression of light-responsive genes has been well documented; however, the impact of posttranscriptional regulation on conveying light signals has been less addressed. Here, we examined whether optimal photomorphogenesis in Arabidopsis thaliana requires the proper biogenesis of small regulatory RNAs that play pivotal roles in the posttranscriptional regulation of gene expression. Arabidopsis carrying a mutation in HUA ENHANCER1 (HEN1), required for stabilization of small regulatory RNAs, showed defects in multiple aspects of photomorphogenic and skotomorphogenic development. HEN1 negatively regulated Arabidopsis photomorphogenesis. Light-activated HEN1 expression depended on the photoreceptors phytochrome A (phyA), phyB, cryptochrome 1 (cry1), and cry2 and key transcriptional regulators ELONGATED HYPOCOTYL5 (HY5) and HY5-HOMOLOG. We also demonstrate the involvement of the small regulatory RNAs miR157d and miR319 in modulating the expression of a positive regulator, HY5, and negative regulators TEOSINTE BRANCHED1, CYCLOIDEA AND PCF family proteins, respectively, for optimal photomorphogenic development in Arabidopsis.

  5. Triple Negative Breast Cancer and Metabolic Regulation

    DTIC Science & Technology

    2015-08-01

    Warburg- like metabolic reprogramming. The Warburg effect is characterized by increased glycolytic flux with increased biosynthesis of amino acids...HBP1 KD tumors and have used the gene expression and NMR analysis to discover an alteration in lipid metabolism . The results are summarized in...execute a multi- disciplinary analysis and discover new aspects to metabolic regulation by HBP1 and by Wnt signaling. Some recent studies have

  6. Negative Regulation of Violacein Biosynthesis in Chromobacterium violaceum

    PubMed Central

    Devescovi, Giulia; Kojic, Milan; Covaceuszach, Sonia; Cámara, Miguel; Williams, Paul; Bertani, Iris; Subramoni, Sujatha; Venturi, Vittorio

    2017-01-01

    In Chromobacteium violaceum, the purple pigment violacein is under positive regulation by the N-acylhomoserine lactone CviI/R quorum sensing system and negative regulation by an uncharacterized putative repressor. In this study we report that the biosynthesis of violacein is negatively controlled by a novel repressor protein, VioS. The violacein operon is regulated negatively by VioS and positively by the CviI/R system in both C. violaceum and in a heterologous Escherichia coli genetic background. VioS does not regulate the CviI/R system and apart from violacein, VioS, and quorum sensing regulate other phenotypes antagonistically. Quorum sensing regulated phenotypes in C. violaceum are therefore further regulated providing an additional level of control. PMID:28326068

  7. Negative Regulation of Violacein Biosynthesis in Chromobacterium violaceum.

    PubMed

    Devescovi, Giulia; Kojic, Milan; Covaceuszach, Sonia; Cámara, Miguel; Williams, Paul; Bertani, Iris; Subramoni, Sujatha; Venturi, Vittorio

    2017-01-01

    In Chromobacteium violaceum, the purple pigment violacein is under positive regulation by the N-acylhomoserine lactone CviI/R quorum sensing system and negative regulation by an uncharacterized putative repressor. In this study we report that the biosynthesis of violacein is negatively controlled by a novel repressor protein, VioS. The violacein operon is regulated negatively by VioS and positively by the CviI/R system in both C. violaceum and in a heterologous Escherichia coli genetic background. VioS does not regulate the CviI/R system and apart from violacein, VioS, and quorum sensing regulate other phenotypes antagonistically. Quorum sensing regulated phenotypes in C. violaceum are therefore further regulated providing an additional level of control.

  8. Expectancies for Negative Mood Regulation, Coping, and Dysphoria among College Students.

    ERIC Educational Resources Information Center

    Catanzaro, Salvatore J.; Greenwood, Gregory

    1994-01-01

    College students (n=222) completed measures of negative mood regulation (NMR) expectancies, negative life events, coping responses, dysphoria, and somatic symptoms. Weeks later, they completed same questionnaires but with daily hassles replacing life events. NMR expectancies were positively related to active coping and negatively related to…

  9. Study of electrochemically active carbon, Ga2O3 and Bi2O3 as negative additives for valve-regulated lead-acid batteries working under high-rate, partial-state-of-charge conditions

    NASA Astrophysics Data System (ADS)

    Zhao, Li; Chen, Baishuang; Wu, Jinzhu; Wang, Dianlong

    2014-02-01

    Electrochemically active carbon (EAC), Gallium (III) oxide (Ga2O3) and Bismuth (III) oxide (Bi2O3) are used as the negative additives of valve-regulated lead-acid (VRLA) batteries to prolong the cycle life of VRLA batteries under high-rate partial-state-of-charge (HRPSoC) conditions, and their effects on the cycle life of VRLA batteries are investigated. It is found that the addition of EAC in negative active material can restrain the sulfation of the negative plates and prolong the cycle performance of VRLA batteries under HRPSoC conditions. It is also observed that the addition of Ga2O3 or Bi2O3 in EAC can effectively increase the overpotential of hydrogen evolution on EAC electrodes, and decrease the evolution rate of hydrogen. An appropriate addition amount of Ga2O3 or Bi2O3 in the negative plates of VRLA batteries can decrease the cut-off charging voltage, increase the cut-off discharging voltage, and prolong the cycle life of VRLA batteries under HRPSoC conditions. The battery added with 0.5% EAC and 0.01% Ga2O3 in negative active material shows a lowest cut-off charging voltage and a highest cut-off discharging voltage under HRPSoC conditions, and its' cycle life reaches about 8100 cycles which is at least three times longer than that without Ga2O3.

  10. Dynein-mediated trafficking negatively regulates LET-23 EGFR signaling

    PubMed Central

    Skorobogata, Olga; Meng, Jassy; Gauthier, Kimberley; Rocheleau, Christian E.

    2016-01-01

    Epidermal growth factor receptor (EGFR) signaling is essential for animal development, and increased signaling underlies many human cancers. Identifying the genes and cellular processes that regulate EGFR signaling in vivo will help to elucidate how this pathway can become inappropriately activated. Caenorhabditis elegans vulva development provides an in vivo model to genetically dissect EGFR signaling. Here we identified a mutation in dhc-1, the heavy chain of the cytoplasmic dynein minus end–directed microtubule motor, in a genetic screen for regulators of EGFR signaling. Despite the many cellular functions of dynein, DHC-1 is a strong negative regulator of EGFR signaling during vulva induction. DHC-1 is required in the signal-receiving cell and genetically functions upstream or in parallel to LET-23 EGFR. LET-23 EGFR accumulates in cytoplasmic foci in dhc-1 mutants, consistent with mammalian cell studies in which dynein is shown to regulate late endosome trafficking of EGFR with the Rab7 GTPase. However, we found different distributions of LET-23 EGFR foci in rab-7 versus dhc-1 mutants, suggesting that dynein functions at an earlier step of LET-23 EGFR trafficking to the lysosome than RAB-7. Our results demonstrate an in vivo role for dynein in limiting LET-23 EGFR signaling via endosomal trafficking. PMID:27654944

  11. Cytochrome c Negatively Regulates NLRP3 Inflammasomes

    PubMed Central

    Shi, Chong-Shan; Kehrl, John H.

    2016-01-01

    The release of cytochrome c from the inner mitochondrial membrane, where it is anchored by caridolipin, triggers the formation of the Apaf-1 apoptosome. Cardiolipin also interacts with NLRP3 recruiting NLRP3 to mitochondria and facilitating inflammasome assembly. In this study we investigated whether cytosolic cytochrome c impacts NLRP3 inflammasome activation in macrophages. We report that cytochrome c binds to the LRR domain of NLRP3 and that cytochrome c reduces the interactions between NLRP3 and cardiolipin and between NLRP3 and NEK7, a recently recognized component of the NLRP3 inflammasome needed for NLRP3 oligomerization. Protein transduction of cytochrome c impairs NLRP3 inflammasome activation, while partially silencing cytochrome c expression enhances it. The addition of cytochrome c to an in vitro inflammasome assay severely limited caspase-1 activation. We propose that there is a crosstalk between the NLRP3 inflammasome and apoptosome pathways mediated by cytochrome c, whose release during apoptosis acts to limit NLRP3 inflammasome activation. PMID:28030552

  12. Focal adhesion kinase negatively regulates neuronal insulin resistance.

    PubMed

    Gupta, Amit; Bisht, Bharti; Dey, Chinmoy Sankar

    2012-06-01

    Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics.

  13. A delayed, gonadotropin-dependent and growth-factor mediated activation of the ERK1/2 cascade negatively regulates aromatase expression in granulosa cells*

    PubMed Central

    Andric, Nebojsa; Ascoli, Mario

    2006-01-01

    Human CG and hFSH elicit a transient increase in ERK1/2 phosphorylation lasting less than 60 min in immature granulosa cells expressing a low density of gonadotropin receptors. In cells expressing a high density of receptors hCG and hFSH elicit this fast transient increase in ERK1/2 phosphorylation and also a delayed and more sustained increase that is detectable after 6–9 h. Both, the early and delayed increases in ERK1/2 phosphorylation can be blocked with inhibitors of PKA, the epidermal growth factor receptor (EGFR) kinase, metalloproteases and MEK. The delayed effect, but not the early effect, can also be blocked with an inhibitor of protein kinase C (PKC). Since the delayed increase in ERK1/2 phosphorylation correlates with low aromatase expression in response to gonadotropins we tested the effects of the inhibitors mentioned on aromatase expression. These inhibitors had little or no effect on gonadotropin-induced aromatase expression in cells expressing a low density of receptors but they enhanced gonadotropin-induced aromatase expression in cells expressing a high density of receptors. Phorbol esters also induced a prolonged increase in ERK1/2 phosphorylation and when added together with hFSH, blocked the induction of aromatase expression by hFSH in cells expressing a low density of hFSHR. A MEK inhibitor reversed the inhibitory effect of the phorbol ester on aromatase induction. We conclude that the effects of gonadotropins on ERK1/2 phosphorylation are mediated by EGF-like growth factors and that the delayed effect is partially mediated by PKC and acts as a negative regulator of aromatase expression. PMID:16973759

  14. Autophagy Negatively Regulates Transmissible Gastroenteritis Virus Replication.

    PubMed

    Guo, Longjun; Yu, Haidong; Gu, Weihong; Luo, Xiaolei; Li, Ren; Zhang, Jian; Xu, Yunfei; Yang, Lijun; Shen, Nan; Feng, Li; Wang, Yue

    2016-03-31

    Autophagy is an evolutionarily ancient pathway that has been shown to be important in the innate immune defense against several viruses. However, little is known about the regulatory role of autophagy in transmissible gastroenteritis virus (TGEV) replication. In this study, we found that TGEV infection increased the number of autophagosome-like double- and single-membrane vesicles in the cytoplasm of host cells, a phenomenon that is known to be related to autophagy. In addition, virus replication was required for the increased amount of the autophagosome marker protein LC3-II. Autophagic flux occurred in TGEV-infected cells, suggesting that TGEV infection triggered a complete autophagic response. When autophagy was pharmacologically inhibited by wortmannin or LY294002, TGEV replication increased. The increase in virus yield via autophagy inhibition was further confirmed by the use of siRNA duplexes, through which three proteins required for autophagy were depleted. Furthermore, TGEV replication was inhibited when autophagy was activated by rapamycin. The antiviral response of autophagy was confirmed by using siRNA to reduce the expression of gene p300, which otherwise inhibits autophagy. Together, the results indicate that TGEV infection activates autophagy and that autophagy then inhibits further TGEV replication.

  15. Negative regulation of RelA phosphorylation: emerging players and their roles in cancer.

    PubMed

    Lu, Xinyuan; Yarbrough, Wendell G

    2015-02-01

    NF-κB signaling contributes to human disease processes, notably inflammatory diseases and cancer. Many advances have been made in understanding mechanisms responsible for abnormal NF-κB activation with RelA post-translational modification, particularly phosphorylation, proven to be critical for RelA function. While the majority of studies have focused on identifying kinases responsible for NF-κB phosphorylation and pathway activation, recently progress has also been made in understanding the negative regulators important for restraining RelA activity. Here we summarize negative regulators of RelA phosphorylation, their targeting sites in RelA and biological functions through negative regulation of RelA activation. Finally, we emphasize the tumor suppressor-like roles that these negative regulators can assume in human cancers.

  16. Intracellular LINGO-1 negatively regulates Trk neurotrophin receptor signaling.

    PubMed

    Meabon, James S; de Laat, Rian; Ieguchi, Katsuaki; Serbzhinsky, Dmitry; Hudson, Mark P; Huber, B Russel; Wiley, Jesse C; Bothwell, Mark

    2016-01-01

    Neurotrophins, essential regulators of many aspects of neuronal differentiation and function, signal via four receptors, p75, TrkA, TrkB and TrkC. The three Trk paralogs are members of the LIG superfamily of membrane proteins, which share extracellular domains consisting of leucine-rich repeat and C2 Ig domains. Another LIG protein, LINGO-1 has been reported to bind and influence signaling of p75 as well as TrkA, TrkB and TrkC. Here we examine the manner in which LINGO-1 influences the function of TrkA, TrkB and TrkC. We report that Trk activation promotes Trk association with LINGO-1, and that this association promotes Trk degradation by a lysosomal mechanism. This mechanism resembles the mechanism by which another LIG protein, LRIG1, promotes lysosomal degradation of receptor tyrosine kinases such as the EGF receptor. We present evidence indicating that the Trk/LINGO-1 interaction occurs, in part, within recycling endosomes. We show that a mutant form of LINGO-1, with much of the extracellular domain deleted, has the capacity to enhance TrkA signaling in PC12 cells, possibly by acting as an inhibitor of Trk down-regulation by full length LINGO-1. We propose that LINGO-1 functions as a negative feedback regulator of signaling by cognate receptor tyrosine kinases including TrkA, TrkB and TrkC.

  17. CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

    PubMed Central

    Li, Xueni; Huang, Mei; Zheng, Huiling; Wang, Yinyin; Ren, Fangli; Shang, Yu; Zhai, Yonggong; Irwin, David M.; Shi, Yuguang; Chen, Di; Chang, Zhijie

    2008-01-01

    Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage. PMID:18541707

  18. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    PubMed Central

    Desfougères, Yann; Vavassori, Stefano; Rompf, Maria; Gerasimaite, Ruta; Mayer, Andreas

    2016-01-01

    The V-ATPase is a proton pump consisting of a membrane-integral V0 sector and a peripheral V1 sector, which carries the ATPase activity. In vitro studies of yeast vacuole fusion and evidence from worms, flies, zebrafish and mice suggested that V0 interacts with the SNARE machinery for membrane fusion, that it promotes the induction of hemifusion and that this activity requires physical presence of V0 rather than its proton pump activity. A recent in vivo study in yeast has challenged these interpretations, concluding that fusion required solely lumenal acidification but not the V0 sector itself. Here, we identify the reasons for this discrepancy and reconcile it. We find that acute pharmacological or physiological inhibition of V-ATPase pump activity de-acidifies the vacuole lumen in living yeast cells within minutes. Time-lapse microscopy revealed that de-acidification induces vacuole fusion rather than inhibiting it. Cells expressing mutated V0 subunits that maintain vacuolar acidity were blocked in this fusion. Thus, proton pump activity of the V-ATPase negatively regulates vacuole fusion in vivo. Vacuole fusion in vivo does, however, require physical presence of a fusion-competent V0 sector. PMID:27363625

  19. Overexpression of the zinc finger protein MZF1 inhibits hematopoietic development from embryonic stem cells: correlation with negative regulation of CD34 and c-myb promoter activity.

    PubMed Central

    Perrotti, D; Melotti, P; Skorski, T; Casella, I; Peschle, C; Calabretta, B

    1995-01-01

    Zinc finger genes encode proteins that act as transcription factors. The myeloid zinc finger 1 (MZF1) gene encodes a zinc finger protein with two DNA-binding domains that recognize two distinct consensus sequences, is preferentially expressed in hematopoietic cells, and may be involved in the transcriptional regulation of hematopoiesis-specific genes. Reverse transcription-PCR analysis of human peripheral blood CD34+ cells cultured under lineage-restricted conditions demonstrated MZF1 expression during both myeloid and erythroid differentiation. Sequence analysis of the 5'-flanking region of the CD34 and c-myb genes, which are a marker of and a transcriptional factor required for hematopoietic proliferation and differentiation, respectively, revealed closely spaced MZF1 consensus binding sites found by electrophoretic mobility shift assays to interact with recombinant MZF1 protein. Transient or constitutive MZF1 expression in different cell types resulted in specific inhibition of chloramphenicol acetyltransferase activity driven by the CD34 or c-myb 5'-flanking region. To determine whether transcriptional modulation by MZF1 activity plays a role in hematopoietic differentiation, constructs containing the MZF1 cDNA under the control of different promoters were transfected into murine embryonic stem cells which, under defined in vitro culture conditions, generate colonies of multiple hematopoietic lineages. Constitutive MZF1 expression interfered with the ability of embryonic stem cells to undergo hematopoietic commitment and erythromyeloid colony formation and prevented the induced expression of CD34 and c-myb mRNAs during differentiation of these cells. These data indicate that MZF1 plays a critical role in hematopoiesis by modulating the expression of genes involved in this process. PMID:7565760

  20. Stress and Coping Activity: Reframing Negative Thoughts

    ERIC Educational Resources Information Center

    Hughes, Jamie S.; Gourley, Mary K.; Madson, Laura; Le Blanc, Katya

    2011-01-01

    Stress management and coping techniques are not only relevant in many psychology courses but also personally relevant for undergraduate students. In this article, the authors describe an activity designed to provide students with practice evaluating and challenging negative self-talk. Students responded to scenarios individually, were paired with…

  1. Bone marrow adipocytes as negative regulators of the hematopoietic microenvironment

    PubMed Central

    Naveiras, Olaia; Nardi, Valentina; Wenzel, Pamela L.; Fahey, Frederic; Daley, George Q.

    2009-01-01

    Osteoblasts and endothelium constitute functional niches that support hematopoietic stem cells (HSC) in mammalian bone marrow (BM) 1,2,3 . Adult BM also contains adipocytes, whose numbers correlate inversely with the hematopoietic activity of the marrow. Fatty infiltration of hematopoietic red marrow follows irradiation or chemotherapy and is a diagnostic feature in biopsies from patients with marrow aplasia 4. To explore whether adipocytes influence hematopoiesis or simply fill marrow space, we compared the hematopoietic activity of distinct regions of the mouse skeleton that differ in adiposity. By flow cytometry, colony forming activity, and competitive repopulation assay, HSCs and short-term progenitors are reduced in frequency in the adipocyte-rich vertebrae of the mouse tail relative to the adipocyte-free vertebrae of the thorax. In lipoatrophic A-ZIP/F1 “fatless” mice, which are genetically incapable of forming adipocytes8, and in mice treated with the PPARγ inhibitor Bisphenol-A-DiGlycidyl-Ether (BADGE), which inhibits adipogenesis9, post-irradiation marrow engraftment is accelerated relative to wild type or untreated mice. These data implicate adipocytes as predominantly negative regulators of the bone marrow microenvironment, and suggest that antagonizingmarrow adipogenesis may enhance hematopoietic recovery in clinical bone marrow transplantation. PMID:19516257

  2. How Novice EFL Teachers Regulate Their Negative Emotions

    ERIC Educational Resources Information Center

    Arizmendi Tejeda, Silvia; Gillings de González, Barbara Scholes; López Martínez, Cecilio Luis de Jesús

    2016-01-01

    This research report shares the findings that emerged from a qualitative study in which the main objective was to discover whether or not novice English as a foreign language teachers regulate their negative emotions during their initial teaching practice, and if so, how they do this. The data were collected by semi-structured interviews and…

  3. A CONSTANS-like transcriptional activator, OsCOL13, functions as a negative regulator of flowering downstream of OsphyB and upstream of Ehd1 in rice.

    PubMed

    Sheng, Peike; Wu, Fuqing; Tan, Junjie; Zhang, Huan; Ma, Weiwei; Chen, Liping; Wang, Jiachang; Wang, Jie; Zhu, Shanshan; Guo, Xiuping; Wang, Jiulin; Zhang, Xin; Cheng, Zhijun; Bao, Yiqun; Wu, Chuanyin; Liu, Xuanming; Wan, Jianmin

    2016-09-01

    Flowering time determines the adaptability of crop plants to different local environments, thus being one of the most important agronomic traits targeted in breeding programs. Photoperiod is one of the key factors that control flowering in plant. A number of genes that participate in the photoperiod pathway have been characterized in long-day plants such as Arabidopsis, as well as in short-day plants such as Oryza sativa. Of those, CONSTANS (CO) as a floral integrator promotes flowering in Arabidopsis under long day conditions. In rice, Heading date1 (Hd1), a homologue of CO, functions in an opposite way, which inhibits flowering under long day conditions and induces flowering under short day conditions. Here, we show that another CONSTANS-like (COL) gene, OsCOL13, negatively regulates flowering in rice under both long and short day conditions. Overexpression of OsCOL13 delays flowering regardless of day length. We also demonstrated that OsCOL13 has a constitutive and rhythmic expression pattern, and that OsCOL13 is localized to the nucleus. OsCOL13 displays transcriptional activation activity in the yeast assays and likely forms homodimers in vivo. OsCOL13 suppresses the florigen genes Hd3a and RFT1 by repressing Ehd1, but has no relationship with other known Ehd1 regulators as determined by using mutants or near isogenic lines. In addition, the transcriptional level of OsCOL13 significantly decreased in the osphyb mutant, but remained unchanged in the osphya and osphyc mutants. Thus, we conclude that OsCOL13 functions as a negative regulator downstream of OsphyB and upstream of Ehd1 in the photoperiodic flowering in rice.

  4. Regulating positive and negative emotions in daily life.

    PubMed

    Nezlek, John B; Kuppens, Peter

    2008-06-01

    The present study examined how people regulate their emotions in daily life and how such regulation is related to their daily affective experience and psychological adjustment. Each day for an average of 3 weeks, participants described how they had regulated their emotions in terms of the reappraisal and suppression (inhibiting the expression) of positive and negative emotions, and they described their emotional experience, self-esteem, and psychological adjustment in terms of Beck's triadic model of depression. Reappraisal was used more often than suppression, and suppressing positive emotions was used less than the other three strategies. In general, regulation through reappraisal was found to be beneficial, whereas regulation by suppression was not. Reappraisal of positive emotions was associated with increases in positive affect, self-esteem, and psychological adjustment, whereas suppressing positive emotions was associated with decreased positive emotion, self-esteem, and psychological adjustment, and increased negative emotions. Moreover, relationships between reappraisal and psychological adjustment and self-esteem were mediated by experienced positive affect, whereas relationships between suppression of positive emotions and self-esteem adjustment were mediated by negative affect.

  5. Histone deacetylase 9 is a negative regulator of adipogenic differentiation.

    PubMed

    Chatterjee, Tapan K; Idelman, Gila; Blanco, Victor; Blomkalns, Andra L; Piegore, Mark G; Weintraub, Daniel S; Kumar, Santosh; Rajsheker, Srinivas; Manka, David; Rudich, Steven M; Tang, Yaoliang; Hui, David Y; Bassel-Duby, Rhonda; Olson, Eric N; Lingrel, Jerry B; Ho, Shuk-Mei; Weintraub, Neal L

    2011-08-05

    Differentiation of preadipocytes into mature adipocytes capable of efficiently storing lipids is an important regulatory mechanism in obesity. Here, we examined the involvement of histone deacetylases (HDACs) and histone acetyltransferases (HATs) in the regulation of adipogenesis. We find that among the various members of the HDAC and HAT families, only HDAC9 exhibited dramatic down-regulation preceding adipogenic differentiation. Preadipocytes from HDAC9 gene knock-out mice exhibited accelerated adipogenic differentiation, whereas HDAC9 overexpression in 3T3-L1 preadipocytes suppressed adipogenic differentiation, demonstrating its direct role as a negative regulator of adipogenesis. HDAC9 expression was higher in visceral as compared with subcutaneous preadipocytes, negatively correlating with their potential to undergo adipogenic differentiation in vitro. HDAC9 localized in the nucleus, and its negative regulation of adipogenesis segregates with the N-terminal nuclear targeting domain, whereas the C-terminal deacetylase domain is dispensable for this function. HDAC9 co-precipitates with USF1 and is recruited with USF1 at the E-box region of the C/EBPα gene promoter in preadipocytes. Upon induction of adipogenic differentiation, HDAC9 is down-regulated, leading to its dissociation from the USF1 complex, whereas p300 HAT is up-regulated to allow its association with USF1 and accumulation at the E-box site of the C/EBPα promoter in differentiated adipocytes. This reciprocal regulation of HDAC9 and p300 HAT in the USF1 complex is associated with increased C/EBPα expression, a master regulator of adipogenic differentiation. These findings provide new insights into mechanisms of adipogenic differentiation and document a critical regulatory role for HDAC9 in adipogenic differentiation through a deacetylase-independent mechanism.

  6. STING Negatively Regulates Double-Stranded DNA-Activated JAK1-STAT1 Signaling via SHP-1/2 in B Cells.

    PubMed

    Dong, Guanjun; You, Ming; Ding, Liang; Fan, Hongye; Liu, Fei; Ren, Deshan; Hou, Yayi

    2015-05-01

    Recognition of cytosolic DNA initiates a series of innate immune responses by inducing IFN-I production and subsequent triggering JAK1-STAT1 signaling which plays critical roles in the pathogenesis of infection, inflammation and autoimmune diseases through promoting B cell activation and antibody responses. The stimulator of interferon genes protein (STING) has been demonstrated to be a critical hub of type I IFN induction in cytosolic DNA-sensing pathways. However, it still remains unknown whether cytosolic DNA can directly activate the JAK1-STAT1 signaling or not. And the role of STING is also unclear in this response. In the present study, we found that dsDNA directly triggered the JAK1-STAT1 signaling by inducing phosphorylation of the Lyn kinase. Moreover, this response is not dependent on type I IFN receptors. Interestingly, STING could inhibit dsDNA-triggered activation of JAK1-STAT1 signaling by inducing SHP-1 and SHP-2 phosphorylation. In addition, compared with normal B cells, the expression of STING was significantly lower and the phosphorylation level of JAK1 was significantly higher in B cells from MRL/lpr lupus-prone mice, highlighting the close association between STING low-expression and JAK1-STAT1 signaling activation in B cells in autoimmune diseases. Our data provide a molecular insight into the novel role of STING in dsDNA-mediated inflammatory disorders.

  7. A lower-order oligomer form of phage shock protein A (PspA) stably associates with the hexameric AAA(+) transcription activator protein PspF for negative regulation.

    PubMed

    Joly, Nicolas; Burrows, Patricia C; Engl, Christoph; Jovanovic, Goran; Buck, Martin

    2009-12-11

    To survive and colonise their various environments, including those used during infection, bacteria have developed a variety of adaptive systems. Amongst these is phage shock protein (Psp) response, which can be induced in Escherichia coli upon filamentous phage infection (specifically phage secretin pIV) and by other membrane-damaging agents. The E. coli Psp system comprises seven proteins, of which PspA is the central component. PspA is a bifunctional protein that is directly involved in (i) the negative regulation of the psp-specific transcriptional activator PspF and (ii) the maintenance of membrane integrity in a mechanism proposed to involve the formation of a 36-mer ring complex. Here we established that the PspA negative regulation of PspF ATPase activity is the result of a cooperative inhibition. We present biochemical evidence showing that an inhibitory PspA-PspF regulatory complex, which has significantly reduced PspF ATPase activity, is composed of around six PspF subunits and six PspA subunits, suggesting that PspA exists in at least two different oligomeric assemblies. We now establish that all four putative helical domains of PspA are critical for the formation of the 36-mer. In contrast, not all four helical domains are required for the formation of the inhibitory PspA-PspF complex. Since a range of initial PspF oligomeric states permit formation of the apparent PspA-PspF dodecameric assembly, we conclude that PspA and PspF demonstrate a strong propensity to self-assemble into a single defined heteromeric regulatory complex.

  8. The neural correlates of regulating positive and negative emotions in medication-free major depression.

    PubMed

    Greening, Steven G; Osuch, Elizabeth A; Williamson, Peter C; Mitchell, Derek G V

    2014-05-01

    Depressive cognitive schemas play an important role in the emergence and persistence of major depressive disorder (MDD). The current study adapted emotion regulation techniques to reflect elements of cognitive behavioural therapy (CBT) and related psychotherapies to delineate neurocognitive abnormalities associated with modulating the negative cognitive style in MDD. Nineteen non-medicated patients with MDD and 19 matched controls reduced negative or enhanced positive feelings elicited by emotional scenes while undergoing functional magnetic resonance imaging. Although both groups showed significant emotion regulation success as measured by subjective ratings of affect, the controls were significantly better at modulating both negative and positive emotion. Both groups recruited regions of dorsolateral prefrontal cortex and ventrolateral prefrontal cortex (VLPFC) when regulating negative emotions. Only in controls was this accompanied by reduced activity in sensory cortices and amygdala. Similarly, both groups showed enhanced activity in VLPFC and ventral striatum when enhancing positive affect; however, only in controls was ventral striatum activity correlated with regulation efficacy. The results suggest that depression is associated with both a reduced capacity to achieve relief from negative affect despite recruitment of ventral and dorsal prefrontal cortical regions implicated in emotion regulation, coupled with a disconnect between activity in reward-related regions and subjective positive affect.

  9. Rab35, acting through ACAP2 switching off Arf6, negatively regulates oligodendrocyte differentiation and myelination

    PubMed Central

    Miyamoto, Yuki; Yamamori, Natsuki; Torii, Tomohiro; Tanoue, Akito; Yamauchi, Junji

    2014-01-01

    Oligodendrocyte precursor cells differentiate to produce myelin sheaths that insulate axons to ensure fast propagation of action potentials. Many aspects of differentiation are regulated by multiple extracellular signals. However, their intracellular signalings remain elusive. We show that Rab35 and its effector, ACAP2, a GTPase-activating protein that switches off Arf6 activity, negatively regulate oligodendrocyte morphological differentiation. Knockdown of Rab35 or ACAP2 with their respective small interfering RNAs promotes differentiation. As differentiation initiates, the activities of Rab35 and ACAP2 are down-regulated. The activity of Arf6, in contrast, is up-regulated. Arf6 knockdown inhibits differentiation, indicating that Rab35 and ACAP2 negatively regulate differentiation by down-regulating Arf6. Importantly, as differentiation proceeds, the activity of cytohesin-2, a guanine nucleotide exchange factor that switches on Arf6 activity, is up-regulated. Pharmacological inhibition of cytohesin-2 inhibits differentiation, suggesting that cytohesin-2 promotes differentiation by activating Arf6. Furthermore, using oligodendrocyte-neuronal cocultures, we find that knockdown of Rab35 or ACAP2 promotes myelination, whereas inhibition of cytohesin-2 or knockdown of Arf6 inhibits myelination. Thus Rab35/ACAP2 and cytohesin-2 antagonistically control oligodendrocyte differentiation and myelination through Arf6 regulation, presenting a unique small GTPase on/off switching mechanism. PMID:24600047

  10. NUMB negatively regulates the epithelial-mesenchymal transition of triple-negative breast cancer by antagonizing Notch signaling

    PubMed Central

    Zhang, Jianchao; Shao, Ximing; Sun, Haiyan; Liu, Ke; Ding, Zhihao; Chen, Juntao; Fang, Lijing; Su, Wu; Hong, Yang; Li, Huashun; Li, Hongchang

    2016-01-01

    Triple-negative breast cancer (TNBC), an aggressive subtype of breast cancer with higher rates of early relapse and metastasis, is frequently associated with aberrant activation of epithelial-mesenchymal transition (EMT). Nonetheless, how EMT is initiated and regulated during TNBC progression is not well understood. Here, we report that NUMB is a negative regulator of EMT in both human mammary epithelial cells and breast cancer cells. Reduced NUMB expression was significantly associated with elevated EMT in TNBC. Conversely, overexpression of NUMB strongly attenuated the EMT program and metastasis of TNBC cell lines. Interestingly, we showed that NUMB employs different molecular mechanisms to regulate EMT. In normal mammary epithelial cells and breast cancer cells expressing wild-type p53, NUMB suppressed EMT by stabilizing p53. However, in TNBC cells, loss of NUMB facilitated the EMT program by activating Notch signaling. Consistent with these findings, low NUMB expression and high Notch activity were significantly correlated with the TNBC subtype in patients. Collectively, these findings reveal novel molecular mechanisms of NUMB in the regulation of breast tumor EMT, especially in TNBC. PMID:27506933

  11. Epstein-Barr virus-negative aggressive natural killer-cell leukaemia with high P-glycoprotein activity and phosphorylated extracellular signal-regulated protein kinases 1 and 2.

    PubMed

    Perkovic, Sanja; Basic-Kinda, Sandra; Gasparovic, Vladimir; Krznaric, Zeljko; Babel, Jaksa; Ilic, Ivana; Aurer, Igor; Batinic, Drago

    2012-07-11

    Aggressive natural killer-cell leukaemia (ANKL) is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV) and P-glycoprotein (P-gp) expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56(+) NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

  12. Regulation of positive and negative emotion: effects of sociocultural context

    PubMed Central

    Snyder, Sara A.; Heller, S. Megan; Lumian, Daniel S.; McRae, Kateri

    2013-01-01

    Previous research has demonstrated that the use of emotion regulation strategies can vary by sociocultural context. In a previous study, we reported changes in the use of two different emotion regulation strategies at an annual alternative cultural event, Burning Man (McRae et al., 2011). In this sociocultural context, as compared to typically at home, participants reported less use of expressive suppression (a strategy generally associated with maladaptive outcomes), and greater use of cognitive reappraisal (a strategy generally associated with adaptive outcomes). What remained unclear was whether these changes in self-reported emotion regulation strategy use were characterized by changes in the regulation of positive emotion, negative emotion, or both. We addressed this issue in the current study by asking Burning Man participants separate questions about positive and negative emotion. Using multiple datasets, we replicated our previous findings, and found that the decreased use of suppression is primarily driven by reports of decreased suppression of positive emotion at Burning Man. By contrast, the increased use of reappraisal is not characterized by differential reappraisal of positive and negative emotion at Burning Man. Moreover, we observed novel individual differences in the magnitude of these effects. The contextual changes in self-reported suppression that we observe are strongest for men and younger participants. For those who had previously attended Burning Man, we observed lower levels of self-reported suppression in both sociocultural contexts: Burning Man and typically at home. These findings have implications for understanding the ways in which certain sociocultural contexts may decrease suppression, and possibly minimize its associated maladaptive effects. PMID:23840191

  13. N-WASP Is Essential for the Negative Regulation of B Cell Receptor Signaling

    PubMed Central

    Liu, Chaohong; Bai, Xiaoming; Wu, Junfeng; Sharma, Shruti; Upadhyaya, Arpita; Dahlberg, Carin I. M.; Westerberg, Lisa S.; Snapper, Scott B.; Zhao, Xiaodong; Song, Wenxia

    2013-01-01

    Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott–Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell–specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation. PMID:24223520

  14. Factors regulating microglia activation

    PubMed Central

    Kierdorf, Katrin; Prinz, Marco

    2013-01-01

    Microglia are resident macrophages of the central nervous system (CNS) that display high functional similarities to other tissue macrophages. However, it is especially important to create and maintain an intact tissue homeostasis to support the neuronal cells, which are very sensitive even to minor changes in their environment. The transition from the “resting” but surveying microglial phenotype to an activated stage is tightly regulated by several intrinsic (e.g., Runx-1, Irf8, and Pu.1) and extrinsic factors (e.g., CD200, CX3CR1, and TREM2). Under physiological conditions, minor changes of those factors are sufficient to cause fatal dysregulation of microglial cell homeostasis and result in severe CNS pathologies. In this review, we discuss recent achievements that gave new insights into mechanisms that ensure microglia quiescence. PMID:23630462

  15. miRNA863-3p sequentially targets negative immune regulator ARLPKs and positive regulator SERRATE upon bacterial infection

    PubMed Central

    Niu, Dongdong; Lii, Yifan E.; Chellappan, Padmanabhan; Lei, Lei; Peralta, Karl; Jiang, Chunhao; Guo, Jianhua; Coaker, Gitta; Jin, Hailing

    2016-01-01

    Plant small RNAs play important roles in gene regulation during pathogen infection. Here we show that miR863-3p is induced by the bacterial pathogen Pseudomonas syringae carrying various effectors. Early during infection, miR863-3p silences two negative regulators of plant defence, atypical receptor-like pseudokinase1 (ARLPK1) and ARLPK2, both lacking extracellular domains and kinase activity, through mRNA degradation to promote immunity. ARLPK1 associates with, and may function through another negative immune regulator ARLPK1-interacting receptor-like kinase 1 (AKIK1), an active kinase with an extracellular domain. Later during infection, miR863-3p silences SERRATE, which is essential for miRNA accumulation and positively regulates defence, through translational inhibition. This results in decreased miR863-3p levels, thus forming a negative feedback loop to attenuate immune responses after successful defence. This is an example of a miRNA that sequentially targets both negative and positive regulators of immunity through two modes of action to fine-tune the timing and amplitude of defence responses. PMID:27108563

  16. FlbD has a DNA-binding activity near its carboxy terminus that recognizes ftr sequences involved in positive and negative regulation of flagellar gene transcription in Caulobacter crescentus.

    PubMed Central

    Mullin, D A; Van Way, S M; Blankenship, C A; Mullin, A H

    1994-01-01

    G. Our results demonstrate that FlbD contains a sequence-specific DNA-binding activity within the 87 amino acids at its carboxy terminus, and the results suggest that FlbD exerts its effect as a positive and negative regulator of C. crescentus flagellar genes by binding to ftr sequences. Images PMID:7928958

  17. Kinase activity profiling of gram-negative pneumonia.

    PubMed

    Hoogendijk, Arie J; Diks, Sander H; Peppelenbosch, Maikel P; Van Der Poll, Tom; Wieland, Catharina W

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential to immune cells. The current study explored signal transduction events during murine Gram-negative pneumonia using a systems biology approach. Kinase activity arrays enable the analysis of 1,024 consensus sequences of protein kinase substrates. Using a kinase activity array on whole lung lysates, cellular kinase activities were determined in a mouse model of K. pneumoniae pneumonia. Notable kinase activities also were validated with phospho-specific Western blots. On the basis of the profiling data, mitogen-activated protein kinase (MAPK) signaling via p42 mitogen-activated protein kinase (p42) and p38 mitogen-activated protein kinase (p38) and transforming growth factor β (TGFβ) activity were reduced during infection, whereas v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) activity generally was enhanced. AKT signaling was represented in both metabolic and inflammatory (mitogen-activated protein kinase kinase 2 [MKK], apoptosis signal-regulating kinase/mitogen-activated protein kinase kinase kinase 5 [ASK] and v-raf murine sarcoma viral oncogene homolog B1 [b-RAF]) context. This study reaffirms the importance of classic inflammation pathways, such as MAPK and TGFβ signaling and reveals less known involvement of glycogen synthase kinase 3β (GSK-3β), AKT and SRC signaling cassettes in pneumonia.

  18. Intrinsic and extrinsic negative regulators of nuclear protein transport processes

    PubMed Central

    Sekimoto, Toshihiro; Yoneda, Yoshihiro

    2012-01-01

    The nuclear–cytoplasmic protein transport is a critical process in cellular events. The identification of transport signals (nuclear localization signal and nuclear export signal) and their receptors has facilitated our understanding of this expanding field. Nuclear transport must be appropriately regulated to deliver proteins through the nuclear pore when their functions are required in the nucleus, and to export them into the cytoplasm when they are not needed in the nucleus. Altered nuclear transport processes have been observed in stressed cells, which would change gene expressions. Some viruses interfere with nuclear transport in host cells to evade immune defense. Moreover, certain transport factors negatively regulate nuclear protein transport in cells. Understanding the regulatory mechanisms of nuclear–cytoplasmic trafficking not only provides important information about cellular processes, but also is of use for developing specific inhibitors for transport pathways. PMID:22672474

  19. 1,4-Naphthoquinone activates the HSP90/HSF1 pathway through the S-arylation of HSP90 in A431 cells: Negative regulation of the redox signal transduction pathway by persulfides/polysulfides.

    PubMed

    Abiko, Yumi; Sha, Liang; Shinkai, Yasuhiro; Unoki, Takamitsu; Luong, Nho Cong; Tsuchiya, Yukihiro; Watanabe, Yasuo; Hirose, Reiko; Akaike, Takaaki; Kumagai, Yoshito

    2017-03-01

    The current consensus is that environmental electrophiles activate redox signal transduction pathways through covalent modification of sensor proteins with reactive thiol groups at low concentrations, while they cause cell damage at higher concentrations. We previously exposed human carcinoma A431 cells to the atmospheric electrophile 1,4-naphthoquinone (1,4-NQ) and found that heat shock protein 90 (HSP90), a negative regulator of heat shock factor 1 (HSF1), was a target of 1,4-NQ. In the study presented here, we determined whether 1,4-NQ activates HSF1. We also examined whether such redox signaling could be regulated by nucleophilic sulfur species. Exposure of A431 cells to 1,4-NQ covalently modified cellular HSP90, resulting in repression of the association between HSF1 with HSP90, thereby enhancing HSF1 translocation into the nuclei. Liquid chromatography-tandem mass spectrometry analysis with recombinant HSP90 revealed that the modifications site were Cys412 and Cys564. We found that HSF1 activation mediated by 1,4-NQ upregulated downstream genes, such as HSPA6. HSF1 knockdown accelerated 1,4-NQ-mediated cytotoxicity in the cells. While simultaneous treatment with reactive persulfide and polysulfide, Na2S2 and Na2S4, blocked 1,4-NQ-dependent protein modification and HSF1 activation in A431 cells, the knockdown of Cys persulfide producing enzymes cystathionine β-synthase (CBS) and/or cystathionine γ-lyase (CSE) enhanced these phenomena. 1,4-NQ-thiol adduct and 1,4-NQ-S-1,4-NQ adduct were produced during the enzymatic reaction of recombinant CSE in the presence of 1,4-NQ. The results suggest that activation of the HSP90-HSF1 signal transduction pathway mediated by 1,4-NQ protects cells against 1,4-NQ and that per/polysulfides can diminish the reactivity of 1,4-NQ by forming sulfur adducts.

  20. p53 regulates thymic Notch1 activation.

    PubMed

    Laws, Amy M; Osborne, Barbara A

    2004-03-01

    Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP)/CD8+ single positive (SP) transition, but regulation of Notchactivation is not well understood. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. We hypothesize that p53 regulates Notch activity during T cell development. We found that Notch1 expression and activation were negatively regulated by p53in several thymoma lines. Additionally, NIC was elevated in Trp53(-/-) thymocytes as compared to Trp53(+/+) thymocytes. To determine if elevated Notch1 activation in Trp53(-/-) thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP/CD8+ SP T cell ratio was decreased in Trp53(-/-) splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8+SP T cells in Trp53(-/-) mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, we suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation.

  1. 15 CFR 930.35 - Negative determinations for proposed activities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Negative determinations for proposed... Federal Agency Activities § 930.35 Negative determinations for proposed activities. (a) If a Federal... agencies with a negative determination for a Federal agency activity: (1) Identified by a State agency...

  2. Interaction between a Domain of the Negative Regulator of the Ras-ERK Pathway, SPRED1 Protein, and the GTPase-activating Protein-related Domain of Neurofibromin Is Implicated in Legius Syndrome and Neurofibromatosis Type 1*♦

    PubMed Central

    Hirata, Yasuko; Brems, Hilde; Suzuki, Mayu; Kanamori, Mitsuhiro; Okada, Masahiro; Morita, Rimpei; Llano-Rivas, Isabel; Ose, Toyoyuki; Messiaen, Ludwine; Legius, Eric; Yoshimura, Akihiko

    2016-01-01

    Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple café-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120GAP. Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome. PMID:26635368

  3. Ikaros dominant negative isoform (Ik6) induces IL-3-independent survival of murine pro-B lymphocytes by activating JAK-STAT and up-regulating Bcl-xl levels.

    PubMed

    Kano, Gen; Morimoto, Akira; Takanashi, Mami; Hibi, Shigeyoshi; Sugimoto, Tohru; Inaba, Tohru; Yagi, Tomohito; Imashuku, Shinsaku

    2008-05-01

    Ikaros is an essential regulator of lymphocyte differentiation. Mice transgenic for the Ikaros dominant negative (DN) mutation rapidly develop lymphoid malignancies. Various human leukemias have also been reported to express Ikaros DN isoforms, but its role in leukemogenesis is yet to be defined. We demonstrate that overexpressed Ikaros DN (Ik6) prolonged the survival of two different murine pro-B cell lines in cytokine deprived condition, and this was associated with increased expression of Bcl-xl. A survey of the upstream controller(s) of Bcl-xl expression revealed Ik6 overexpression enhanced the phosphorylation of JAK2 and STAT5. Interestingly, the Ik6 expressing cell lines showed reduced expression of B-cell differentiation surface marker CD45R (B220), which is also known as a JAK2 inhibitor. Although further evaluation with human clinical materials are required, these results propose a putative role of Ik6 in the development of B-lineage acute lymphoblastic leukemia, by activating the JAK2-STAT5 pathway and thus stimulating the production of Bcl-xl.

  4. MiR-143-3p controls TGF-β1-induced cell proliferation and extracellular matrix production in airway smooth muscle via negative regulation of the nuclear factor of activated T cells 1.

    PubMed

    Cheng, Wei; Yan, Kun; Xie, Li-Yi; Chen, Feng; Yu, Hong-Chuan; Huang, Yan-Xia; Dang, Cheng-Xue

    2016-10-01

    MicroRNAs (miRNAs) are small noncoding RNAs that function in diverse biological processes. However, little is known about the precise role of microRNAs in the functioning of airway smooth muscle cells (ASMCs). Here, we investigated the potential role and mechanisms of the miR-143 -3p on proliferation and the extracellular matrix (ECM) protein production of ASMCs. We demonstrated that miR-143-3p was aberrantly lower in ASMCs isolated from individuals with asthma than in individuals without asthma. Meanwhile, TGF-β1 caused a marked decrease in a time-dependent manner in miR-143-3p expression in ASMCs from asthmatics. Additionally, the overexpression of miR- 143-3p robustly reduced TGF-β1-induced ASMCs proliferation and downregulated CDK and cyclin expression, whereas the inhibition of miR-143-3p significantly enhanced ASMCs proliferation and upregulated the level of CDKs and cyclins. Re-expression of miR-143-3p attenuated ECM protein deposition reflected as a marked decrease in the expression of type I collagen and fibronectin, whereas miR-143-3p downregulation caused an opposite effect on the expression of type I collagen and fibronectin. Moreover, qRT-PCR and western blot analysis indicated that miR-143-3p negatively regulated the expression of nuclear factor of activated T cells 1 (NFATc1). Subsequent analyses demonstrated that NFATc1 was a direct and functional target of miR-143-3p, which was validated by the dual luciferase reporter assay. Most importantly, the overexpression of NFATc1 effectively reversed the inhibition of miR-143-3p on TGF-β1-induced proliferation, and strikingly abrogated the effect of miR-143-3p on the expression of CDK4 and Cyclin D1. Together, miR-143-3p may function as an inhibitor of asthma airway remodeling by suppressing proliferation and ECM protein deposition in TGF-β1-mediated ASMCs via the negative regulation of NFATc1 signaling, suggesting miR-143-3p as a potential therapeutic target for asthma.

  5. Negative regulation of ErbB family receptor tyrosine kinases.

    PubMed

    Sweeney, C; Carraway, K L

    2004-01-26

    Receptors of the EGF receptor or ErbB family of growth factor receptor tyrosine kinases are frequently overexpressed in a variety of solid tumours, and the aberrant activation of their tyrosine kinase activities is thought to contribute to tumour growth and progression. Much effort has been put into developing inhibitors of ErbB receptors, and both antibody and small-molecule approaches have exhibited clinical success. Recently, a number of endogenous negative regulatory proteins have been identified that suppress the signalling activity of ErbB receptors in cells. These include intracellular RING finger E3 ubiquitin ligases such as cbl and Nrdp1 that mediate ErbB receptor degradation, and may include a wide variety of secreted and transmembrane proteins that suppress receptor activation by growth factor ligands. It will be of interest to determine the extent to which tumour cells suppress these pathways to promote their progression, and whether restoration of endogenous receptor-negative regulatory pathways may be exploited for therapeutic benefit.

  6. Runx3 negatively regulates Osterix expression in dental pulp cells.

    PubMed

    Zheng, Li; Iohara, Koichiro; Ishikawa, Masaki; Into, Takeshi; Takano-Yamamoto, Teruko; Matsushita, Kenji; Nakashima, Misako

    2007-07-01

    Osterix, a zinc-finger-containing transcription factor, is required for osteoblast differentiation and bone formation. Osterix is also expressed in dental mesenchymal cells of the tooth germ. However, transcriptional regulation by Osterix in tooth development is not clear. Genetic studies in osteogenesis place Osterix downstream of Runx2 (Runt-related 2). The expression of Osterix in odontoblasts overlaps with Runx3 during terminal differentiation in vivo. Runx3 down-regulates Osterix expression in mouse DPCs (dental pulp cells). Therefore the regulatory role of Runx3 on Osterix expression in tooth development was investigated. Enforced expression of Runx3 down-regulated the activity of the Osterix promoter in the human embryonic kidney 293 cell line. When the Runx3 responsive element on the Osterix promoter, located at -713 to -707 bp (site 3, AGTGGTT) relative to the cap site, was mutated, this down-regulation was abrogated. Furthermore, electrophoretic mobility-shift assay and chromatin immunoprecipitation assays in mouse DPCs demonstrated direct functional binding of Runx3 to the Osterix promoter. These results demonstrate the transcriptional regulation of Osterix expression by Runx3 during differentiation of dental pulp cells into odontoblasts during tooth development.

  7. CARD9 negatively regulates NLRP3-induced IL-1β production on Salmonella infection of macrophages

    PubMed Central

    Pereira, Milton; Tourlomousis, Panagiotis; Wright, John; P. Monie, Tom; Bryant, Clare E.

    2016-01-01

    Interleukin-1β (IL-1β) is a proinflammatory cytokine required for host control of bacterial infections, and its production must be tightly regulated to prevent excessive inflammation. Here we show that caspase recruitment domain-containing protein 9 (CARD9), a protein associated with induction of proinflammatory cytokines by fungi, has a negative role on IL-1β production during bacterial infection. Specifically, in response to activation of the nucleotide oligomerization domain receptor pyrin-domain containing protein 3 (NLRP3) by Salmonella infection, CARD9 negatively regulates IL-1β by fine-tuning pro-IL-1β expression, spleen tyrosine kinase (SYK)-mediated NLRP3 activation and repressing inflammasome-associated caspase-8 activity. CARD9 is suppressed during Salmonella enterica serovar Typhimurium infection, facilitating increased IL-1β production. CARD9 is, therefore, a central signalling hub that coordinates a pathogen-specific host inflammatory response. PMID:27670879

  8. CARD9 negatively regulates NLRP3-induced IL-1β production on Salmonella infection of macrophages.

    PubMed

    Pereira, Milton; Tourlomousis, Panagiotis; Wright, John; P Monie, Tom; Bryant, Clare E

    2016-09-27

    Interleukin-1β (IL-1β) is a proinflammatory cytokine required for host control of bacterial infections, and its production must be tightly regulated to prevent excessive inflammation. Here we show that caspase recruitment domain-containing protein 9 (CARD9), a protein associated with induction of proinflammatory cytokines by fungi, has a negative role on IL-1β production during bacterial infection. Specifically, in response to activation of the nucleotide oligomerization domain receptor pyrin-domain containing protein 3 (NLRP3) by Salmonella infection, CARD9 negatively regulates IL-1β by fine-tuning pro-IL-1β expression, spleen tyrosine kinase (SYK)-mediated NLRP3 activation and repressing inflammasome-associated caspase-8 activity. CARD9 is suppressed during Salmonella enterica serovar Typhimurium infection, facilitating increased IL-1β production. CARD9 is, therefore, a central signalling hub that coordinates a pathogen-specific host inflammatory response.

  9. Phosphofructokinase-1 Negatively Regulates Neurogenesis from Neural Stem Cells.

    PubMed

    Zhang, Fengyun; Qian, Xiaodan; Qin, Cheng; Lin, Yuhui; Wu, Haiyin; Chang, Lei; Luo, Chunxia; Zhu, Dongya

    2016-06-01

    Phosphofructokinase-1 (PFK-1), a major regulatory glycolytic enzyme, has been implicated in the functions of astrocytes and neurons. Here, we report that PFK-1 negatively regulates neurogenesis from neural stem cells (NSCs) by targeting pro-neural transcriptional factors. Using in vitro assays, we found that PFK-1 knockdown enhanced, and PFK-1 overexpression inhibited the neuronal differentiation of NSCs, which was consistent with the findings from NSCs subjected to 5 h of hypoxia. Meanwhile, the neurogenesis induced by PFK-1 knockdown was attributed to the increased proliferation of neural progenitors and the commitment of NSCs to the neuronal lineage. Similarly, in vivo knockdown of PFK-1 also increased neurogenesis in the dentate gyrus of the hippocampus. Finally, we demonstrated that the neurogenesis mediated by PFK-1 was likely achieved by targeting mammalian achaete-scute homologue-1 (Mash 1), neuronal differentiation factor (NeuroD), and sex-determining region Y (SRY)-related HMG box 2 (Sox2). All together, our results reveal PFK-1 as an important regulator of neurogenesis.

  10. TRIM45 negatively regulates NF-{kappa}B-mediated transcription and suppresses cell proliferation

    SciTech Connect

    Shibata, Mio; Sato, Tomonobu; Nukiwa, Ryota; Ariga, Tadashi; Hatakeyama, Shigetsugu

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer NF-{kappa}B plays an important role in cell survival and carcinogenesis. Black-Right-Pointing-Pointer TRIM45 negatively regulates TNF{alpha}-induced NF-{kappa}B-mediated transcription. Black-Right-Pointing-Pointer TRIM45 overexpression suppresses cell growth. Black-Right-Pointing-Pointer TRIM45 acts as a repressor for the NF-{kappa}B signal and regulates cell growth. -- Abstract: The NF-{kappa}B signaling pathway plays an important role in cell survival, immunity, inflammation, carcinogenesis, and organogenesis. Activation of NF-{kappa}B is regulated by several posttranslational modifications including phosphorylation, neddylation and ubiquitination. The NF-{kappa}B signaling pathway is activated by two distinct signaling mechanisms and is strictly modulated by the ubiquitin-proteasome system. It has been reported that overexpression of TRIM45, one of the TRIM family ubiquitin ligases, suppresses transcriptional activities of Elk-1 and AP-1, which are targets of the MAPK signaling pathway. In this study, we showed that TRIM45 also negatively regulates TNF{alpha}-induced NF-{kappa}B-mediated transcription by a luciferase reporter assay and that TRIM45 lacking a RING domain also has an activity to inhibit the NF-{kappa}B signal. Moreover, we found that TRIM45 overexpression suppresses cell growth. These findings suggest that TRIM45 acts as a repressor for the NF-{kappa}B signal and regulates cell growth.

  11. Necdin, a negative growth regulator, is a novel STAT3 target gene down-regulated in human cancer.

    PubMed

    Haviland, Rachel; Eschrich, Steven; Bloom, Gregory; Ma, Yihong; Minton, Susan; Jove, Richard; Cress, W Douglas

    2011-01-01

    Cytokine and growth factor signaling pathways involving STAT3 are frequently constitutively activated in many human primary tumors, and are known for the transcriptional role they play in controlling cell growth and cell cycle progression. However, the extent of STAT3's reach on transcriptional control of the genome as a whole remains an important question. We predicted that this persistent STAT3 signaling affects a wide variety of cellular functions, many of which still remain to be characterized. We took a broad approach to identify novel STAT3 regulated genes by examining changes in the genome-wide gene expression profile by microarray, using cells expressing constitutively-activated STAT3. Using computational analysis, we were able to define the gene expression profiles of cells containing activated STAT3 and identify candidate target genes with a wide range of biological functions. Among these genes we identified Necdin, a negative growth regulator, as a novel STAT3 target gene, whose expression is down-regulated at the mRNA and protein levels when STAT3 is constitutively active. This repression is STAT3 dependent, since inhibition of STAT3 using siRNA restores Necdin expression. A STAT3 DNA-binding site was identified in the Necdin promoter and both EMSA and chromatin immunoprecipitation confirm binding of STAT3 to this region. Necdin expression has previously been shown to be down-regulated in a melanoma and a drug-resistant ovarian cancer cell line. Further analysis of Necdin expression demonstrated repression in a STAT3-dependent manner in human melanoma, prostate and breast cancer cell lines. These results suggest that STAT3 coordinates expression of genes involved in multiple metabolic and biosynthetic pathways, integrating signals that lead to global transcriptional changes and oncogenesis. STAT3 may exert its oncogenic effect by up-regulating transcription of genes involved in promoting growth and proliferation, but also by down-regulating expression

  12. LINGO-1 negatively regulates TrkB phosphorylation after ocular hypertension.

    PubMed

    Fu, Qing-Ling; Hu, Bing; Li, Xin; Shao, Zhaohui; Shi, Jian-Bo; Wu, Wutian; So, Kwok-Fai; Mi, Sha

    2010-03-01

    The antagonism of LINGO-1, a CNS-specific negative regulator of neuronal survival, was shown to promote short-term survival of retinal ganglion cell (RGC) in an ocular hypertension model. LINGO-1 antagonists, combined with brain-derived neurotrophic factor (BDNF), can increase the length of neuron survival through an unclear molecular mechanism. To determine the relationship between LINGO-1 and BDNF/TrkB receptor in neuronal protection, we show here that LINGO-1 forms a receptor complex with TrkB and negatively regulates its activation in the retina after ocular hypertension injury. LINGO-1 antagonist antibody 1A7 or soluble LINGO-1 (LINGO-1-Fc) treatment upregulates phospho-TrkB phosphorylation and leads to RGC survival after high intraocular pressure injury. This neuronal protective effect was blocked by anti-BDNF antibody. LINGO-1 antagonism therefore promotes RGC survival by regulating the BDNF and TrkB signaling pathway after ocular hypertension.

  13. Negative regulation of neuronal cell differentiation by INHAT subunit SET/TAF-Iβ.

    PubMed

    Kim, Dong-Wook; Kim, Kee-Beom; Kim, Ji-Young; Lee, Kyu-Sun; Seo, Sang-Beom

    2010-09-24

    Epigenetic modification plays an important role in transcriptional regulation. As a subunit of the INHAT (inhibitor of histone acetyltransferases) complex, SET/TAF-Iβ evidences transcriptional repression activity. In this study, we demonstrate that SET/TAF-Iβ is abundantly expressed in neuronal tissues of Drosophila embryos. It is expressed at high levels prior to and in early stages of neuronal development, and gradually reduced as differentiation proceeds. SET/TAF-Iβ binds to the promoters of a subset of neuronal development markers and negatively regulates the transcription of these genes. The results of this study show that the knockdown of SET/TAF-Iβ by si-RNA induces neuronal cell differentiation, thus implicating SET/TAF-Iβ as a negative regulator of neuronal development.

  14. Positive and negative elements regulate a melanocyte-specific promoter.

    PubMed Central

    Lowings, P; Yavuzer, U; Goding, C R

    1992-01-01

    Melanocytes are specialized cells residing in the hair follicles, the eye, and the basal layer of the human epidermis whose primary function is the production of the pigment melanin, giving rise to skin, hair, and eye color. Melanogenesis, a process unique to melanocytes that involves the processing of tyrosine by a number of melanocyte-specific enzymes, including tyrosinase and tyrosinase-related protein 1 (TRP-1), occurs only after differentiation from the melanocyte precursor, the melanoblast. In humans, melanogenesis is inducible by UV irradiation, with melanin being transferred from the melanocyte in the epidermis to the surrounding keratinocytes as protection from UV-induced damage. Excessive exposure to UV, however, is the primary cause of malignant melanoma, an increasingly common and highly aggressive disease. As an initial approach to understanding the regulation of melanocyte differentiation and melanocyte-specific transcription, we have isolated the gene encoding TRP-1 and examined the cis- and trans-acting factors required for cell-type-specific expression. We find that the TRP-1 promoter comprises both positive and negative regulatory elements which confer efficient expression in a TRP-1-expressing, pigmented melanoma cell line but not in NIH 3T3 or JEG3 cells and that a minimal promoter extending between -44 and +107 is sufficient for cell-type-specific expression. Assays for DNA-protein interactions coupled with extensive mutagenesis identified three factors, whose binding correlated with the function of two positive and one negative regulatory element. One of these factors, termed M-box-binding factor 1, binds to an 11-bp motif, the M box, which acts as a positive regulatory element both in TRP-1-expressing and -nonexpressing cell lines, despite being entirely conserved between the melanocyte-specific tyrosinase and TRP-1 promoters. The possible mechanisms underlying melanocyte-specific gene expression are discussed. Images PMID:1321344

  15. Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis

    PubMed Central

    Hu, Chi-Kuo; Özlü, Nurhan; Coughlin, Margaret; Steen, Judith J.; Mitchison, Timothy J.

    2012-01-01

    To achieve mitosis and cytokinesis, microtubules must assemble into distinct structures at different stages of cell division—mitotic spindles to segregate the chromosomes before anaphase and midzones to keep sister genomes apart and guide the cleavage furrow after anaphase. This temporal regulation is believed to involve Cdk1 kinase, which is inactivated in a switch-like way after anaphase. We found that inhibiting Plk1 caused premature assembly of midzones in cells still in metaphase, breaking the temporal regulation of microtubules. The antiparallel microtubule-bundling protein PRC1 plays a key role in organizing the midzone complex. We found that Plk1 negatively regulates PRC1 through phosphorylation of a single site, Thr-602, near the C-terminus of PRC1. We also found that microtubules stimulated Thr-602 phosphorylation by Plk1. This creates a potential negative feedback loop controlling PRC1 activity. It also made the extent of Thr-602 phosphorylation during mitotic arrest dependent on the mechanism of the arresting drug. Unexpectedly, we could not detect a preanaphase regulatory role for Cdk1 sites on PRC1. We suggest that PRC1 is regulated by Plk1, rather than Cdk1 as previously proposed, because its activity must be spatiotemporally regulated both preanaphase and postanaphase, and Cdk1 activity is too binary for this purpose. PMID:22621898

  16. Autophagy triggered by magnolol derivative negatively regulates angiogenesis

    PubMed Central

    Kumar, S; Guru, S K; Pathania, A S; Kumar, A; Bhushan, S; Malik, F

    2013-01-01

    Angiogenesis has a key role in the tumor progression and metastasis; targeting endothelial cell proliferation has emerged as a promising therapeutic strategy for the prevention of cancer. Previous studies have revealed a complex association between the process of angiogenesis and autophagy and its outcome on tumorigenesis. Autophagy, also known as type-II cell death, has been identified as an alternative way of cell killing in apoptotic-resistant cancer cells. However, its involvement in chemoresistance and tumor promotion is also well known. In this study, we used a derivate of natural product magnolol (Ery5), a potent autophagy inducer, to study the association between the autophagy and angiogenesis in both in vitro and in vivo model system. We found that the robust autophagy triggered by Ery5, inhibited angiogenesis and caused cell death independent of the apoptosis in human umbilical cord vein endothelial cells and PC-3 cells. Ery5 induced autophagy effectively inhibited cell proliferation, migration, invasion and tube formation. We further demonstrated that Ery5-mediated autophagy and subsequent inhibition of angiogenesis was reversed when autophagy was inhibited through 3-methyl adenine and knocking down of key autophagy proteins ATG7 and microtubule-associated protein light chain 3. While evaluating the negative regulation of autophagy on angiogenesis, it was interesting to find that angiogenic environment produced by the treatment of VEGF and CoCl2 remarkably downregulated the autophagy and autophagic cell death induced by Ery5. These studies, while disclosing the vital role of autophagy in the regulation of angiogenesis, also suggest that the potent modulators of autophagy can lead to the development of effective therapeutics in apoptosis-resistant cancer. PMID:24176847

  17. Human VAP-C negatively regulates hepatitis C virus propagation.

    PubMed

    Kukihara, Hiroshi; Moriishi, Kohji; Taguwa, Shuhei; Tani, Hideki; Abe, Takayuki; Mori, Yoshio; Suzuki, Tetsuro; Fukuhara, Takasuke; Taketomi, Akinobu; Maehara, Yoshihiko; Matsuura, Yoshiharu

    2009-08-01

    Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.

  18. Plexin-A4 negatively regulates T lymphocyte responses.

    PubMed

    Yamamoto, Midori; Suzuki, Kazuhiro; Okuno, Tatsusada; Ogata, Takehiro; Takegahara, Noriko; Takamatsu, Hyota; Mizui, Masayuki; Taniguchi, Masahiko; Chédotal, Alain; Suto, Fumikazu; Fujisawa, Hajime; Kumanogoh, Atsushi; Kikutani, Hitoshi

    2008-03-01

    Semaphorins and their receptors play crucial roles not only in axon guidance during neuronal development but also in the regulation of immune responses. Plexin-A4, a member of the plexin-A subfamily, forms a receptor complex with neuropilins and transduces signals for class III semaphorins in the nervous system. Although plexin-A4 is also expressed in the lymphoid tissues, the involvement of plexin-A4 in immune responses remains unknown. To explore the role of plexin-A4 in the immune system, we analyzed immune responses in plexin-A4-deficient (plexin-A4-/-) mice. Among immune cells, plexin-A4 mRNA was detected in T cells, dendritic cells and macrophages but not in B cells and NK cells. Plexin-A4-/- mice had normal numbers and cell surface markers for each lymphocyte subset, suggesting that plexin-A4 is not essential for lymphocyte development. However, plexin-A4-/- mice exhibited enhanced antigen-specific T cell responses and heightened sensitivity to experimental autoimmune encephalomyelitis. Plexin-A4-/- T cells exhibited hyperproliferative responses to anti-CD3 stimulation and to allogeneic dendritic cells in vitro. Furthermore, this hyperproliferation was also observed in both T cells from neuropilin-1 mutant (npn-1(Sema-)) mice, in which the binding site of class III semaphorins is disrupted, and T cells from Sema3A-deficient (Sema-3A-/-) mice. Collectively, these results suggest that plexin-A4, as a component of the receptor complex for class III semaphorins, negatively regulates T cell-mediated immune responses.

  19. The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.

    PubMed

    Carty, Michael; Goodbody, Rory; Schröder, Martina; Stack, Julianne; Moynagh, Paul N; Bowie, Andrew G

    2006-10-01

    Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll-interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction 'downstream' of TRIF but not of MyD88. SARM associated with TRIF, and 'knockdown' of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

  20. Wheat CBL-interacting protein kinase 25 negatively regulates salt tolerance in transgenic wheat

    PubMed Central

    Jin, Xia; Sun, Tao; Wang, Xiatian; Su, Peipei; Ma, Jingfei; He, Guangyuan; Yang, Guangxiao

    2016-01-01

    CBL-interacting protein kinases are involved in plant responses to abiotic stresses, including salt stress. However, the negative regulating mechanism of this gene family in response to salinity is less reported. In this study, we evaluated the role of TaCIPK25 in regulating salt response in wheat. Under conditions of high salinity, TaCIPK25 expression was markedly down-regulated in roots. Overexpression of TaCIPK25 resulted in hypersensitivity to Na+ and superfluous accumulation of Na+ in transgenic wheat lines. TaCIPK25 expression did not decline in transgenic wheat and remained at an even higher level than that in wild-type wheat controls under high-salinity treatment. Furthermore, transmembrane Na+/H+ exchange was impaired in the root cells of transgenic wheat. These results suggested that TaCIPK25 negatively regulated salt response in wheat. Additionally, yeast-one-hybrid, β-glucuronidase activity and DNA-protein-interaction-enzyme-linked-immunosorbent assays showed that the transcription factor TaWRKY9 bound W-box in the TaCIPK25 promoter region. Quantitative real-time polymerase chain reaction assays showed concomitantly inverted expression patterns of TaCIPK25 and TaWRKY9 in wheat roots under salt treatment, ABA application and inhibition of endogenous ABA condition. Overall, based on our results, in a salt stress condition, the negative salt response in wheat involved TaCIPK25 with the expression regulated by TaWRKY9. PMID:27358166

  1. CREB is a key negative regulator of carbonic anhydrase IX (CA9) in gastric cancer.

    PubMed

    Wang, Guanqiao; Cheng, Zhenguo; Liu, Funan; Zhang, Hongyan; Li, Jiabin; Li, Feng

    2015-07-01

    Carbonic anhydrase IX(CA9)is a member of the carbonic anhydrase family that catalyzes the reversible hydration of carbon dioxide, and plays a key role in the regulation of pH. Although a large number of studies have shown that CA9 is strongly up-regulated by HIF1-α, little is known about the negative regulation mechanism of CA9 in cancer cells. Here we find that CREB is a key negative regulator of CA9 in gastric cancer. Over-expression of CREB can significantly repress the expression of CA9. Treating with anisomycin (ANS), an activator of p38, the phosphorylation and nuclear translocation of CREB are both promoted, while the transcription of CA9 is repressed. Besides, our results firstly identify that CREB can recruit SIRT1 (class III HDACS) by adaptor protein p300, then repress the expression of CA9. These findings may contribute to understand the negative regulation mechanisms of CA9 in gastric cancer.

  2. AMPK: positive and negative regulation, and its role in whole-body energy homeostasis.

    PubMed

    Hardie, D Grahame

    2015-04-01

    The AMP-activated protein kinase (AMPK) is a sensor of energy status that, when activated by metabolic stress, maintains cellular energy homeostasis by switching on catabolic pathways and switching off ATP-consuming processes. Recent results suggest that activation of AMPK by the upstream kinase LKB1 in response to nutrient lack occurs at the surface of the lysosome. AMPK is also crucial in regulation of whole body energy balance, particularly by mediating effects of hormones acting on the hypothalamus. Recent crystal structures of complete AMPK heterotrimers have illuminated its complex mechanisms of activation, involving both allosteric activation and increased net phosphorylation mediated by effects on phosphorylation and dephosphorylation. Finally, AMPK is negatively regulated by phosphorylation of the 'ST loop' within the catalytic subunit.

  3. MEIS1 functions as a potential AR negative regulator

    SciTech Connect

    Cui, Liang; Yang, Yutao; Hang, Xingyi; Cui, Jiajun; Gao, Jiangping

    2014-10-15

    The androgen receptor (AR) plays critical roles in human prostate carcinoma progression and transformation. However, the activation of AR is regulated by co-regulators. MEIS1 protein, the homeodomain transcription factor, exhibited a decreased level in poor-prognosis prostate tumors. In this study, we investigated a potential interaction between MEIS1 and AR. We found that overexpression of MEIS1 inhibited the AR transcriptional activity and reduced the expression of AR target gene. A potential protein–protein interaction between AR and MEIS1 was identified by the immunoprecipitation and GST pull-down assays. Furthermore, MEIS1 modulated AR cytoplasm/nucleus translocation and the recruitment to androgen response element in prostate specific antigen (PSA) gene promoter sequences. In addition, MEIS1 promoted the recruitment of NCoR and SMRT in the presence of R1881. Finally, MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells. Taken together, our data suggests that MEIS1 functions as a novel AR co-repressor. - Highlights: • A potential interaction was identified between MEIS1 and AR signaling. • Overexpression of MEIS1 reduced the expression of AR target gene. • MEIS1 modulated AR cytoplasm/nucleus translocation. • MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells.

  4. Architecture and regulation of negative-strand viral enzymatic machinery

    PubMed Central

    Kranzusch, Philip J.; Whelan, Sean P.J.

    2012-01-01

    Negative-strand (NS) RNA viruses initiate infection with a unique polymerase complex that mediates both mRNA transcription and subsequent genomic RNA replication. For nearly all NS RNA viruses, distinct enzymatic domains catalyzing RNA polymerization and multiple steps of 5′ mRNA cap formation are contained within a single large polymerase protein (L). While NS RNA viruses include a variety of emerging human and agricultural pathogens, the enzymatic machinery driving viral replication and gene expression remains poorly understood. Recent insights with Machupo virus and vesicular stomatitis virus have provided the first structural information of viral L proteins, and revealed how the various enzymatic domains are arranged into a conserved architecture shared by both segmented and nonsegmented NS RNA viruses. In vitro systems reconstituting RNA synthesis from purified components provide new tools to understand the viral replicative machinery, and demonstrate the arenavirus matrix protein regulates RNA synthesis by locking a polymerase–template complex. Inhibition of gene expression by the viral matrix protein is a distinctive feature also shared with influenza A virus and nonsegmented NS RNA viruses, possibly illuminating a conserved mechanism for coordination of viral transcription and polymerase packaging PMID:22767259

  5. Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide.

    PubMed

    Józefowski, Szczepan; Czerkies, Maciej; Łukasik, Anna; Bielawska, Alicja; Bielawski, Jacek; Kwiatkowska, Katarzyna; Sobota, Andrzej

    2010-12-01

    LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.

  6. DjlA negatively regulates the Rcs signal transduction system in Escherichia coli.

    PubMed

    Shiba, Yasuhiro; Matsumoto, Kouji; Hara, Hiroshi

    2006-02-01

    The Rcs signal transduction system of Escherichia coli regulating capsular polysaccharide synthesis (cps) genes is activated by overexpression of the djlA gene encoding a cytoplasmic membrane-anchored DnaJ-like protein. However, by monitoring the expression of a cpsB'-lac fusion in pgsA- and mdoH-null mutants in which the Rcs system is activated, we found that the Rcs activity was further increased by deletion of djlA and decreased by low-level extrachromosomal expression of djlA. Furthermore, deletion of djlA in a wild-type strain led to small but significant increase of the basal-level activity of the Rcs system. These results demonstrate that DjlA functions as a negative regulator of the Rcs system unless abnormally overproduced.

  7. Regulation of inflammasome activation.

    PubMed

    Man, Si Ming; Kanneganti, Thirumala-Devi

    2015-05-01

    Inflammasome biology is one of the most exciting and rapidly growing areas in immunology. Over the past 10 years, inflammasomes have been recognized for their roles in the host defense against invading pathogens and in the development of cancer, auto-inflammatory, metabolic, and neurodegenerative diseases. Assembly of an inflammasome complex requires cytosolic sensing of pathogen-associated molecular patterns or danger-associated molecular patterns by a nucleotide-binding domain and leucine-rich repeat receptor (NLR) or absent in melanoma 2 (AIM2)-like receptors (ALR). NLRs and ALRs engage caspase-1, in most cases requiring the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC), to catalyze proteolytic cleavage of pro-interleukin-1β (pro-IL-1β) and pro-IL-18 and drive pyroptosis. Recent studies indicate that caspase-8, caspase-11, IL-1R-associated kinases (IRAK), and receptor-interacting protein (RIP) kinases contribute to inflammasome functions. In addition, post-translational modifications, including ubiquitination, deubiquitination, phosphorylation, and degradation control almost every aspect of inflammasome activities. Genetic studies indicate that mutations in NLRP1, NLRP3, NLRC4, and AIM2 are linked with the development of auto-inflammatory diseases, enterocolitis, and cancer. Overall, these findings transform our understanding of the basic biology and clinical relevance of inflammasomes. In this review, we provide an overview of the latest development of inflammasome research and discuss how inflammasome activities govern health and disease.

  8. ExsE Is a Negative Regulator for T3SS Gene Expression in Vibrio alginolyticus

    PubMed Central

    Liu, Jinxin; Lu, Shao-Yeh; Orfe, Lisa H.; Ren, Chun-Hua; Hu, Chao-Qun; Call, Douglas R.; Avillan, Johannetsy J.; Zhao, Zhe

    2016-01-01

    Type III secretion systems (T3SSs) contribute to microbial pathogenesis of Vibrio species, but the regulatory mechanisms are complex. We determined if the classic ExsACDE protein-protein regulatory model from Pseudomonas aeruginosa applies to Vibrio alginolyticus. Deletion mutants in V. alginolyticus demonstrated that, as expected, the T3SS is positively regulated by ExsA and ExsC and negatively regulated by ExsD and ExsE. Interestingly, deletion of exsE enhanced the ability of V. alginolyticus to induce host-cell death while cytotoxicity was inhibited by in trans complementation of this gene in a wild-type strain, a result that differs from a similar experiment with Vibrio parahaemolyticus ExsE. We further showed that ExsE is a secreted protein that does not contribute to adhesion to Fathead minnow epithelial cells. An in vitro co-immunoprecipitation assay confirmed that ExsE binds to ExsC to exert negative regulatory effect on T3SS genes. T3SS in V. alginolyticus can be activated in the absence of physical contact with host cells and a separate regulatory pathway appears to contribute to the regulation of ExsA. Consequently, like ExsE from P. aeruginosa, ExsE is a negative regulator for T3SS gene expression in V. alginolyticus. Unlike the V. parahaemolyticus orthologue, however, deletion of exsE from V. alginolyticus enhanced in vitro cytotoxicity. PMID:27999769

  9. RNase L is a negative regulator of cell migration.

    PubMed

    Banerjee, Shuvojit; Li, Geqiang; Li, Yize; Gaughan, Christina; Baskar, Danika; Parker, Yvonne; Lindner, Daniel J; Weiss, Susan R; Silverman, Robert H

    2015-12-29

    RNase L is a regulated endoribonuclease that functions in the interferon antiviral response. Activation of RNase L by 2', 5'-oligoadenylates has been linked to apoptosis, autophagy and inflammation. Genetic studies have also suggested the possible involvement of the RNase L gene (RNASEL) on chromosome 1q25.3 in several types of cancer. Here we report that ablation of RNase L in human prostate cancer PC3 cells by CRISPR/Cas9 gene editing technology enhanced cell migration as determined both by transwell assays and scratch wound healing assays. In addition, RNase L knockdown by means of RNAi increased migration of PC3 and DU145 cells in response to either fibronectin or serum stimulation, as did homozygous disruption of the RNase L gene in mouse embryonic fibroblasts. Serum or fibronectin stimulation of focal adhesion kinase (FAK) autophosphorylation on tyrosine-397 was increased by either knockdown or ablation of RNase L. In contrast, a missense mutant RNase L (R667A) lacking catalytic activity failed to suppress cell migration in PC3 cells. However, a nuclease-inactive mutant mouse RNase L (W630A) was able to partially inhibit migration of mouse fibroblasts. Consistent with a role for the catalytic activity of RNase L, transfection of PC3 cells with the RNase L activator, 2', 5'-oligoadenylate, suppressed cell migration. RNase L knockdown in PC3 cells enhanced tumor growth and metastasis following implantation in the mouse prostate. Our results suggest that naturally occurring mutations in the RNase L gene might promote enhanced cell migration and metastasis.

  10. RNase L is a negative regulator of cell migration

    PubMed Central

    Banerjee, Shuvojit; Li, Geqiang; Li, Yize; Gaughan, Christina; Baskar, Danika; Parker, Yvonne; Lindner, Daniel J.; Weiss, Susan R.; Silverman, Robert H.

    2015-01-01

    RNase L is a regulated endoribonuclease that functions in the interferon antiviral response. Activation of RNase L by 2′, 5′-oligoadenylates has been linked to apoptosis, autophagy and inflammation. Genetic studies have also suggested the possible involvement of the RNase L gene (RNASEL) on chromosome 1q25.3 in several types of cancer. Here we report that ablation of RNase L in human prostate cancer PC3 cells by CRISPR/Cas9 gene editing technology enhanced cell migration as determined both by transwell assays and scratch wound healing assays. In addition, RNase L knockdown by means of RNAi increased migration of PC3 and DU145 cells in response to either fibronectin or serum stimulation, as did homozygous disruption of the RNase L gene in mouse embryonic fibroblasts. Serum or fibronectin stimulation of focal adhesion kinase (FAK) autophosphorylation on tyrosine-397 was increased by either knockdown or ablation of RNase L. In contrast, a missense mutant RNase L (R667A) lacking catalytic activity failed to suppress cell migration in PC3 cells. However, a nuclease-inactive mutant mouse RNase L (W630A) was able to partially inhibit migration of mouse fibroblasts. Consistent with a role for the catalytic activity of RNase L, transfection of PC3 cells with the RNase L activator, 2′, 5′-oligoadenylate, suppressed cell migration. RNase L knockdown in PC3 cells enhanced tumor growth and metastasis following implantation in the mouse prostate. Our results suggest that naturally occurring mutations in the RNase L gene might promote enhanced cell migration and metastasis. PMID:26517238

  11. Enterovirus 71 Infection Cleaves a Negative Regulator for Viral Internal Ribosomal Entry Site-Driven Translation

    PubMed Central

    Chen, Li-Lien; Kung, Yu-An; Weng, Kuo-Feng; Lin, Jing-Yi; Horng, Jim-Tong

    2013-01-01

    Far-upstream element-binding protein 2 (FBP2) is an internal ribosomal entry site (IRES) trans-acting factor (ITAF) that negatively regulates enterovirus 71 (EV71) translation. This study shows that EV71 infection cleaved FBP2. Live EV71 and the EV71 replicon (but not UV-inactivated virus particles) induced FBP2 cleavage, suggesting that viral replication results in FBP2 cleavage. The results also showed that virus-induced proteasome, autophagy, and caspase activity co-contribute to EV71-induced FBP2 cleavage. Using FLAG-fused FBP2, we mapped the potential cleavage fragments of FBP2 in infected cells. We also found that FBP2 altered its function when its carboxyl terminus was cleaved. This study presents a mechanism for virus-induced cellular events to cleave a negative regulator for viral IRES-driven translation. PMID:23345520

  12. Negative regulators of the PHO system in Saccharomyces cerevisiae: isolation and structural characterization of PHO85.

    PubMed Central

    Uesono, Y; Tanaka, K; Toh-e, A

    1987-01-01

    One of the negative regulators of the PHO system of Saccharomyces cerevisiae, PHO85, has been isolated by transformation and complementation of a pho85 strain. The complementing activity was delimited within a 1258 bp DNA segment and this region has been sequenced. The largest open reading frame found in this region can encode a protein of 302 amino acid residues. A pho85 mutant resulted from disruption of the chromosomal counterpart of the open reading frame described above. Therefore, we concluded that the gene we have cloned is PHO85. This result also indicates that PHO85 is nonessential. Northern analysis revealed that the size of the PHO85 message is 1.1 kb. No similarity was found between the putative amino acid sequences of two negative regulators, the PHO80 and PHO85 proteins. Images PMID:3320965

  13. Sensitivity control through attenuation of signal transfer efficiency by negative regulation of cellular signalling.

    PubMed

    Toyoshima, Yu; Kakuda, Hiroaki; Fujita, Kazuhiro A; Uda, Shinsuke; Kuroda, Shinya

    2012-03-13

    Sensitivity is one of the hallmarks of biological and pharmacological responses. However, the principle of controlling sensitivity remains unclear. Here we theoretically analyse a simple biochemical reaction and find that the signal transfer efficiency of the transient peak amplitude attenuates depending on the strength of negative regulation. We experimentally find that many signalling pathways in various cell lines, including the Akt and ERK pathways, can be approximated by simple biochemical reactions and that the same property of the attenuation of signal transfer efficiency was observed for such pathways. Because of this property, a downstream molecule should show higher sensitivity to an activator and lower sensitivity to an inhibitor than an upstream molecule. Indeed, we experimentally verify that S6, which lies downstream of Akt, shows lower sensitivity to an epidermal growth factor receptor inhibitor than Akt. Thus, cells can control downstream sensitivity through the attenuation of signal transfer efficiency by changing the expression level of negative regulators.

  14. Pebble/ECT2 RhoGEF negatively regulates the Wingless/Wnt signaling pathway.

    PubMed

    Greer, Elisabeth R; Chao, Anna T; Bejsovec, Amy

    2013-12-01

    Wingless (Wg)/Wnt signaling is essential for patterning invertebrate and vertebrate embryos, and inappropriate Wnt activity is associated with a variety of human cancers. Despite intensive study, Wnt pathway mechanisms are not fully understood. We have discovered a new mechanism for regulating the Wnt pathway: activity of a Rho guanine nucleotide exchange factor (GEF) encoded by pebble (pbl) in Drosophila and ECT2 in humans. This RhoGEF has an essential role in cytokinesis, but also plays an unexpected, conserved role in inhibiting Wg/Wnt activity. Loss and gain of pbl function in Drosophila embryos cause pattern defects that indicate altered Wg activity. Both Pbl and ECT2 repress Wg/Wnt target gene expression in cultured Drosophila and human cells. The GEF activity is required for Wnt regulation, whereas other protein domains important for cytokinesis are not. Unlike most negative regulators of Wnt activity, Pbl/ECT2 functions downstream of Armadillo (Arm)/beta-catenin stabilization. Our results indicate GTPase regulation at a novel point in Wg/Wnt signal transduction, and provide new insight into the categorization of ECT2 as a human proto-oncogene.

  15. Cut! that’s a wrap: regulating negative emotion by ending emotion-eliciting situations

    PubMed Central

    Vujovic, Lara; Opitz, Philipp C.; Birk, Jeffrey L.; Urry, Heather L.

    2014-01-01

    Little is known about the potentially powerful set of emotion regulation (ER) processes that target emotion-eliciting situations. We thus studied the decision to end emotion-eliciting situations in the laboratory. We hypothesized that people would try to end negative situations more frequently than neutral situations to regulate distress. In addition, motivated by the selection, optimization, and compensation with ER framework, we hypothesized that failed attempts to end the situation would prompt either (a) greater negative emotion or (b) compensatory use of a different ER process, attentional deployment (AD). Fifty-eight participants (18–26 years old, 67% women) viewed negative and neutral pictures and pressed a key whenever they wished to stop viewing them. After key press, the picture disappeared (“success”) or stayed (“failure”) on screen. To index emotion, we measured corrugator and electrodermal activity, heart rate, and self-reported arousal. To index overt AD, we measured eye gaze. As their reason for ending the situation, participants more frequently reported being upset by high- than low-arousal negative pictures; they more frequently reported being bored by low- than high-arousal neutral pictures. Nevertheless, participants’ negative emotional responding did not increase in the context of ER failure nor did they use overt AD as a compensatory ER strategy. We conclude that situation-targeted ER processes are used to regulate emotional responses to high-arousal negative and low-arousal neutral situations; ER processes other than overt AD may be used to compensate for ER failure in this context. PMID:24592251

  16. Negative feedback regulation of Homer 1a on norepinephrine-dependent cardiac hypertrophy

    SciTech Connect

    Chiarello, Carmelina; Bortoloso, Elena; Carpi, Andrea; Furlan, Sandra; Volpe, Pompeo

    2013-07-15

    Homers are scaffolding proteins that modulate diverse cell functions being able to assemble signalling complexes. In this study, the presence, sub-cellular distribution and function of Homer 1 was investigated. Homer 1a and Homer 1b/c are constitutively expressed in cardiac muscle of both mouse and rat and in HL-1 cells, a cardiac cell line. As judged by confocal immunofluorescence microscopy, Homer 1a displays sarcomeric and peri-nuclear localization. In cardiomyocytes and cultured HL-1 cells, the hypertrophic agonist norepinephrine (NE) induces α{sub 1}-adrenergic specific Homer 1a over-expression, with a two-to-three-fold increase within 1 h, and no up-regulation of Homer 1b/c, as judged by Western blot and qPCR. In HL-1 cells, plasmid-driven over-expression of Homer 1a partially antagonizes activation of ERK phosphorylation and ANF up-regulation, two well-established, early markers of hypertrophy. At the morphometric level, NE-induced increase of cell size is likewise and partially counteracted by exogenous Homer 1a. Under the same experimental conditions, Homer 1b/c does not have any effect on ANF up-regulation nor on cell hypertrophy. Thus, Homer 1a up-regulation is associated to early stages of cardiac hypertrophy and appears to play a negative feedback regulation on molecular transducers of hypertrophy. -- Highlights: • Homer 1a is constitutively expressed in cardiac tissue. • In HL-1 cells, norepinephrine activates signaling pathways leading to hypertrophy. • Homer 1a up-regulation is an early event of norepinephrine-induced hypertrophy. • Homer 1a plays a negative feedback regulation modulating pathological hypertrophy. • Over-expression of Homer 1a per se does not induce hypertrophy.

  17. Expression of Tyrosine Hydroxylase is Negatively Regulated Via Prion Protein.

    PubMed

    da Luz, Marcio Henrique Mello; Glezer, Isaias; Xavier, Andre Machado; da Silva, Marcelo Alberti Paiva; Pino, Jessica Monteiro Volejnik; Zamith, Thiago Panaro; Vieira, Taynara Fernanda; Antonio, Bruno Brito; Antunes, Hanna Karen Moreira; Martins, Vilma Regina; Lee, Kil Sun

    2016-07-01

    Cellular prion protein (PrP(C)) is a glycoprotein of the plasma membrane that plays pleiotropic functions by interacting with multiple signaling complexes at the cell surface. Recently, a number of studies have reported the involvement of PrP(C) in dopamine metabolism and signaling, including its interactions with tyrosine hydroxylase (TH) and dopamine receptors. However, the outcomes reported by independent studies are still debatable. Therefore in this study, we investigated the effects of PrP(C) on the TH expression during the differentiation of N2a cells with dibutyryl-cAMP, a well-known cAMP analog that activates TH transcription. Upon differentiation, TH was induced with concomitant reduction of PrP(C) at protein level, but not at mRNA level. shRNA-mediated PrP(C) reduction increased the basal level of TH at both mRNA and protein levels without dibutyryl-cAMP treatment. This phenotype was reversed by re-expression of PrP(C). PrP(C) knockdown also potentiated the effect of dibutyryl-cAMP on TH expression. Our findings suggest that PrP(C) has suppressive effects on TH expression. As a consequence, altered PrP(C) functions may affect the regulation of dopamine metabolism and related neurological disorders.

  18. NLK-mediated phosphorylation of HDAC1 negatively regulates Wnt signaling

    PubMed Central

    Masoumi, Katarzyna Chmielarska; Daams, Renée; Sime, Wondossen; Siino, Valentina; Ke, Hengning; Levander, Fredrik; Massoumi, Ramin

    2017-01-01

    The Wnt signaling pathway is essential in regulating various cellular processes. Different mechanisms of inhibition for Wnt signaling have been proposed. Besides β-catenin degradation through the proteasome, nemo-like kinase (NLK) is another molecule that is known to negatively regulate Wnt signaling. However, the mechanism by which NLK mediates the inhibition of Wnt signaling was not known. In the present study, we used primary embryonic fibroblast cells isolated from NLK-deficient mice and showed that these cells proliferate faster and have a shorter cell cycle than wild-type cells. In NLK-knockout cells, we observed sustained interaction between Lef1 and β-catenin, leading to elevated luciferase reporter of β-catenin/Lef1–mediated transcriptional activation. The mechanism for the reduced β-catenin/Lef1 promoter activation was explained by phosphorylation of HDAC1 at serine 421 via NLK. The phosphorylation of HDAC1 was achieved only in the presence of wild-type NLK because a catalytically inactive mutant of NLK was unable to phosphorylate HDAC1 and reduced the luciferase reporter of β-catenin/Lef1–mediated transcriptional activation. This result suggests that NLK and HDAC1 together negatively regulate Wnt signaling, which is vital in preventing aberrant proliferation of nontransformed primary fibroblast cells. PMID:27903773

  19. Mindfulness in schizophrenia: Associations with self-reported motivation, emotion regulation, dysfunctional attitudes, and negative symptoms

    PubMed Central

    Tabak, Naomi T.; Horan, William P.; Green, Michael F.

    2015-01-01

    Mindfulness-based interventions are gaining empirical support as alternative or adjunctive treatments for a variety of mental health conditions, including anxiety, depression, and substance use disorders. Emerging evidence now suggests that mindfulness-based treatments may also improve clinical features of schizophrenia, including negative symptoms. However, no research has examined the construct of mindfulness and its correlates in schizophrenia. In this study, we examined self-reported mindfulness in patients (n=35) and controls (n=25) using the Five-Facet Mindfulness Questionnaire. We examined correlations among mindfulness, negative symptoms, and psychological constructs associated with negative symptoms and adaptive functioning, including motivation, emotion regulation, and dysfunctional attitudes. As hypothesized, patients endorsed lower levels of mindfulness than controls. In patients, mindfulness was unrelated to negative symptoms, but it was associated with more adaptive emotion regulation (greater reappraisal) and beliefs (lower dysfunctional attitudes). Some facets of mindfulness were also associated with self-reported motivation (behavioral activation and inhibition). These patterns of correlations were similar in patients and controls. Findings from this initial study suggest that schizophrenia patients may benefit from mindfulness-based interventions because they (a) have lower self-reported mindfulness than controls and (b) demonstrate strong relationships between mindfulness and psychological constructs related to adaptive functioning. PMID:26232242

  20. Mindfulness in schizophrenia: Associations with self-reported motivation, emotion regulation, dysfunctional attitudes, and negative symptoms.

    PubMed

    Tabak, Naomi T; Horan, William P; Green, Michael F

    2015-10-01

    Mindfulness-based interventions are gaining empirical support as alternative or adjunctive treatments for a variety of mental health conditions, including anxiety, depression, and substance use disorders. Emerging evidence now suggests that mindfulness-based treatments may also improve clinical features of schizophrenia, including negative symptoms. However, no research has examined the construct of mindfulness and its correlates in schizophrenia. In this study, we examined self-reported mindfulness in patients (n=35) and controls (n=25) using the Five-Facet Mindfulness Questionnaire. We examined correlations among mindfulness, negative symptoms, and psychological constructs associated with negative symptoms and adaptive functioning, including motivation, emotion regulation, and dysfunctional attitudes. As hypothesized, patients endorsed lower levels of mindfulness than controls. In patients, mindfulness was unrelated to negative symptoms, but it was associated with more adaptive emotion regulation (greater reappraisal) and beliefs (lower dysfunctional attitudes). Some facets of mindfulness were also associated with self-reported motivation (behavioral activation and inhibition). These patterns of correlations were similar in patients and controls. Findings from this initial study suggest that schizophrenia patients may benefit from mindfulness-based interventions because they (a) have lower self-reported mindfulness than controls and (b) demonstrate strong relationships between mindfulness and psychological constructs related to adaptive functioning.

  1. Negative mood regulation expectancies moderate the relationship between psychological abuse and avoidant coping.

    PubMed

    Shepherd-McMullen, Cassandra; Mearns, Jack; Stokes, Julie E; Mechanic, Mindy B

    2015-05-01

    This study explored the relationships among psychological abuse, attitudes about intimate partner violence (IPV), negative mood regulation expectancies (NMRE), and coping. Participants were 126 female college students in dating, cohabitating, or married relationships within the previous year. In one single session, they completed self-report scales measuring IPV, NMRE, and coping. Results indicated that women reporting higher levels of psychological abuse reported less negative attitudes toward IPV, engaged in less-active coping responses, and had lower NMRE. Psychological abuse was a significant predictor of avoidant coping, while NMRE significantly predicted both active and avoidant coping. In addition, the interaction of NMRE × Psychological abuse added incremental prediction of avoidant coping. Implications for research and practice are discussed.

  2. PP6 controls T cell development and homeostasis by negatively regulating distal TCR signaling.

    PubMed

    Ye, Jian; Shi, Hao; Shen, Ye; Peng, Chao; Liu, Yan; Li, Chenyu; Deng, Kejing; Geng, Jianguo; Xu, Tian; Zhuang, Yuan; Zheng, Biao; Tao, Wufan

    2015-02-15

    T cell development and homeostasis are both regulated by TCR signals. Protein phosphorylation and dephosphorylation, which are catalyzed by protein kinases and phosphatases, respectively, serve as important switches controlling multiple downstream pathways triggered by TCR recognition of Ags. It has been well documented that protein tyrosine phosphatases are involved in negative regulation of proximal TCR signaling. However, how TCR signals are terminated or attenuated in the distal TCR signaling pathways is largely unknown. We investigated the function of Ser/Thr protein phosphatase (PP) 6 in TCR signaling. T cell lineage-specific ablation of PP6 in mice resulted in enhanced thymic positive and negative selection, and preferential expansion of fetal-derived, IL-17-producing Vγ6Vδ1(+) T cells. Both PP6-deficient peripheral CD4(+) helper and CD8(+) cytolytic cells could not maintain a naive state and became fast-proliferating and short-lived effector cells. PP6 deficiency led to profound hyperactivation of multiple distal TCR signaling molecules, including MAPKs, AKT, and NF-κB. Our studies demonstrate that PP6 acts as a critical negative regulator, not only controlling both αβ and γδ lineage development, but also maintaining naive T cell homeostasis by preventing their premature activation before Ag stimulation.

  3. Down-Regulation of Negative Emotional Processing by Transcranial Direct Current Stimulation: Effects of Personality Characteristics

    PubMed Central

    Peña-Gómez, Cleofé; Vidal-Piñeiro, Dídac; Clemente, Immaculada C.; Pascual-Leone, Álvaro; Bartrés-Faz, David

    2011-01-01

    Evidence from neuroimaging and electrophysiological studies indicates that the left dorsolateral prefrontal cortex (DLPFC) is a core region in emotional processing, particularly during down-regulation of negative emotional conditions. However, emotional regulation is a process subject to major inter-individual differences, some of which may be explained by personality traits. In the present study we used transcranial direct current stimulation (tDCS) over the left DLPFC to investigate whether transiently increasing the activity of this region resulted in changes in the ratings of positive, neutral and negative emotional pictures. Results revealed that anodal, but not cathodal, tDCS reduced the perceived degree of emotional valence for negative stimuli, possibly due to an enhancement of cognitive control of emotional expression. We also aimed to determine whether personality traits (extraversion and neuroticism) might condition the impact of tDCS. We found that individuals with higher scores on the introversion personality dimension were more permeable than extraverts to the modulatory effects of the stimulation. The present study underlines the role of the left DLPFC in emotional regulation, and stresses the importance of considering individual personality characteristics as a relevant variable, although replication is needed given the limited sample size of our study. PMID:21829522

  4. RIP1 negatively regulates basal autophagic flux through TFEB to control sensitivity to apoptosis

    PubMed Central

    Yonekawa, Tohru; Gamez, Graciela; Kim, Jihye; Tan, Aik Choon; Thorburn, Jackie; Gump, Jacob; Thorburn, Andrew; Morgan, Michael J

    2015-01-01

    In a synthetic lethality/viability screen, we identified the serine–threonine kinase RIP1 (RIPK1) as a gene whose knockdown is highly selected against during growth in normal media, in which autophagy is not critical, but selected for in conditions that increase reliance on basal autophagy. RIP1 represses basal autophagy in part due to its ability to regulate the TFEB transcription factor, which controls the expression of autophagy-related and lysosomal genes. RIP1 activates ERK, which negatively regulates TFEB though phosphorylation of serine 142. Thus, in addition to other pro-death functions, RIP1 regulates cellular sensitivity to pro-death stimuli by modulating basal autophagy. PMID:25908842

  5. Spontaneous Emotion Regulation to Positive and Negative Stimuli

    ERIC Educational Resources Information Center

    Volokhov, Rachael N.; Demaree, Heath A.

    2010-01-01

    The ability to regulate one's emotions is an integral part of human social behavior. One antecedent emotion regulation strategy, known as reappraisal, is characterized by cognitively evaluating an emotional stimulus to alter its emotional impact and one response-focused strategy, suppression, is aimed at reducing behavioral output. People are…

  6. Protein phosphatase AP2C1 negatively regulates basal resistance and defense responses to Pseudomonas syringae.

    PubMed

    Shubchynskyy, Volodymyr; Boniecka, Justyna; Schweighofer, Alois; Simulis, Justinas; Kvederaviciute, Kotryna; Stumpe, Michael; Mauch, Felix; Balazadeh, Salma; Mueller-Roeber, Bernd; Boutrot, Freddy; Zipfel, Cyril; Meskiene, Irute

    2017-01-06

    Mitogen-activated protein kinases (MAPKs) mediate plant immune responses to pathogenic bacteria. However, less is known about the cell autonomous negative regulatory mechanism controlling basal plant immunity. We report the biological role of Arabidopsis thaliana MAPK phosphatase AP2C1 as a negative regulator of plant basal resistance and defense responses to Pseudomonas syringae AP2C2, a closely related MAPK phosphatase, also negatively controls plant resistance. Loss of AP2C1 leads to enhanced pathogen-induced MAPK activities, increased callose deposition in response to pathogen-associated molecular patterns or to P. syringae pv. tomato (Pto) DC3000, and enhanced resistance to bacterial infection with Pto. We also reveal the impact of AP2C1 on the global transcriptional reprogramming of transcription factors during Pto infection. Importantly, ap2c1 plants show salicylic acid-independent transcriptional reprogramming of several defense genes and enhanced ethylene production in response to Pto This study pinpoints the specificity of MAPK regulation by the different MAPK phosphatases AP2C1 and MKP1, which control the same MAPK substrates, nevertheless leading to different downstream events. We suggest that precise and specific control of defined MAPKs by MAPK phosphatases during plant challenge with pathogenic bacteria can strongly influence plant resistance.

  7. Development and Pilot Investigation of Behavioral Activation for Negative Symptoms

    ERIC Educational Resources Information Center

    Mairs, Hilary; Lovell, Karina; Campbell, Malcolm; Keeley, Philip

    2011-01-01

    Negative symptoms cause functional impairment and impede recovery from psychosis, not least, because of limited developments in empirically validated treatments. This article details a pilot evaluation of a behavioral activation (BA) treatment with eight people presenting with psychosis and marked negative symptoms. The rationale for this…

  8. The Emerging Regulation of VEGFR-2 in Triple-Negative Breast Cancer

    PubMed Central

    Zhu, Xiaoxia; Zhou, Wen

    2015-01-01

    Vascular endothelial growth factor-A (VEGF) signals vascular development and angiogenesis mainly by binding to VEGF receptor family member 2 (VEGFR-2). Adaptor proteins mediate many VEGFR-2’s functions in the development of blood vessels. Cancer cells secrete VEGF to activate VEGFR-2 pathway in their neighboring endothelial cells in the process of cancer-related angiogenesis. Interestingly, activation of VEGFR-2 signaling is found in breast cancer cells, but its role and regulation are not clear. We highlighted research advances of VEGFR-2, with a focus on VEGFR-2’s regulation by mutant p53 in breast cancer. In addition, we reviewed recent Food and Drug Administration-approved tyrosine kinase inhibitor drugs that can inhibit the function of VEGFR-2. Ongoing preclinical and clinical studies might prove that pharmaceutically targeting VEGFR-2 could be an effective therapeutic strategy in treating triple-negative breast cancer. PMID:26500608

  9. Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

    PubMed

    Sinha, Siddhartha S; Cameron, Jody; Brooks, James C; Leifer, Cynthia A

    2016-08-15

    TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.

  10. MecA Protein Acts as a Negative Regulator of Genetic Competence in Streptococcus mutans

    PubMed Central

    Tian, Xiao-Lin; Dong, Gaofeng; Liu, Tianlei; Gomez, Zubelda A.; Wahl, Astrid; Hols, Pascal

    2013-01-01

    Streptococcus mutans develops competence for genetic transformation through a complex network that receives inputs from at least two signaling peptides, competence-stimulating peptide (CSP) and sigX-inducing peptide (XIP). The key step of competence induction is the transcriptional activation of comX, which encodes an alternative sigma factor, SigX (σX), controlling the expression of late competence genes essential for DNA uptake and recombination. In this study, we provide evidence that MecA acts as a negative regulator in the posttranslational regulation of SigX in S. mutans. Using luxAB transcriptional reporter strains, we demonstrate that MecA represses the expression of late competence genes in S. mutans grown in a complex medium that is subpermissive for competence induction by CSP. The negative regulation of competence by MecA requires the presence of a functional SigX. Accordingly, inactivation of MecA results in a prolonged competence state of S. mutans under this condition. We have also found that the AAA+ protease ClpC displays a similar repressing effect on late competence genes, suggesting that both MecA and ClpC function coordinately to regulate competence in the same regulatory circuit in S. mutans. This suggestion is strongly supported by the results of bacterial two-hybrid assays, which demonstrate that MecA interacts with both SigX and ClpC, forming a ternary SigX-MecA-ClpC complex. Western blot analysis also confirms that inactivation of MecA or ClpC results in the intracellular accumulation of the SigX in S. mutans. Together, our data support the notion that MecA mediates the formation of a ternary SigX-MecA-ClpC complex that sequesters SigX and thereby negatively regulates genetic competence in S. mutans. PMID:24039267

  11. Zinc Oxide Nanoparticles Suppress LPS-Induced NF-κB Activation by Inducing A20, a Negative Regulator of NF-κB, in RAW 264.7 Macrophages.

    PubMed

    Kim, Min-Ho; Jeong, Hyun-Ja

    2015-09-01

    Zinc contained in solar salt and bamboo salt plays a critical role in various immune responses. Zinc oxide is a source of zinc, and recently it has been reported that zinc oxide nanoparticles (ZO-NP) more effectively decrease allergic inflammatory reactions than zinc oxide bulk material. The aim of this work was to investigate the regulatory effect of ZO-NP on interferon (IFN)-γ plus lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. ZO-NP (0.1-10 μg/mL) did not affect cell viability but toxicity was evident at a ZO-NP concentration of 100 μg/mL. ZO-NP (10 μg/mL) inhibited the IFN-γ plus LPS-induced production of nitric oxide and the protein expressions of inducible nitric oxide synthase and cyclooxygenase-2. The productions of inflammatory cytokines, such as, interleukin (IL)-1β and tumor necrosis factor (TNF)-α were increased by IFN-γ plus LPS but down-regulated by ZO-NP treatment. Furthermore, the up-regulations of IL-1β and TNF-α mRNAs by IFN-γ plus LPS were reduced by ZO-NP at low (0.1 μg/mL) and high (10 μg/mL) concentrations. ZO-NP (0.1, 1, and 10 μg/mL) inhibited the nuclear translocation of nuclear factor-κB by blocking IκBα phosphorylation and degradation. In addition, ZO-NP induced the expression of A20, a zinc finger protein and negative regulator of NF-κB. In conclusion, the present study demonstrated that ZO-NP offer a potential means of treating inflammatory diseases.

  12. OsGF14b Positively Regulates Panicle Blast Resistance but Negatively Regulates Leaf Blast Resistance in Rice.

    PubMed

    Liu, Qing; Yang, Jianyuan; Zhang, Shaohong; Zhao, Junliang; Feng, Aiqing; Yang, Tifeng; Wang, Xiaofei; Mao, Xinxue; Dong, Jingfang; Zhu, Xiaoyuan; Leung, Hei; Leach, Jan E; Liu, Bin

    2016-01-01

    Although 14-3-3 proteins have been reported to be involved in responses to biotic stresses in plants, their functions in rice blast, the most destructive disease in rice, are largely unknown. Only GF14e has been confirmed to negatively regulate leaf blast. We report that GF14b is highly expressed in seedlings and panicles during blast infection. Rice plants overexpressing GF14b show enhanced resistance to panicle blast but are susceptible to leaf blast. In contrast, GF14b-silenced plants show increased susceptibility to panicle blast but enhanced resistance to leaf blast. Yeast one-hybrid assays demonstrate that WRKY71 binds to the promoter of GF14b and modulates its expression. Overexpression of GF14b induces expression of jasmonic acid (JA) synthesis-related genes but suppresses expression of salicylic acid (SA) synthesis-related genes. In contrast, suppressed GF14b expression causes decreased expression of JA synthesis-related genes but activation of SA synthesis-related genes. These results suggest that GF14b positively regulates panicle blast resistance but negatively regulates leaf blast resistance, and that GF14b-mediated disease resistance is associated with the JA- and SA-dependent pathway. The different functions for 14-3-3 proteins in leaf and panicle blast provide new evidence that leaf and panicle blast resistance are controlled by different mechanisms.

  13. Resveratrol suppresses NTHi-induced inflammation via up-regulation of the negative regulator MyD88 short

    PubMed Central

    Andrews, Carla S.; Matsuyama, Shingo; Lee, Byung-Cheol; Li, Jian-Dong

    2016-01-01

    Upper respiratory tract inflammatory diseases such as asthma and chronic obstructive pulmonary diseases (COPD) affect more than one-half billion people globally and are characterized by chronic inflammation that is often exacerbated by respiratory pathogens such as nontypeable Haemophilus influenzae (NTHi). The increasing numbers of antibiotic-resistant bacterial strains and the limited success of currently available pharmaceuticals used to manage the symptoms of these diseases present an urgent need for the development of novel anti-inflammatory therapeutic agents. Resveratrol has long been thought as an interesting therapeutic agent for various diseases including inflammatory diseases. However, the molecular mechanisms underlying its anti-inflammatory properties remain largely unknown. Here we show for the first time that resveratrol decreases expression of pro-inflammatory mediators in airway epithelial cells and in the lung of mice by enhancing NTHi-induced MyD88 short, a negative regulator of inflammation, via inhibition of ERK1/2 activation. Furthermore, resveratrol inhibits NTHi-induced ERK1/2 phosphorylation by increasing MKP-1 expression via a cAMP-PKA-dependent signaling pathway. Finally, we show that resveratrol has anti-inflammatory effects post NTHi infection, thereby demonstrating its therapeutic potential. Together these data reveal a novel mechanism by which resveratrol alleviates NTHi-induced inflammation in airway disease by up-regulating the negative regulator of inflammation MyD88s. PMID:27677845

  14. Mechanisms of JAK/STAT pathway negative regulation by the short coreceptor Eye Transformer/Latran.

    PubMed

    Fisher, Katherine H; Stec, Wojciech; Brown, Stephen; Zeidler, Martin P

    2016-02-01

    Transmembrane receptors interact with extracellular ligands to transduce intracellular signaling cascades, modulate target gene expression, and regulate processes such as proliferation, apoptosis, differentiation, and homeostasis. As a consequence, aberrant signaling events often underlie human disease. Whereas the vertebrate JAK/STAT signaling cascade is transduced via multiple receptor combinations, the Drosophila pathway has only one full-length signaling receptor, Domeless (Dome), and a single negatively acting receptor, Eye Transformer/Latran (Et/Lat). Here we investigate the molecular mechanisms underlying Et/Lat activity. We demonstrate that Et/Lat negatively regulates the JAK/STAT pathway activity and can bind to Dome, thus reducing Dome:Dome homodimerization by creating signaling-incompetent Dome:Et/Lat heterodimers. Surprisingly, we find that Et/Lat is able to bind to both JAK and STAT92E but, despite the presence of putative cytokine-binding motifs, does not detectably interact with pathway ligands. We find that Et/Lat is trafficked through the endocytic machinery for lysosomal degradation but at a much slower rate than Dome, a difference that may enhance its ability to sequester Dome into signaling-incompetent complexes. Our data offer new insights into the molecular mechanism and regulation of Et/Lat in Drosophila that may inform our understanding of how short receptors function in other organisms.

  15. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1.

    PubMed

    Couto, Daniel; Niebergall, Roda; Liang, Xiangxiu; Bücherl, Christoph A; Sklenar, Jan; Macho, Alberto P; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Maclean, Dan; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min; Zipfel, Cyril

    2016-08-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component.

  16. The Arabidopsis Protein Phosphatase PP2C38 Negatively Regulates the Central Immune Kinase BIK1

    PubMed Central

    Liang, Xiangxiu; Bücherl, Christoph A.; Sklenar, Jan; Macho, Alberto P.; Ntoukakis, Vardis; Derbyshire, Paul; Altenbach, Denise; Robatzek, Silke; Uhrig, Joachim; Menke, Frank; Zhou, Jian-Min

    2016-01-01

    Plants recognize pathogen-associated molecular patterns (PAMPs) via cell surface-localized pattern recognition receptors (PRRs), leading to PRR-triggered immunity (PTI). The Arabidopsis cytoplasmic kinase BIK1 is a downstream substrate of several PRR complexes. How plant PTI is negatively regulated is not fully understood. Here, we identify the protein phosphatase PP2C38 as a negative regulator of BIK1 activity and BIK1-mediated immunity. PP2C38 dynamically associates with BIK1, as well as with the PRRs FLS2 and EFR, but not with the co-receptor BAK1. PP2C38 regulates PAMP-induced BIK1 phosphorylation and impairs the phosphorylation of the NADPH oxidase RBOHD by BIK1, leading to reduced oxidative burst and stomatal immunity. Upon PAMP perception, PP2C38 is phosphorylated on serine 77 and dissociates from the FLS2/EFR-BIK1 complexes, enabling full BIK1 activation. Together with our recent work on the control of BIK1 turnover, this study reveals another important regulatory mechanism of this central immune component. PMID:27494702

  17. Positive and negative regulation of a SNARE protein by control of intracellular localization.

    PubMed

    Nakanishi, Hideki; de los Santos, Pablo; Neiman, Aaron M

    2004-04-01

    In Saccharomyces cerevisiae, the developmentally regulated Soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) protein Spo20p mediates the fusion of vesicles with the prospore membrane, which is required for the formation of spores. Spo20p is subject to both positive and negative regulation by separate sequences in its aminoterminal domain. We report that the positive activity is conferred by a short, amphipathic helix that is sufficient to confer plasma membrane or prospore membrane localization to green fluorescent protein. In vitro, this helix binds to acidic phospholipids, and mutations that reduce or eliminate phospholipid binding in vitro inactivate Spo20p in vivo. Genetic manipulation of phospholipid pools indicates that the likely in vivo ligand of this domain is phosphatidic acid. The inhibitory activity is a nuclear targeting signal, which confers nuclear localization in vegetative cells and in cells entering meiosis. However, as cells initiate spore formation, fusions containing the inhibitory domain exit the nucleus and localize to the nascent prospore membrane. Thus, the SNARE Spo20p is both positively and negatively regulated by control of its intracellular localization.

  18. Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

    SciTech Connect

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim

    2014-07-18

    Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated that TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.

  19. Fused-Ring Oxazolopyrrolopyridopyrimidine Systems with Gram-Negative Activity

    PubMed Central

    Chen, Yiyuan; Moloney, Jonathan G.; Christensen, Kirsten E.; Moloney, Mark G.

    2017-01-01

    Fused polyheterocyclic derivatives are available by annulation of a tetramate scaffold, and been shown to have antibacterial activity against a Gram-negative, but not a Gram-positive, bacterial strain. While the activity is not potent, these systems are structurally novel showing, in particular, a high level of polarity, and offer potential for the optimization of antibacterial activity. PMID:28098784

  20. PKC{eta} is a negative regulator of AKT inhibiting the IGF-I induced proliferation

    SciTech Connect

    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta

    2012-04-15

    The PI3K-AKT pathway is frequently activated in human cancers, including breast cancer, and its activation appears to be critical for tumor maintenance. Some malignant cells are dependent on activated AKT for their survival; tumors exhibiting elevated AKT activity show sensitivity to its inhibition, providing an Achilles heel for their treatment. Here we show that the PKC{eta} isoform is a negative regulator of the AKT signaling pathway. The IGF-I induced phosphorylation on Ser473 of AKT was inhibited by the PKC{eta}-induced expression in MCF-7 breast adenocarcinoma cancer cells. This was further confirmed in shRNA PKC{eta}-knocked-down MCF-7 cells, demonstrating elevated phosphorylation on AKT Ser473. While PKC{eta} exhibited negative regulation on AKT phosphorylation it did not alter the IGF-I induced ERK phosphorylation. However, it enhanced ERK phosphorylation when stimulated by PDGF. Moreover, its effects on IGF-I/AKT and PDGF/ERK pathways were in correlation with cell proliferation. We further show that both PKC{eta} and IGF-I confer protection against UV-induced apoptosis and cell death having additive effects. Although the protective effect of IGF-I involved activation of AKT, it was not affected by PKC{eta} expression, suggesting that PKC{eta} acts through a different route to increase cell survival. Hence, our studies show that PKC{eta} provides negative control on AKT pathway leading to reduced cell proliferation, and further suggest that its presence/absence in breast cancer cells will affect cell death, which could be of therapeutic value.

  1. Negative regulation of geminin by CDK-dependent ubiquitination controls replication licensing.

    PubMed

    Li, Anatoliy; Blow, J Julian

    2004-04-01

    The replication licensing system ensures the precise duplication of chromosomal DNA in each cell cycle. In metazoans, a small protein called geminin plays a central role in negatively regulating licensing late in the cell cycle. Recent work using Xenopus egg extracts shows how geminin activity is downregulated on exit from metaphase in a process that requires mitotic cyclin-dependent kinases (CDKs). Geminin is polyubiquitinated by the Anaphase Promoting Complex, but instead of being proteolysed-the normal fate of polyubiquitinated proteins-much of the geminin is deubiquitinated, leaving it inactive. These results suggest a simple model for how precise chromosome duplication is ensured in the Xenopus model system.

  2. On the possibility of negative activation energies in bimolecular reactions

    NASA Technical Reports Server (NTRS)

    Jaffe, R. L.

    1978-01-01

    The temperature dependence of the rate constants for model reacting systems was studied to understand some recent experimental measurements which imply the existence of negative activation energies. A collision theory model and classical trajectory calculations are used to demonstrate that the reaction probability can vary inversely with collision energy for bimolecular reactions occurring on attractive potential energy surfaces. However, this is not a sufficient condition to ensure that the rate constant has a negative temperature dependence. On the basis of these calculations, it seems unlikely that a true bimolecular reaction between neutral molecules will have a negative activation energy.

  3. Children's Negative Emotionality Combined with Poor Self-Regulation Affects Allostatic Load in Adolescence

    ERIC Educational Resources Information Center

    Dich, Nadya; Doan, Stacey; Evans, Gary

    2015-01-01

    The present study examined the concurrent and prospective, longitudinal effects of childhood negative emotionality and self-regulation on allostatic load (AL), a physiological indicator of chronic stress. We hypothesized that negative emotionality in combination with poor self-regulation would predict elevated AL. Mothers reported on children's…

  4. [The regulation of negative and positive emotions during picture viewing: an ERP study].

    PubMed

    Reva, N V; Pavlov, S V; Korenek, V V; Loktev, K V; Tumialis, A V; Brak, I V; Aftanas, L I

    2015-01-01

    The study examines the effects of cognitive reappraisal on the event-related potentials (ERPs) to affective stimuli. Participants (n = 53) were asked either to attend affective images, or to down-regulate negative affect, or to up-regulate positive affect. Reappraisal of negative images was associated with attenuation of the P300 and late positive potential (LPP) over parietal regions, whereas reappraisal of positive images had no significant effect on ERP components. The weak P300 reduction correlated with high personality scores of negative affectivity. We assume that only down-regulation of negative emotions is associated with the changes in primary appraisals, and so far reflected in ERP modulation.

  5. Control and regulation of pathways via negative feedback

    PubMed Central

    2017-01-01

    The biochemical networks found in living organisms include a huge variety of control mechanisms at multiple levels of organization. While the mechanistic and molecular details of many of these control mechanisms are understood, their exact role in driving cellular behaviour is not. For example, yeast glycolysis has been studied for almost 80 years but it is only recently that we have come to understand the systemic role of the multitude of feedback and feed-forward controls that exist in this pathway. In this article, control theory is discussed as an approach to dissect the control logic of complex pathways. One of the key issues is distinguishing between the terms control and regulation and how these concepts are applied to regulated enzymes such as phosphofructokinase. In doing so, one of the paradoxes in metabolic regulation can be resolved where enzymes such as phosphofructokinase have little control but, nevertheless, possess significant regulatory influence. PMID:28202588

  6. MAP Kinase-Mediated Negative Regulation of Symbiotic Nodule Formation in Medicago truncatula

    PubMed Central

    Ryu, Hojin; Laffont, Carole; Frugier, Florian; Hwang, Ildoo

    2017-01-01

    Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events in plants, including stress responses, innate immunity, hormone signaling, and cell specificity. MAPK-mediated stress signaling is also known to negatively regulate nitrogen-fixing symbiotic interactions, but the molecular mechanism of the MAPK signaling cascades underlying the symbiotic nodule development remains largely unknown. We show that the MtMKK5-MtMPK3/6 signaling module negatively regulates the early symbiotic nodule formation, probably upstream of ERN1 (ERF Required for Nodulation 1) and NSP1 (Nod factor Signaling Pathway 1) in Medicago truncatula. The overexpression of MtMKK5 stimulated stress and defense signaling pathways but also reduced nodule formation in M. truncatula roots. Conversely, a MAPK specific inhibitor, U0126, enhanced nodule formation and the expression of an early nodulation marker gene, MtNIN. We found that MtMKK5 directly activates MtMPK3/6 by phosphorylating the TEY motif within the activation loop and that the MtMPK3/6 proteins physically interact with the early nodulation-related transcription factors ERN1 and NSP1. These data suggest that the stress signaling-mediated MtMKK5/MtMPK3/6 module suppresses symbiotic nodule development via the action of early nodulation transcription factors. PMID:28152300

  7. KIF14 negatively regulates Rap1a–Radil signaling during breast cancer progression

    PubMed Central

    Ahmed, Syed M.; Thériault, Brigitte L.; Uppalapati, Maruti; Chiu, Catherine W.N.; Gallie, Brenda L.; Sidhu, Sachdev S.

    2012-01-01

    The small GTPase Rap1 regulates inside-out integrin activation and thereby influences cell adhesion, migration, and polarity. Several Rap1 effectors have been described to mediate the cellular effects of Rap1 in a context-dependent manner. Radil is emerging as an important Rap effector implicated in cell spreading and migration, but the molecular mechanisms underlying its functions are unclear. We report here that the kinesin KIF14 associates with the PDZ domain of Radil and negatively regulates Rap1-mediated inside-out integrin activation by tethering Radil on microtubules. The depletion of KIF14 led to increased cell spreading, altered focal adhesion dynamics, and inhibition of cell migration and invasion. We also show that Radil is important for breast cancer cell proliferation and for metastasis in mice. Our findings provide evidence that the concurrent up-regulation of Rap1 activity and increased KIF14 levels in several cancers is needed to reach optimal levels of Rap1–Radil signaling, integrin activation, and cell–matrix adhesiveness required for tumor progression. PMID:23209302

  8. Quantifying negative feedback regulation by micro-RNAs

    NASA Astrophysics Data System (ADS)

    Wang, Shangying; Raghavachari, Sridhar

    2011-10-01

    Micro-RNAs (miRNAs) play a crucial role in post-transcriptional gene regulation by pairing with target mRNAs to repress protein production. It has been shown that over one-third of human genes are targeted by miRNA. Although hundreds of miRNAs have been identified in mammalian genomes, the function of miRNA-based repression in the context of gene regulation networks still remains unclear. In this study, we explore the functional roles of feedback regulation by miRNAs. In a model where repression of translation occurs by sequestration of mRNA by miRNA, we find that miRNA and mRNA levels are anti-correlated, resulting in larger fluctuation in protein levels than theoretically expected assuming no correlation between miRNA and mRNA levels. If miRNA repression is due to a catalytic suppression of translation rates, we analytically show that the protein fluctuations can be strongly repressed with miRNA regulation. We also discuss how either of these modes may be relevant for cell function.

  9. Chromatin associated SETD3 negatively regulates VEGF expression

    PubMed Central

    Cohn, Ofir; Feldman, Michal; Weil, Lital; Kublanovsky, Margarita; Levy, Dan

    2016-01-01

    SETD3 is a member of the protein lysine methyltransferase (PKMT) family, which catalyzes the addition of methyl group to lysine residues. Accumulating data suggest that PKMTs are involved in the regulation of a broad spectrum of biological processes by targeting histone and non-histone proteins. Using a proteomic approach, we have identified 172 new SETD3 interacting proteins. We show that SETD3 binds and methylates the transcription factor FoxM1, which has been previously shown to be associated with the regulation of VEGF expression. We further demonstrate that under hypoxic conditions SETD3 is down-regulated. Mechanistically, we find that under basal conditions, SETD3 and FoxM1 are enriched on the VEGF promoter. Dissociation of both SETD3 and FoxM1 from the VEGF promoter under hypoxia correlates with elevated expression of VEGF. Taken together, our data reveal a new SETD3-dependent methylation-based signaling pathway at chromatin that regulates VEGF expression under normoxic and hypoxic conditions. PMID:27845446

  10. The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination.

    PubMed

    Yamauchi, Junji; Miyamoto, Yuki; Hamasaki, Hajime; Sanbe, Atsushi; Kusakawa, Shinji; Nakamura, Akane; Tsumura, Hideki; Maeda, Masahiro; Nemoto, Noriko; Kawahara, Katsumasa; Torii, Tomohiro; Tanoue, Akito

    2011-08-31

    In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination.

  11. Procyanidin dimer B2-mediated IRAK-M induction negatively regulates TLR4 signaling in macrophages

    SciTech Connect

    Sung, Nak-Yun; Yang, Mi-So; Song, Du-Sub; Kim, Jae-Kyung; Park, Jong-Heum; Song, Beom-Seok; Park, Sang-Hyun; Lee, Ju-Woon; Park, Hyun-Jin; Kim, Jae-Hun; Byun, Eui-Baek; Byun, Eui-Hong

    2013-08-16

    Highlights: •Pro B2 elevated the expression of IRAK-M, a negative regulator of TLR signaling. •LPS-induced expression of cell surface molecules was inhibited by Pro B2. •LPS-induced production of pro-inflammatory cytokines was inhibited by Pro B2. •Pro B2 inhibited LPS-induced activation of MAPKs and NF-κB through IRAK-M. •Pro B2 inactivated naïve T cells by inhibiting LPS-induced cytokines via IRAK-M. -- Abstract: Polyphenolic compounds have been found to possess a wide range of physiological activities that may contribute to their beneficial effects against inflammation-related diseases; however, the molecular mechanisms underlying this anti-inflammatory activity are not completely characterized, and many features remain to be elucidated. In this study, we investigated the molecular basis for the down-regulation of toll-like receptor 4 (TLR4) signal transduction by procyanidin dimer B2 (Pro B2) in macrophages. Pro B2 markedly elevated the expression of the interleukin (IL)-1 receptor-associated kinase (IRAK)-M protein, a negative regulator of TLR signaling. Lipopolysaccharide (LPS)-induced expression of cell surface molecules (CD80, CD86, and MHC class I/II) and production of pro-inflammatory cytokines (tumor necrosis factor-α, IL-1β, IL-6, and IL-12p70) were inhibited by Pro B2, and this action was prevented by IRAK-M silencing. In addition, Pro B2-treated macrophages inhibited LPS-induced activation of mitogen-activated protein kinases such as extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase and the translocation of nuclear factor κB and p65 through IRAK-M. We also found that Pro B2-treated macrophages inactivated naïve T cells by inhibiting LPS-induced interferon-γ and IL-2 secretion through IRAK-M. These novel findings provide new insights into the understanding of negative regulatory mechanisms of the TLR4 signaling pathway and the immune-pharmacological role of Pro B2 in the immune response against the development

  12. TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    PubMed Central

    Zurek, Birte; Schoultz, Ida; Neerincx, Andreas; Napolitano, Luisa M.; Birkner, Katharina; Bennek, Eveline; Sellge, Gernot; Lerm, Maria; Meroni, Germana; Söderholm, Johan D.; Kufer, Thomas A.

    2012-01-01

    NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases. PMID:22829933

  13. PvRbohB negatively regulates Rhizophagus irregularis colonization in Phaseolus vulgaris.

    PubMed

    Arthikala, Manoj-Kumar; Montiel, Jesús; Nava, Noreide; Santana, Olivia; Sánchez-López, Rosana; Cárdenas, Luis; Quinto, Carmen

    2013-08-01

    Plant NADPH oxidases (RBOHs) regulate the early stages of rhizobial infection in Phaseolus vulgaris and affect nodule function in Medicago truncatula. In contrast, the role of RBOHs in the plant-arbuscular mycorrhizal (AM) symbiosis and in the regulation of reactive oxygen species (ROS) production during the establishment of the AM interaction is largely unknown. In this study, we assessed the role of P. vulgaris Rboh (PvRbohB) during the symbiosis with the AM fungus, Rhizophagus irregularis. Our results indicate that the PvRbohB transcript is significantly up-regulated in the mycorrhized roots of P. vulgaris. Further, the PvRbohB promoter was found to be active during the invasion of R. irregularis. Down-regulation of PvRbohB transcription by RNAi (RNA interference) silencing resulted in diminished ROS levels in the transgenic mycorrhized roots and induced early hyphal root colonization. Interestingly, the size of appressoria increased in PvRbohB-RNAi roots (760 ± 70.1 µm) relative to controls (251 ± 73.2 µm). Finally, the overall level of mycorrhizal colonization significantly increased in PvRbohB-RNAi roots [48.1 ± 3.3% root length colonization (RLC)] compared with controls (29.4 ± 1.9% RLC). We propose that PvRbohB negatively regulates AM colonization in P. vulgaris.

  14. Checkpoint kinase 2 (CHK2) negatively regulates androgen sensitivity and prostate cancer cell growth

    PubMed Central

    Ta, Huy Q; Ivey, Melissa L; Frierson, Henry F; Conaway, Mark R; Dziegielewski, Jaroslaw; Larner, James M; Gioeli, Daniel

    2015-01-01

    Prostate cancer (PCa) is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated PCa initially respond to androgen deprivation therapy (ADT), but virtually all patient will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating PCa cell growth led to our discovery that Checkpoint Kinase 2 (CHK2) knockdown dramatically increased PCa growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects PCa proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. Additionally, we provide evidence that CHK2 physically associates with the AR, and that cell cycle inhibition increased this association. Finally, immunohistochemical analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and PCa growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation. PMID:26573794

  15. Hfq negatively regulates type III secretion in EHEC and several other pathogens

    PubMed Central

    Shakhnovich, Elizabeth A.; Davis, Brigid M.; Waldor, Matthew K.

    2009-01-01

    Summary Hfq is a conserved RNA-binding protein that regulates diverse cellular processes through post-transcriptional control of gene expression, often by functioning as a chaperone for regulatory sRNAs. Here, we explored the role of Hfq in enterohaemorrhagic E. coli (EHEC), a group of non-invasive intestinal pathogens. EHEC virulence is dependent on a Type III secretion system encoded in the LEE pathogenicity island. The abundance of transcripts for all 41 LEE genes and more than half of confirmed non-LEE-encoded T3 effectors were elevated in an EHEC hfq deletion mutant. Thus, Hfq promotes coordinated expression of the LEE-encoded T3S apparatus and both LEE- and non-LEE-encoded effectors. Increased transcript levels led to the formation of functional secretion complexes capable of secreting high quantities of effectors into the supernatant. The increase in LEE-derived transcripts and proteins was dependent on Ler, the LEE-encoded transcriptional activator, and the ler transcript appears to be a direct target of Hfq-mediated negative regulation. Finally, we found that Hfq contributes to the negative regulation of T3SSs in several other pathogens, suggesting that Hfq, potentially along with species-specific sRNAs, underlies a common means to prevent unfettered expression of T3SSs. PMID:19703108

  16. Lack of Csk-mediated negative regulation in a unicellular SRC kinase.

    PubMed

    Schultheiss, Kira P; Suga, Hiroshi; Ruiz-Trillo, Iñaki; Miller, W Todd

    2012-10-16

    Phosphotyrosine-based signaling plays a vital role in cellular communication in multicellular organisms. Unexpectedly, unicellular choanoflagellates (the closest phylogenetic group to metazoans) possess numbers of tyrosine kinases that are comparable to those in complex metazoans. Here, we have characterized tyrosine kinases from the filasterean Capsaspora owczarzaki, a unicellular protist representing the sister group to choanoflagellates and metazoans. Two Src-like tyrosine kinases have been identified in C. owczarzaki (CoSrc1 and CoSrc2), both of which have the arrangement of SH3, SH2, and catalytic domains seen in mammalian Src kinases. In Capsaspora cells, CoSrc1 and CoSrc2 localize to punctate structures in filopodia that may represent primordial focal adhesions. We have cloned, expressed, and purified both enzymes. CoSrc1 and CoSrc2 are active tyrosine kinases. Mammalian Src kinases are normally regulated in a reciprocal fashion by autophosphorylation in the activation loop (which increases activity) and by Csk-mediated phosphorylation of the C-terminal tail (which inhibits activity). Similar to mammalian Src kinases, the enzymatic activities of CoSrc1 and CoSrc2 are increased by autophosphorylation in the activation loop. We have identified a Csk-like kinase (CoCsk) in the genome of C. owczarzaki. We cloned, expressed, and purified CoCsk and found that it has no measurable tyrosine kinase activity. Furthermore, CoCsk does not phosphorylate or regulate CoSrc1 or CoSrc2 in cells or in vitro, and CoSrc1 and CoSrc2 are active in Capsaspora cell lysates. Thus, the function of Csk as a negative regulator of Src family kinases appears to have arisen with the emergence of metazoans.

  17. E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53

    PubMed Central

    Jung, J H; Bae, S; Lee, J Y; Woo, S R; Cha, H J; Yoon, Y; Suh, K-S; Lee, S-J; Park, I-C; Jin, Y-W; Lee, K-H; An, S; Lee, J H

    2011-01-01

    Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53. PMID:21597459

  18. E3 ubiquitin ligase Hades negatively regulates the exonuclear function of p53.

    PubMed

    Jung, J H; Bae, S; Lee, J Y; Woo, S R; Cha, H J; Yoon, Y; Suh, K-S; Lee, S-J; Park, I-C; Jin, Y-W; Lee, K-H; An, S; Lee, J H

    2011-12-01

    Following DNA damage, p53 translocates to the cytoplasm and mitochondria, where it triggers transcription-independent apoptosis by binding to Bcl-2 family proteins. However, little is known about how this exonuclear function of p53 is regulated. Here, we identify and characterize a p53-interacting protein called Hades, an E3 ligase that interacts with p53 in the mitochondria. Hades reduces p53 stability via a mechanism that requires its RING-finger domain with ubiquitin ligase activity. Hades polyubiquitinates p53 in vitro independent of Mdm2 and targets a critical lysine residue in p53 (lysine 24) distinct from those targeted by Mdm2. Hades inhibits a p53-dependent mitochondrial cell death pathway by inhibiting p53 and Bcl-2 interactions. These findings show that Hades-mediated p53 ubiquitination is a novel mechanism for negatively regulating the exonuclear function of p53.

  19. Mesolimbic leptin signaling negatively regulates cocaine-conditioned reward.

    PubMed

    Shen, M; Jiang, C; Liu, P; Wang, F; Ma, L

    2016-12-06

    The regulatory mechanisms underlying the response to addictive drugs are complex, and increasing evidence indicates that there is a role for appetite-regulating pathways in substance abuse. Leptin, an important adipose hormone that regulates energy balance and appetite, exerts its physiological functions via leptin receptors. However, the role of leptin signaling in regulating the response to cocaine remains unclear. Here we examined the potential role of leptin signaling in cocaine reward using a conditioned place preference (CPP) procedure. Our results showed that inhibition of leptin signaling by intracerebroventricular infusion of the leptin receptor (LepR) antagonist SMLA during cocaine conditioning increased the cocaine-CPP and upregulated the level of dopamine and its metabolites in the nucleus accumbens (NAc). We then selectively knocked down the LepR in the mesolimbic ventral tegmental area (VTA), NAc core and central amygdala (CeA) by injecting AAV-Cre into Lepr(flox/flox) mice. LepR deletion in the VTA increased the dopamine levels in the NAc and enhanced the cocaine-conditioned reward. LepR deletion in the NAc core enhanced the cocaine-conditioned reward and impaired the effect of the D2-dopamine receptor on cocaine-CPP, whereas LepR deletion in the CeA had no effect on cocaine-CPP but increased the anxiety level of mice. In addition, prior exposure to saccharin increased LepR mRNA and STAT3 phosphorylation in the NAc and VTA and impaired cocaine-CPP. These results indicate that leptin signaling is critically involved in cocaine-conditioned reward and the regulation of drug reward by a natural reward and that these effects are dependent on mesolimbic LepR.

  20. Mesolimbic leptin signaling negatively regulates cocaine-conditioned reward

    PubMed Central

    Shen, M; Jiang, C; Liu, P; Wang, F; Ma, L

    2016-01-01

    The regulatory mechanisms underlying the response to addictive drugs are complex, and increasing evidence indicates that there is a role for appetite-regulating pathways in substance abuse. Leptin, an important adipose hormone that regulates energy balance and appetite, exerts its physiological functions via leptin receptors. However, the role of leptin signaling in regulating the response to cocaine remains unclear. Here we examined the potential role of leptin signaling in cocaine reward using a conditioned place preference (CPP) procedure. Our results showed that inhibition of leptin signaling by intracerebroventricular infusion of the leptin receptor (LepR) antagonist SMLA during cocaine conditioning increased the cocaine-CPP and upregulated the level of dopamine and its metabolites in the nucleus accumbens (NAc). We then selectively knocked down the LepR in the mesolimbic ventral tegmental area (VTA), NAc core and central amygdala (CeA) by injecting AAV-Cre into Leprflox/flox mice. LepR deletion in the VTA increased the dopamine levels in the NAc and enhanced the cocaine-conditioned reward. LepR deletion in the NAc core enhanced the cocaine-conditioned reward and impaired the effect of the D2-dopamine receptor on cocaine-CPP, whereas LepR deletion in the CeA had no effect on cocaine-CPP but increased the anxiety level of mice. In addition, prior exposure to saccharin increased LepR mRNA and STAT3 phosphorylation in the NAc and VTA and impaired cocaine-CPP. These results indicate that leptin signaling is critically involved in cocaine-conditioned reward and the regulation of drug reward by a natural reward and that these effects are dependent on mesolimbic LepR. PMID:27922639

  1. Galangin Abrogates Ovalbumin-Induced Airway Inflammation via Negative Regulation of NF-κB.

    PubMed

    Zha, Wang-Jian; Qian, Yan; Shen, Yi; Du, Qiang; Chen, Fei-Fei; Wu, Zhen-Zhen; Li, Xiao; Huang, Mao

    2013-01-01

    Persistent activation of nuclear factor κB (NF-κB) has been associated with the development of asthma. Galangin, the active pharmacological ingredient from Alpinia galanga, is reported to have a variety of anti-inflammatory properties in vitro via negative regulation of NF-κB. This study aimed to investigate whether galangin can abrogate ovalbumin- (OVA-) induced airway inflammation by negative regulation of NF-κB. BALB/c mice sensitized and challenged with OVA developed airway hyperresponsiveness (AHR) and inflammation. Galangin dose dependently inhibited OVA-induced increases in total cell counts, eosinophil counts, and interleukin-(IL-) 4, IL-5, and IL-13 levels in bronchoalveolar lavage fluid, and reduced serum level of OVA-specific IgE. Galangin also attenuated AHR, reduced eosinophil infiltration and goblet cell hyperplasia, and reduced expression of inducible nitric oxide synthase and vascular cell adhesion protein-1 (VCAM-1) levels in lung tissue. Additionally, galangin blocked inhibitor of κB degradation, phosphorylation of the p65 subunit of NF-κB, and p65 nuclear translocation from lung tissues of OVA-sensitized mice. Similarly, in normal human airway smooth muscle cells, galangin blocked tumor necrosis factor-α induced p65 nuclear translocation and expression of monocyte chemoattractant protein-1, eotaxin, CXCL10, and VCAM-1. These results suggest that galangin can attenuate ovalbumin-induced airway inflammation by inhibiting the NF-κB pathway.

  2. Plasma glutamate carboxypeptidase is a negative regulator in liver cancer metastasis

    PubMed Central

    Lee, Jae-Hye; Cho, Hyun-Soo; Lee, Jeong-Ju; Jun, Soo Young; Ahn, Jun-Ho; Min, Ju-Sik; Yoon, Ji-Yong; Choi, Min-Hyuk; Jeon, Su-Jin; Lim, Jung Hwa; Jung, Cho-Rok; Kim, Dae-Soo; Kim, Hyun-Taek; Factor, Valentina M.; Lee, Yun-Han; Thorgeirsson, Snorri S.; Kim, Cheol-Hee; Kim, Nam-Soon

    2016-01-01

    Tumor metastasis is the leading cause of cancer death. In the metastatic process, EMT is a unique phenotypic change that plays an important role in cell invasion and changes in cell morphology. Despite the clinical significance, the mechanism underlying tumor metastasis is still poorly understood. Here we report a novel mechanism by which secreted plasma glutamate carboxypeptidase(PGCP) negatively involves Wnt/β-catenin signaling by DKK4 regulation in liver cancer metastasis. Pathway analysis of the RNA sequencing data showed that PGCP knockdown in liver cancer cell lines enriched the functions of cell migration, motility and mesenchymal cell differentiation. Depletion of PGCP promoted cell migration and invasion via activation of Wnt/β-catenin signaling pathway components such as phospho-LRP6 and β-catenin. Also, addition of DKK4 antagonized the Wnt/β-catenin signaling cascade in a thyroxine (T4)-dependent manner. In an in vivo study, metastatic nodules were observed in the lungs of the mice after injection of shPGCP stable cell lines. Our findings suggest that PGCP negatively associates with Wnt/β-catenin signaling during metastasis. Targeting this regulation may represent a novel and effective therapeutic option for liver cancer by preventing metastatic activity of primary tumor cells. PMID:27806330

  3. The inositol phosphatase SHIP-1 is negatively regulated by Fli-1 and its loss accelerates leukemogenesis.

    PubMed

    Lakhanpal, Gurpreet K; Vecchiarelli-Federico, Laura M; Li, You-Jun; Cui, Jiu-Wei; Bailey, Monica L; Spaner, David E; Dumont, Daniel J; Barber, Dwayne L; Ben-David, Yaacov

    2010-07-22

    The activation of Fli-1, an Ets transcription factor, is the critical genetic event in Friend murine leukemia virus (F-MuLV)-induced erythroleukemia. Fli-1 overexpression leads to erythropoietin-dependent erythroblast proliferation, enhanced survival, and inhibition of terminal differentiation, through activation of the Ras pathway. However, the mechanism by which Fli-1 activates this signal transduction pathway has yet to be identified. Down-regulation of the Src homology 2 (SH2) domain-containing inositol-5-phosphatase-1 (SHIP-1) is associated with erythropoietin-stimulated erythroleukemic cells and correlates with increased proliferation of transformed cells. In this study, we have shown that F-MuLV-infected SHIP-1 knockout mice display accelerated erythroleukemia progression. In addition, RNA interference (RNAi)-mediated suppression of SHIP-1 in erythroleukemia cells activates the phosphatidylinositol 3-kinase (PI 3-K) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathways, blocks erythroid differentiation, accelerates erythropoietin-induced proliferation, and leads to PI 3-K-dependent Fli-1 up-regulation. Chromatin immunoprecipitation and luciferase assays confirmed that Fli-1 binds directly to an Ets DNA binding site within the SHIP-1 promoter and suppresses SHIP-1 transcription. These data provide evidence to suggest that SHIP-1 is a direct Fli-1 target, SHIP-1 and Fli-1 regulate each other in a negative feedback loop, and the suppression of SHIP-1 by Fli-1 plays an important role in the transformation of erythroid progenitors by F-MuLV.

  4. Negative Regulators of JAK/STAT Signaling in Rheumatoid Arthritis and Osteoarthritis

    PubMed Central

    Malemud, Charles J.

    2017-01-01

    Elevated levels of pro-inflammatory cytokines are generally thought to be responsible for driving the progression of synovial joint inflammation in rheumatoid arthritis (RA) and osteoarthritis (OA). These cytokines activate several signal transduction pathways, including the Janus kinase/Signal Transducers and Activators of Transcription (JAK/STAT), Stress-Activated/Mitogen-Activated Protein Kinase (SAPK/MAPK) and phosphatidylinositol-3-kinase/Akt/mechanistic target of rapamycin (PI3K/Akt/mTOR) pathways which regulate numerous cellular responses. However, cytokine gene expression, matrix metalloproteinase gene expression and aberrant immune cell and synoviocyte survival via reduced apoptosis are most critical in the context of inflammation characteristic of RA and OA. Negative regulation of JAK/STAT signaling is controlled by Suppressor of Cytokine Signaling (SOCS) proteins. SOCS is produced at lower levels in RA and OA. In addition, gaining further insight into the role played in RA and OA pathology by the inhibitors of the apoptosis protein family, cellular inhibitor of apoptosis protein-1, -2 (c-IAP1, c-IAP2), X (cross)-linked inhibitor of apoptosis protein (XIAP), protein inhibitor of activated STAT (PIAS), and survivin (human) as well as SOCS appears to be a worthy endeavor going forward. PMID:28245561

  5. Parental reactions to children's negative emotions: relationships with emotion regulation in children with an anxiety disorder.

    PubMed

    Hurrell, Katherine E; Hudson, Jennifer L; Schniering, Carolyn A

    2015-01-01

    Research has demonstrated that parental reactions to children's emotions play a significant role in the development of children's emotion regulation (ER) and adjustment. This study compared parent reactions to children's negative emotions between families of anxious and non-anxious children (aged 7-12) and examined associations between parent reactions and children's ER. Results indicated that children diagnosed with an anxiety disorder had significantly greater difficulty regulating a range of negative emotions and were regarded as more emotionally negative and labile by their parents. Results also suggested that mothers of anxious children espoused less supportive parental emotional styles when responding to their children's negative emotions. Supportive and non-supportive parenting reactions to children's negative emotions related to children's emotion regulation skills, with father's non-supportive parenting showing a unique relationship to children's negativity/lability.

  6. N-acyl homoserinelactone-mediated gene regulation in gram-negative bacteria.

    PubMed

    Eberl, L

    1999-12-01

    The view of bacteria as unicellular organisms has strong roots in the tradition of culturing bacteria in liquid media. However, in nature microbial activity is mainly associated with surfaces where bacteria form highly structured and cooperative consortia which are commonly referred to as biofilms. The ability of bacteria to organize structurally and to distribute metabolic activities between the different members of the consortium demands a high degree of coordinated cell-cell interaction. Recent work has established that many bacteria employ sophisticated intercellular communication systems that rely on small signal molecules to control the expression of multiple target genes. In Gram-negative bacteria, the most intensively investigated signal molecules are N-acyl-L-homoserine lactones (AHLs), which are utilized by the bacteria to monitor their own population densities in a process known as 'quorum sensing'. These density-dependent regulatory systems rely on two proteins, an AHL synthase, usually a member of the LuxI family of proteins, and an AHL receptor protein belonging to the LuxR family of transcriptional regulators. At low population densities cells produce a basal level of AHL via the activity of an AHL synthase. As the cell density increases, AHL accumulates in the growth medium. On reaching a critical threshold concentration, the AHL molecule binds to its cognate receptor which in turn leads to the induction/repression of AHL-regulated genes. To date, AHL-dependent quorum sensing circuits have been identified in a wide range of gram-negative bacteria where they regulate various functions including bioluminescence, plasmid conjugal transfer, biofilm formation, motility, antibiotic biosynthesis, and the production of virulence factors in plant and animal pathogens. Moreover, AHL signal molecules appear to play important roles in the ecology of complex consortia as they allow bacterial populations to interact with each other as well as with their

  7. The Temporal Deployment of Emotion Regulation Strategies During Negative Emotional Episodes.

    PubMed

    Kalokerinos, Elise K; Résibois, Maxime; Verduyn, Philippe; Kuppens, Peter

    2016-11-07

    Time is given a central place in theoretical models of emotion regulation (Gross, 1998, 2015), but key questions regarding the role of time remain unanswered. We investigated 2 such unanswered questions. First, we explored when different emotion regulation strategies were used within the course of an emotional episode in daily life. Second, we investigated the association between the temporal deployment of strategies and negative emotional experience. We conducted a daily diary study in which participants (N = 74) drew an intensity profile depicting the temporal unfolding of their negative emotional experience across daily events (N = 480), and mapped their usage of emotion regulation strategies onto this intensity profile. Strategies varied in their temporal deployment, with suppression and rumination occurring more at the beginning of the episode, and reappraisal and distraction occurring more toward the end of the episode. Strategies also varied in their association with negative emotion: rumination was positively associated with negative emotion, and reappraisal and distraction were negatively associated with negative emotion. Finally, both rumination and reappraisal interacted with time to predict negative emotional experience. Rumination was more strongly positively associated with negative emotions at the end of the episode than the beginning, but reappraisal was more strongly negatively associated with negative emotion at the beginning of the episode than the end. These findings highlight the importance of accounting for timing in the study of emotion regulation, as well as the necessity of studying these temporal processes in daily life. (PsycINFO Database Record

  8. CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6

    PubMed Central

    Chen, Bo; Zhao, Ruiying; Su, Chun-Hui; Linan, Monica; Tseng, Chieh; Phan, Liem; Fang, Lekuan; Yang, Heng-Yin; Yang, Huiling; Wang, Wenqian; Xu, Xiaoyin; Jiang, Nan; Cai, Shouliang; Jin, Feng; Yeung, Sai-Ching J.; Lee, Mong-Hong

    2012-01-01

    Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2 and result in promoted cancer cell growth. PMID:23187808

  9. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    SciTech Connect

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  10. SREBP-2 negatively regulates FXR-dependent transcription of FGF19 in human intestinal cells.

    PubMed

    Miyata, Masaaki; Hata, Tatsuya; Yamazoe, Yasushi; Yoshinari, Kouichi

    2014-01-10

    Sterol regulatory element-binding protein-2 (SREBP-2) is a basic helix-loop-helix-leucine zipper transcription factor that positively regulates transcription of target genes involved in cholesterol metabolism. In the present study, we have investigated a possible involvement of SREBP-2 in human intestinal expression of fibroblast growth factor (FGF)19, which is an endocrine hormone involved in the regulation of lipid and glucose metabolism. Overexpression of constitutively active SREBP-2 decreased FGF19 mRNA levels in human colon-derived LS174T cells. In reporter assays, active SREBP-2 overexpression suppressed GW4064/FXR-mediated increase in reporter activities in regions containing the IR-1 motif (+848 to +5200) in the FGF19 gene. The suppressive effect disappeared in reporter activities in the region containing the IR-1 motif when the mutation was introduced into the IR-1 motif. In electrophoretic mobility shift assays, binding of the FXR/retinoid X receptor α heterodimer to the IR-1 motif was attenuated by adding active SREBP-2, but SREBP-2 binding to the IR-1 motif was not observed. In chromatin immunoprecipitation assays, specific binding of FXR to the IR-1-containing region of the FGF19 gene (+3214 to +3404) was increased in LS174T cells by treatment with cholesterol and 25-hydroxycholesterol. Specific binding of SREBP-2 to FXR was observed in glutathione-S-transferase (GST) pull-down assays. These results suggest that SREBP-2 negatively regulates the FXR-mediated transcriptional activation of the FGF19 gene in human intestinal cells.

  11. NRROS Negatively Regulates Osteoclast Differentiation by Inhibiting RANKL-Mediated NF-κB and Reactive Oxygen Species Pathways

    PubMed Central

    Kim, Jung Ha; Kim, Kabsun; Kim, Inyoung; Seong, Semun; Kim, Nacksung

    2015-01-01

    Negative regulator of reactive oxygen species (NRROS) is known to repress ROS generation in phagocytes. In this study, we examined the roles of NRROS in both osteoclasts and osteoblasts. Our results demonstrate that NRROS negatively regulates the differentiation of osteoclasts, but not osteoblasts. Further, overexpression of NRROS in osteoclast precursor cells attenuates RANKL-induced osteoclast differentiation. Conversely, osteoclast differentiation is enhanced upon siRNA-mediated knockdown of NRROS. Additionally, NRROS attenuates RANKL-induced NF-κB activation, as well as degradation of the NOX1 and NOX2 proteins, which are required for ROS generation. Based on our observations, we present NRROS as a novel negative regulator of RANKL-induced osteoclastogenesis. PMID:26442864

  12. Characterization of active metamaterials based on negative impedance converters

    NASA Astrophysics Data System (ADS)

    Rajab, K. Z.; Fan, Y. F.; Hao, Y.

    2012-11-01

    Negative impedance converters (NICs) are used to create impedance loads that can effectively cancel the inductive properties of magnetic dipoles, resulting in active metamaterials with increased bandwidth and reduced loss for μ-near-zero (MNZ) and negative-Re(μ) (MNG) media. We demonstrate techniques for analyzing the stability and characterizing the magnetic properties of effective media loaded with NICs. Specifically, we apply the Nyquist criterion to validate the stability of sample active metamaterials. It is shown that the practical NIC-loaded metamaterial may maintain stability and reduce dispersion, albeit with reduced performance as compared to the ideal NIC load.

  13. IRTKS negatively regulates antiviral immunity through PCBP2 sumoylation-mediated MAVS degradation

    PubMed Central

    Xia, Pengyan; Wang, Shuo; Xiong, Zhen; Ye, Buqing; Huang, Li-Yu; Han, Ze-Guang; Fan, Zusen

    2015-01-01

    RNA virus infection is recognized by the RIG-I family of receptors that activate the mitochondrial adaptor MAVS, leading to the clearance of viruses. Antiviral signalling activation requires strict modulation to avoid damage to the host from exacerbated inflammation. Insulin receptor tyrosine kinase substrate (IRTKS) participates in actin bundling and insulin signalling and its deficiency causes insulin resistance. However, whether IRTKS is involved in the regulation of innate immunity remains elusive. Here we show that IRTKS deficiency causes enhanced innate immune responses against RNA viruses. IRTKS-mediated suppression of antiviral responses depends on the RIG-I-MAVS signalling pathway. IRTKS recruits the E2 ligase Ubc9 to sumoylate PCBP2 in the nucleus, which causes its cytoplasmic translocation during viral infection. The sumoylated PCBP2 associates with MAVS to initiate its degradation, leading to downregulation of antiviral responses. Thus, IRTKS functions as a negative modulator of excessive inflammation. PMID:26348439

  14. ERK8 is a negative regulator of O-GalNAc glycosylation and cell migration

    PubMed Central

    Chia, Joanne; Tham, Keit Min; Gill, David James; Bard-Chapeau, Emilie Anne; Bard, Frederic A

    2014-01-01

    ER O-glycosylation can be induced through relocalisation GalNAc-Transferases from the Golgi. This process markedly stimulates cell migration and is constitutively activated in more than 60% of breast carcinomas. How this activation is achieved remains unclear. Here, we screened 948 signalling genes using RNAi and imaging. We identified 12 negative regulators of O-glycosylation that all control GalNAc-T sub-cellular localisation. ERK8, an atypical MAPK with high basal kinase activity, is a strong hit and is partially localised at the Golgi. Its inhibition induces the relocation of GalNAc-Ts, but not of KDEL receptors, revealing the existence of two separate COPI-dependent pathways. ERK8 down-regulation, in turn, activates cell motility. In human breast and lung carcinomas, ERK8 expression is reduced while ER O-glycosylation initiation is hyperactivated. In sum, ERK8 appears as a constitutive brake on GalNAc-T relocalisation, and the loss of its expression could drive cancer aggressivity through increased cell motility. DOI: http://dx.doi.org/10.7554/eLife.01828.001 PMID:24618899

  15. Soybean Homologs of MPK4 Negatively Regulate Defense Responses and Positively Regulate Growth and Development1[W][OA

    PubMed Central

    Liu, Jian-Zhong; Horstman, Heidi D.; Braun, Edward; Graham, Michelle A.; Zhang, Chunquan; Navarre, Duroy; Qiu, Wen-Li; Lee, Yeunsook; Nettleton, Dan; Hill, John H.; Whitham, Steven A.

    2011-01-01

    Mitogen-activated protein kinase (MAPK) cascades play important roles in disease resistance in model plant species such as Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum). However, the importance of MAPK signaling pathways in the disease resistance of crops is still largely uninvestigated. To better understand the role of MAPK signaling pathways in disease resistance in soybean (Glycine max), 13, nine, and 10 genes encoding distinct MAPKs, MAPKKs, and MAPKKKs, respectively, were silenced using virus-induced gene silencing mediated by Bean pod mottle virus. Among the plants silenced for various MAPKs, MAPKKs, and MAPKKKs, those in which GmMAPK4 homologs (GmMPK4s) were silenced displayed strong phenotypes including stunted stature and spontaneous cell death on the leaves and stems, the characteristic hallmarks of activated defense responses. Microarray analysis showed that genes involved in defense responses, such as those in salicylic acid (SA) signaling pathways, were significantly up-regulated in GmMPK4-silenced plants, whereas genes involved in growth and development, such as those in auxin signaling pathways and in cell cycle and proliferation, were significantly down-regulated. As expected, SA and hydrogen peroxide accumulation was significantly increased in GmMPK4-silenced plants. Accordingly, GmMPK4-silenced plants were more resistant to downy mildew and Soybean mosaic virus compared with vector control plants. Using bimolecular fluorescence complementation analysis and in vitro kinase assays, we determined that GmMKK1 and GmMKK2 might function upstream of GmMPK4. Taken together, our results indicate that GmMPK4s negatively regulate SA accumulation and defense response but positively regulate plant growth and development, and their functions are conserved across plant species. PMID:21878550

  16. Genomic regulation of invasion by STAT3 in triple negative breast cancer.

    PubMed

    McDaniel, Joy M; Varley, Katherine E; Gertz, Jason; Savic, Daniel S; Roberts, Brian S; Bailey, Sarah K; Shevde, Lalita A; Ramaker, Ryne C; Lasseigne, Brittany N; Kirby, Marie K; Newberry, Kimberly M; Partridge, E Christopher; Jones, Angela L; Boone, Braden; Levy, Shawn E; Oliver, Patsy G; Sexton, Katherine C; Grizzle, William E; Forero, Andres; Buchsbaum, Donald J; Cooper, Sara J; Myers, Richard M

    2017-01-31

    Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as "triple negative breast cancer" (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis.

  17. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

    PubMed Central

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  18. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-08-16

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

  19. Interferon Regulatory Factor 7 Functions as a Novel Negative Regulator of Pathological Cardiac Hypertrophy

    PubMed Central

    Jiang, Ding-Sheng; Liu, Yu; Zhou, Heng; Zhang, Yan; Zhang, Xiao-Dong; Zhang, Xiao-Fei; Chen, Ke; Gao, Lu; Peng, Juan; Gong, Hui; Chen, Yingjie; Yang, Qinglin; Liu, Peter P.; Fan, Guo-Chang; Zou, Yunzeng; Li, Hongliang

    2017-01-01

    Cardiac hypertrophy is a complex pathological process that involves multiple factors including inflammation and apoptosis. Interferon regulatory factor 7 (IRF7) is a multifunctional regulator that participates in immune regulation, cell differentiation, apoptosis, and oncogenesis. However, the role of IRF7 in cardiac hypertrophy remains unclear. We performed aortic banding in cardiac-specific IRF7 transgenic mice, IRF7 knockout mice, and the wild-type littermates of these mice. Our results demonstrated that IRF7 was downregulated in aortic banding–induced animal hearts and cardiomyocytes that had been treated with angiotensin II or phenylephrine for 48 hours. Accordingly, heart-specific overexpression of IRF7 significantly attenuated pressure overload–induced cardiac hypertrophy, fibrosis, and dysfunction, whereas loss of IRF7 led to opposite effects. Moreover, IRF7 protected against angiotensin II–induced cardiomyocyte hypertrophy in vitro. Mechanistically, we identified that IRF7-dependent cardioprotection was mediated through IRF7 binding to inhibitor of κB kinase-β, and subsequent nuclear factor-κB inactivation. In fact, blocking nuclear factor-κB signaling with cardiac-specific inhibitors of κBαS32A/S36A super-repressor transgene counteracted the adverse effect of IRF7 deficiency. Conversely, activation of nuclear factor-κB signaling via a cardiac-specific conditional inhibitor of κB kinase-βS177E/S181E (constitutively active) transgene negated the antihypertrophic effect of IRF7 overexpression. Our data demonstrate that IRF7 acts as a novel negative regulator of pathological cardiac hypertrophy by inhibiting nuclear factor-κB signaling and may constitute a potential therapeutic target for pathological cardiac hypertrophy. PMID:24396025

  20. MIP-T3 is a negative regulator of innate type I IFN response.

    PubMed

    Ng, Ming-Him James; Ho, Ting-Hin; Kok, Kin-Hang; Siu, Kam-Leung; Li, Jun; Jin, Dong-Yan

    2011-12-15

    TNFR-associated factor (TRAF) 3 is an important adaptor that transmits upstream activation signals to protein kinases that phosphorylate transcription factors to induce the production of type I IFNs, the important effectors in innate antiviral immune response. MIP-T3 interacts specifically with TRAF3, but its function in innate IFN response remains unclear. In this study, we demonstrated a negative regulatory role of MIP-T3 in type I IFN production. Overexpression of MIP-T3 inhibited RIG-I-, MDA5-, VISA-, TBK1-, and IKKε-induced transcriptional activity mediated by IFN-stimulated response elements and IFN-β promoter. MIP-T3 interacted with TRAF3 and perturbed in a dose-dependent manner the formation of functional complexes of TRAF3 with VISA, TBK1, IKKε, and IFN regulatory factor 3. Consistent with this finding, retinoic acid-inducible gene I- and TBK1-induced phosphorylation of IFN regulatory factor 3 was significantly diminished when MIP-T3 was overexpressed. Depletion of MIP-T3 facilitated Sendai virus-induced activation of IFN production and attenuated the replication of vesicular stomatitis virus. In addition, MIP-T3 was found to be dissociated from TRAF3 during the course of Sendai virus infection. Our findings suggest that MIP-T3 functions as a negative regulator of innate IFN response by preventing TRAF3 from forming protein complexes with critical downstream transducers and effectors.

  1. Mood regulation and quality of life in social anxiety disorder: an examination of generalized expectancies for negative mood regulation.

    PubMed

    Sung, Sharon C; Porter, Eliora; Robinaugh, Donald J; Marks, Elizabeth H; Marques, Luana M; Otto, Michael W; Pollack, Mark H; Simon, Naomi M

    2012-04-01

    The present study examined negative mood regulation expectancies, anxiety symptom severity, and quality of life in a sample of 167 patients with social anxiety disorder (SAD) and 165 healthy controls with no DSM-IV Axis I disorders. Participants completed the Generalized Expectancies for Negative Mood Regulation Scale (NMR), the Beck Anxiety Inventory, and the Quality of Life Enjoyment and Satisfaction Questionnaire. SAD symptom severity was assessed using the Liebowitz Social Anxiety Scale. Individuals with SAD scored significantly lower than controls on the NMR. Among SAD participants, NMR scores were negatively correlated with anxiety symptoms and SAD severity, and positively correlated with quality of life. NMR expectancies positively predicted quality of life even after controlling for demographic variables, comorbid diagnoses, anxiety symptoms, and SAD severity. Individuals with SAD may be less likely to engage in emotion regulating strategies due to negative beliefs regarding their effectiveness, thereby contributing to poorer quality of life.

  2. Abnormal brain activation during directed forgetting of negative memory in depressed patients.

    PubMed

    Yang, Wenjing; Chen, Qunlin; Liu, Peiduo; Cheng, Hongsheng; Cui, Qian; Wei, Dongtao; Zhang, Qinglin; Qiu, Jiang

    2016-01-15

    The frequent occurrence of uncontrollable negative thoughts and memories is a troubling aspect of depression. Thus, knowledge on the mechanism underlying intentional forgetting of these thoughts and memories is crucial to develop an effective emotion regulation strategy for depressed individuals. Behavioral studies have demonstrated that depressed participants cannot intentionally forget negative memories. However, the neural mechanism underlying this process remains unclear. In this study, participants completed the directed forgetting task in which they were instructed to remember or forget neutral or negative words. Standard univariate analysis based on the General Linear Model showed that the depressed participants have higher activation in the inferior frontal gyrus (IFG), superior frontal gyrus (SFG), superior parietal gyrus (SPG), and inferior temporal gyrus (ITG) than the healthy individuals. The results indicated that depressed participants recruited more frontal and parietal inhibitory control resources to inhibit the TBF items, but the attempt still failed because of negative bias. We also used the Support Vector Machine to perform multivariate pattern classification based on the brain activation during directed forgetting. The pattern of brain activity in directed forgetting of negative words allowed correct group classification with an overall accuracy of 75% (P=0.012). The brain regions which are critical for this discrimination showed abnormal activation when depressed participants were attempting to forget negative words. These results indicated that the abnormal neural circuitry when depressed individuals tried to forget the negative words might provide neurobiological markers for depression.

  3. The Heme Oxygenase-1 Inducer THI-56 Negatively Regulates iNOS Expression and HMGB1 Release in LPS-Activated RAW 264.7 Cells and CLP-Induced Septic Mice

    PubMed Central

    Kim, Young Min; Park, Sang Won; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl

    2013-01-01

    The nuclear DNA binding protein high mobility group box 1 (HMGB1) has recently been suggested to act as a late mediator of septic shock. The effect of ((S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid, also known as THI-56, in an experimental model of sepsis was investigated. THI-56 exhibited potent anti-inflammatory properties in response to LPS in RAW 264.7 cells. In particular, THI-56 significantly inhibited the expression of inducible nitric oxide synthase (iNOS) and the release of HMGB1 in activated macrophages. THI-56 activated NE-F2-regulated factor 2 (Nrf-2)/heme oxygenase 1 (HO-1). The specific knockdown of the HO-1 gene by HO-1 siRNA significantly reversed the inhibitory effects of THI-56 on iNOS expression and HMGB1 release in LPS-stimulated macrophages. Importantly, THI-56 administration protected animals from death induced by either a lethal dose of LPS or cecal ligation and puncture (CLP). Furthermore, the ALT, AST, BUN, creatinine, and HMGB1 levels in the blood were significantly increased in CLP-induced septic mice, and the administration of THI-56 reduced these levels in a concentration-dependent and zinc protoporphyrin IX (ZnPPIX)-sensitive manner. In addition, the administration of THI-56 significantly ameliorated not only lung damage but also macrophage infiltration in the livers of CLP-induced septic mice, and these effects were also abrogated in the presence of ZnPPIX. Thus, we conclude that THI-56 significantly attenuates the proinflammatory response induced by LPS and reduces organ damage in a CLP-induced sepsis model through the upregulation of Nrf-2/HO-1. PMID:24098466

  4. PTPN2 negatively regulates oncogenic JAK1 in T-cell acute lymphoblastic leukemia.

    PubMed

    Kleppe, Maria; Soulier, Jean; Asnafi, Vahid; Mentens, Nicole; Hornakova, Tekla; Knoops, Laurent; Constantinescu, Stefan; Sigaux, François; Meijerink, Jules P; Vandenberghe, Peter; Tartaglia, Marco; Foa, Robin; Macintyre, Elizabeth; Haferlach, Torsten; Cools, Jan

    2011-06-30

    We have recently reported inactivation of the tyrosine phosphatase PTPN2 (also known as TC-PTP) through deletion of the entire gene locus in ∼ 6% of T-cell acute lymphoblastic leukemia (T-ALL) cases. T-ALL is an aggressive disease of the thymocytes characterized by the stepwise accumulation of chromosomal abnormalities and gene mutations. In the present study, we confirmed the strong association of the PTPN2 deletion with TLX1 and NUP214-ABL1 expression. In addition, we found cooperation between PTPN2 deletion and activating JAK1 gene mutations. Activating mutations in JAK1 kinase occur in ∼ 10% of human T-ALL cases, and aberrant kinase activity has been shown to confer proliferation and survival advantages. Our results reveal that some JAK1 mutation-positive T-ALLs harbor deletions of the tyrosine phosphatase PTPN2, a known negative regulator of the JAK/STAT pathway. We provide evidence that down-regulation of Ptpn2 sensitizes lymphoid cells to JAK1-mediated transformation and reduces their sensitivity to JAK inhibition.

  5. JMJD6 Promotes Colon Carcinogenesis through Negative Regulation of p53 by Hydroxylation

    PubMed Central

    Wang, Feng; He, Lin; Huangyang, Peiwei; Liang, Jing; Si, Wenzhe; Yan, Ruorong; Han, Xiao; Liu, Shumeng; Gui, Bin; Li, Wanjin; Miao, Di; Jing, Chao; Liu, Zhihua; Pei, Fei; Sun, Luyang; Shang, Yongfeng

    2014-01-01

    Jumonji domain-containing 6 (JMJD6) is a member of the Jumonji C domain-containing family of proteins. Compared to other members of the family, the cellular activity of JMJD6 is still not clearly defined and its biological function is still largely unexplored. Here we report that JMJD6 is physically associated with the tumor suppressor p53. We demonstrated that JMJD6 acts as an α-ketoglutarate– and Fe(II)-dependent lysyl hydroxylase to catalyze p53 hydroxylation. We found that p53 indeed exists as a hydroxylated protein in vivo and that the hydroxylation occurs mainly on lysine 382 of p53. We showed that JMJD6 antagonizes p53 acetylation, promotes the association of p53 with its negative regulator MDMX, and represses transcriptional activity of p53. Depletion of JMJD6 enhances p53 transcriptional activity, arrests cells in the G1 phase, promotes cell apoptosis, and sensitizes cells to DNA damaging agent-induced cell death. Importantly, knockdown of JMJD6 represses p53-dependent colon cell proliferation and tumorigenesis in vivo, and significantly, the expression of JMJD6 is markedly up-regulated in various types of human cancer especially in colon cancer, and high nuclear JMJD6 protein is strongly correlated with aggressive clinical behaviors of colon adenocarcinomas. Our results reveal a novel posttranslational modification for p53 and support the pursuit of JMJD6 as a potential biomarker for colon cancer aggressiveness and a potential target for colon cancer intervention. PMID:24667498

  6. A competitive peptide inhibitor KIDARI negatively regulates HFR1 by forming nonfunctional heterodimers in Arabidopsis photomorphogenesis.

    PubMed

    Hong, Shin-Young; Seo, Pil Joon; Ryu, Jae Yong; Cho, Shin-Hae; Woo, Je-Chang; Park, Chung-Mo

    2013-01-01

    Dynamic dimer formation is an elaborate means of modulating transcription factor activities in diverse cellular processes. The basic helix-loop-helix (bHLH) transcription factor LONG HYPOCOTYL IN FAR-RED 1 (HFR1), for example, plays a role in plant photomorphogenesis by forming non-DNA binding heterodimers with PHYTOCHROMEINTERACTING FACTORS (PIFs). Recent studies have shown that a small HLH protein KIDARI (KDR) negatively regulates the HFR1 activity in the process. However, molecular mechanisms underlying the KDR control of the HFR1 activity are unknown. Here, we demonstrate that KDR attenuates the HFR1 activity by competitively forming nonfunctional heterodimers, causing liberation of PIF4 from the transcriptionally inactive HFR1-PIF4 complex. Accordingly, the photomorphogenic hypocotyl growth of the HFR1-overexpressing plants can be suppressed by KDR coexpression, as observed in the HFR1-deficient hfr1-201 mutant. These results indicate that the PIF4 activity is modulated through a double layer of competitive inhibition by HFR1 and KDR, which could in turn ensure fine-tuning of the PIF4 activity under fluctuating light conditions.

  7. Prediction of Elementary School Children's Externalizing Problem Behaviors from Attentional and Behavioral Regulation and Negative Emotionality.

    ERIC Educational Resources Information Center

    Eisenberg, Nancy; Guthrie, Ivanna K.; Fabes, Richard A.; Shepard, Stephanie; Losoya, Sandra; Murphy, Bridget C.; Jones, Sarah; Paulin, Rick; Reiser, Mark

    2000-01-01

    Examined the moderating role of individual differences in negative emotionality in the relations of behavioral and attentional regulation to externalizing problem behaviors. Found that at two ages behavioral dysregulation predicted externalizing behavior problems for children both high and low in negative emotionality, whereas prediction of…

  8. Toddler Emotion Regulation with Mothers and Fathers: Temporal Associations between Negative Affect and Behavioral Strategies

    ERIC Educational Resources Information Center

    Ekas, Naomi V.; Braungart-Rieker, Julia M.; Lickenbrock, Diane M.; Zentall, Shannon R.; Maxwell, Scott M.

    2011-01-01

    The present study investigated temporal associations between putative emotion regulation strategies and negative affect in 20-month-old toddlers. Toddlers' parent-focused, self-distraction, and toy-focused strategies, as well as negative affect, were rated on a second-by-second basis during laboratory parent-toddler interactions. Longitudinal…

  9. The Role of Depression and Negative Affect Regulation Expectancies in Tobacco Smoking among College Students

    ERIC Educational Resources Information Center

    Schleicher, Holly E.; Harris, Kari Jo; Catley, Delwyn; Nazir, Niaman

    2009-01-01

    Objective: Expectancies about nicotine's ability to alleviate negative mood states may play a role in the relationship between smoking and depression. The authors examined the role of negative affect regulation expectancies as a potential mediator of depression (history of depression and depressive symptoms) and smoking among college students.…

  10. Re-appraisal of negative emotions in cocaine dependence: dysfunctional corticolimbic activation and connectivity.

    PubMed

    Albein-Urios, Natalia; Verdejo-Román, Juan; Asensio, Samuel; Soriano-Mas, Carles; Martínez-González, José M; Verdejo-García, Antonio

    2014-05-01

    Cocaine dependence is associated with pronounced elevations of negative affect and deficient regulation of negative emotions. We aimed to investigate the neural substrates of negative emotion regulation in cocaine-dependent individuals (CDI), as compared to non-drug-using controls, using functional magnetic resonance imaging (fMRI) during a re-appraisal task. Seventeen CDI abstinent for at least 15 days and without other psychiatric co-morbidities and 18 intelligence quotient-matched non-drug-using controls participated in the study. Participants performed the re-appraisal task during fMRI scanning: they were exposed to 24 blocks of negative affective or neutral pictures that they should Observe (neutral pictures), Maintain (sustain the emotion elicited by negative pictures) or Suppress (regulate the emotion elicited by negative pictures through previously trained re-appraisal techniques). Task-related activations during two conditions of interest (Maintain>Observe and Suppress>Maintain) were analyzed using the general linear model in SPM8 software. We also performed psychophysiological interaction (PPI) seed-based analyses based on one region from each condition: the dorsolateral prefrontal cortex (dlPFC-Maintain>Observe) and the inferior frontal gyrus (IFG-Suppress>Maintain). Results showed that cocaine users had increased right dlPFC and bilateral temporoparietal junction activations during Maintain>Observe, whereas they showed decreased right IFG, posterior cingulate cortex, insula and fusiform gyrus activations during Suppress>Maintain. PPI analyses showed that cocaine users had increased functional coupling between the dlPFC and emotion-related regions during Maintain>Observe, whereas they showed decreased functional coupling between the right IFG and the amygdala during Suppress>Maintain. These findings indicate that CDI have dysfunctional corticolimbic activation and connectivity during negative emotion experience and re-appraisal.

  11. Mothers' responses to children's negative emotions and child emotion regulation: the moderating role of vagal suppression.

    PubMed

    Perry, Nicole B; Calkins, Susan D; Nelson, Jackie A; Leerkes, Esther M; Marcovitch, Stuart

    2012-07-01

    The current study examined the moderating effect of children's cardiac vagal suppression on the association between maternal socialization of negative emotions (supportive and nonsupportive responses) and children's emotion regulation behaviors. One hundred and ninety-seven 4-year-olds and their mothers participated. Mothers reported on their reactions to children's negative emotions and children's regulatory behaviors. Observed distraction, an adaptive self-regulatory strategy, and vagal suppression were assessed during a laboratory task designed to elicit frustration. Results indicated that children's vagal suppression moderated the association between mothers' nonsupportive emotion socialization and children's emotion regulation behaviors such that nonsupportive reactions to negative emotions predicted lower observed distraction and lower reported emotion regulation behaviors when children displayed lower levels of vagal suppression. No interaction was found between supportive maternal emotion socialization and vagal suppression for children's emotion regulation behaviors. Results suggest physiological regulation may serve as a buffer against nonsupportive emotion socialization.

  12. Corrugator activity confirms immediate negative affect in surprise

    PubMed Central

    Topolinski, Sascha; Strack, Fritz

    2015-01-01

    The emotion of surprise entails a complex of immediate responses, such as cognitive interruption, attention allocation to, and more systematic processing of the surprising stimulus. All these processes serve the ultimate function to increase processing depth and thus cognitively master the surprising stimulus. The present account introduces phasic negative affect as the underlying mechanism responsible for this switch in operating mode. Surprising stimuli are schema-discrepant and thus entail cognitive disfluency, which elicits immediate negative affect. This affect in turn works like a phasic cognitive tuning switching the current processing mode from more automatic and heuristic to more systematic and reflective processing. Directly testing the initial elicitation of negative affect by surprising events, the present experiment presented high and low surprising neutral trivia statements to N = 28 participants while assessing their spontaneous facial expressions via facial electromyography. High compared to low surprising trivia elicited higher corrugator activity, indicative of negative affect and mental effort, while leaving zygomaticus (positive affect) and frontalis (cultural surprise expression) activity unaffected. Future research shall investigate the mediating role of negative affect in eliciting surprise-related outcomes. PMID:25762956

  13. Silver enhances antibiotic activity against gram-negative bacteria.

    PubMed

    Morones-Ramirez, Jose Ruben; Winkler, Jonathan A; Spina, Catherine S; Collins, James J

    2013-06-19

    A declining pipeline of clinically useful antibiotics has made it imperative to develop more effective antimicrobial therapies, particularly against difficult-to-treat Gram-negative pathogens. Silver has been used as an antimicrobial since antiquity, yet its mechanism of action remains unclear. We show that silver disrupts multiple bacterial cellular processes, including disulfide bond formation, metabolism, and iron homeostasis. These changes lead to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria that can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain. We show both in vitro and in a mouse model of urinary tract infection that the ability of silver to induce oxidative stress can be harnessed to potentiate antibiotic activity. Additionally, we demonstrate in vitro and in two different mouse models of peritonitis that silver sensitizes Gram-negative bacteria to the Gram-positive-specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. Finally, we used silver and antibiotic combinations in vitro to eradicate bacterial persister cells, and show both in vitro and in a mouse biofilm infection model that silver can enhance antibacterial action against bacteria that produce biofilms. This work shows that silver can be used to enhance the action of existing antibiotics against Gram-negative bacteria, thus strengthening the antibiotic arsenal for fighting bacterial infections.

  14. Protein Kinase C isoform epsilon (ε) negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets

    PubMed Central

    Bynagari-Settipalli, Yamini S; Lakhani, Parth; Jin, Jianguo; Bhavaraju, Kamala; Rico, Mario C.; Kim, Soochong; Woulfe, Donna; Kunapuli, Satya P

    2012-01-01

    Objective Members of Protein Kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. In this study we investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. Methods and Results A pan-PKC inhibitor GF109203X potentiated ADP-induced cPLA2 phosphorylation and thromboxane generation, as well as ERK activation and intracellular calcium (Ca2+i) mobilization, two signaling molecules, upstream of cPLA2 activation. Thus, PKCs inhibit cPLA2 activation by inhibiting ERK and Ca2+i mobilization. Since, the inhibitor of Classical PKC isoforms, GO-6976 did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP- induced thromboxane generation, calcium mobilization and ERK phosphorylation were potentiated in PKCε null murine platelets compared to platelets from wild type (WT) littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε KO and WT was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in tail bleeding experiments. Conclusion We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis. PMID:22362759

  15. PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor

    PubMed Central

    Bucur, Octavian; Stancu, Andreea Lucia; Muraru, Maria Sinziana; Melet, Armelle; Petrescu, Stefana Maria; Khosravi-Far, Roya

    2015-01-01

    FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ’s development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor. PMID:26280018

  16. PLK1 is a binding partner and a negative regulator of FOXO3 tumor suppressor.

    PubMed

    Bucur, Octavian; Stancu, Andreea Lucia; Muraru, Maria Sinziana; Melet, Armelle; Petrescu, Stefana Maria; Khosravi-Far, Roya

    2014-01-01

    FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ's development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor.

  17. Induction of Posttranslational Modifications of Mitochondrial Proteins by ATP Contributes to Negative Regulation of Mitochondrial Function

    PubMed Central

    Zhang, Yong; Zhao, Zhiyun; Ke, Bilun; Wan, Lin; Wang, Hui; Ye, Jianping

    2016-01-01

    It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH) phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2) and 4 (PDK4). In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function. PMID:26930489

  18. TPL-2 negatively regulates interferon-β production in macrophages and myeloid dendritic cells

    PubMed Central

    Kaiser, Frank; Cook, Dorthe; Papoutsopoulou, Stamatia; Rajsbaum, Ricardo; Wu, Xuemei; Yang, Huei-Ting; Grant, Susan; Ricciardi-Castagnoli, Paola; Tsichlis, Philip N.; O'Garra, Anne

    2009-01-01

    Stimulation of Toll-like receptors (TLRs) on macrophages and dendritic cells (DCs) by pathogen-derived products induces the production of cytokines, which play an important role in immune responses. Here, we investigated the role of the TPL-2 signaling pathway in TLR induction of interferon-β (IFN-β) and interleukin-10 (IL-10) in these cell types. It has previously been suggested that IFN-β and IL-10 are coordinately regulated after TLR stimulation. However, in the absence of TPL-2 signaling, lipopolysaccharide (TLR4) and CpG (TLR9) stimulation resulted in increased production of IFN-β while decreasing IL-10 production by both macrophages and myeloid DCs. In contrast, CpG induction of both IFN-α and IFN-β by plasmacytoid DCs was decreased in the absence of TPL-2, although extracellular signal-regulated kinase (ERK) activation was blocked. Extracellular signal-related kinase–dependent negative regulation of IFN-β in macrophages was IL-10–independent, required protein synthesis, and was recapitulated in TPL-2–deficient myeloid DCs by retroviral transduction of the ERK-dependent transcription factor c-fos. PMID:19667062

  19. Ikaros Is a Negative Regulator of B1 Cell Development and Function.

    PubMed

    Macias-Garcia, Alejandra; Heizmann, Beate; Sellars, MacLean; Marchal, Patricia; Dali, Hayet; Pasquali, Jean-Louis; Muller, Sylviane; Kastner, Philippe; Chan, Susan

    2016-04-22

    B1 B cells secrete most of the circulating natural antibodies and are considered key effector cells of the innate immune response. However, B1 cell-associated antibodies often cross-react with self-antigens, which leads to autoimmunity, and B1 cells have been implicated in cancer. How B1 cell activity is regulated remains unclear. We show that the Ikaros transcription factor is a major negative regulator of B1 cell development and function. Using conditional knock-out mouse models to delete Ikaros at different locations, we show that Ikaros-deficient mice exhibit specific and significant increases in splenic and bone marrow B1 cell numbers, and that the B1 progenitor cell pool is increased ∼10-fold in the bone marrow. Ikaros-null B1 cells resemble WT B1 cells at the molecular and cellular levels, but show a down-regulation of signaling components important for inhibiting proliferation and immunoglobulin production. Ikaros-null B1 cells hyper-react to TLR4 stimulation and secrete high amounts of IgM autoantibodies. These results indicate that Ikaros is required to limit B1 cell homeostasis in the adult.

  20. Integrated expression analysis of muscle hypertrophy identifies Asb2 as a negative regulator of muscle mass

    PubMed Central

    Davey, Jonathan R.; Watt, Kevin I.; Parker, Benjamin L.; Chaudhuri, Rima; Ryall, James G.; Cunningham, Louise; Qian, Hongwei; Sartorelli, Vittorio; Chamberlain, Jeffrey; James, David E.

    2016-01-01

    The transforming growth factor-β (TGF-β) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-β network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature. Quantitative proteomic and transcriptomic analyses of young-adult muscles identified a transcription/translation signature elicited by follistatin exposure, which included repression of ankyrin repeat and SOCS box protein 2 (Asb2). Increasing expression of ASB2 reduced muscle mass, thereby demonstrating that Asb2 is a TGF-β network–responsive negative regulator of muscle mass. In contrast to young-adult muscles, sarcopenic muscles do not exhibit reduced ASB2 abundance with follistatin exposure. Moreover, preventing repression of ASB2 in young-adult muscles diminished follistatin-induced muscle hypertrophy. These findings provide insight into the program of transcription and translation events governing follistatin-mediated adaptation of skeletal muscle attributes and identify Asb2 as a regulator of muscle mass implicated in the potential mechanistic dysfunction between follistatin-mediated muscle growth in young and old muscles. PMID:27182554

  1. Evidence for the negative regulation of phytase gene expression in Streptomyces lividans and Streptomyces coelicolor.

    PubMed

    Boukhris, Ines; Dulermo, Thierry; Chouayekh, Hichem; Virolle, Marie-Joëlle

    2016-01-01

    Sco7697, a gene encoding a phytase, enzyme able to degrade phytate (myo-inositol 1,2,3,4,5,6-hexakis phosphate), the most abundant phosphorus storing compound in plants is present in the genome of S. coelicolor, a soil born bacteria with a saprophytic lifestyle. The expression of this gene was previously shown to be induced in conditions of Pi limitation by the response regulator PhoP binding to an operator sequence, the PHO box, located upstream of the -35 promoter sequence. A close examination of the promoter region of sco7697 revealed the presence of another putative operator site, a Direct Repeat (DR), located downstream of the -10 promoter sequence. In order to determine whether this DR played a role in regulation of sco7697 expression, different variants of the phytase gene promoter region were transcriptionally fused to the ß-glucuronidase reporter gene (GUS). As expected, deletion of the PHO box led to abolition of sco7697 induction in conditions of Pi limitation. Interestingly, alteration of the DR correlated with a dramatic increase of GUS expression but only when PhoP was present. These results demonstrated that this DR is the site of strong negative regulation by an unknown repressor. The latter would impede the necessary activation of phytase expression by PhoP.

  2. Piwi maintains germline stem cells and oogenesis in Drosophila through negative regulation of Polycomb group proteins.

    PubMed

    Peng, Jamy C; Valouev, Anton; Liu, Na; Lin, Haifan

    2016-03-01

    The Drosophila melanogaster Piwi protein regulates both niche and intrinsic mechanisms to maintain germline stem cells, but its underlying mechanism remains unclear. Here we report that Piwi interacts with Polycomb group complexes PRC1 and PRC2 in niche and germline cells to regulate ovarian germline stem cells and oogenesis. Piwi physically interacts with the PRC2 subunits Su(z)12 and Esc in the ovary and in vitro. Chromatin coimmunoprecipitation of Piwi, the PRC2 enzymatic subunit E(z), histone H3 trimethylated at lysine 27 (H3K27me3) and RNA polymerase II in wild-type and piwi mutant ovaries demonstrates that Piwi binds a conserved DNA motif at ∼ 72 genomic sites and inhibits PRC2 binding to many non-Piwi-binding genomic targets and H3K27 trimethylation. Moreover, Piwi influences RNA polymerase II activities in Drosophila ovaries, likely via inhibiting PRC2. We hypothesize that Piwi negatively regulates PRC2 binding by sequestering PRC2 in the nucleoplasm, thus reducing PRC2 binding to many targets and influencing transcription during oogenesis.

  3. Induction of Posttranslational Modifications of Mitochondrial Proteins by ATP Contributes to Negative Regulation of Mitochondrial Function.

    PubMed

    Zhang, Yong; Zhao, Zhiyun; Ke, Bilun; Wan, Lin; Wang, Hui; Ye, Jianping

    2016-01-01

    It is generally accepted that ATP regulates mitochondrial function through the AMPK signaling pathway. However, the AMPK-independent pathway remains largely unknown. In this study, we investigated ATP surplus in the negative regulation of mitochondrial function with a focus on pyruvate dehydrogenase (PDH) phosphorylation and protein acetylation. PDH phosphorylation was induced by a high fat diet in the liver of obese mice, which was associated with ATP elevation. In 1c1c7 hepatoma cells, the phosphorylation was induced by palmitate treatment through induction of ATP production. The phosphorylation was associated with a reduction in mitochondria oxygen consumption after 4 h treatment. The palmitate effect was blocked by etomoxir, which inhibited ATP production through suppression of fatty acid β-oxidation. The PDH phosphorylation was induced by incubation of mitochondrial lysate with ATP in vitro without altering the expression of PDH kinase 2 (PDK2) and 4 (PDK4). In addition, acetylation of multiple mitochondrial proteins was induced by ATP in the same conditions. Acetyl-CoA exhibited a similar activity to ATP in induction of the phosphorylation and acetylation. These data suggest that ATP elevation may inhibit mitochondrial function through induction of the phosphorylation and acetylation of mitochondrial proteins. The results suggest an AMPK-independent mechanism for ATP regulation of mitochondrial function.

  4. A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus

    USGS Publications Warehouse

    Leisy, D.J.; Rasmussen, C.; Owusu, E.O.; Rohrmann, G.F.

    1997-01-01

    The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with Bg/II sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (ple-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1- specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.

  5. Mothers' Socialization of Emotion Regulation: The Moderating Role of Children's Negative Emotional Reactivity

    ERIC Educational Resources Information Center

    Mirabile, Scott P.; Scaramella, Laura V.; Sohr-Preston, Sara L.; Robison, Sarah D.

    2009-01-01

    During the toddler period, children begin to shift from being primarily dependent on parents to regulate their emotions to managing their emotions independently. The present study considers how children's propensity towards negative emotional arousal interacts with mothers' efforts to socialize emotion regulation. Fifty-five low income mothers and…

  6. PIMS modulates immune tolerance by negatively regulating Drosophila innate immune signaling.

    PubMed

    Lhocine, Nouara; Ribeiro, Paulo S; Buchon, Nicolas; Wepf, Alexander; Wilson, Rebecca; Tenev, Tencho; Lemaitre, Bruno; Gstaiger, Matthias; Meier, Pascal; Leulier, François

    2008-08-14

    Metazoans tolerate commensal-gut microbiota by suppressing immune activation while maintaining the ability to launch rapid and balanced immune reactions to pathogenic bacteria. Little is known about the mechanisms underlying the establishment of this threshold. We report that a recently identified Drosophila immune regulator, which we call PGRP-LC-interacting inhibitor of Imd signaling (PIMS), is required to suppress the Imd innate immune signaling pathway in response to commensal bacteria. pims expression is Imd (immune deficiency) dependent, and its basal expression relies on the presence of commensal flora. In the absence of PIMS, resident bacteria trigger constitutive expression of antimicrobial peptide genes (AMPs). Moreover, pims mutants hyperactivate AMPs upon infection with Gram-negative bacteria. PIMS interacts with the peptidoglycan recognition protein (PGRP-LC), causing its depletion from the plasma membrane and shutdown of Imd signaling. Therefore, PIMS is required to establish immune tolerance to commensal bacteria and to maintain a balanced Imd response following exposure to bacterial infections.

  7. NK Cells Alleviate Lung Inflammation by Negatively Regulating Group 2 Innate Lymphoid Cells.

    PubMed

    Bi, Jiacheng; Cui, Lulu; Yu, Guang; Yang, Xiaolu; Chen, Youhai; Wan, Xiaochun

    2017-03-08

    Group 2 innate lymphoid cells (ILC2s) play an important role in orchestrating type II immune responses. However, the cellular mechanisms of group 2 innate lymphoid cell regulation remain poorly understood. In this study, we found that activated NK cells inhibited the proliferation of, as well as IL-5 and IL-13 production by, ILC2s in vitro via IFN-γ. In addition, in a murine model of ILC2 expansion in the liver, polyinosinic-polycytidylic acid, an NK cell-activating agent, inhibited ILC2 proliferation, IL-5 and IL-13 production, and eosinophil recruitment. Such effects of polyinosinic-polycytidylic acid were abrogated in NK cell-depleted mice and in IFN-γ-deficient mice. Adoptively transferring wild-type NK cells into NK cell-depleted mice resulted in fewer ILC2s induced by IL-33 compared with the transfer of IFN-γ-deficient NK cells. Importantly, during the early stage of papain- or bleomycin-induced lung inflammation, depletion of NK cells resulted in increased ILC2 numbers and enhanced cytokine production by ILC2s, as well as aggravated eosinophilia and goblet cell hyperplasia. Collectively, these data show that NK cells negatively regulate ILC2s during the early stage of lung inflammation, which represents the novel cellular interaction between two family members of ILCs.

  8. NCoR negatively regulates adipogenic differentiation of mesenchymal stem cells.

    PubMed

    Hong-Wei, Gao; Lan, Liu; De-Guo, Xing; Zhong-Hao, Liu; Peng, Ren; Zhi-Qiang, Li; Guo-Qiang, Shan; Ming-Zhi, Gong

    2015-08-01

    The nuclear receptor corepressor (NCoR) regulates the activities of gene transcription. Mesenchymal stem cells (MSCs) derived from bone marrow are multipotent cells which can differentiate into osteoblasts and adipocytes. This study was conducted to investigate the effects of NCoR on adipogenic differentiation of MSCs isolated from the rats. The results suggested that rat MSCs could differentiate into adipocytes successfully after cultured in adipogenic medium. NCoR protein determined by Western blot showed a lower expression in MSC-derived adipocytes, indicating that NCoR was involved in adipocyte differentiation of rat MSCs. It further proved that small interfering RNA (siRNA)-mediated knockdown of NCoR could promote cell viability and differentiation and enhance messenger RNA (mRNA) expression of lipoprotein lipase (LPL) and protein expression of CCAAT/enhancer binding protein-α (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ). However, over-expression of NCoR exerted its functions in contrary to NCoR knockdown. It indicated that NCoR could negatively regulate adipogenic differentiation of rat MSCs.

  9. SOCS1 Mimetics and Antagonists: A Complementary Approach to Positive and Negative Regulation of Immune Function

    PubMed Central

    Ahmed, Chulbul M. I.; Larkin, Joseph; Johnson, Howard M.

    2015-01-01

    Suppressors of cytokine signaling (SOCS) are inducible intracellular proteins that play essential regulatory roles in both immune and non-immune function. Of the eight known members, SOCS1 and SOCS3 in conjunction with regulatory T cells play key roles in regulation of the immune system. Molecular tools such as gene transfections and siRNA have played a major role in our functional understanding of the SOCS proteins where a key functional domain of 12-amino acid residues called the kinase inhibitory region (KIR) has been identified on SOCS1 and SOCS3. KIR plays a key role in inhibition of the JAK2 tyrosine kinase, which in turn plays a key role in cytokine signaling. A peptide corresponding to KIR (SOCS1-KIR) bound to the activation loop of JAK2 and inhibited tyrosine phosphorylation of STAT1α transcription factor by JAK2. Cell internalized SOCS1-KIR is a potent therapeutic in the experimental allergic encephalomyelitis (EAE) mouse model of multiple sclerosis and showed promise in a psoriasis model and a model of diabetes-associated cardiovascular disease. By contrast, a peptide, pJAK2(1001–1013), that corresponds to the activation loop of JAK2 is a SOCS1 antagonist. The antagonist enhanced innate and adaptive immune response against a broad range of viruses including herpes simplex virus, vaccinia virus, and an EMC picornavirus. SOCS mimetics and antagonists are thus potential therapeutics for negative and positive regulation of the immune system. PMID:25954276

  10. RUNX3 is a novel negative regulator of oncogenic TEAD-YAP complex in gastric cancer.

    PubMed

    Qiao, Y; Lin, S J; Chen, Y; Voon, D C-C; Zhu, F; Chuang, L S H; Wang, T; Tan, P; Lee, S C; Yeoh, K G; Sudol, M; Ito, Y

    2016-05-19

    Runt-related transcription factor 3 (RUNX3) is a well-documented tumour suppressor that is frequently inactivated in gastric cancer. Here, we define a novel mechanism by which RUNX3 exerts its tumour suppressor activity involving the TEAD-YAP complex, a potent positive regulator of proliferative genes. We report that the TEAD-YAP complex is not only frequently hyperactivated in liver and breast cancer, but also confers a strong oncogenic activity in gastric epithelial cells. The increased expression of TEAD-YAP in tumour tissues significantly correlates with poorer overall survival of gastric cancer patients. Strikingly, RUNX3 physically interacts with the N-terminal region of TEAD through its Runt domain. This interaction markedly reduces the DNA-binding ability of TEAD that attenuates the downstream signalling of TEAD-YAP complex. Mutation of RUNX3 at Arginine 122 to Cysteine, which was previously identified in gastric cancer, impairs the interaction between RUNX3 and TEAD. Our data reveal that RUNX3 acts as a tumour suppressor by negatively regulating the TEAD-YAP oncogenic complex in gastric carcinogenesis.

  11. Automatic control of negative emotions: evidence that structured practice increases the efficiency of emotion regulation.

    PubMed

    Christou-Champi, Spyros; Farrow, Tom F D; Webb, Thomas L

    2015-01-01

    Emotion regulation (ER) is vital to everyday functioning. However, the effortful nature of many forms of ER may lead to regulation being inefficient and potentially ineffective. The present research examined whether structured practice could increase the efficiency of ER. During three training sessions, comprising a total of 150 training trials, participants were presented with negatively valenced images and asked either to "attend" (control condition) or "reappraise" (ER condition). A further group of participants did not participate in training but only completed follow-up measures. Practice increased the efficiency of ER as indexed by decreased time required to regulate emotions and increased heart rate variability (HRV). Furthermore, participants in the ER condition spontaneously regulated their negative emotions two weeks later and reported being more habitual in their use of ER. These findings indicate that structured practice can facilitate the automatic control of negative emotions and that these effects persist beyond training.

  12. Metacognitive emotion regulation: children's awareness that changing thoughts and goals can alleviate negative emotions.

    PubMed

    Davis, Elizabeth L; Levine, Linda J; Lench, Heather C; Quas, Jodi A

    2010-08-01

    Metacognitive emotion regulation strategies involve deliberately changing thoughts or goals to alleviate negative emotions. Adults commonly engage in this type of emotion regulation, but little is known about the developmental roots of this ability. Two studies were designed to assess whether 5- and 6-year-old children can generate such strategies and, if so, the types of metacognitive strategies they use. In Study 1, children described how story protagonists could alleviate negative emotions. In Study 2, children recalled times that they personally had felt sad, angry, and scared and described how they had regulated their emotions. In contrast to research suggesting that young children cannot use metacognitive regulation strategies, the majority of children in both studies described such strategies. Children were surprisingly sophisticated in their suggestions for how to cope with negative emotions and tailored their regulatory responses to specific emotional situations.

  13. The Immunophilin-Like Protein XAP2 Is a Negative Regulator of Estrogen Signaling through Interaction with Estrogen Receptor α

    PubMed Central

    Berg, Petra; Korbonits, Marta; Pongratz, Ingemar

    2011-01-01

    XAP2 (also known as aryl hydrocarbon receptor interacting protein, AIP) is originally identified as a negative regulator of the hepatitis B virus X-associated protein. Recent studies have expanded the range of XAP2 client proteins to include the nuclear receptor family of transcription factors. In this study, we show that XAP2 is recruited to the promoter of ERα regulated genes like the breast cancer marker gene pS2 or GREB1 and negatively regulate the expression of these genes in MCF-7 cells. Interestingly, we show that XAP2 downregulates the E2-dependent transcriptional activation in an estrogen receptor (ER) isoform-specific manner: XAP2 inhibits ERα but not ERβ-mediated transcription. Thus, knockdown of intracellular XAP2 levels leads to increased ERα activity. XAP2 proteins, carrying mutations in their primary structures, loose the ability of interacting with ERα and can no longer regulate ER target gene transcription. Taken together, this study shows that XAP2 exerts a negative effect on ERα transcriptional activity and may thus prevent ERα-dependent events. PMID:21984905

  14. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells.

    PubMed

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-04-04

    Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA1-LPA6) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA1 and LPA5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA1.

  15. Asymmetric frontal cortical activity and negative affective responses to ostracism.

    PubMed

    Peterson, Carly K; Gravens, Laura C; Harmon-Jones, Eddie

    2011-06-01

    Ostracism arouses negative affect. However, little is known about variables that influence the intensity of these negative affective responses. Two studies fill this void by incorporating work on approach- and withdrawal-related emotional states and their associated cortical activations. Study 1 found that following ostracism anger related directly to relative left frontal cortical activation. Study 2 used unilateral hand contractions to manipulate frontal cortical activity prior to an ostracizing event. Right-hand contractions, compared to left-hand contractions, caused greater relative left frontal cortical activation during the hand contractions as well as ostracism. Also, right-hand contractions caused more self-reported anger in response to being ostracized. Within-condition correlations revealed patterns of associations between ostracism-induced frontal asymmetry and emotive responses to ostracism consistent with Study 1. Taken together, these results suggest that asymmetrical frontal cortical activity is related to angry responses to ostracism, with greater relative left frontal cortical activity being associated with increased anger.

  16. Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6.

    PubMed

    Min, Yoon; Wi, Sae Mi; Kang, Jung-Ah; Yang, Taewoo; Park, Chul-Seung; Park, Sung-Gyoo; Chung, Sungkwon; Shim, Jae-Hyuck; Chun, Eunyoung; Lee, Ki-Young

    2016-07-28

    Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1β in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn(-/-) mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination.

  17. Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6

    PubMed Central

    Min, Yoon; Wi, Sae Mi; Kang, Jung-Ah; Yang, Taewoo; Park, Chul-Seung; Park, Sung-Gyoo; Chung, Sungkwon; Shim, Jae-Hyuck; Chun, Eunyoung; Lee, Ki-Young

    2016-01-01

    Cereblon (CRBN) is a substrate receptor protein for the CRL4A E3 ubiquitin ligase complex. In this study, we report on a new regulatory role of CRBN in TLR4 signaling. CRBN overexpression leads to suppression of NF-κB activation and production of pro-inflammatory cytokines including IL-6 and IL-1β in response to TLR4 stimulation. Biochemical studies revealed interactions between CRBN and TAK1, and TRAF6 proteins. The interaction between CRBN and TAK1 did not affect the association of the TAB1 and TAB2 proteins, which have pivotal roles in the activation of TAK1, whereas the CRBN-TRAF6 interaction critically affected ubiquitination of TRAF6 and TAB2. Binding mapping results revealed that CRBN interacts with the Zinc finger domain of TRAF6, which contains the ubiquitination site of TRAF6, leading to attenuation of ubiquitination of TRAF6 and TAB2. Functional studies revealed that CRBN-knockdown THP-1 cells show enhanced NF-κB activation and p65- or p50-DNA binding activities, leading to up-regulation of NF-κB-dependent gene expression and increased pro-inflammatory cytokine levels in response to TLR4 stimulation. Furthermore, Crbn−/− mice exhibit decreased survival in response to LPS challenge, accompanied with marked enhancement of pro-inflammatory cytokines, such as TNF-α and IL-6. Taken together, our data demonstrate that CRBN negatively regulates TLR4 signaling via attenuation of TRAF6 and TAB2 ubiquitination. PMID:27468689

  18. The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity

    PubMed Central

    Heinz, Leonhard X.; Baumann, Christoph L.; Köberlin, Marielle S.; Snijder, Berend; Gawish, Riem; Shui, Guanghou; Sharif, Omar; Aspalter, Irene M.; Müller, André C.; Kandasamy, Richard K.; Breitwieser, Florian P.; Pichlmair, Andreas; Bruckner, Manuela; Rebsamen, Manuele; Blüml, Stephan; Karonitsch, Thomas; Fauster, Astrid; Colinge, Jacques; Bennett, Keiryn L.; Knapp, Sylvia; Wenk, Markus R.; Superti-Furga, Giulio

    2015-01-01

    Summary Lipid metabolism and receptor-mediated signaling are highly intertwined processes that cooperate to fulfill cellular functions and safeguard cellular homeostasis. Activation of Toll-like receptors (TLRs) leads to a complex cellular response, orchestrating a diverse range of inflammatory events that need to be tightly controlled. Here, we identified the GPI-anchored Sphingomyelin Phosphodiesterase, Acid-Like 3B (SMPDL3B) in a mass spectrometry screening campaign for membrane proteins co-purifying with TLRs. Deficiency of Smpdl3b in macrophages enhanced responsiveness to TLR stimulation and profoundly changed the cellular lipid composition and membrane fluidity. Increased cellular responses could be reverted by re-introducing affected ceramides, functionally linking membrane lipid composition and innate immune signaling. Finally, Smpdl3b-deficient mice displayed an intensified inflammatory response in TLR-dependent peritonitis models, establishing its negative regulatory role in vivo. Taken together, our results identify the membrane-modulating enzyme SMPDL3B as a negative regulator of TLR signaling that functions at the interface of membrane biology and innate immunity. PMID:26095358

  19. Negative regulation of Jak2 by its auto-phosphorylation at tyrosine 913 via the Epo signaling pathway.

    PubMed

    Funakoshi-Tago, Megumi; Tago, Kenji; Kasahara, Tadashi; Parganas, Evan; Ihle, James N

    2008-11-01

    Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.

  20. The master switch gene Sex-lethal promotes female development by negatively regulating the N signaling pathway

    PubMed Central

    Penn, Jill K M; Schedl, Paul

    2011-01-01

    Summary Notch (N) signaling is used for cell fate determination in many different developmental contexts. Here we show that the master control gene for sex determination in Drosophila melanogaster, Sex-lethal (Sxl), negatively regulates the N signaling pathway in females. In genetic assays, reducing Sxl activity suppresses the phenotypic effects of N mutations while increasing Sxl activity enhances the effects. Sxl appears to negatively regulate the pathway by reducing N protein accumulation and higher levels of N are found in Sxl−clones than in adjacent wild type cells. The inhibition of N expression does not depend on the known downstream targets of Sxl; however we find that Sxl protein can bind to N mRNAs. Finally our results indicate that downregulation of the N pathway by Sxl contributes to sex specific differences in morphology and suggest that it may also play an important role in follicle cell specification during oogenesis. PMID:17276344

  1. Comparing Models for Generating a System of Activation and Inhibition of Self-Regulated Learning

    ERIC Educational Resources Information Center

    Magno, Carlo

    2008-01-01

    The study investigated the effect of activation and negative affect on self-regulation. The activation factors are self-determination, disengagement, initiative, and persistence while negative affect is composed of worry, anxiety, thought suppression, and fear of negative evaluation. Separate measures were used for each factor and administered to…

  2. Insulin receptor substrate 2 is a negative regulator of memory formation

    PubMed Central

    Irvine, Elaine E.; Drinkwater, Laura; Radwanska, Kasia; Al-Qassab, Hind; Smith, Mark A.; O’Brien, Melissa; Kielar, Catherine; Choudhury, Agharul I; Krauss, Stefan; Cooper, Jonathan D.; Withers, Dominic J.; Giese, K. Peter

    2015-01-01

    Insulin has been shown to impact on learning and memory in both humans and animals, but the downstream signaling mechanisms involved are poorly characterized. Insulin receptor substrate-2 (Irs2) is an adaptor protein that couples activation of insulin- and insulin-like growth factor-1- receptors to downstream signaling pathways. Here, we have deleted Irs2, either in the whole brain or selectively in the forebrain, using the nestin Cre- or D6 Cre- deleter mouse lines respectively. We show that brain- and forebrain-specific Irs2 knockout mice have enhanced hippocampal spatial reference memory. Furthermore, NesCreIrs2KO mice have enhanced spatial working memory and contextual- and cued-fear memory. Deletion of Irs2 in the brain also increases PSD-95 expression and the density of dendritic spines in hippocampal area CA1, possibly reflecting an increase in the number of excitatory synapses per neuron in the hippocampus that can become activated during memory formation. This increase in activated excitatory synapses might underlie the improved hippocampal memory formation observed in NesCreIrs2KO mice. Overall, these results suggest that Irs2 acts as a negative regulator on memory formation by restricting dendritic spine generation. PMID:21597043

  3. Egr2 induced during DC development acts as an intrinsic negative regulator of DC immunogenicity.

    PubMed

    Miah, Mohammad Alam; Byeon, Se Eun; Ahmed, Md Selim; Yoon, Cheol-Hee; Ha, Sang-Jun; Bae, Yong-Soo

    2013-09-01

    Early growth response gene 2 (Egr2), which encodes a zinc finger transcription factor, is rapidly and transiently induced in various cell types independently of de novo protein synthesis. Although a role for Egr2 is well established in T-cell development, Egr2 expression and its biological function in dendritic cells (DCs) have not yet been described. Here, we demonstrate Egr2 expression during DC development, and its role in DC-mediated immune responses. Egr2 is expressed in the later stage of DC development from BM precursor cells. Even at steady state, Egr2 is highly expressed in mouse splenic DCs. Egr2-knockdown (Egr2-KD) DCs showed increased levels of major histocompatability complex (MHC) class I and II and co-stimulatory molecules, and enhanced antigen uptake and migratory capacities. Furthermore, Egr2-KD abolished SOCS1 expression and signal transducer and activator of transcription 5 (STAT5) activation during DC development, probably resulting in the enhancement of IL-12 expression and Th1 immunogenicity of a DC vaccine. DC-mediated cytotoxic T lymphocyte (CTL) activation and antitumor immunity were significantly enhanced by Egr2-KD, and impaired by Egr2 overexpression in antigen-pulsed DC vaccines. These data suggest that Egr2 acts as an intrinsic negative regulator of DC immunogenicity and can be an attractive molecular target for DC vaccine development.

  4. Neuronal leucine-rich repeat 1 negatively regulates anaplastic lymphoma kinase in neuroblastoma

    PubMed Central

    Satoh, Shunpei; Takatori, Atsushi; Ogura, Atsushi; Kohashi, Kenichi; Souzaki, Ryota; Kinoshita, Yoshiaki; Taguchi, Tomoaki; Hossain, Md. Shamim; Ohira, Miki; Nakamura, Yohko; Nakagawara, Akira

    2016-01-01

    In neuroblastoma (NB), one of the most common paediatric solid tumours, activation of anaplastic lymphoma kinase (ALK) is often associated with poor outcomes. Although genetic studies have identified copy number alteration and nonsynonymous mutations of ALK, the regulatory mechanism of ALK signalling at protein levels is largely elusive. Neuronal leucine-rich repeat 1 (NLRR1) is a type 1 transmembrane protein that is highly expressed in unfavourable NB and potentially influences receptor tyrosine kinase signalling. Here, we showed that NLRR1 and ALK exhibited a mutually exclusive expression pattern in primary NB tissues by immunohistochemistry. Moreover, dorsal root ganglia of Nlrr1+/+ and Nlrr1−/− mice displayed the opposite expression patterns of Nlrr1 and Alk. Of interest, NLRR1 physically interacted with ALK in vitro through its extracellular region. Notably, the NLRR1 ectodomain impaired ALK phosphorylation and proliferation of ALK-mutated NB cells. A newly identified cleavage of the NLRR1 ectodomain also supported NLRR1-mediated ALK signal regulation in trans. Thus, we conclude that NLRR1 appears to be an extracellular negative regulator of ALK signalling in NB and neuronal development. Our findings may be beneficial to comprehend NB heterogeneity and to develop a novel therapy against unfavourable NB. PMID:27604320

  5. SOCS3 is a critical physiological negative regulator of G-CSF signaling and emergency granulopoiesis.

    PubMed

    Croker, Ben A; Metcalf, Donald; Robb, Lorraine; Wei, Wei; Mifsud, Sandra; DiRago, Ladina; Cluse, Leonie A; Sutherland, Kate D; Hartley, Lynne; Williams, Emily; Zhang, Jian-Guo; Hilton, Douglas J; Nicola, Nicos A; Alexander, Warren S; Roberts, Andrew W

    2004-02-01

    To determine the importance of suppressor of cytokine signaling-3 (SOCS3) in the regulation of hematopoietic growth factor signaling generally, and of G-CSF-induced cellular responses specifically, we created mice in which the Socs3 gene was deleted in all hematopoietic cells. Although normal until young adulthood, these mice then developed neutrophilia and a spectrum of inflammatory pathologies. When stimulated with G-CSF in vitro, SOCS3-deficient cells of the neutrophilic granulocyte lineage exhibited prolonged STAT3 activation and enhanced cellular responses to G-CSF, including an increase in cloning frequency, survival, and proliferative capacity. Consistent with the in vitro findings, mutant mice injected with G-CSF displayed enhanced neutrophilia, progenitor cell mobilization, and splenomegaly, but unexpectedly also developed inflammatory neutrophil infiltration into multiple tissues and consequent hind-leg paresis. We conclude that SOCS3 is a key negative regulator of G-CSF signaling in myeloid cells and that this is of particular significance during G-CSF-driven emergency granulopoiesis.

  6. Regulated Breathing Effect of Silicon Negative Electrode for Dramatically Enhanced Performance of Li-Ion Battery

    SciTech Connect

    Xiao, Xingcheng; Zhou, Weidong; Kim, Youngnam; Ryu, Ill; Gu, Meng; Wang, Chong M.; Liu, Gao; Liu, Zhongyi; Gao, Huajian

    2015-03-01

    Si is an attractive negative electrode material for lithium ion batteries due to its high specifi c capacity (≈3600 mAh g –1 ). However, the huge volume swelling and shrinking during cycling, which mimics a breathing effect at the material/electrode/cell level, leads to several coupled issues including fracture of Si particles, unstable solid electrolyte interphase, and low Coulombic effi ciency. In this work, the regulation of the breathing effect is reported by using Si–C yolk–shell nanocomposite which has been well-developed by other researchers. The focus is on understanding how the nanoscaled materials design impacts the mechanical and electrochemical response at electrode level. For the fi rst time, it is possible to observe one order of magnitude of reduction on breathing effect at the electrode level during cycling: the electrode thickness variation reduced down to 10%, comparing with 100% in the electrode with Si nanoparticles as active materials. The Si–C yolk–shell nanocomposite electrode exhibits excellent capacity retention and high cycle effi ciency. In situ transmission electron microscopy and fi nite element simulations consistently reveals that the dramatically enhanced performance is associated with the regulated breathing of the Si in the new composite, therefore the suppression of the overall electrode expansion.

  7. TDP-43 regulates its mRNA levels through a negative feedback loop

    PubMed Central

    Ayala, Youhna M; De Conti, Laura; Avendaño-Vázquez, S Eréndira; Dhir, Ashish; Romano, Maurizio; D'Ambrogio, Andrea; Tollervey, James; Ule, Jernej; Baralle, Marco; Buratti, Emanuele; Baralle, Francisco E

    2011-01-01

    TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3′ UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43–hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3′ UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis. PMID:21131904

  8. Purification and Crystallization of Murine Myostatin: A Negative Regulator of Muscle Mass

    NASA Technical Reports Server (NTRS)

    Hong, Young S.; Adamek, Daniel; Bridge, Kristi; Malone, Christine C.; Young, Ronald B.; Miller, Teresa; Karr, Laurel

    2004-01-01

    Myostatin (MSTN) has been crystallized and its preliminary X-ray diffraction data were collected. MSTN is a negative regulator of muscle growt/differentiation and suppressor of fat accumulation. It is a member of TGF-b family of proteins. Like other members of this family, the regulation of MSTN is critically tied to its process of maturation. This process involves the formation of a homodimer followed by two proteolytic steps. The first proteolytic cleavage produces a species where the n-terminal portion of the dimer is covalently separated from, but remains non-covalently bound to, the c-terminal, functional, portion of the protein. The protein is activated upon removal of the n-terminal "pro-segment" by a second n-terminal proteolytic cut by BMP-1 in vivo, or by acid treatment in vitro. Understanding the structural nature and physical interactions involved in these regulatory processes is the objective of our studies. Murine MSTN was purified from culture media of genetically engineered Chinese Hamster Ovary cells by multicolumn purification process and crystallized using the vapor diffusion method.

  9. Rethinking emotion: cognitive reappraisal is an effective positive and negative emotion regulation strategy in bipolar disorder.

    PubMed

    Gruber, June; Hay, Aleena C; Gross, James J

    2014-04-01

    Bipolar disorder involves difficulties with emotion regulation, yet the precise nature of these emotion regulatory difficulties is unclear. The current study examined whether individuals with remitted bipolar I disorder (n = 23) and healthy controls (n = 23) differ in their ability to use one effective and common form of emotion regulation, cognitive reappraisal. Positive, negative, and neutral films were used to elicit emotion, and participants were cued to watch the film carefully (i.e., uninstructed condition) or reappraise while measures of affect, behavior, and psychophysiology were obtained. Results showed that reappraisal was associated with reductions in emotion reactivity across subjective (i.e., positive and negative affect), behavioral (i.e., positive facial displays), and physiological (i.e., skin conductance) response domains across all participants. Results suggest that reappraisal may be an effective regulation strategy for both negative and positive emotion across both healthy adults and individuals with bipolar disorder. Discussion focuses on clinical and treatment implications for bipolar disorder.

  10. Transcription factor Foxo1 is a negative regulator of natural killer cell maturation and function.

    PubMed

    Deng, Youcai; Kerdiles, Yann; Chu, Jianhong; Yuan, Shunzong; Wang, Youwei; Chen, Xilin; Mao, Hsiaoyin; Zhang, Lingling; Zhang, Jianying; Hughes, Tiffany; Deng, Yafei; Zhang, Qi; Wang, Fangjie; Zou, Xianghong; Liu, Chang-Gong; Freud, Aharon G; Li, Xiaohui; Caligiuri, Michael A; Vivier, Eric; Yu, Jianhua

    2015-03-17

    Little is known about the role of negative regulators in controlling natural killer (NK) cell development and effector functions. Foxo1 is a multifunctional transcription factor of the forkhead family. Using a mouse model of conditional deletion in NK cells, we found that Foxo1 negatively controlled NK cell differentiation and function. Immature NK cells expressed abundant Foxo1 and little Tbx21 relative to mature NK cells, but these two transcription factors reversed their expression as NK cells proceeded through development. Foxo1 promoted NK cell homing to lymph nodes by upregulating CD62L expression and inhibited late-stage maturation and effector functions by repressing Tbx21 expression. Loss of Foxo1 rescued the defect in late-stage NK cell maturation in heterozygous Tbx21(+/-) mice. Collectively, our data reveal a regulatory pathway by which the negative regulator Foxo1 and the positive regulator Tbx21 play opposing roles in controlling NK cell development and effector functions.

  11. Negative control in two-component signal transduction by transmitter phosphatase activity.

    PubMed

    Huynh, TuAnh Ngoc; Stewart, Valley

    2011-10-01

    Bifunctional sensor transmitter modules of two-component systems exert both positive and negative control on the receiver domain of the cognate response regulator. In negative control, the transmitter module accelerates the rate of phospho-receiver dephosphorylation. This transmitter phosphatase reaction serves the important physiological functions of resetting response regulator phosphorylation level and suppressing cross-talk. Although the biochemical reactions underlying positive control are reasonably well understood, the mechanism for transmitter phosphatase activity has been unknown. A recent hypothesis is that the transmitter phosphatase reaction is catalysed by a conserved Gln, Asn or Thr residue, via a hydrogen bond between the amide or hydroxyl group and the nucleophilic water molecule in acyl-phosphate hydrolysis. This hypothetical mechanism closely resembles the established mechanisms of auxiliary phosphatases such as CheZ and CheX, and may be widely conserved in two-component signal transduction. In addition to the proposed catalytic residues, transmitter phosphatase activity also requires the correct transmitter conformation and appropriate interactions with the receiver. Evidence suggests that the phosphatase-competent and autokinase-competent states are mutually exclusive, and the corresponding negative and positive activities are likely to be reciprocally regulated through dynamic control of transmitter conformations.

  12. Negatively charged liposomes show potent adjuvant activity when simply admixed with protein antigens

    PubMed Central

    Yanasarn, Nijaporn; Sloat, Brian R.; Cui, Zhengrong

    2011-01-01

    Liposomes have been investigated extensively as a vaccine delivery system. Herein the adjuvant activities of liposomes with different net surface charges (neutral, positive, or negative) were evaluated when admixed with protein antigens, ovalbumin (OVA, pI = 4.7), Bacillus anthracis protective antigen protein (PA, pI = 5.6), or cationized OVA (cOVA). Mice immunized subcutaneously with OVA admixed with different liposomes generated different antibody responses. Interestingly, OVA admixed with net negatively charged liposomes prepared with DOPA was as immunogenic as OVA admixed with positively charged liposomes prepared with DOTAP. Immunization of mice with the anthrax PA protein admixed with the net negatively charged DOPA liposomes also induced a strong and functional anti-PA antibody response. When the cationized OVA was used as a model antigen, liposomes with net neutral, negative, or positive charges showed comparable adjuvant activities. Immunization of mice with the OVA admixed with DOPA liposomes also induced OVA-specific CD8+ cytotoxic T lymphocyte responses and significantly delayed the growth of OVA-expressing B16-OVA tumors in mice. However, not all net negatively charged liposomes showed a strong adjuvant activity. The adjuvant activity of the negatively charged liposomes may be related to the liposome’s ability (i) to up-regulate the expression of molecules related to the activation and maturation of antigen-presenting cells and (ii) to slightly facilitate the uptake of the antigens by antigen-presenting cells. Simply admixing certain negatively charged liposomes with certain protein antigens of interest may represent a novel platform for vaccine development. PMID:21615153

  13. Negative Regulation of the Endocytic Adaptor Disabled-2 (Dab2) in Mitosis*

    PubMed Central

    Chetrit, David; Barzilay, Lior; Horn, Galit; Bielik, Tom; Smorodinsky, Nechama I.; Ehrlich, Marcelo

    2011-01-01

    Mitotic cells undergo extensive changes in shape and size through the altered regulation and function of their membrane trafficking machinery. Disabled 2 (Dab2), a multidomain cargo-specific endocytic adaptor and a mediator of signal transduction, is a potential integrator of trafficking and signaling. Dab2 binds effectors of signaling and trafficking that localize to different intracellular compartments. Thus, differential localization is a putative regulatory mechanism of Dab2 function. Furthermore, Dab2 is phosphorylated in mitosis and is thus regulated in the cell cycle. However, a detailed description of the intracellular localization of Dab2 in the different phases of mitosis and an understanding of the functional consequences of its phosphorylation are lacking. Here, we show that Dab2 is progressively displaced from the membrane in mitosis. This phenomenon is paralleled by a loss of co-localization with clathrin. Both phenomena culminate in metaphase/anaphase and undergo partial recovery in cytokinesis. Treatment with 2-methoxyestradiol, which arrests cells at the spindle assembly checkpoint, induces the same effects observed in metaphase cells. Moreover, 2-methoxyestradiol also induced Dab2 phosphorylation and reduced Dab2/clathrin interactions, endocytic vesicle motility, clathrin exchange dynamics, and the internalization of a receptor endowed with an NPXY endocytic signal. Serine/threonine to alanine mutations, of residues localized to the central region of Dab2, attenuated its phosphorylation, reduced its membrane displacement, and maintained its endocytic abilities in mitosis. We propose that the negative regulation of Dab2 is part of an accommodation of the cell to the altered physicochemical conditions prevalent in mitosis, aimed at allowing endocytic activity throughout the cell cycle. PMID:21097498

  14. Spontaneous emotion regulation during evaluated speaking tasks: associations with negative affect, anxiety expression, memory, and physiological responding.

    PubMed

    Egloff, Boris; Schmukle, Stefan C; Burns, Lawrence R; Schwerdtfeger, Andreas

    2006-08-01

    In these studies, the correlates of spontaneously using expressive suppression and cognitive reappraisal during stressful speeches were examined. Spontaneous emotion regulation means that there were no instructions of how to regulate emotions during the speech. Instead, participants indicated after the speech to what extent they used self-motivated expressive suppression or reappraisal during the task. The results show that suppression is associated with less anxiety expression, greater physiological responding, and less memory for the speech while having no impact on negative affect. In contrast, reappraisal has no impact on physiology and memory while leading to less expression and affect. Taken together, spontaneous emotion regulation in active coping tasks has similar consequences as experimentally induced emotion regulation in passive tasks.

  15. The Transcription Activity of Gis1 Is Negatively Modulated by Proteasome-mediated Limited Proteolysis*

    PubMed Central

    Zhang, Nianshu; Oliver, Stephen G.

    2010-01-01

    The transcriptional response to environmental changes has to be prompt but appropriate. Previously, it has been shown that the Gis1 transcription factor is responsible for regulating the expression of postdiauxic shift genes in response to nutrient starvation, and this transcription regulation is dependent upon the Rim15 kinase. Here we demonstrate that the activity of Gis1 is negatively modulated by proteasome-mediated limited proteolysis. Limited degradation of Gis1 by the proteasome leads to the production of smaller variants, which have weaker transcription activities than the full-length protein. The coiled-coil domain, absent from the smaller variants, is part of the second transcription activation domain in Gis1 and is essential for both the limited proteolysis of Gis1 and its full activity. Endogenous Gis1 and its variants, regardless of their transcription capabilities, activate transcription in a Rim15-dependent manner. However, when the full-length Gis1 accumulates in cells due to overexpression or inhibition of the proteasome function, transcription activation by Gis1 is no longer solely controlled by Rim15. Together, these data strongly indicate that the function of the limited degradation is to ensure that Gis1-dependent transcription is strictly regulated by the Rim15 kinase. Furthermore, we have revealed that the kinase activity of Rim15 is essential for this regulation. PMID:20022953

  16. Negative regulation of NaF-induced apoptosis by Bad-CAII complex.

    PubMed

    Otsuki, S; Sugiyama, K; Amano, O; Yasui, T; Sakagami, H

    2011-09-05

    Fluoride is used to prevent caries in dentistry. However, its mechanism of cytotoxicity induction is unclear. This study was undertaken to determine whether sodium fluoride (NaF) induces apoptosis in human oral cells and if so, whether Bad protein is involved in the process. NaF showed higher cytotoxicity and apoptosis-inducing activity against human oral squamous cell carcinoma cells (HSC-2) than against human gingival fibroblasts (HGF). Western blot analysis showed that NaF enhanced the expression and dephosphorylation of Bad protein. This study demonstrates for the first time that Bad protein forms a complex with carbonic anhydrase II (CAII), and NaF stimulates the detachment of CAII from the Bad-CAII complex and the replacement by the formation of Bad-Bcl-2 complex. Knockdown of Bad and CAII mRNA by siRNA inhibited and enhanced the NaF-induced caspase activation, respectively. The present study suggests that CAII negatively regulates the NaF-induced apoptosis by forming a complex with Bad.

  17. PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells

    PubMed Central

    Vaid, Samir; Ariz, Mohd; Chaturbedi, Amaresh; Kumar, Ganga Anil; Subramaniam, Kuppuswamy

    2013-01-01

    Signals that promote germ cell self-renewal by preventing premature meiotic entry are well understood. However, signals that control mitotic proliferation to promote meiotic differentiation have not been well characterized. In Caenorhabditis elegans, GLP-1 Notch signalling promotes the proliferative fate by preventing premature meiotic entry. The germline niche cell, which is the source of the ligand for GLP-1, spatially restricts GLP-1 signalling and thus enables the germ cells that have moved away from the niche to enter meiosis. Here, we show that the suppression of RAS/MAP kinase signalling in the mitotic and meiotic-entry regions is essential for the regulation of the mitosis-meiosis switch by niche signalling. We provide evidence that the conserved PUF family RNA-binding protein PUF-8 and the RAS GAP protein GAP-3 function redundantly to suppress the LET-60 RAS in the mitotic and meiotic entry regions. Germ cells missing both PUF-8 and GAP-3 proliferate in an uncontrolled fashion and fail to undergo meiotic development. MPK-1, the MAP kinase downstream of the LET-60 RAS, is prematurely activated in these cells; downregulation of MPK-1 activation eliminates tumours and restores differentiation. Our results further reveal that PUF-8 negatively regulates LET-60 expression at a post-transcriptional step. LET-60 is misexpressed in the puf-8(-) mutant germlines and PUF-8 physically interacts with the let-60 3′ UTR. Furthermore, PUF-8 suppresses let-60 3′ UTR-mediated expression in the germ cells that are transitioning from the mitotic to meiotic fate. These results reveal that PUF-8-mediated inhibition of the RAS/MAPK pathway is essential for mitotic-to-meiotic fate transition. PMID:23487310

  18. G protein-coupled receptor signaling through Gq and JNK negatively regulates neural progenitor cell migration

    PubMed Central

    Mizuno, Norikazu; Kokubu, Hiroshi; Sato, Maiko; Nishimura, Akiyuki; Yamauchi, Junji; Kurose, Hitoshi; Itoh, Hiroshi

    2005-01-01

    In the early development of the central nervous system, neural progenitor cells divide in an asymmetric manner and migrate along the radial glia cells. The radial migration is an important process for the proper lamination of the cerebral cortex. Recently, a new mode of the radial migration was found at the intermediate zone where the neural progenitor cells become multipolar and reduce the migration rate. However, the regulatory signals for the radial migration are unknown. Using the migration assay in vitro, we examined how neural progenitor cell migration is regulated. Neural progenitor cells derived from embryonic mouse telencephalon migrated on laminin-coated dishes. Endothelin (ET)-1 inhibited the neural progenitor cell migration. This ET-1 effect was blocked by BQ788, a specific inhibitor of the ETB receptor, and by the expression of a carboxyl-terminal peptide of Gαq but not Gαi. The expression of constitutively active mutant of Gαq, GαqR183C, inhibited the migration of neural progenitor cells. Moreover, the inhibitory effect of ET-1 was suppressed by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the expression of the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of the JNK pathway. Using the slice culture system of embryonic brain, we demonstrated that ET-1 and the constitutively active mutant of Gαq caused the retention of the neural progenitor cells in the intermediate zone and JNK-binding domain of JNK-interacting protein-1 abrogated the effect of ET-1. These results indicated that G protein-coupled receptor signaling negatively regulates neural progenitor cell migration through Gq and JNK. PMID:16116085

  19. SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana

    PubMed Central

    Sadanandom, Ari; Ádám, Éva; Orosa, Beatriz; Viczián, András; Klose, Cornelia; Zhang, Cunjin; Josse, Eve-Marie; Kozma-Bognár, László; Nagy, Ferenc

    2015-01-01

    The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyBLys996Arg-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases. PMID:26283376

  20. Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences

    PubMed Central

    2014-01-01

    Background Mitogen-activated protein kinases (MAPKs) are key regulators of immune responses in animals and plants. In Arabidopsis, perception of microbe-associated molecular patterns (MAMPs) activates the MAPKs MPK3, MPK4 and MPK6. Increasing information depicts the molecular events activated by MAMPs in plants, but the specific and cooperative contributions of the MAPKs in these signalling events are largely unclear. Results In this work, we analyse the behaviour of MPK3, MPK4 and MPK6 mutants in early and late immune responses triggered by the MAMP flg22 from bacterial flagellin. A genome-wide transcriptome analysis reveals that 36% of the flg22-upregulated genes and 68% of the flg22-downregulated genes are affected in at least one MAPK mutant. So far MPK4 was considered as a negative regulator of immunity, whereas MPK3 and MPK6 were believed to play partially redundant positive functions in defence. Our work reveals that MPK4 is required for the regulation of approximately 50% of flg22-induced genes and we identify a negative role for MPK3 in regulating defence gene expression, flg22-induced salicylic acid accumulation and disease resistance to Pseudomonas syringae. Among the MAPK-dependent genes, 27% of flg22-upregulated genes and 76% of flg22-downregulated genes require two or three MAPKs for their regulation. The flg22-induced MAPK activities are differentially regulated in MPK3 and MPK6 mutants, both in amplitude and duration, revealing a highly interdependent network. Conclusions These data reveal a new set of distinct functions for MPK3, MPK4 and MPK6 and indicate that the plant immune signalling network is choreographed through the interplay of these three interwoven MAPK pathways. PMID:24980080

  1. Attachment's Links With Adolescents' Social Emotions: The Roles of Negative Emotionality and Emotion Regulation.

    PubMed

    Murphy, Tia Panfile; Laible, Deborah J; Augustine, Mairin; Robeson, Lindsay

    2015-01-01

    Recent research has attempted to explain the mechanisms through which parental attachment affects social and emotional outcomes (e.g., Burnette, Taylor, Worthington, & Forsyth, 2007 ; Panfile & Laible, 2012 ). The authors' goal was to examine negative emotionality and emotion regulation as mediators of the associations that attachment has with empathy, forgiveness, guilt, and jealousy. One hundred forty-eight adolescents reported their parental attachment security, general levels of negative emotionality and abilities to regulate emotional responses, and tendencies to feel empathy, forgiveness, guilt, and jealousy. Results revealed that attachment security was associated with higher levels of empathy, forgiveness, and guilt, but lower levels of jealousy. In addition, emotion regulation mediated the links attachment shared with both empathy and guilt, such that higher levels of attachment security were linked with greater levels of emotion regulation, which led to greater levels of empathy and guilt. Alternatively, negative emotionality mediated the links attachment shared with both forgiveness and jealousy, such that higher levels of attachment security were associated with lower levels of negative emotionality, which in turn was linked to lower levels of forgiveness and higher levels of jealousy. This study provides a general picture of how attachment security may play a role in shaping an individual's levels of social emotions.

  2. Evidence that dendritic mitochondria negatively regulate dendritic branching in pyramidal neurons in the neocortex.

    PubMed

    Kimura, Toshiya; Murakami, Fujio

    2014-05-14

    The precise branching patterns of dendritic arbors have a profound impact on information processing in individual neurons and the brain. These patterns are established by positive and negative regulation of the dendritic branching. Although the mechanisms for positive regulation have been extensively investigated, little is known about those for negative regulation. Here, we present evidence that mitochondria located in developing dendrites are involved in the negative regulation of dendritic branching. We visualized mitochondria in pyramidal neurons of the mouse neocortex during dendritic morphogenesis using in utero electroporation of a mitochondria-targeted fluorescent construct. We altered the mitochondrial distribution in vivo by overexpressing Mfn1, a mitochondrial shaping protein, or the Miro-binding domain of TRAK2 (TRAK2-MBD), a truncated form of a motor-adaptor protein. We found that dendritic mitochondria were preferentially targeted to the proximal portion of dendrites only during dendritic morphogenesis. Overexpression of Mfn1 or TRAK2-MBD depleted mitochondria from the dendrites, an effect that was accompanied by increased branching of the proximal portion of the dendrites. This dendritic abnormality cannot be accounted for by changes in the distribution of membrane trafficking organelles since the overexpression of Mfn1 did not alter the distributions of the endoplasmic reticulum, Golgi, or endosomes. Additionally, neither did these constructs impair neuronal viability or mitochondrial function. Therefore, our results suggest that dendritic mitochondria play a critical role in the establishment of the precise branching pattern of dendritic arbors by negatively affecting dendritic branching.

  3. Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli

    PubMed Central

    2011-01-01

    Background Gene regulation networks are made of recurring regulatory patterns, called network motifs. One of the most common network motifs is negative auto-regulation, in which a transcription factor represses its own production. Negative auto-regulation has several potential functions: it can shorten the response time (time to reach halfway to steady-state), stabilize expression against noise, and linearize the gene's input-output response curve. This latter function of negative auto-regulation, which increases the range of input signals over which downstream genes respond, has been studied by theory and synthetic gene circuits. Here we ask whether negative auto-regulation preserves this function also in the context of a natural system, where it is embedded within many additional interactions. To address this, we studied the negative auto-regulation motif in the arabinose utilization system of Escherichia coli, in which negative auto-regulation is part of a complex regulatory network. Results We find that when negative auto-regulation is disrupted by placing the regulator araC under constitutive expression, the input dynamic range of the arabinose system is reduced by 10-fold. The apparent Hill coefficient of the induction curve changes from about n = 1 with negative auto-regulation, to about n = 2 when it is disrupted. We present a mathematical model that describes how negative auto-regulation can increase input dynamic-range, by coupling the transcription factor protein level to the input signal. Conclusions Here we demonstrate that the negative auto-regulation motif in the native arabinose system of Escherichia coli increases the range of arabinose signals over which the system can respond. In this way, negative auto-regulation may help to increase the input dynamic-range while maintaining the specificity of cooperative regulatory systems. This function may contribute to explaining the common occurrence of negative auto-regulation in biological systems. PMID

  4. Positive And Negative Feedback Loops Coupled By Common Transcription Activator And Repressor

    NASA Astrophysics Data System (ADS)

    Sielewiesiuk, Jan; Łopaciuk, Agata

    2015-03-01

    Dynamical systems consisting of two interlocked loops with negative and positive feedback have been studied using the linear analysis of stability and numerical solutions. Conditions for saddle-node bifurcation were formulated in a general form. Conditions for Hopf bifurcations were found in a few symmetrical cases. Auto-oscillations, when they exist, are generated by the negative feedback repressive loop. This loop determines the frequency and amplitude of oscillations. The positive feedback loop of activation slightly modifies the oscillations. Oscillations are possible when the difference between Hilll's coefficients of the repression and activation is sufficiently high. The highly cooperative activation loop with a fast turnover slows down or even makes the oscillations impossible. The system under consideration can constitute a component of epigenetic or enzymatic regulation network.

  5. Individual differences in positive and negative emotion regulation: Which strategies explain variability in loneliness?

    PubMed

    Kearns, Sinead M; Creaven, Ann-Marie

    2017-02-01

    Loneliness is the distressing feeling accompanying the perception that one's social needs are not being met by one's social relationships. Conceptual models point to a role for cognitive factors in this experience. Because research on determinants of loneliness is sparse, this study investigates associations between individual differences in emotion regulation (ER) and loneliness. Participants (N = 116) completed measures of loneliness, and a vignette-based measure of adaptive and maladaptive ER in response to positive and negative scenarios. Regression analyses indicated that the regulation of positive and negative emotions explained comparable variance in loneliness, and associations were only partially reduced by the inclusion of social support. The specific strategies positive reappraisal, being present and negative mental time travel explained the most variance in loneliness. The findings are consistent with both the cognitive and the social needs models of loneliness and suggest that variability in ER strategies should be considered relevant to loneliness. Copyright © 2016 John Wiley & Sons, Ltd.

  6. Merlin negative regulation by miR-146a promotes cell transformation.

    PubMed

    Pérez-García, Erick I; Meza-Sosa, Karla F; López-Sevilla, Yaxem; Camacho-Concha, Nohemi; Sánchez, Nilda C; Pérez-Martínez, Leonor; Pedraza-Alva, Gustavo

    2015-12-25

    Inactivation of the tumor suppressor Merlin, by deleterious mutations or by protein degradation via sustained growth factor receptor signaling-mediated mechanisms, results in cell transformation and tumor development. In addition to these mechanisms, here we show that, miRNA-dependent negative regulation of Merlin protein levels also promotes cell transformation. We provide experimental evidences showing that miR-146a negatively regulates Merlin protein levels through its interaction with an evolutionary conserved sequence in the 3´ untranslated region of the NF2 mRNA. Merlin downregulation by miR-146a in A549 lung epithelial cells resulted in enhanced cell proliferation, migration and tissue invasion. Accordingly, stable miR-146a-transfectant cells formed tumors with metastatic capacity in vivo. Together our results uncover miRNAs as yet another negative mechanism controlling Merlin tumor suppressor functions.

  7. Mitophagy: a balance regulator of NLRP3 inflammasome activation

    PubMed Central

    Kim, Min-Ji; Yoon, Joo-Heon; Ryu, Ji-Hwan

    2016-01-01

    The NLRP3 inflammasome is activated by a variety of external or host-derived stimuli and its activation initiates an inflammatory response through caspase-1 activation, resulting in inflammatory cytokine IL-1β maturation and secretion. The NLRP3 inflammasome activation is a kind of innate immune response, most likely mediated by myeloid cells acting as a host defense mechanism. However, if this activation is not properly regulated, excessive inflammation induced by overactivated NLRP3 inflammasome can be detrimental to the host, causing tissue damage and organ dysfunction, eventually causing several diseases. Previous studies have suggested that mitochondrial damage may be a cause of NLRP3 inflammasome activation and autophagy, which is a conserved self-degradation process that negatively regulates NLRP3 inflammasome activation. Recently, mitochondria-selective autophagy, termed mitophagy, has emerged as a central player for maintaining mitochondrial homeostasis through the elimination of damaged mitochondria, leading to the prevention of hyperinflammation triggered by NLRP3 inflammasome activation. In this review, we will first focus on the molecular mechanisms of NLRP3 inflammasome activation and NLRP3 inflammasome-related diseases. We will then discuss autophagy, especially mitophagy, as a negative regulator of NLPP3 inflammasome activation by examining recent advances in research. [BMB Reports 2016; 49(10): 529-535] PMID:27439607

  8. Improved wound management by regulated negative pressure-assisted wound therapy and regulated, oxygen- enriched negative pressure-assisted wound therapy through basic science research and clinical assessment.

    PubMed

    Topaz, Moris

    2012-05-01

    Regulated negative pressure-assisted wound therapy (RNPT) should be regarded as a state-of-the-art technology in wound treatment and the most important physical, nonpharmaceutical, platform technology developed and applied for wound healing in the last two decades. RNPT systems maintain the treated wound's environment as a semi-closed, semi-isolated system applying external physical stimulations to the wound, leading to biological and biochemical effects, with the potential to substantially influence wound-host interactions, and when properly applied may enhance wound healing. RNPT is a simple, safe, and affordable tool that can be utilized in a wide range of acute and chronic conditions, with reduced need for complicated surgical procedures, and antibiotic treatment. This technology has been shown to be effective and safe, saving limbs and lives on a global scale. Regulated, oxygen-enriched negative pressure-assisted wound therapy (RO-NPT) is an innovative technology, whereby supplemental oxygen is concurrently administered with RNPT for their synergistic effect on treatment and prophylaxis of anaerobic wound infection and promotion of wound healing. Understanding the basic science, modes of operation and the associated risks of these technologies through their fundamental clinical mechanisms is the main objective of this review.

  9. Fbxw7β, E3 ubiquitin ligase, negative regulation of primary myoblast differentiation, proliferation and migration.

    PubMed

    Shin, Kyungshin; Hwang, Sang-Gu; Choi, Ik Joon; Ko, Young-Gyu; Jeong, Jaemin; Kwon, Heechung

    2017-04-01

    Satellite cells attached to skeletal muscle fibers play a crucial role in skeletal muscle regeneration. During regeneration, the satellite cells proliferate, migrate to the damaged region, and fuse to each other. Although it is important to determine the cellular mechanisms controlling myoblast behavior, their regulators are not well understood. In this study, we evaluated the roles of Fbxw7 in primary myoblasts and determined its potential as a therapeutic target for muscle disease. We originally found that Fbxw7β, one of the E3 ubiquitin ligase Fbxw7 subtypes, negatively regulates differentiation, proliferation and migration of myoblasts and satellite cells on muscle fiber. However, these phenomena were not observed in myoblasts expressing a dominant-negative, F-box deleted Fbxw7β, mutant. Our results suggest that myoblast differentiation potential and muscle regeneration can be regulated by Fbxw7β.

  10. When death is not a problem: Regulating implicit negative affect under mortality salience.

    PubMed

    Lüdecke, Christina; Baumann, Nicola

    2015-12-01

    Terror management theory assumes that death arouses existential anxiety in humans which is suppressed in focal attention. Whereas most studies provide indirect evidence for negative affect under mortality salience by showing cultural worldview defenses and self-esteem strivings, there is only little direct evidence for implicit negative affect under mortality salience. In the present study, we assume that this implicit affective reaction towards death depends on people's ability to self-regulate negative affect as assessed by the personality dimension of action versus state orientation. Consistent with our expectations, action-oriented participants judged artificial words to express less negative affect under mortality salience compared to control conditions whereas state-oriented participants showed the reversed pattern.

  11. Staufen Negatively Modulates MicroRNA Activity in Caenorhabditis elegans

    PubMed Central

    Ren, Zhiji; Veksler-Lublinsky, Isana; Morrissey, David; Ambros, Victor

    2016-01-01

    The double-stranded RNA-binding protein Staufen has been implicated in various posttranscriptional gene regulatory processes. Here, we demonstrate that the Caenorhabditis elegans homolog of Staufen, STAU-1, functionally interacts with microRNAs. Loss-of-function mutations of stau-1 significantly suppress phenotypes of let-7 family microRNA mutants, a hypomorphic allele of dicer, and a lsy-6 microRNA partial loss-of-function mutant. Furthermore, STAU-1 modulates the activity of lin-14, a target of lin-4 and let-7 family microRNAs, and this modulation is abolished when the 3′ untranslated region of lin-14 is removed. Deep sequencing of small RNA cDNA libraries reveals no dramatic change in the levels of microRNAs or other small RNA populations between wild-type and stau-1 mutants, with the exception of certain endogenous siRNAs in the WAGO pathway. The modulation of microRNA activity by STAU-1 does not seem to be associated with the previously reported enhanced exogenous RNAi (Eri) phenotype of stau-1 mutants, since eri-1 exhibits the opposite effect on microRNA activity. Altogether, our results suggest that STAU-1 negatively modulates microRNA activity downstream of microRNA biogenesis, possibly by competing with microRNAs for binding on the 3′ untranslated region of target mRNAs. PMID:26921297

  12. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    SciTech Connect

    Iimori, Makoto; Ozaki, Kanako; Chikashige, Yuji; Habu, Toshiyuki; Hiraoka, Yasushi; Maki, Takahisa; Hayashi, Ikuko; Obuse, Chikashi; Matsumoto, Tomohiro

    2012-02-01

    Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+ TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly. The mutant Mal3 is no longer a + TIP, but binds strongly the microtubule lattice. Live cell imaging has revealed that while the wild type Mal3 proteins dissociate from the tip of the growing microtubules before the onset of shrinkage, the mutant Mal3 proteins persist on microtubules and reduces a rate of shrinkage after a longer pausing period. Consequently, the mutant Mal3 proteins cause abnormal elongation of microtubules composing the spindle and aster. Mal3 is phosphorylated at a cluster of serine/threonine residues in the linker connecting the CH and EB1-like C-terminal motif domains. The phosphorylation occurs in a microtubule-dependent manner and reduces the affinity of Mal3 to microtubules. We propose that because the 89R mutation is resistant to the effect of phosphorylation, it can associate persistently with microtubules and confers a stronger stability of microtubules likely by reinforcing the cylindrical structure. -- Highlights: Black-Right-Pointing-Pointer We characterize a mutation (mal3-89R) in fission yeast homolog of EB1. Black-Right-Pointing-Pointer The mutation enhances the activity to assemble microtubules. Black-Right-Pointing-Pointer Mal3 is phosphorylated in a microtubule-dependent manner. Black-Right-Pointing-Pointer The phosphorylation negatively regulates the Mal3 activity.

  13. STAT1 negatively regulates spatial memory formation and mediates the memory-impairing effect of Aβ.

    PubMed

    Hsu, Wei-Lun; Ma, Yun-Li; Hsieh, Ding-You; Liu, Yen-Chen; Lee, Eminy Hy

    2014-02-01

    Signal transducer and activator of transcription-1 (STAT1) has an important role in inflammation and the innate immune response, but its role in the central nervous system is less well understood. Here, we examined the role of STAT1 in spatial learning and memory, and assessed the involvement of STAT1 in mediating the memory-impairing effect of amyloid-beta (Aβ). We found that water maze training downregulated STAT1 expression in the rat hippocampal CA1 area, and spatial learning and memory function was enhanced in Stat1-knockout mice. Conversely, overexpression of STAT1 impaired water maze performance. STAT1 strongly upregulated the expression of the extracellular matrix protein laminin β1 (LB1), which also impaired water maze performance in rats. Furthermore, Aβ impaired spatial learning and memory in association with a dose-dependent increase in STAT1 and LB1 expression, but knockdown of STAT1 and LB1 both reversed this effect of Aβ. This Aβ-induced increase in STAT1 and LB1 expression was also associated with a decrease in the expression of the N-methyl-D-aspartate receptor (NMDAR) subunits, NR1, and NR2B. Overexpression of NR1 or NR2B or exogenous application of NMDA reversed Aβ-induced learning and memory deficits as well as Aβ-induced STAT1 and LB1 expression. Our results demonstrate that STAT1 negatively regulates spatial learning and memory through transcriptional regulation of LB1 expression. We also identified a novel mechanism for Aβ pathogenesis through STAT1 induction. Notably, impairment of spatial learning and memory by this STAT1-mediated mechanism is independent of cAMP responsive element-binding protein signaling.

  14. Syndecan-4 negatively regulates antiviral signalling by mediating RIG-I deubiquitination via CYLD

    PubMed Central

    Lin, Wei; Zhang, Jing; Lin, Haiyan; Li, Zexing; Sun, Xiaofeng; Xin, Di; Yang, Meng; Sun, Liwei; Li, Lin; Wang, Hongmei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Retinoic acid-inducible gene I (RIG-I) plays important roles in pathogen recognition and antiviral signalling transduction. Here we show that syndecan-4 (SDC4) is a RIG-I-interacting partner identified in a yeast two-hybrid screen. We find that SDC4 negatively regulates the RIG-I-mediated antiviral signalling in a feedback-loop control manner. The genetic evidence obtained by using knockout mice further emphasizes this biological role of SDC4 in antiviral signalling. Mechanistically, we show that SDC4 interacts with both RIG-I and deubiquitinase CYLD via its carboxyl-terminal intracellular region. SDC4 likely promotes redistribution of RIG-I and CYLD in a perinuclear pattern post viral infection, and thus enhances the RIG-I–CYLD interaction and potentiates the K63-linked deubiquitination of RIG-I. Collectively, our findings uncover a mechanism by which SDC4 antagonizes the activation of RIG-I in a CYLD-mediated deubiquitination-dependent process, thereby balancing antiviral signalling to avoid deleterious effects on host cells. PMID:27279133

  15. Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation.

    PubMed

    Ageta-Ishihara, Natsumi; Miyata, Takaki; Ohshima, Chika; Watanabe, Masahiko; Sato, Yoshikatsu; Hamamura, Yuki; Higashiyama, Tetsuya; Mazitschek, Ralph; Bito, Haruhiko; Kinoshita, Makoto

    2013-01-01

    Neurite growth requires two guanine nucleotide-binding protein polymers of tubulins and septins. However, whether and how those cytoskeletal systems are coordinated was unknown. Here we show that the acute knockdown or knockout of the pivotal septin subunit SEPT7 from cerebrocortical neurons impairs their interhemispheric and cerebrospinal axon projections and dendritogenesis in perinatal mice, when the microtubules are severely hyperacetylated. The resulting hyperstabilization and growth retardation of microtubules are demonstrated in vitro. The phenotypic similarity between SEPT7 depletion and the pharmacological inhibition of α-tubulin deacetylase HDAC6 reveals that HDAC6 requires SEPT7 not for its enzymatic activity, but to associate with acetylated α-tubulin. These and other findings indicate that septins provide a physical scaffold for HDAC6 to achieve efficient microtubule deacetylation, thereby negatively regulating microtubule stability to an optimal level for neuritogenesis. Our findings shed light on the mechanisms underlying the HDAC6-mediated coupling of the two ubiquitous cytoskeletal systems during neural development.

  16. Schizosaccharomyces pombe mst2+ Encodes a MYST Family Histone Acetyltransferase That Negatively Regulates Telomere Silencing†

    PubMed Central

    Gómez, Eliana B.; Espinosa, Joaquín M.; Forsburg, Susan L.

    2005-01-01

    Histone acetylation and deacetylation are associated with transcriptional activity and the formation of constitutively silent heterochromatin. Increasingly, histone acetylation is also implicated in other chromosome transactions, including replication and segregation. We have cloned the only Schizosaccharomyces pombe MYST family histone acetyltransferase genes, mst1+ and mst2+. Mst1p, but not Mst2p, is essential for viability. Both proteins are localized to the nucleus and bound to chromatin throughout the cell cycle. Δmst2 genetically interacts with mutants that affect heterochromatin, cohesion, and telomere structure. Mst2p is a negative regulator of silencing at the telomere but does not affect silencing in the centromere or mating type region. We generated a census of proteins and histone modifications at wild-type telomeres. A histone acetylation gradient at the telomeres is lost in Δmst2 cells without affecting the distribution of Taz1p, Swi6p, Rad21p, or Sir2p. We propose that the increased telomeric silencing is caused by histone hypoacetylation and/or an increase in the ratio of methylated to acetylated histones. Although telomere length is normal, meiosis is aberrant in Δmst2 diploid homozygote mutants, suggesting that telomeric histone acetylation contributes to normal meiotic progression. PMID:16199868

  17. NLRP12 negatively regulates proinflammatory cytokine production and host defense against Brucella abortus.

    PubMed

    Silveira, Tatiana N; Gomes, Marco Túlio R; Oliveira, Luciana S; Campos, Priscila C; Machado, Gabriela G; Oliveira, Sergio C

    2017-01-01

    Brucella abortus is the causative agent of brucellosis, which causes abortion in domestic animals and undulant fever in humans. This bacterium infects and proliferates mainly in macrophages and dendritic cells, where it is recognized by pattern recognition receptors (PRRs) including Nod-like receptors (NLRs). Our group recently demonstrated the role of AIM2 and NLRP3 in Brucella recognition. Here, we investigated the participation of NLRP12 in innate immune response to B. abortus. We show that NLRP12 inhibits the early production of IL-12 by bone marrow-derived macrophages upon B. abortus infection. We also observed that NLRP12 suppresses in vitro NF-κB and MAPK signaling in response to Brucella. Moreover, we show that NLRP12 modulates caspase-1 activation and IL-1β secretion in B. abortus infected-macrophages. Furthermore, we show that mice lacking NLRP12 are more resistant in the early stages of B. abortus infection: NLRP12(-/-) infected-mice have reduced bacterial burdens in the spleens and increased production of IFN-γ and IL-1β compared with wild-type controls. In addition, NLRP12 deficiency leads to reduction in granuloma number and size in mouse livers. Altogether, our findings suggest that NLRP12 plays an important role in negatively regulating the early inflammatory responses against B. abortus.

  18. Cyclooxygenase-2 regulates TGFβ-induced cancer stemness in triple-negative breast cancer

    PubMed Central

    Tian, Jun; Hachim, Mahmood Y.; Hachim, Ibrahim Y.; Dai, Meiou; Lo, Chieh; Raffa, Fatmah Al; Ali, Suhad; Lebrun, Jean Jacques

    2017-01-01

    Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, display poor prognosis and exhibit resistance to conventional therapies, partly due to an enrichment in breast cancer stem cells (BCSCs). Here, we investigated the role of the cyclooxygenase-2 (COX-2), a downstream target of TGFβ, in regulating BCSCs in TNBC. Bioinformatics analysis revealed that COX-2 is highly expressed in TNBC and that its expression correlated with poor survival outcome in basal subtype of breast cancer. We also found TGFβ-mediated COX-2 expression to be Smad3-dependent and to be required for BCSC self-renewal and expansion in TNBCs. Knocking down COX-2 expression strikingly blocked TGFβ-induced tumorsphere formation and TGFβ-induced enrichment of the two stem-like cell populations, CD24lowCD44high and ALDH+ BCSCs. Blocking COX-2 activity, using a pharmacological inhibitor also prevented TGFβ-induced BCSC self-renewal. Moreover, we found COX-2 to be required for TGFβ-induced expression of mesenchymal and basal breast cancer markers. In particular, we found that TGFβ-induced expression of fibronectin plays a central role in TGFβ-mediated breast cancer stemness. Together, our results describe a novel role for COX-2 in mediating the TGFβ effects on BCSC properties and imply that targeting the COX-2 pathway may prove useful for the treatment of TNBC by eliminating BCSCs. PMID:28054666

  19. Negative regulation of bacterial killing and inflammation by two novel CD16 ligands.

    PubMed

    Beppler, Jaqueline; Mkaddem, Sanae Ben; Michaloski, Jussara; Honorato, Rodrigo Vargas; Velasco, Irineu Tadeu; de Oliveira, Paulo Sérgio Lopes; Giordano, Ricardo José; Monteiro, Renato C; Pinheiro da Silva, Fabiano

    2016-08-01

    Sepsis, a leading cause of death worldwide, involves exacerbated proinflammatory responses and inefficient bacterial clearance. Phagocytic cells play a crucial part in the prevention of sepsis by clearing bacteria through host innate receptors. Here, we used a phage display library to identify two peptides in Escherichia coli that interact with host innate receptors. One of these peptides, encoded by the wzxE gene of E. coli K-12, was involved in the transbilayer movement of a trisaccharide-lipid intermediate in the assembly of enterobacterial common antigen. Peptide-receptor interactions induced CD16-mediated inhibitory immunoreceptor tyrosine-based activating motif signaling, blocking the production of ROS and bacterial killing. This CD16-mediated inhibitory signaling was abrogated in a WzxE(-/-) mutant of E. coli K-12, restoring the production of ROS and bacterial killing. Taken together, the two novel CD16 ligands identified negatively regulate bacterial killing and inflammation. Our findings may contribute toward the development of new immunotherapies for E. coli-mediated infectious diseases and inflammation.

  20. Maternal Attachment Style and Responses to Adolescents’ Negative Emotions: The Mediating Role of Maternal Emotion Regulation

    PubMed Central

    Jones, Jason D.; Brett, Bonnie E.; Ehrlich, Katherine B.; Lejuez, Carl W.; Cassidy, Jude

    2014-01-01

    SYNOPSIS Objective Previous research has examined the developmental consequences, particularly in early childhood, of parents’ supportive and unsupportive responses to children’s negative emotions. Much less is known about factors that explain why parents respond in ways that may support or undermine their children’s emotions, and even less is known about how these parenting processes unfold with adolescents. We examined the associations between mothers’ attachment styles and their distress, harsh, and supportive responses to their adolescents’ negative emotions two years later and whether these links were mediated by maternal emotion regulation difficulties. Design Mothers in a longitudinal study (n = 230) reported on their attachment style, difficulties regulating their emotions, and their hypothetical responses to their adolescents’ negative emotions, respectively, at consecutive laboratory visits one year apart. Results Mothers who reported greater attachment-related avoidance and anxiety reported having greater difficulties with emotion regulation one year later. Emotion dysregulation, in turn, predicted more distressed, harsher, and less supportive maternal responses to adolescents’ negative emotions the following year. In addition, greater avoidance directly predicted harsher maternal responses two years later. Conclusions These findings extend previous research by identifying maternal attachment style as a predictor of responses to adolescent distress and by documenting the underlying role of emotion dysregulation in the link between adult attachment style and parenting. PMID:25568638

  1. [Regulation of Positive and Negative Emotions as Mediator between Maternal Emotion Socialization and Child Problem Behavior].

    PubMed

    Fäsche, Anika; Gunzenhauser, Catherine; Friedlmeier, Wolfgang; von Suchodoletz, Antje

    2015-01-01

    The present study investigated five to six year old children's ability to regulate negative and positive emotions in relation to psychosocial problem behavior (N=53). It was explored, whether mothers' supportive and nonsupportive strategies of emotion socialization influence children's problem behavior by shaping their emotion regulation ability. Mothers reported on children's emotion regulation and internalizing and externalizing problem behavior via questionnaire, and were interviewed about their preferences for socialization strategies in response to children's expression of negative affect. Results showed that children with more adaptive expression of adequate positive emotions had less internalizing behavior problems. When children showed more control of inadequate negative emotions, children were less internalizing as well as externalizing in their behavior. Furthermore, results indicated indirect relations of mothers' socialization strategies with children's problem behavior. Control of inadequate negative emotions mediated the link between non-supportive strategies on externalizing problem behavior. Results suggest that emotion regulatory processes should be part of interventions to reduce the development of problematic behavior in young children. Parents should be trained in dealing with children's emotions in a constructive way.

  2. Instrumental Motives in Negative Emotion Regulation in Daily Life: Frequency, Consistency, and Predictors.

    PubMed

    Kalokerinos, Elise K; Tamir, Maya; Kuppens, Peter

    2016-12-19

    People regulate their emotions not only for hedonic reasons but also for instrumental reasons, to attain the potential benefits of emotions beyond pleasure and pain. However, such instrumental motives have rarely been examined outside the laboratory as they naturally unfold in daily life. To assess whether and how instrumental motives operate outside the laboratory, it is necessary to examine them in response to real and personally relevant stimuli in ecologically valid contexts. In this research, we assessed the frequency, consistency, and predictors of instrumental motives in negative emotion regulation in daily life. Participants (N = 114) recalled the most negative event of their day each evening for 7 days and reported their instrumental motives and negative emotion goals in that event. Participants endorsed performance motives in approximately 1 in 3 events and social, eudaimonic, and epistemic motives in approximately 1 in 10 events. Instrumental motives had substantially higher within- than between-person variance, indicating that they were context-dependent. Indeed, although we found few associations between instrumental motives and personality traits, relationships between instrumental motives and contextual variables were more extensive. Performance, social, and eudaimonic motives were each predicted by a unique pattern of contextual appraisals. Our data demonstrate that instrumental motives play a role in daily negative emotion regulation as people encounter situations that pose unique regulatory demands. (PsycINFO Database Record

  3. E3 ubiquitin ligase Cbl-b negatively regulates C-type lectin receptor–mediated antifungal innate immunity

    PubMed Central

    Zhu, Le-Le; Xu, Xia; Zhao, Xue-Qiang; Wang, Ting-Ting; Tang, Bing; Jiang, Yuan-Ying

    2016-01-01

    Activation of various C-type lectin receptors (CLRs) initiates potent proinflammatory responses against various microbial infections. However, how activated CLRs are negatively regulated remains unknown. In this study, we report that activation of CLRs Dectin-2 and Dectin-3 by fungi infections triggers them for ubiquitination and degradation in a Syk-dependent manner. Furthermore, we found that E3 ubiquitin ligase Casitas B–lineage lymphoma protein b (Cbl-b) mediates the ubiquitination of these activated CLRs through associating with each other via adapter protein FcR-γ and tyrosine kinase Syk, and then the ubiquitinated CLRs are sorted into lysosomes for degradation by an endosomal sorting complex required for transport (ESCRT) system. Therefore, the deficiency of either Cbl-b or ESCRT subunits significantly decreases the degradation of activated CLRs, thereby resulting in the higher expression of proinflammatory cytokines and inflammation. Consistently, Cbl-b–deficient mice are more resistant to fungi infections compared with wild-type controls. Together, our study indicates that Cbl-b negatively regulates CLR-mediated antifungal innate immunity, which provides molecular insight for designing antifungal therapeutic agents. PMID:27432944

  4. CDK4 regulates cancer stemness and is a novel therapeutic target for triple-negative breast cancer

    PubMed Central

    Dai, Meiou; Zhang, Chenjing; Ali, Ayad; Hong, Xinyuan; Tian, Jun; Lo, Chieh; Fils-Aimé, Nadège; Burgos, Sergio A.; Ali, Suhad; Lebrun, Jean-Jacques

    2016-01-01

    Triple negative breast cancers exhibit very aggressive features and poor patient outcomes. These tumors are enriched in cancer stem cells and exhibit resistance to most treatments and chemotherapy. In this study, we found the cyclin-dependent kinase (CDK4) to act as a cancer stem cell regulator and novel prognostic marker in triple negative breast cancers. We found CDK4 to be highly expressed in these tumors and its expression to correlate with poor overall and relapse free survival outcomes, high tumor grade and poor prognostic features of triple negative breast cancer patients. Moreover, we found that blocking CDK4 expression or kinase activity, using a pharmacological inhibitor prevented breast cancer stem cell self-renewal. Interestingly, suppression of CDK4 expression or kinase activity reversed the basal-B TNBC mesenchymal phenotype to an epithelial- and luminal-like phenotype which correlates with better clinical prognosis. Finally, blocking CDK4 activity efficiently eliminated both normal and chemotherapy-resistant cancer cells in triple negative breast cancers, highlighting CDK4 as a promising novel therapeutic target for these aggressive breast tumors. PMID:27759034

  5. Maternal self-regulation, relationship adjustment, and home chaos: contributions to infant negative emotionality.

    PubMed

    Bridgett, David J; Burt, Nicole M; Laake, Lauren M; Oddi, Kate B

    2013-12-01

    There has been increasing interest in the direct and indirect effects of parental self-regulation on children's outcomes. In the present investigation, the effects of maternal self-regulation, home chaos, and inter-parental relationship adjustment on broad and specific indicators of infant negative emotionality (NE) were examined. A sample of maternal caregivers and their 4-month-old infants (N = 85) from a rural community participated. Results demonstrated that better maternal self-regulation was associated with lower infant NE broadly, as well as with lower infant sadness and distress to limitations/frustration and better falling reactivity (i.e., emotion regulation), specifically. Maternal self-regulation also predicted less chaotic home environments and better maternal inter-parental relationship adjustment. Findings also supported the indirect effects of maternal self-regulation on broad and specific indicators of infant NE through home chaos and maternal relationship adjustment. Some differential effects were also identified. Elevated home chaos appeared to specifically affect infant frustration/distress to limitations whereas maternal relationship adjustment affected broad infant NE, as well as several specific indicators of infant NE: frustration/distress to limitations, sadness, and falling reactivity. In conjunction with other recent investigations that have reported the effects of maternal self-regulation on parenting, the findings in the present investigation suggest that parental self-regulation may influence children's outcomes through several proximal environmental pathways.

  6. miR-34a negatively regulates efferocytosis by tissue macrophages in part via SIRT1

    PubMed Central

    McCubbrey, Alexandra L; Nelson, Joshua D.; Stolberg, Valerie R.; Blakely, Pennelope K.; McCloskey, Lisa; Janssen, William J.; Freeman, Christine M.; Curtis, Jeffrey L.

    2015-01-01

    Apoptotic cell (AC) clearance (“efferocytosis”) is an evolutionarily conserved process essential for immune health, particularly to maintain self-tolerance. Despite identification of many recognition receptors and intracellular signaling components of efferocytosis, its negative regulation remains incompletely understood, and has not previously been known to involve microRNAs (miRs). Here we show that miR-34a (gene ID 407040), well-recognized as a p53-dependent tumor suppressor, mediates coordinated negative regulation of efferocytosis by resident murine and human tissue macrophages (Mø). miR-34a expression varied greatly between Mø from different tissues, correlating inversely with their capacity for AC uptake. Transient or genetic knockdown of miR-34a increased efferocytosis, whereas miR-34a over-expression decreased efferocytosis, without altering recognition of live, necrotic or Ig-opsonized cells. The inhibitory effect of miR-34a was mediated both by reduced expression of Axl, a receptor tyrosine kinase known to recognize AC, and of the deacetylase SIRT1, which had not previously been linked to efferocytosis by tissue Mø. Exposure to AC down-regulated Mø miR-34a expression, resulting in a positive feedback loop that increased subsequent capacity to engulf AC. These findings demonstrate that miR-34a both specifically regulates and is regulated by efferocytosis. Given the ability of efferocytosis to polarize ingesting Mø uniquely and to reduce their host-defense functions, dynamic negative regulation by miR-34a provides one means of fine-tuning Mø behavior towards AC in specific tissue environments with differing potentials for microbial exposure. PMID:26718338

  7. The plasminogen activator system: biology and regulation.

    PubMed

    Irigoyen, J P; Muñoz-Cánoves, P; Montero, L; Koziczak, M; Nagamine, Y

    1999-10-01

    The regulation of plasminogen activation involves genes for two plasminogen activators (tissue type and urokinase type), two specific inhibitors (type 1 and type 2), and a membrane-anchored urokinase-type plasminogen-activator-specific receptor. This system plays an important role in various biological processes involving extracellular proteolysis. Recent studies have revealed that the system, through interplay with integrins and the extracellular matrix protein vitronectin, is also involved in the regulation of cell migration and proliferation in a manner independent of proteolytic activity. The genes are expressed in many different cell types and their expression is under the control of diverse extracellular signals. Gene expression reflects the levels of the corresponding mRNA, which should be the net result of synthesis and degradation. Thus, modulation of mRNA stability is an important factor in overall regulation. This review summarizes current understanding of the biology and regulation of genes involved in plasminogen activation at different levels.

  8. Forskolin-inducible cAMP pathway negatively regulates T-cell proliferation by uncoupling the interleukin-2 receptor complex.

    PubMed

    Rodriguez, Georgialina; Ross, Jeremy A; Nagy, Zsuzsanna S; Kirken, Robert A

    2013-03-08

    Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γc chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.

  9. Annexin A2 binds to endosomes and negatively regulates TLR4-triggered inflammatory responses via the TRAM-TRIF pathway

    PubMed Central

    Zhang, Shuang; Yu, Min; Guo, Qiang; Li, Rongpeng; Li, Guobo; Tan, Shirui; Li, Xuefeng; Wei, Yuquan; Wu, Min

    2015-01-01

    Lipopolysaccharide (LPS) derived from Gram-negative bacteria activates plasma membrane signaling via Toll-like receptor 4 (TLR4) on host cells and triggers innate inflammatory responses, but the underlying mechanisms remain to be fully elucidated. Here we reveal a role for annexin A2 (AnxA2) in host defense against infection as anxa2−/− mice were highly susceptible to Gram-negative bacteria-induced sepsis with enhanced inflammatory responses. Computing analysis and biochemical experiments identified that constitutive AnxA2 expression facilitated TLR4 internalization and its subsequent translocation into early endosomal membranes. It activated the TRAM-dependent endosomal signaling, leading to the release of anti-inflammatory cytokines. Importantly, AnxA2 deficiency prolonged TLR4-mediated signaling from the plasma membrane, which was attributable to pro-inflammatory cytokine production (IL-6, TNFα and IL-1β). Thus, AnxA2 directly exerted negative regulation of inflammatory responses through TLR4-initiated TRAM-TRIF pathway occurring on endosomes. This study reveals AnxA2 as a critical regulator in infection-initiated inflammation, which protects the host from excessive inflammatory damage. PMID:26527544

  10. Robust activation method for negative electron affinity photocathodes

    DOEpatents

    Mulhollan, Gregory A.; Bierman, John C.

    2011-09-13

    A method by which photocathodes(201), single crystal, amorphous, or otherwise ordered, can be surface modified to a robust state of lowered and in best cases negative, electron affinity has been discovered. Conventional methods employ the use of Cs(203) and an oxidizing agent(207), typically carried by diatomic oxygen or by more complex molecules, for example nitrogen trifluoride, to achieve a lowered electron affinity(404). In the improved activation method, a second alkali, other than Cs(205), is introduced onto the surface during the activation process, either by co-deposition, yo-yo, or sporadic or intermittent application. Best effect for GaAs photocathodes has been found through the use of Li(402) as the second alkali, though nearly the same effect can be found by employing Na(406). Suitable photocathodes are those which are grown, cut from boules, implanted, rolled, deposited or otherwise fabricated in a fashion and shape desired for test or manufacture independently supported or atop a support structure or within a framework or otherwise affixed or suspended in the place and position required for use.

  11. Negative Urgency Mediates the Relationship between Amygdala and Orbitofrontal Cortex Activation to Negative Emotional Stimuli and General Risk-Taking

    PubMed Central

    Cyders, Melissa A.; Dzemidzic, Mario; Eiler, William J.; Coskunpinar, Ayca; Karyadi, Kenny A.; Kareken, David A.

    2015-01-01

    The tendency toward impulsive behavior under emotional duress (negative and positive urgency) predicts a wide range of maladaptive risk-taking and behavioral disorders. However, it remains unclear how urgency relates to limbic system activity as induced from emotional provocation. This study used functional magnetic resonance imaging to examine the relationship between brain responses to visual emotional stimuli and urgency traits. Twenty-seven social drinkers (mean age = 25.2, 14 males) viewed negative (Neg), neutral (Neu), and positive (Pos) images during 6 fMRI scans. Brain activation was extracted from a priori limbic regions previously identified in studies of emotional provocation. The right posterior orbitofrontal cortex (OFC) and left amygdala were activated in the [Neg>Neu] contrast, whereas the left posterior OFC was activated in the [Pos>Neu] contrast. Negative urgency was related to the right lateral OFC (r = 0.43, P = 0.03) and the left amygdala (r = 0.39, P = 0.04) [Neg>Neu] activation. Negative urgency also mediated the relationship between [Neg>Neu] activation and general risk-taking (regression weights = 3.42 for right OFC and 2.75 for the left amygdala). Emotional cue-induced activation in right lateral OFC and left amygdala might relate to emotion-based risk-taking through negative urgency. PMID:24904065

  12. Negative regulation of IL-17-mediated signaling and inflammation by ubiquitin-specific protease 25

    PubMed Central

    Zhong, Bo; Liu, Xikui; Wang, Xiaohu; Chang, Seon Hee; Liu, Xindong; Wang, Aibo; Reynolds, Joseph M.; Dong, Chen

    2012-01-01

    Interleukin 17 (IL-17) plays an important role in infection and autoimmunity; how it signals remains poorly understood. In this study, we identified ubiquitin-specific protease 25 (USP25) as a negative regulator of IL-17-mediated signaling and inflammation. Overexpression of USP25 inhibited IL-17-triggered signaling, while USP25 deficiency resulted in increased phosphorylation of IκBα and Jnk, increased expression of chemokines and cytokines as well as prolonged half-life of Cxcl1 mRNA following IL-17 treatment. Consistently, Usp25-/- mice exhibited increased sensitivity to IL-17-dependent inflammation and autoimmunity in vivo. Mechanistically, IL-17 stimulation induced the association of USP25 with TRAF5 and TRAF6 and USP25 induced removal of Act1-mediated K63-linked ubiquitination in TRAF5 and TRAF6. Thus, our results demonstrate that USP25 is a deubiquitinating enzyme (DUB) that negatively regulates IL-17-triggered signaling. PMID:23042150

  13. Cell type-specific expression of JC virus early promoter is determined by positive and negative regulation.

    PubMed

    Tada, H; Lashgari, M; Rappaport, J; Khalili, K

    1989-01-01

    We analyzed control sequences of the human papovavirus JC virus (JCV) to define the cis-acting elements that regulate specific expression of the viral early region genes in glial cells. Nuclear run-on transcription, S1 analysis, and chloramphenicol acetyltransferase enzyme activity in a transient transfection assay established that the cell type-specific expression of JCV early genes is determined at the transcriptional level. Using DNase footprinting analysis of nuclear proteins prepared from glial and nonglial cells, we located four regions within the JCV control sequences that specifically interacted with the proteins. In glial cells, all four domains contributed to the specific expression of a heterologous promoter, whereas in nonglial cells, two protein-binding regions showed no effect on basal transcriptional activity and the other two domains significantly downregulated transcription of the promoter. We conclude that cell type-specific transcription of the JCV early promoter is under both positive and negative regulation in eucaryotic cells.

  14. Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Regulation of Oncogenic Properties in Triple Negative Breast Cancer

    PubMed Central

    Park, Jun Hyoung; Vithayathil, Sajna; Kumar, Santosh; Sung, Pi-Lin; Dobrolecki, Lacey Elizabeth; Putluri, Vasanta; Bhat, Vadiraja B.; Bhowmik, Salil Kumar; Gupta, Vineet; Arora, Kavisha; Wu, Danli; Tsouko, Efrosini; Zhang, Yiqun; Maity, Suman; Donti, Taraka R.; Graham, Brett H.; Frigo, Daniel E.; Coarfa, Cristian; Yotnda, Patricia; Putluri, Nagireddy; Sreekumar, Arun; Lewis, Michael T.; Creighton, Chad J.; Wong, Lee-Jun C.; Kaipparettu, Benny Abraham

    2016-01-01

    Summary Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid β-oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1 (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models, confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis. PMID:26923594

  15. The Potato ERF Transcription Factor StERF3 Negatively Regulates Resistance to Phytophthora infestans and Salt Tolerance in Potato.

    PubMed

    Tian, Zhendong; He, Qin; Wang, Haixia; Liu, Ying; Zhang, Ying; Shao, Fang; Xie, Conghua

    2015-05-01

    Ethylene response factors (ERFs) are unique to the plant kingdom and play crucial roles in plant response to various biotic and abiotic stresses. We show here that a potato StERF3, which contains an ERF-associated amphiphilic repression (EAR) motif in its C-terminal region, negatively regulates resistance to Phytophthora infestans and salt tolerance in potato. The StERF3 promoter responds to induction by salicylic acid, ABA ethylene and NaCl, as well as P. infestans, the causal agent of potato late blight disease. StERF3 could bind to the GCC box element of the HIS3 promoter and activate transcription of HIS3 in yeast cells. Importantly, silencing of StERF3 in potato produced an enhanced foliage resistance to P. infestans and elevated plant tolerance to NaCl stress accompanied by the activation of defense-related genes (PR1, NPR1 and WRKY1). In contrast, StERF3-overexpressing plants showed reduced expression of these defense-related genes and enhanced susceptibility to P. infestans, suggesting that StERF3 functions as a negative regulator of downstream defense- and/or stress-related genes in potato. StERF3 is localized to the nucleus. Interestingly, yeast two-hybrid assay and a bimolecular fluorescence complementation (BiFC) test clarified that StERF3 could interact with other proteins in the cytoplasm which may lead to its re-localization between the nucleus and cytoplasm, revealing a novel means of StERF3 regulation. Taken together, these data provide new insights into the mechanism underlying how StERF3 negatively regulates late blight resistance and abiotic tolerance in potato and may have a potential use in engineering late blight resistance in potato.

  16. Negative feedback regulation of thyrotropin subunits and pituitary deiodinases in red drum, Sciaenops ocellatus.

    PubMed

    Jones, R A; Cohn, W B; Wilkes, A A; MacKenzie, D S

    2017-01-01

    Thyroxine (T4) undergoes dynamic daily cycles in the perciform fish the red drum, Sciaenops ocellatus, that are inversely timed to cycles of thyrotropin (TSH) subunit mRNA expression in the pituitary gland. We have proposed that these daily cycles are regulated by negative feedback of circulating T4 on expression of pituitary thyroid hormone deiodinase type 3 (Dio3), such that elevated circulating T4 results in diminished pituitary thyroid hormone catabolism and consequent increased negative feedback on expression of TSH subunits during the day. To determine whether thyroid hormones function to modulate expression of pituitary deiodinase enzymes we developed an immersion technique to administer physiological doses of T3 and T4in vivo. Immersion in T4 or T3 significantly inhibited the mRNA expression of the TSH α and β subunits from 4 to 66h of immersion. Pituitary Dio3 expression was significantly diminished by T3 and T4 at 22h. These results indicate that both T4 and T3 are capable of negative feedback regulation of TSH subunit expression in red drum at physiological concentrations and on a time scale consistent with the T4 daily cycle. Furthermore, thyroid hormones negatively regulate Dio3 expression in the pituitary in a manner suggesting that negative thyroxine feedback on Dio3 promotes the release of TSH subunits from TH inhibition and may be an important mechanism for generating daily thyroid hormone cycles. These results highlight a potentially important role for D3 in mediating thyroid hormone feedback on TSH expression, not previously described in other species.

  17. Glycosylation regulates prestin cellular activity.

    PubMed

    Rajagopalan, Lavanya; Organ-Darling, Louise E; Liu, Haiying; Davidson, Amy L; Raphael, Robert M; Brownell, William E; Pereira, Fred A

    2010-03-01

    Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by

  18. 100 s extraction of negative ion beams by using actively temperature-controlled plasma grid

    SciTech Connect

    Kojima, A. Hanada, M.; Yoshida, M.; Tobari, H.; Kashiwagi, M.; Umeda, N.; Watanabe, K.; Grisham, L. R.

    2014-02-15

    Long pulse beam extraction with a current density of 120 A/m{sup 2} for 100 s has been achieved with a newly developed plasma grid (PG) for the JT-60SA negative ion source which is designed to produce high power and long pulse beams with a negative ion current of 130 A/m{sup 2} (22 A) and a pulse length of 100 s. The PG temperature is regulated by fluorinated fluids in order to keep the high PG temperature for the cesium-seeded negative ion production. The time constant for temperature controllability of the PG was measured to be below 10 s, which was mainly determined by the heat transfer coefficient of the fluorinated fluid. The measured decay time of the negative ion current extracted from the actively temperature-controlled PG was 430 s which was sufficient for the JT-60SA requirement, and much longer than that by inertial-cooling PG of 60 s. Obtained results of the long pulse capability are utilized to design the full size PG for the JT-60SA negative ion source.

  19. 50 CFR 404.7 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... Wildlife and Fisheries JOINT REGULATIONS (UNITED STATES FISH AND WILDLIFE SERVICE, DEPARTMENT OF THE INTERIOR AND NATIONAL MARINE FISHERIES SERVICE, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT... MARINE NATIONAL MONUMENT § 404.7 Regulated activities. Except as provided in §§ 404.8, 404.9 and...

  20. A loss-of-function screen for phosphatases that regulate neurite outgrowth identifies PTPN12 as a negative regulator of TrkB tyrosine phosphorylation.

    PubMed

    Ambjørn, Malene; Dubreuil, Véronique; Miozzo, Federico; Nigon, Fabienne; Møller, Bente; Issazadeh-Navikas, Shohreh; Berg, Jacob; Lees, Michael; Sap, Jan

    2013-01-01

    Alterations in function of the neurotrophin BDNF are associated with neurodegeneration, cognitive decline, and psychiatric disorders. BDNF promotes axonal outgrowth and branching, regulates dendritic tree morphology and is important for axonal regeneration after injury, responses that largely result from activation of its tyrosine kinase receptor TrkB. Although intracellular neurotrophin (NT) signaling presumably reflects the combined action of kinases and phosphatases, little is known about the contributions of the latter to TrkB regulation. The issue is complicated by the fact that phosphatases belong to multiple independently evolved families, which are rarely studied together. We undertook a loss-of-function RNA-interference-based screen of virtually all known (254) human phosphatases to understand their function in BDNF/TrkB-mediated neurite outgrowth in differentiated SH-SY5Y cells. This approach identified phosphatases from diverse families, which either positively or negatively modulate BDNF-TrkB-mediated neurite outgrowth, and most of which have little or no previously established function related to NT signaling. "Classical" protein tyrosine phosphatases (PTPs) accounted for 13% of the candidate regulatory phosphatases. The top classical PTP identified as a negative regulator of BDNF-TrkB-mediated neurite outgrowth was PTPN12 (also called PTP-PEST). Validation and follow-up studies showed that endogenous PTPN12 antagonizes tyrosine phosphorylation of TrkB itself, and the downstream activation of ERK1/2. We also found PTPN12 to negatively regulate phosphorylation of p130cas and FAK, proteins with previously described functions related to cell motility and growth cone behavior. Our data provide the first comprehensive survey of phosphatase function in NT signaling and neurite outgrowth. They reveal the complexity of phosphatase control, with several evolutionarily unrelated phosphatase families cooperating to affect this biological response, and hence the

  1. A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis.

    PubMed

    Chien, Pei-Shan; Nam, Hong Gil; Chen, Yet-Ran

    2015-08-01

    High salinity has negative impacts on plant growth through altered water uptake and ion-specific toxicities. Plants have therefore evolved an intricate regulatory network in which plant hormones play significant roles in modulating physiological responses to salinity. However, current understanding of the plant peptides involved in this regulatory network remains limited. Here, we identified a salt-regulated peptide in Arabidopsis. The peptide was 11 aa and was derived from the C terminus of a cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily. This peptide was found by searching homologues in Arabidopsis using the precursor of a tomato CAP-derived peptide (CAPE) that was initially identified as an immune signal. In searching for a CAPE involved in salt responses, we screened CAPE precursor genes that showed salt-responsive expression and found that the PROAtCAPE1 (AT4G33730) gene was regulated by salinity. We confirmed the endogenous Arabidopsis CAP-derived peptide 1 (AtCAPE1) by mass spectrometry and found that a key amino acid residue in PROAtCAPE1 is critical for AtCAPE1 production. Moreover, although PROAtCAPE1 was expressed mainly in the roots, AtCAPE1 was discovered to be upregulated systemically upon salt treatment. The salt-induced AtCAPE1 negatively regulated salt tolerance by suppressing several salt-tolerance genes functioning in the production of osmolytes, detoxification, stomatal closure control, and cell membrane protection. This discovery demonstrates that AtCAPE1, a homologue of tomato immune regulator CAPE1, plays an important role in the regulation of salt stress responses. Our discovery thus suggests that the peptide may function in a trade-off between pathogen defence and salt tolerance.

  2. Indium phosphide negative electron affinity photocathodes: Surface cleaning and activation

    NASA Astrophysics Data System (ADS)

    Sun, Yun

    InP(100) is a very important semi-conductor for many applications. When activated by Cs and oxygen, the InP surface achieves the state of Negative Electron Affinity (NEA) making the Cs+O/InP system a very efficient electron source. Despite many years of study, the chemical cleaning and activation of InP are still not well understood. In our work, we have established an understanding of the basic physics and chemistry for the chemical cleaning and activation of the InP(100) surface. Synchrotron Radiation Photoelectron Spectroscopy is the main technique used in this study because of its high surface sensitivity and ability to identify chemical species present on the surface at each stage of our process. A clean, stoichiometric InP(100) surface is crucial for obtaining high performance of NEA photocathodes. Therefore, the first part of our study focused on the chemical cleaning of InP(100). We found that hydrogen peroxide based solutions alone, originally developed to clean GaAs(100) surfaces and widely used for InP(100), do not result in clean InP(I00) surfaces because oxide is left on the surface. A second cleaning step, which uses acid solutions like HCl or H2SO4, can remove all the oxide and leave a 0.4 ML protective layer of elemental phosphorous on the surface. The elemental phosphorous can be removed by annealing at 330°C and a clean InP(100) surface can be obtained. Cs deposition on InP(100) surface shows clear charge transfer from the Cs ad-atoms to the substrate. When the Cs/InP(100) surface is dosed with oxygen, the charge transfer from the Cs to substrate is reduced and substrate is oxidized. The activation of InP as a NEA photocathode is carried out by an alternating series of steps consisting of Cs deposition and Cs+O co-deposition. Two types of oxygen are found after activation. The first is dissociated oxygen and the other is a di-oxygen species (peroxide or superoxide). The decay of quantum-yield with time and with annealing is studied and changes in

  3. Dopamine transporter regulates the enhancement of novelty processing by a negative emotional context.

    PubMed

    Garcia-Garcia, Manuel; Clemente, Immaculada; Domínguez-Borràs, Judith; Escera, Carles

    2010-04-01

    The dopaminergic (DA) system has been recently related the emotional modulation of cognitive processes. Moreover, patients with midbrain DA depletion, such as Parkinson's Disease (PD), have shown diminished reactivity during unpleasant events. Here, we examined the role of DA in the enhancement of novelty processing during negative emotion. Forty healthy volunteers were genotyped for the dopamine transporter (DAT) gene SLC6A3 or DAT1 and performed an auditory-visual distraction paradigm in negative and neutral emotional context conditions. 9R- individuals, associated to a lesser striatal DA display, failed to show increased distraction during negative emotion, but experienced an enhancement of the early phase of the novelty-P3 brain response, associated to the evaluation of novel events, in the negative relative to the neutral context. However, 9R+ individuals (associated to larger striatal DA display) showed larger distraction during negative emotion and larger amplitudes of the novelty-P3, irrespective of the condition. These results suggest a blunted reactivity to novelty during negative emotion in 9R- individuals due to a lesser DA display and stronger activation of the representation of novel events in the 9R+ group, due to a larger DA availability, thus reaching a ceiling effect in the neutral context condition with no further enhancement during negative emotion. The present results might help to understand the functional implications of dopamine in some neuropsychiatric disorders.

  4. Regulation of TRPM8 channel activity

    PubMed Central

    Yudin, Yevgen; Rohacs, Tibor

    2011-01-01

    Transient Receptor Potential Melastatin 8 (TRPM8) is a Ca2+ permeable non-selective cation channel directly activated by cold temperatures and chemical agonists such as menthol. It is a well established sensor of environmental cold temperatures, found in peripheral sensory neurons, where its activation evokes depolarization and action potentials. The activity of TRPM8 is regulated by a number of cellular signaling pathways, most notably by phosphoinositides and the activation of phospholipase C. This review will summarize current knowledge on the physiological and pathophysiological roles of TRPM8 and its regulation by various intracellular messenger molecules and signaling pathways. PMID:22061619

  5. Cryptococcus neoformans Mediator Protein Ssn8 Negatively Regulates Diverse Physiological Processes and Is Required for Virulence

    PubMed Central

    Wang, Lin-Ing; Lin, Yu-Sheng; Liu, Kung-Hung; Jong, Ambrose Y.; Shen, Wei-Chiang

    2011-01-01

    Cryptococcus neoformans is a ubiquitously distributed human pathogen. It is also a model system for studying fungal virulence, physiology and differentiation. Light is known to inhibit sexual development via the evolutionarily conserved white collar proteins in C. neoformans. To dissect molecular mechanisms regulating this process, we have identified the SSN8 gene whose mutation suppresses the light-dependent CWC1 overexpression phenotype. Characterization of sex-related phenotypes revealed that Ssn8 functions as a negative regulator in both heterothallic a-α mating and same-sex mating processes. In addition, Ssn8 is involved in the suppression of other physiological processes including invasive growth, and production of capsule and melanin. Interestingly, Ssn8 is also required for the maintenance of cell wall integrity and virulence. Our gene expression studies confirmed that deletion of SSN8 results in de-repression of genes involved in sexual development and melanization. Epistatic and yeast two hybrid studies suggest that C. neoformans Ssn8 plays critical roles downstream of the Cpk1 MAPK cascade and Ste12 and possibly resides at one of the major branches downstream of the Cwc complex in the light-mediated sexual development pathway. Taken together, our studies demonstrate that the conserved Mediator protein Ssn8 functions as a global regulator which negatively regulates diverse physiological and developmental processes and is required for virulence in C. neoformans. PMID:21559476

  6. Btg2 is a Negative Regulator of Cardiomyocyte Hypertrophy through a Decrease in Cytosolic RNA

    PubMed Central

    Masumura, Yuki; Higo, Shuichiro; Asano, Yoshihiro; Kato, Hisakazu; Yan, Yi; Ishino, Saki; Tsukamoto, Osamu; Kioka, Hidetaka; Hayashi, Takaharu; Shintani, Yasunori; Yamazaki, Satoru; Minamino, Tetsuo; Kitakaze, Masafumi; Komuro, Issei; Takashima, Seiji; Sakata, Yasushi

    2016-01-01

    Under hypertrophic stimulation, cardiomyocytes enter a hypermetabolic state and accelerate biomass accumulation. Although the molecular pathways that regulate protein levels are well-studied, the functional implications of RNA accumulation and its regulatory mechanisms in cardiomyocytes remain elusive. Here, we have elucidated the quantitative kinetics of RNA in cardiomyocytes through single cell imaging and c-Myc (Myc)-mediated hypermetabolic analytical model using cultured cardiomyocytes. Nascent RNA labeling combined with single cell imaging demonstrated that Myc protein significantly increased the amount of global RNA production per cardiomyocyte. Chromatin immunoprecipitation with high-throughput sequencing clarified that overexpressed Myc bound to a specific set of genes and recruits RNA polymerase II. Among these genes, we identified Btg2 as a novel target of Myc. Btg2 overexpression significantly reduced cardiomyocyte surface area. Conversely, shRNA-mediated knockdown of Btg2 accelerated adrenergic stimulus-induced hypertrophy. Using mass spectrometry analysis, we determined that Btg2 binds a series of proteins that comprise mRNA deadenylation complexes. Intriguingly, Btg2 specifically suppresses cytosolic, but not nuclear, RNA levels. Btg2 knockdown further enhances cytosolic RNA accumulation in cardiomyocytes under adrenergic stimulation, suggesting that Btg2 negatively regulates reactive hypertrophy by negatively regulating RNA accumulation. Our findings provide insight into the functional significance of the mechanisms regulating RNA levels in cardiomyocytes. PMID:27346836

  7. Negative feedback regulation of auxin signaling by ATHB8/ACL5-BUD2 transcription module.

    PubMed

    Baima, Simona; Forte, Valentina; Possenti, Marco; Peñalosa, Andrés; Leoni, Guido; Salvi, Sergio; Felici, Barbara; Ruberti, Ida; Morelli, Giorgio

    2014-06-01

    The role of auxin as main regulator of vascular differentiation is well established, and a direct correlation between the rate of xylem differentiation and the amount of auxin reaching the (pro)cambial cells has been proposed. It has been suggested that thermospermine produced by ACAULIS5 (ACL5) and bushy and dwarf2 (BUD2) is one of the factors downstream to auxin contributing to the regulation of this process in Arabidopsis. Here, we provide an in-depth characterization of the mechanism through which ACL5 modulates xylem differentiation. We show that an increased level of ACL5 slows down xylem differentiation by negatively affecting the expression of homeodomain-leucine zipper (HD-ZIP) III and key auxin signaling genes. This mechanism involves the positive regulation of thermospermine biosynthesis by the HD-ZIP III protein Arabidopsis thaliana homeobox8 tightly controlling the expression of ACL5 and BUD2. In addition, we show that the HD-ZIP III protein REVOLUTA contributes to the increased leaf vascularization and long hypocotyl phenotype of acl5 likely by a direct regulation of auxin signaling genes such as like auxin resistant2 (LAX2) and LAX3. We propose that proper formation and differentiation of xylem depend on a balance between positive and negative feedback loops operating through HD-ZIP III genes.

  8. Platelet-derived thrombospondin-1 is a critical negative regulator and potential biomarker of angiogenesis

    PubMed Central

    Zaslavsky, Alexander; Baek, Kwan-Hyuck; Lynch, Ryan C.; Short, Sarah; Grillo, Jenny; Folkman, Judah; Italiano, Joseph E.

    2010-01-01

    The sequential events leading to tumor progression include a switch to the angiogenic phenotype, dependent on a shift in the balance between positive and negative angiogenic regulators produced by tumor and stromal cells. Although the biologic properties of many angiogenesis regulatory proteins have been studied in detail, the mechanisms of their transport and delivery in vivo during pathologic angiogenesis are not well understood. Here, we demonstrate that expression of one of the most potent angiogenesis inhibitors, thrombospondin-1, is up-regulated in the platelets of tumor-bearing mice. We establish that this up-regulation is a consequence of both increased levels of thrombospondin-1 mRNA in megakaryocytes, as well as increased numbers of megakaryocytes in the bone marrow of tumor-bearing mice. Through the use of mouse tumor models and bone marrow transplantations, we show that platelet-derived thrombospondin-1 is a critical negative regulator during the early stages of tumor angiogenesis. Collectively, our data suggest that the production and delivery of the endogenous angiogenesis inhibitor thrombospondin-1 by platelets may be a critical host response to suppress tumor growth through inhibiting tumor angiogenesis. Further, this work implicates the use of thrombospondin-1 levels in platelets as an indicator of tumor growth and regression. PMID:20086246

  9. Arabidopsis cold shock domain protein 2 influences ABA accumulation in seed and negatively regulates germination.

    PubMed

    Sasaki, Kentaro; Kim, Myung-Hee; Kanno, Yuri; Seo, Mitsunori; Kamiya, Yuji; Imai, Ryozo

    2015-01-02

    The cold shock domain (CSD) is the most conserved nucleic acid binding domain and is distributed from bacteria to animals and plants. CSD proteins are RNA chaperones that destabilize RNA secondary structures to regulate stress tolerance and development. AtCSP2 is one of the four CSD proteins in Arabidopsis and is up-regulated in response to cold. Since AtCSP2 negatively regulates freezing tolerance, it was proposed to be a modulator of freezing tolerance during cold acclimation. Here, we examined the function of AtCSP2 in seed germination. We found that AtCSP2-overexpressing lines demonstrated retarded germination as compared with the wild type, with or without stress treatments. The ABA levels in AtCSP2-overexpressing seeds were higher than those in the wild type. In addition, overexpression of AtCSP2 reduced the expression of an ABA catabolic gene (CYP707A2) and gibberellin biosynthesis genes (GA20ox and GA3ox). These results suggest that AtCSP2 negatively regulates seed germination by controlling ABA and GA levels.

  10. Emotion Regulation and Excess Weight: Impaired Affective Processing Characterized by Dysfunctional Insula Activation and Connectivity.

    PubMed

    Steward, Trevor; Picó-Pérez, Maria; Mata, Fernanda; Martínez-Zalacaín, Ignacio; Cano, Marta; Contreras-Rodríguez, Oren; Fernández-Aranda, Fernando; Yucel, Murat; Soriano-Mas, Carles; Verdejo-García, Antonio

    2016-01-01

    Emotion-regulation strategies are understood to influence food intake. This study examined the neurophysiological underpinnings of negative emotion processing and emotion regulation in individuals with excess weight compared to normal-weight controls. Fifteen participants with excess-weight (body mass index >25) and sixteen normal-weight controls (body mass index 18-25) performed an emotion-regulation task during functional magnetic resonance imaging. Participants were exposed to 24 negative affective or neutral pictures that they were instructed to Observe (neutral pictures), Maintain (sustain the emotion elicited by negative pictures) or Regulate (down-regulate the emotion provoked by negative pictures through previously trained reappraisal techniques). When instructed to regulate negative emotions by means of cognitive reappraisal, participants with excess weight displayed persistently heightened activation in the right anterior insula. Decreased responsivity was also found in right anterior insula, the orbitofrontal cortex and cerebellum during negative emotion experience in participants with excess weight. Psycho-physiological interaction analyses showed that excess-weight participants had decreased negative functional coupling between the right anterior insula and the right dlPFC, and the bilateral dmPFC during cognitive reappraisal. Our findings support contentions that excess weight is linked to an abnormal pattern of neural activation and connectivity during the experience and regulation of negative emotions, with the insula playing a key role in these alterations. We posit that ineffective regulation of emotional states contributes to the acquisition and preservation of excess weight.

  11. Emotion Regulation and Excess Weight: Impaired Affective Processing Characterized by Dysfunctional Insula Activation and Connectivity

    PubMed Central

    Mata, Fernanda; Martínez-Zalacaín, Ignacio; Cano, Marta; Contreras-Rodríguez, Oren; Fernández-Aranda, Fernando; Yucel, Murat; Soriano-Mas, Carles; Verdejo-García, Antonio

    2016-01-01

    Emotion-regulation strategies are understood to influence food intake. This study examined the neurophysiological underpinnings of negative emotion processing and emotion regulation in individuals with excess weight compared to normal-weight controls. Fifteen participants with excess-weight (body mass index >25) and sixteen normal-weight controls (body mass index 18–25) performed an emotion-regulation task during functional magnetic resonance imaging. Participants were exposed to 24 negative affective or neutral pictures that they were instructed to Observe (neutral pictures), Maintain (sustain the emotion elicited by negative pictures) or Regulate (down-regulate the emotion provoked by negative pictures through previously trained reappraisal techniques). When instructed to regulate negative emotions by means of cognitive reappraisal, participants with excess weight displayed persistently heightened activation in the right anterior insula. Decreased responsivity was also found in right anterior insula, the orbitofrontal cortex and cerebellum during negative emotion experience in participants with excess weight. Psycho-physiological interaction analyses showed that excess-weight participants had decreased negative functional coupling between the right anterior insula and the right dlPFC, and the bilateral dmPFC during cognitive reappraisal. Our findings support contentions that excess weight is linked to an abnormal pattern of neural activation and connectivity during the experience and regulation of negative emotions, with the insula playing a key role in these alterations. We posit that ineffective regulation of emotional states contributes to the acquisition and preservation of excess weight. PMID:27003840

  12. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  13. A comparison of autonomous regulation and negative self-evaluative emotions as predictors of smoking behavior change among college students.

    PubMed

    Lee, Hyoung S; Catley, Delwyn; Harris, Kari Jo

    2012-05-01

    This study compared autonomous self-regulation and negative self-evaluative emotions as predictors of smoking behavior change in college student smokers (N = 303) in a smoking cessation intervention study. Although the two constructs were moderately correlated, latent growth curve modeling revealed that only autonomous regulation, but not negative self-evaluative emotions, was negatively related to the number of days smoked. Results suggest that the two variables tap different aspects of motivation to change smoking behaviors, and that autonomous regulation predicts smoking behavior change better than negative self-evaluative emotions.

  14. EFA6 controls Arf1 and Arf6 activation through a negative feedback loop.

    PubMed

    Padovani, Dominique; Folly-Klan, Marcia; Labarde, Audrey; Boulakirba, Sonia; Campanacci, Valérie; Franco, Michel; Zeghouf, Mahel; Cherfils, Jacqueline

    2014-08-26

    Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.

  15. DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins.

    PubMed

    Nguyen, Hung Thanh; Kugler, Jan-Michael; Cohen, Stephen M

    2017-01-01

    The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity.

  16. DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

    PubMed Central

    2017-01-01

    The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity. PMID:28061504

  17. TaMDAR6 acts as a negative regulator of plant cell death and participates indirectly in stomatal regulation during the wheat stripe rust-fungus interaction.

    PubMed

    Abou-Attia, Mohamed Awaad; Wang, Xiaojie; Nashaat Al-Attala, Mohamed; Xu, Qiang; Zhan, Gangming; Kang, Zhensheng

    2016-03-01

    We identified a new monodehydroascorbate reductase (MDAR) gene from wheat, designated TaMDAR6, which is differentially affected by wheat-Puccinia striiformis f. sp. tritici (Pst) interactions. TaMDAR6 is a negative regulator of plant cell death (PCD) triggered by the Bax gene and Pst. Transcript levels of TaMDAR6 are significantly upregulated during a compatible wheat-Pst interaction, indicating that TaMDAR6 may contribute to plant susceptibility. In addition, H2 O2 production and PCD are significantly induced and initial pathogen development is significantly reduced in the TaMDAR6 knocked-down plants upon Pst infection. Thus, the suppression of TaMDAR6 enhances wheat resistance to Pst. Besides, the suppression of TaMDAR6 during an incompatible interaction induces a change in the morphology of stomata, which leads to poor stoma recognition and as a consequence to reduced infection efficiency. The percentage of infection sites that develop substomatal vesicles decreases in the TaMDAR6 knocked-down plants during the incompatible interaction presumably due to the increase in ROS accumulation, which is likely to activate other resistance mechanisms that have a negative effect on substomatal vesicle formation. TaMDAR6 can therefore be considered a negative regulator of PCD and of wheat defense to Pst.

  18. A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

    PubMed

    Düwel, Michael; Welteke, Verena; Oeckinghaus, Andrea; Baens, Mathijs; Kloo, Bernhard; Ferch, Uta; Darnay, Bryant G; Ruland, Jürgen; Marynen, Peter; Krappmann, Daniel

    2009-06-15

    The Carma1-Bcl10-Malt1 signaling module bridges TCR signaling to the canonical IkappaB kinase (IKK)/NF-kappaB pathway. Covalent attachment of regulatory ubiquitin chains to Malt1 paracaspase directs TCR signaling to IKK activation. Further, the ubiquitin-editing enzyme A20 was recently suggested to suppress T cell activation, but molecular targets for A20 remain elusive. In this paper, we show that A20 regulates the strength and duration of the IKK/NF-kappaB response upon TCR/CD28 costimulation. By catalyzing the removal of K63-linked ubiquitin chains from Malt1, A20 prevents sustained interaction between ubiquitinated Malt1 and the IKK complex and thus serves as a negative regulator of inducible IKK activity. Upon T cell stimulation, A20 is rapidly removed and paracaspase activity of Malt1 has been suggested to cleave A20. Using antagonistic peptides or reconstitution of Malt1(-/-) T cells, we show that Malt1 paracaspase activity is required for A20 cleavage and optimal IL-2 production, but dispensable for initial IKK/NF-kappaB signaling in CD4(+) T cells. However, proteasomal inhibition impairs A20 degradation and impedes TCR/CD28-induced IKK activation. Taken together, A20 functions as a Malt1 deubiquitinating enzyme and proteasomal degradation and de novo synthesis of A20 contributes to balance TCR/CD28-induced IKK/NF-kappaB signaling.

  19. The RNA-binding protein Tristetraprolin (TTP) is a critical negative regulator of the NLRP3 inflammasome.

    PubMed

    Haneklaus, Moritz; O'Neil, John D; Clark, Andrew R; Masters, Seth L; O'Neill, Luke A J

    2017-03-16

    The NLRP3 inflammasome is a central regulator of inflammation in many common diseases, including atherosclerosis and Type 2 diabetes, driving the production of pro-inflammatory mediators such as IL-1β and IL-18. Due to its function as an inflammatory gatekeeper, expression and activation of NLRP3 need to be tightly regulated. In this study, we highlight novel post-transcriptional mechanisms that can modulate NLRP3 expression. We have identified the RNA-binding protein Tristetraprolin (TTP) as a negative regulator of NLRP3 in human macrophages. TTP targets AU-rich elements in the NLRP3 3' untranslated region (UTR) and represses NLRP3 expression. Knocking down TTP in primary macrophages leads to an increased induction of NLRP3 by LPS, which is also accompanied by increased Caspase-1 and IL-1β cleavage upon NLRP3, but not AIM2 or NLRC4 inflammasome activation. Furthermore, we found that human NLRP3 can be alternatively polyadenylated, producing a short 3'UTR isoform that excludes regulatory elements, including the TTP and miRNA-223 binding sites. Since TTP also represses IL-1β expression, it is a dual inhibitor of the IL-1β system, regulating expression of the cytokine and the upstream controller NLRP3.

  20. Small leucine zipper protein (sLZIP) negatively regulates skeletal muscle differentiation via interaction with α-actinin-4.

    PubMed

    An, Hyoung-Tae; Kim, Jeonghan; Yoo, Seungmin; Ko, Jesang

    2014-02-21

    The small leucine zipper protein (sLZIP) plays a role in transcriptional regulation in various types of cells. However, the role of sLZIP in myogenesis is unknown. We identified α-actinin-4 (ACTN4) as a sLZIP-binding protein. ACTN4 functions as a transcriptional regulator of myocyte enhancer factor (MEF)2, which plays a critical role in expression of muscle-specific genes during skeletal muscle differentiation. We found that ACTN4 translocates to the nucleus, induces myogenic gene expression, and promotes myotube formation during myogenesis. The myogenic process is controlled by an association between myogenic factors and MEF2 transcription factors. ACTN4 increased expression of muscle-specific proteins via interaction with MEF2. However, sLZIP decreased myogenic gene expression and myotube formation during myogenesis via disruption of the association between ACTN4 and MEF2. ACTN4 increased the promoter activities of myogenic genes, whereas sLZIP abrogated the effect of ACTN4 on transcriptional activation of myogenic genes in myoblasts. The C terminus of sLZIP is required for interaction with the C terminus of ACTN4, based on deletion mutant analysis, and sLZIP plays a role in regulation of MEF2 transactivation via interaction with ACTN4. Our results indicate that sLZIP negatively regulates skeletal muscle differentiation via interaction with ACTN4 and that sLZIP can be used as a therapeutic target molecule for treatment of muscle hypertrophy and associated diseases.

  1. Overlapping neural substrates between intentional and incidental down-regulation of negative emotions.

    PubMed

    Payer, Doris E; Baicy, Kate; Lieberman, Matthew D; London, Edythe D

    2012-04-01

    Emotion regulation can be achieved in various ways, but few studies have evaluated the extent to which the neurocognitive substrates of these distinct operations overlap. In the study reported here, functional magnetic resonance imaging (fMRI) was used to measure activity in the amygdala and prefrontal cortex of 10 participants who completed two independent tasks of emotion regulation-reappraisal, measuring intentional emotion regulation, and affect labeling, measuring incidental emotion regulation-with the objective of identifying potential overlap in the neural substrates underlying each task. Analyses focused on a priori regions of interest in the amygdala and inferior frontal gyrus (IFG). For both tasks, fMRI showed decreased amygdala activation during emotion regulation compared with emotion conditions. During reappraisal, this decrease in amygdala activation was accompanied by a proportional decrease in emotional intensity ratings; during affect labeling, the decrease in amygdala activation correlated with self-reported aggression. Importantly, across participants, the magnitude of decrease in amygdala activation during reappraisal correlated with the magnitude of decrease during affect labeling, even though the tasks were administered on separate days, and values indexing amygdala activation during each task were extracted independently of one another. In addition, IFG-amygdala connectivity, assessed via psychophysiological interaction analysis, overlapped between tasks in two regions within the right IFG. The results suggest that the two tasks recruit overlapping regions of prefrontal cortex, resulting in similar reductions in amygdala activation, regardless of the strategy employed. Intentional and incidental forms of emotion regulation, despite their phenomenological differences, may therefore converge on a common neurocognitive pathway.

  2. Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium.

    PubMed

    Schneider, Fabian T; Schänzer, Anne; Czupalla, Cathrin J; Thom, Sonja; Engels, Knut; Schmidt, Mirko H H; Plate, Karl H; Liebner, Stefan

    2010-07-01

    Wnt/beta-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/beta-catenin pathway activation in reporter mice and by nuclear beta-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. beta-Catenin activation in APC(min/+) mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses beta-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate beta-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear beta-catenin in the tongue epithelium of Patched(+/-) mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear beta-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce beta-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on beta-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/beta-catenin targets Shh and JAG2 control beta-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC.

  3. Sonic Hedgehog Acts as a Negative Regulator of β-Catenin Signaling in the Adult Tongue Epithelium

    PubMed Central

    Schneider, Fabian T.; Schänzer, Anne; Czupalla, Cathrin J.; Thom, Sonja; Engels, Knut; Schmidt, Mirko H.H.; Plate, Karl H.; Liebner, Stefan

    2010-01-01

    Wnt/β-catenin signaling has been implicated in taste papilla development; however, its role in epithelial maintenance and tumor progression in the adult tongue remains elusive. We show Wnt/β-catenin pathway activation in reporter mice and by nuclear β-catenin staining in the epithelium and taste papilla of adult mouse and human tongues. β-Catenin activation in APCmin/+ mice, which carry a mutation in adenomatous poliposis coli (APC), up-regulates Sonic hedgehog (Shh) and Jagged-2 (JAG2) in the tongue epithelium without formation of squamous cell carcinoma (SCC). We demonstrate that Shh suppresses β-catenin transcriptional activity in a signaling-dependent manner in vitro and in vivo. A similar regulation and function was observed for JAG2, suggesting that both pathways negatively regulate β-catenin, thereby preventing SCC formation in the tongue. This was supported by reduced nuclear β-catenin in the tongue epithelium of Patched+/− mice, exhibiting dominant active Shh signaling. At the invasive front of human tongue cancer, nuclear β-catenin and Shh were increased, suggesting their participation in tumor progression. Interestingly, Shh but not JAG2 was able to reduce β-catenin signaling in SCC cells, arguing for a partial loss of negative feedback on β-catenin transcription in tongue cancer. We show for the first time that the putative Wnt/β-catenin targets Shh and JAG2 control β-catenin signaling in the adult tongue epithelium, a function that is partially lost in lingual SCC. PMID:20508033

  4. The negative cell cycle regulator, Tob (transducer of ErbB-2), is involved in motor skill learning

    SciTech Connect

    Wang Xinming; Gao Xiang; Zhang Xuehan; Tu Yanyang; Jin Meilei; Zhao Guoping; Yu Lei; Jing Naihe; Li Baoming . E-mail: bmli@fudan.edu.cn

    2006-02-24

    Tob (transducer of ErbB-2) is a negative cell cycle regulator with anti-proliferative activity in peripheral tissues. Our previous study identified Tob as a protein involved in hippocampus-dependent memory consolidation (M.L. Jin, X.M. Wang, Y.Y. Tu, X.H. Zhang, X. Gao, N. Guo, Z.Q. Xie, G.P. Zhao, N.H. Jing, B.M. Li, Y.Yu, The negative cell cycle regulator, Tob (Transducer of ErbB-2), is a multifunctional protein involved in hippocampus-dependent learning and memory, Neuroscience 131 (2005) 647-659). Here, we provide evidence that Tob in the central nervous system is engaged in acquisition of motor skill. Tob has a relatively high expression in the cerebellum. Tob expression is up-regulated in the cerebellum after rats receive training on a rotarod-running task. Rats infused with Tob antisense oligonucleotides into the 4th ventricle exhibit a severe deficit in running on a rotating rod or walking across a horizontally elevated beam.

  5. miR-375 Negatively Regulates the Synthesis and Secretion of Catecholamines by Targeting Sp1 in Rat Adrenal Medulla.

    PubMed

    Gai, Yedan; Zhang, Jinglin; Wei, Chao; Cao, Wei; Cui, Yan; Cui, Sheng

    2017-03-29

    Adrenal gland is a crucial endocrine gland, and the most important function is to synthesize and secrete catecholamines (CATs) which play crucial roles in balancing homeostasis and the responding to stress. microRNA-375 (miR-375) has been detected to highly express in the adrenal, however its role and underlying mechanism are currently unclear. Herein, our results showed that miR-375 was specifically localized to the rat adrenal medulla chromaffin cells, and miR-375 expressing level decreased, when the rats were exposed to stress. The further functional studies demonstrated that the inhibition of endogenous miR-375 induced the secretion of CATs in primary rat medulla chromaffin cells and in PC12 cells, and over-expression of miR-375 resulted in decline of the CATs secretion. Furthermore, the results showed that miR-375 negatively regulated tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) and mediated adrenomedullary CATs biosynthesis. Sp1(a transcriptional activator of TH and DBH) was involved in mediating the regulation of TH and DBH as miR-375 direct target gene. These novel findings suggest that miR-375 acts as a potent negative mediator in regulating the synthesis and secretion of CATs in the adrenal medulla during the maintenance of homeostasis under stress.

  6. Active Negative Pressure Peritoneal Therapy After Abbreviated Laparotomy

    PubMed Central

    Roberts, Derek J.; Faris, Peter D.; Ball, Chad G.; Kubes, Paul; Tiruta, Corina; Xiao, Zhengwen; Holodinsky, Jessalyn K.; McBeth, Paul B.; Doig, Christopher J.; Jenne, Craig N.

    2015-01-01

    Objective: To determine whether active negative pressure peritoneal therapy with the ABThera temporary abdominal closure device reduces systemic inflammation after abbreviated laparotomy. Background: Excessive systemic inflammation after abdominal injury or intra-abdominal sepsis is associated with poor outcomes. Methods: We conducted a single-center, randomized controlled trial. Forty-five adults with abdominal injury (46.7%) or intra-abdominal sepsis (52.3%) were randomly allocated to the ABThera (n = 23) or Barker's vacuum pack (n = 22). On study days 1, 2, 3, 7, and 28, blood and peritoneal fluid were collected. The primary endpoint was the difference in the plasma concentration of interleukin-6 (IL-6) 24 and 48 hours after temporary abdominal closure application. Results: There was a significantly lower peritoneal fluid drainage from the ABThera at 48 hours after randomization. Despite this, there was no difference in plasma concentration of IL-6 at baseline versus 24 (P = 0.52) or 48 hours (P = 0.82) between the groups. There was also no significant intergroup difference in the plasma concentrations of IL-1β, −8, −10, or −12 p70 or tumor necrosis factor α between these time points. The cumulative incidence of primary fascial closure at 90 days was similar between groups (hazard ratio, 1.6; 95% confidence interval, 0.82–3.0; P = 0.17). However, 90-day mortality was improved in the ABThera group (hazard ratio, 0.32; 95% confidence interval, 0.11–0.93; P = 0.04). Conclusions: This trial observed a survival difference between patients randomized to the ABThera versus Barker's vacuum pack that did not seem to be mediated by an improvement in peritoneal fluid drainage, fascial closure rates, or markers of systemic inflammation. Trial Registration: ClinicalTrials.gov identifier NCT01355094. PMID:25536308

  7. Integral role of transcription factor 8 in the negative regulation of tumor angiogenesis.

    PubMed

    Inuzuka, Takayuki; Tsuda, Masumi; Tanaka, Shinya; Kawaguchi, Hideaki; Higashi, Yujiro; Ohba, Yusuke

    2009-02-15

    Angiogenesis is involved in various physiologic and pathological conditions, including tumor growth, and is tightly regulated by the orchestration of proangiogenic and antiangiogenic factors. Inhibition of vascular endothelial growth factor (VEGF), the best-established antiangiogenic treatment in cancer, has shown some effectiveness; however, the identification of novel regulators, whose function is independent of VEGF, is required to achieve better outcomes. Here, we show that transcription factor 8 (TCF8) is up-regulated in endothelial cells during angiogenesis, acting as a negative regulator. Furthermore, TCF8 is specifically expressed in the endothelium of tumor vessels. Tcf8-heterozygous knockout mice are more permissive than wild-type mice to the formation of tumor blood vessels in s.c. implanted melanoma, which seems to contribute to the more aggressive growth and the lung metastases of the tumor in mutant mice. Suppression of TCF8 facilitates angiogenesis in both in vitro and ex vivo models, and displays comprehensive cellular phenotypes, including enhanced cell invasion, impaired cell adhesion, and increased cell monolayer permeability due to, at least partly, MMP1 overexpression, attenuation of focal adhesion formation, and insufficient VE-cadherin recruitment, respectively. Taken together, our findings define a novel, integral role for TCF8 in the regulation of pathologic angiogenesis, and propose TCF8 as a target for therapeutic intervention in cancer.

  8. Suppressor of IKKɛ is an essential negative regulator of pathological cardiac hypertrophy

    PubMed Central

    Deng, Ke-Qiong; Wang, Aibing; Ji, Yan-Xiao; Zhang, Xiao-Jing; Fang, Jing; Zhang, Yan; Zhang, Peng; Jiang, Xi; Gao, Lu; Zhu, Xue-Yong; Zhao, Yichao; Gao, Lingchen; Yang, Qinglin; Zhu, Xue-Hai; Wei, Xiang; Pu, Jun; Li, Hongliang

    2016-01-01

    Although pathological cardiac hypertrophy represents a leading cause of morbidity and mortality worldwide, our understanding of the molecular mechanisms underlying this disease is still poor. Here, we demonstrate that suppressor of IKKɛ (SIKE), a negative regulator of the interferon pathway, attenuates pathological cardiac hypertrophy in rodents and non-human primates in a TANK-binding kinase 1 (TBK1)/AKT-dependent manner. Sike-deficient mice develop cardiac hypertrophy and heart failure, whereas Sike-overexpressing transgenic (Sike-TG) mice are protected from hypertrophic stimuli. Mechanistically, SIKE directly interacts with TBK1 to inhibit the TBK1-AKT signalling pathway, thereby achieving its anti-hypertrophic action. The suppression of cardiac remodelling by SIKE is further validated in rats and monkeys. Collectively, these findings identify SIKE as a negative regulator of cardiac remodelling in multiple animal species due to its inhibitory regulation of the TBK1/AKT axis, suggesting that SIKE may represent a therapeutic target for the treatment of cardiac hypertrophy and heart failure. PMID:27249321

  9. Social anxiety and emotion regulation in daily life: spillover effects on positive and negative social events.

    PubMed

    Farmer, Antonina Savostyanova; Kashdan, Todd B

    2012-01-01

    To minimize the possibility of scrutiny, people with social anxiety difficulties exert great effort to manage their emotions, particularly during social interactions. We examined how the use of two emotion regulation strategies, emotion suppression and cognitive reappraisal, predict the generation of emotions and social events in daily life. Over 14 consecutive days, 89 participants completed daily diary entries on emotions, positive and negative social events, and their regulation of emotions. Using multilevel modeling, we found that when people high in social anxiety relied more on positive emotion suppression, they reported fewer positive social events and less positive emotion on the subsequent day. In contrast, people low in social anxiety reported fewer negative social events on days subsequent to using cognitive reappraisal to reduce distress; the use of cognitive reappraisal did not influence the daily lives of people high in social anxiety. Our findings support theories of emotion regulation difficulties associated with social anxiety. In particular, for people high in social anxiety, maladaptive strategy use contributed to diminished reward responsiveness.

  10. The ciliary protein nephrocystin-4 translocates the canonical Wnt regulator Jade-1 to the nucleus to negatively regulate β-catenin signaling.

    PubMed

    Borgal, Lori; Habbig, Sandra; Hatzold, Julia; Liebau, Max C; Dafinger, Claudia; Sacarea, Ilinca; Hammerschmidt, Matthias; Benzing, Thomas; Schermer, Bernhard

    2012-07-20

    Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease and represents the most common genetic cause for end-stage renal disease in children and adolescents. It can be caused by the mutation of genes encoding for the nephrocystin proteins (NPHPs). All NPHPs localize to primary cilia, classifying this disease as a "ciliopathy." The primary cilium is a critical regulator of several cell signaling pathways. Cystogenesis in the kidney is thought to involve overactivation of canonical Wnt signaling, which is negatively regulated by the primary cilium and several NPH proteins, although the mechanism remains unclear. Jade-1 has recently been identified as a novel ubiquitin ligase targeting the canonical Wnt downstream effector β-catenin for proteasomal degradation. Here, we identify Jade-1 as a novel component of the NPHP protein complex. Jade-1 colocalizes with NPHP1 at the transition zone of primary cilia and interacts with NPHP4. Furthermore, NPHP4 stabilizes protein levels of Jade-1 and promotes the translocation of Jade-1 to the nucleus. Finally, NPHP4 and Jade-1 additively inhibit canonical Wnt signaling, and this genetic interaction is conserved in zebrafish. The stabilization and nuclear translocation of Jade-1 by NPHP4 enhances the ability of Jade-1 to negatively regulate canonical Wnt signaling. Loss of this repressor function in nephronophthisis might be an important factor promoting Wnt activation and contributing to cyst formation.

  11. Impact of physical maltreatment on the regulation of negative affect and aggression.

    PubMed

    Shackman, Jessica E; Pollak, Seth D

    2014-11-01

    Physically maltreated children are at risk for developing externalizing behavioral problems characterized by reactive aggression. The current experiment tested the relationships between individual differences in a neural index of social information processing, histories of child maltreatment, child negative affect, and aggressive behavior. Fifty boys (17 maltreated) performed an emotion recognition task while the P3b component of the event-related potential was recorded to index attention allocation to angry faces. Children then participated in a peer-directed aggression task. Negative affect was measured by recording facial electromyography, and aggression was indexed by the feedback that children provided to a putative peer. Physically maltreated children exhibited greater negative affect and more aggressive behavior, compared to nonmaltreated children, and this relationship was mediated by children's allocation of attention to angry faces. These data suggest that physical maltreatment leads to inappropriate regulation of both negative affect and aggression, which likely place maltreated children at increased risk for the development and maintenance of externalizing behavior disorders.

  12. Impact of physical maltreatment on the regulation of negative affect and aggression

    PubMed Central

    SHACKMAN, JESSICA E.; POLLAK, SETH D.

    2015-01-01

    Physically maltreated children are at risk for developing externalizing behavioral problems characterized by reactive aggression. The current experiment tested the relationships between individual differences in a neural index of social information processing, histories of child maltreatment, child negative affect, and aggressive behavior. Fifty boys (17 maltreated) performed an emotion recognition task while the P3b component of the event-related potential was recorded to index attention allocation to angry faces. Children then participated in a peer-directed aggression task. Negative affect was measured by recording facial electromyography, and aggression was indexed by the feedback that children provided to a putative peer. Physically maltreated children exhibited greater negative affect and more aggressive behavior, compared to nonmaltreated children, and this relationship was mediated by children’s allocation of attention to angry faces. These data suggest that physical maltreatment leads to inappropriate regulation of both negative affect and aggression, which likely place maltreated children at increased risk for the development and maintenance of externalizing behavior disorders. PMID:24914736

  13. The circadian clock-related gene pex regulates a negative cis element in the kaiA promoter region.

    PubMed

    Kutsuna, Shinsuke; Kondo, Takao; Ikegami, Haruki; Uzumaki, Tatsuya; Katayama, Mitsunori; Ishiura, Masahiro

    2007-11-01

    In the cyanobacterium Synechococcus sp. strain PCC 7942, a circadian clock-related gene, pex, was identified as the gene prolonging the period of the clock. A PadR domain, which is a newly classified transcription factor domain, and the X-ray crystal structure of the Pex protein suggest a role for Pex in transcriptional regulation in the circadian system. However, the regulatory target of the Pex protein is unknown. To determine the role of Pex, we monitored bioluminescence rhythms that reported the expression activity of the kaiA gene or the kaiBC operon in pex deficiency, pex constitutive expression, and the wild-type genotype. The expression of kaiA in the pex-deficient or constitutive expression genotype was 7 or 1/7 times that of the wild type, respectively, suggesting that kaiA is the target of negative regulation by Pex. In contrast, the expression of the kaiBC gene in the two pex-related genotypes was the same as that in the wild type, suggesting that Pex specifically regulates kaiA expression. We used primer extension analysis to map the transcription start site for the kaiA gene 66 bp upstream of the translation start codon. Mapping with deletion and base pair substitution of the kaiA upstream region revealed that a 5-bp sequence in this region was essential for the regulation of kaiA. The repression or constitutive expression of the kaiA transgene caused the prolongation or shortening of the circadian period, respectively, suggesting that the Pex protein changes the period via the negative regulation of kaiA.

  14. Regulation of negative affect in schizophrenia: the effectiveness of acceptance versus reappraisal and suppression.

    PubMed

    Perry, Yael; Henry, Julie D; Nangle, Matthew R; Grisham, Jessica R

    2012-01-01

    Although general emotion coping difficulties are well documented in schizophrenia, there has been limited study of specific regulatory strategies such as suppression, reappraisal, and acceptance. In the present study, clinical and control participants were asked to watch video clips selected to elicit negative affect while engaging in one of these three different emotion regulation strategies (counterbalanced), versus a passive viewing condition. The experiential and expressive components of emotion were quantified using self-report and facial electromyography, respectively. A major finding was that, in contrast to control participants, individuals with schizophrenia did not report a greater willingness to reexperience negative emotion after engaging in acceptance. These data are discussed in the context of evidence highlighting the potentially important role of acceptance in understanding affective abnormalities in clinical conditions such as schizophrenia.

  15. An H-NS-like protein involved in the negative regulation of hrp genes in Xanthomonas oryzae pv. oryzae.

    PubMed

    Kametani-Ikawa, Yumi; Tsuge, Seiji; Furutani, Ayako; Ochiai, Hirokazu

    2011-06-01

    hrp genes encode components of a type III secretion (T3S) system and play crucial roles in the pathogenicity of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). A histone-like nucleoid-structuring (H-NS) protein binds DNA and acts as a global transcriptional repressor. Here, we investigated the involvement of an h-ns-like gene, named xrvB, in the expression of hrp genes in Xoo. Under the hrp-inducing culture condition, the expression of a key hrp regulator HrpG increased in the XrvB mutant, followed by activation of the downstream gene expression. Also, in planta, the secretion of a T3S protein (XopR) was activated by the mutation in xrvB. Gel retardation assay indicated that XrvB has DNA-binding activity, but without a preference for the promoter region of hrpG. The results suggest that XrvB negatively regulates hrp gene expression and that an unknown factor(s) mediates the regulation of hrpG expression by XrvB.

  16. Negative Regulation of Anthocynanin Biosynthesis in Arabidopsis by a miR156-Targeted SPL Transcription Factor

    SciTech Connect

    Gou, J.Y.; Liu, C.; Felippes, F. F.; Weigel, D.; Wang, J.-W.

    2011-04-01

    Flavonoids are synthesized through an important metabolic pathway that leads to the production of diverse secondary metabolites, including anthocyanins, flavonols, flavones, and proanthocyanidins. Anthocyanins and flavonols are derived from Phe and share common precursors, dihydroflavonols, which are substrates for both flavonol synthase and dihydroflavonol 4-reductase. In the stems of Arabidopsis thaliana, anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. We show here that this accumulation pattern is under the regulation of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are deeply conserved and known to have important roles in regulating phase change and flowering. Increased miR156 activity promotes accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols. We further provide evidence that at least one of the miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Our results reveal a direct link between the transition to flowering and secondary metabolism and provide a potential target for manipulation of anthocyanin and flavonol content in plants.

  17. A Serine Protease Homolog Negatively Regulates TEP1 Consumption in Systemic Infections of the Malaria Vector Anopheles gambiae

    PubMed Central

    Yassine, Hassan; Kamareddine, Layla; Chamat, Soulaima; Christophides, George K.; Osta, Mike A.

    2016-01-01

    Clip domain serine protease homologs are widely distributed in insect genomes and play important roles in regulating insect immune responses, yet their exact functions remain poorly understood. Here, we show that CLIPA2, a clip domain serine protease homolog of Anopheles gambiae, regulates the consumption of the mosquito complement-like protein TEP1 during systemic bacterial infections. We provide evidence that CLIPA2 localizes to microbial surfaces in a TEP1-dependent manner whereby it negatively regulates the activity of a putative TEP1 convertase, which converts the full-length TEP1-F form into active TEP1cut. CLIPA2 silencing triggers an exacerbated TEP1-mediated response that significantly enhances mosquito resistance to infections with a broad class of microorganisms including Plasmodium berghei, Escherichia coli and the entomopathogenic fungus Beauveria bassiana. We also provide further evidence for the existence of a functional link between TEP1 and activation of hemolymph prophenoloxidase during systemic infections. Interestingly, the enhanced TEP1-mediated immune response in CLIPA2 knockdown mosquitoes correlated with a significant reduction in fecundity, corroborating the existence of a trade-off between immunity and reproduction. In sum, CLIPA2 is an integral regulatory component of the mosquito complement-like pathway which functions to prevent an overwhelming response by the host in response to systemic infections. PMID:25012124

  18. Positive and negative regulation of T cell responses by fibroblastic reticular cells within paracortical regions of lymph nodes

    PubMed Central

    Siegert, Stefanie; Luther, Sanjiv A.

    2012-01-01

    Fibroblastic reticular cells (FRC) form the structural backbone of the T cell rich zones in secondary lymphoid organs (SLO), but also actively influence the adaptive immune response. They provide a guidance path for immigrating T lymphocytes and dendritic cells (DC) and are the main local source of the cytokines CCL19, CCL21, and IL-7, all of which are thought to positively regulate T cell homeostasis and T cell interactions with DC. Recently, FRC in lymph nodes (LN) were also described to negatively regulate T cell responses in two distinct ways. During homeostasis they express and present a range of peripheral tissue antigens, thereby participating in peripheral tolerance induction of self-reactive CD8+ T cells. During acute inflammation T cells responding to foreign antigens presented on DC very quickly release pro-inflammatory cytokines such as interferon γ. These cytokines are sensed by FRC which transiently produce nitric oxide (NO) gas dampening the proliferation of neighboring T cells in a non-cognate fashion. In summary, we propose a model in which FRC engage in a bidirectional crosstalk with both DC and T cells to increase the efficiency of the T cell response. However, during an acute response, FRC limit excessive expansion and inflammatory activity of antigen-specific T cells. This negative feedback loop may help to maintain tissue integrity and function during rapid organ growth. PMID:22973278

  19. FoxO3a regulates hematopoietic homeostasis through a negative feedback pathway in conditions of stress or aging

    PubMed Central

    Miyamoto, Kana; Kato, Reiko; Yoshimura, Akihiko; Motoyama, Noboru

    2008-01-01

    Stress or aging of tissue-specific stem cells is considered central to the decline of tissue homeostasis in the elderly, although little is known of molecular mechanisms underlying hematopoietic stem cell (HSC) aging and stress resistance. Here, we report that mice lacking the transcription factor forkhead box O3a (FoxO3a) develop neutrophilia associated with inhibition of the up-regulation of negative regulator of cell proliferation, Sprouty-related Ena/VASP homology 1 domain-containing proteins 2 (Spred2) and AKT and ERK activation, in HSCs during hematopoietic recovery following myelosuppressive stress conditions. Compared with aged wild-type mice, more severe neutrophilia was also observed in aged Foxo3a-deficient mice. AKT and ERK activation and inhibition of Spred2 were detected in HSCs from aged FoxO3a-deficient mice. Spred2-deficient mice also developed neutrophilia during hematopoietic recovery following myelosuppressive stress, indicating that FoxO3a plays a pivotal role in maintenance, integrity, and stress resistance of HSCs through negative feedback pathways for proliferation. This will provide new insight into the hematopoietic homeostasis in conditions of aging and stress. PMID:18799725

  20. Long noncoding RNA LINP1 regulates double strand DNA break repair in triple negative breast cancer

    PubMed Central

    Zhang, Youyou; He, Qun; Hu, Zhongyi; Feng, Yi; Fan, Lingling; Tang, Zhaoqing; Yuan, Jiao; Shan, Weiwei; Li, Chunsheng; Hu, Xiaowen; Tanyi, Janos L; Fan, Yi; Huang, Qihong; Montone, Kathleen; Dang, Chi V; Zhang, Lin

    2016-01-01

    Long noncoding RNAs (lncRNAs), which are transcripts that are larger than 200 nucleotides but do not appear to have protein-coding potential, play critical roles during tumorigenesis by functioning as scaffolds to regulate protein-protein, protein-DNA or protein-RNA interactions. Using a clinically guided genetic screening approach, we identified (lncRNA in Non-homologous end joining [NHEJ] pathway 1) as a lncRNA that is overexpressed in human triple-negative breast cancer. We found that LINP1 enhances double-strand DNA break repair by serving as a scaffold that links Ku80 and DNA-PKcs, thereby coordinating the NHEJ pathway. Importantly, blocking LINP1, which is regulated by the p53 and epidermal growth factor receptor (EGFR) signaling, increases sensitivity of tumor cell response to radiotherapy in breast cancer. PMID:27111890

  1. MBSR vs aerobic exercise in social anxiety: fMRI of emotion regulation of negative self-beliefs.

    PubMed

    Goldin, Philippe; Ziv, Michal; Jazaieri, Hooria; Hahn, Kevin; Gross, James J

    2013-01-01

    Mindfulness-based stress reduction (MBSR) is thought to reduce emotional reactivity and enhance emotion regulation in patients with social anxiety disorder (SAD). The goal of this study was to examine the neural correlates of deploying attention to regulate responses to negative self-beliefs using functional magnetic resonance imaging. Participants were 56 patients with generalized SAD in a randomized controlled trial who were assigned to MBSR or a comparison aerobic exercise (AE) stress reduction program. Compared to AE, MBSR yielded greater (i) reductions in negative emotion when implementing regulation and (ii) increases in attention-related parietal cortical regions. Meditation practice was associated with decreases in negative emotion and social anxiety symptom severity, and increases in attention-related parietal cortex neural responses when implementing attention regulation of negative self-beliefs. Changes in attention regulation during MBSR may be an important psychological factor that helps to explain how mindfulness meditation training benefits patients with anxiety disorders.

  2. The R3-MYB gene GhCPC negatively regulates cotton fiber elongation.

    PubMed

    Liu, Bingliang; Zhu, Yichao; Zhang, Tianzhen

    2015-01-01

    Cotton (Gossypium spp.) fibers are single-cell trichomes that arise from the outer epidermal layer of seed coat. Here, we isolated a R3-MYB gene GhCPC, identified by cDNA microarray analysis. The only conserved R3 motif and different expression between TM-1 and fuzzless-lintless mutants suggested that it might be a negative regulator in fiber development. Transgenic evidence showed that GhCPC overexpression not only delayed fiber initiation but also led to significant decreases in fiber length. Interestingly, Yeast two-hybrid analysis revealed an interaction complex, in which GhCPC and GhTTG1/4 separately interacted with GhMYC1. In transgenic plants, Q-PCR analysis showed that GhHOX3 (GL2) and GhRDL1 were significantly down regulated in -1-5 DPA ovules and fibers. In addition, Yeast one-hybrid analysis demonstrated that GhMYC1 could bind to the E-box cis-elements and the promoter of GhHOX3. These results suggested that GhHOX3 (GL2) might be downstream gene of the regulatory complex. Also, overexpression of GhCPC in tobacco led to differential loss of pigmentation. Taken together, the results suggested that GhCPC might negatively regulate cotton fiber initiation and early elongation by a potential CPC-MYC1-TTG1/4 complex. Although the fibers were shorter in transgenic cotton lines than in the wild type, no significant difference was detected in stem or leaf trichomes, even in cotton mutants (five naked seed or fuzzless), suggesting that fiber and trichome development might be regulated by two sets of genes sharing a similar model.

  3. Osa-miR169 Negatively Regulates Rice Immunity against the Blast Fungus Magnaporthe oryzae

    PubMed Central

    Li, Yan; Zhao, Sheng-Li; Li, Jin-Lu; Hu, Xiao-Hong; Wang, He; Cao, Xiao-Long; Xu, Yong-Ju; Zhao, Zhi-Xue; Xiao, Zhi-Yuan; Yang, Nan; Fan, Jing; Huang, Fu; Wang, Wen-Ming

    2017-01-01

    miR169 is a conserved microRNA (miRNA) family involved in plant development and stress-induced responses. However, how miR169 functions in rice immunity remains unclear. Here, we show that miR169 acts as a negative regulator in rice immunity against the blast fungus Magnaporthe oryzae by repressing the expression of nuclear factor Y-A (NF-YA) genes. The accumulation of miR169 was significantly increased in a susceptible accession but slightly fluctuated in a resistant accession upon M. oryzae infection. Consistently, the transgenic lines overexpressing miR169a became hyper-susceptible to different M. oryzae strains associated with reduced expression of defense-related genes and lack of hydrogen peroxide accumulation at the infection site. Consequently, the expression of its target genes, the NF-YA family members, was down-regulated by the overexpression of miR169a at either transcriptional or translational level. On the contrary, overexpression of a target mimicry that acts as a sponge to trap miR169a led to enhanced resistance to M. oryzae. In addition, three of miR169’s target genes were also differentially up-regulated in the resistant accession upon M. oryzae infection. Taken together, our data indicate that miR169 negatively regulates rice immunity against M. oryzae by differentially repressing its target genes and provide the potential to engineer rice blast resistance via a miRNA. PMID:28144248

  4. Importin beta negatively regulates nuclear membrane fusion and nuclear pore complex assembly.

    PubMed

    Harel, Amnon; Chan, Rene C; Lachish-Zalait, Aurelie; Zimmerman, Ella; Elbaum, Michael; Forbes, Douglass J

    2003-11-01

    Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin beta negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin beta is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin beta down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin beta to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin beta 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin beta-regulation of NPC assembly. Excess full-length importin beta and beta 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin beta NPC block, which maps downstream of GTPgammaS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 microM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin beta. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.

  5. Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport

    PubMed Central

    Held, Aaron; Sargeant, John; Thorsen, Kevin; Hay, Jesse C.

    2016-01-01

    Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport—peflin is a negative regulator of transport. PMID:27276012

  6. The veA gene is necessary for the negative regulation of the veA expression in Aspergillus nidulans.

    PubMed

    Kim, Hyoun-Young; Han, Kap-Hoon; Lee, Mimi; Oh, Miae; Kim, Hee-Seo; Zhixiong, Xie; Han, Dong-Min; Jahng, Kwang-Yeop; Kim, Jong Hwa; Chae, Keon-Sang

    2009-08-01

    The veA gene is one of the key genes in regulating sexual development of Aspergillus nidulans. During the study on the veA gene, it was observed that the veA expression level is slightly higher in a veA1 mutant than in a wild type at 37 degrees C, suggesting that the wild type veA gene is necessary for the negative regulation of the veA expression. In the veA1 mutant, the veA expression was higher than in a wild type grown at 42 degrees C but equal at 30 degrees C. Furthermore, in a veA deletion mutant having its own promoter and the N-terminus of the VeA ORF, expression of the N-terminus by the veA promoter was highly up-regulated, supporting the possibility that the veA gene is important for the negative regulation of the veA expression. Analyses of the lacZ transcript and the beta-galactosidase activity from the reporter strains in the veA1 background, which were constructed by transformation of the lacZ reporter plasmids containing the lacZ gene under the control of the intact or the truncated veA promoters from the -943 to +262 bp region, showed that the truncated promoters produced more veA transcript and higher beta-galactosidase activity than the intact one at 30 degrees C, but equal at 42 degrees C. In addition, the serial-deletion analysis of the veA promoter identified a crucial region in the promoter from -943 to -740 bp for this derepression of the veA expression. Taken together, these results indicated that the veA gene is necessary for the negative regulation of the veA expression. Moreover, the veA expression was derepressed in the light-illuminated condition, where the VeA protein is hardly transported into the nucleus.

  7. The citrus flavonoid naringenin confers protection in a murine endotoxaemia model through AMPK-ATF3-dependent negative regulation of the TLR4 signalling pathway

    PubMed Central

    Liu, Xin; Wang, Ning; Fan, Shijun; Zheng, Xinchuan; Yang, Yongjun; Zhu, Yuanfeng; Lu, Yongling; Chen, Qian; Zhou, Hong; Zheng, Jiang

    2016-01-01

    Excessive activation of the TLR4 signalling pathway is critical for inflammation-associated disorders, while negative regulators play key roles in restraining TLR4 from over-activation. Naringenin is a citrus flavonoid with remarkable anti-inflammatory activity, but the mechanisms underlying its inhibition of LPS/TLR4 signalling are less clear. This study investigated the molecular targets and therapeutic effects of naringenin in vitro and in vivo. In LPS-stimulated murine macrophages, naringenin suppressed the expression of TNF-α, IL-6, TLR4, inducible NO synthase (iNOS), cyclo-oxygenase-2 (COX2) and NADPH oxidase-2 (NOX2). Naringenin also inhibited NF-κB and mitogen-activated protein kinase (MAPK) activation. However, it did not affect the IRF3 signalling pathway or interferon production, which upregulate activating transcription factor 3 (ATF3), an inducible negative regulator of TLR4 signalling. Naringenin was demonstrated to directly increase ATF3 expression. Inhibition of AMPK and its upstream calcium-dependent signalling reduced ATF3 expression and dampened the anti-inflammatory activity of naringenin. In murine endotoxaemia models, naringenin ameliorated pro-inflammatory reactions and improved survival. Furthermore, it induced AMPK activation in lung tissues, which was required for ATF3 upregulation and the enhanced anti-inflammatory activity. Overall, this study reveals a novel mechanism of naringenin through AMPK-ATF3-dependent negative regulation of the LPS/TLR4 signalling pathway, which thereby confers protection against murine endotoxaemia. PMID:28004841

  8. microRNAs are differentially regulated between MDM2-positive and negative malignant pleural mesothelioma

    PubMed Central

    Walter, Robert Fred Henry; Vollbrecht, Claudia; Werner, Robert; Wohlschlaeger, Jeremias; Christoph, Daniel Christian; Schmid, Kurt Werner; Mairinger, Fabian Dominik

    2016-01-01

    Background Malignant pleural mesothelioma (MPM) is a highly aggressive tumour first-line treated with a combination of cisplatin and pemetrexed. MDM2 and P14/ARF (CDKN2A) are upstream regulators of TP53 and may contribute to its inactivation. In the present study, we now aimed to define the impact of miRNA expression on this mechanism. Material and Methods 24 formalin-fixed paraffin-embedded (FFPE) tumour specimens were used for miRNA expression analysis of the 800 most important miRNAs using the nCounter technique (NanoString). Significantly deregulated miRNAs were identified before a KEGG-pathway analysis was performed. Results 17 miRNAs regulating TP53, 18 miRNAs regulating MDM2, and 11 miRNAs directly regulating CDKN2A are significantly downregulated in MDM2-expressing mesotheliomas. TP53 is downregulated in MDM2-negative tumours through miRNAs with a miSVR prediction score of 11.67, RB1 with a prediction score of 8.02, MDM2 with a prediction score of 4.50 and CDKN2A with a prediction score of 1.27. Conclusion MDM2 expression seems to impact miRNA expression levels in MPM. Especially, miRNAs involved in TP53-signaling are strongly decreased in MDM2-positive mesotheliomas. A better understanding of its tumour biology may open the chance for new therapeutic approaches and thereby augment patients' outcome. PMID:26918730

  9. Sef is a negative regulator of fiber cell differentiation in the ocular lens.

    PubMed

    Newitt, Peter; Boros, Jessica; Madakashira, Bhavani P; Robinson, Michael L; Reneker, Lixing W; McAvoy, John W; Lovicu, Frank J

    2010-07-01

    Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.

  10. CCAAT/enhancer binding protein β negatively regulates progesterone receptor expression in human glioblastoma cells.

    PubMed

    Hansberg-Pastor, Valeria; González-Arenas, Aliesha; Camacho-Arroyo, Ignacio

    2017-01-05

    Many progesterone (P4) actions are mediated by its intracellular receptor (PR), which has two isoforms (PR-A and PR-B) differentially transcribed from separate promoters of a single gene. In glioblastomas, the most frequent and aggressive brain tumors, PR-B is the predominant isoform. In an in silico analysis we showed putative CCAAT/Enhancer Binding Protein (C/EBP) binding sites at PR-B promoter. We evaluated the role of C/EBPβ in PR-B expression regulation in glioblastoma cell lines, which expressed different ratios of PR and C/EBPβ isoforms (LAP1, LAP2, and LIP). ChIP assays showed a significant basal binding of C/EBPβ, specific protein 1 (Sp1) and estrogen receptor alpha (ERα) to PR-B promoter. C/EBPβ knockdown increased PR-B expression and treatment with estradiol (E2) reduced C/EBPβ binding to the promoter and up-regulated PR-B expression. P4 induced genes were differently regulated when CEBP/β was silenced. These data show that C/EBPβ negatively regulates PR-B expression in glioblastoma cells.

  11. Negative regulation of TCR signaling by ubiquitination of Zap-70 Lys-217.

    PubMed

    Ivanova, Elitza; Carpino, Nick

    2016-05-01

    The tyrosine kinase Zap-70 is a key regulator of T cell receptor (TCR) signaling downstream of antigen presentation, with coordinated regulation of Zap-70 kinase activity critical for proper T cell proliferation, differentiation, and effector function during an immune response. Zap-70 is cytosolic in unstimulated T cells, but is rapidly recruited to the TCR complex following receptor stimulation. Its activity is regulated both by binding to subunits of the TCR and by phosphorylation on multiple tyrosine residues. Zap-70 also has been reported to be ubiquitinated following TCR stimulation. Herein, we confirm the ubiquitination of Zap-70 in T cell lines and in primary human and mouse T cells, and report the identification of nine novel Zap-70 ubiquitination sites. Three sites, including Lys-193, Lys-217, and Lys-376, displayed greater than 20-fold increase in modification levels following TCR stimulation. Abrogation of Lys-217 ubiquitination results in increased kinase activation, enhanced activation of downstream signaling pathways, and elevated IL-2 production following TCR stimulation. These data suggest that Zap-70 ubiquitination contributes to the regulation of Zap-70 signaling following TCR stimulation.

  12. Negative Suppressors of Oncogenic Activation of the Met Receptor Tyrosine Kinase

    DTIC Science & Technology

    2007-03-01

    progesterone (PR) receptor positive), basal ( triple negative : ER/PER/Her2 negative ) and the Her2 (ER/PR negative , Her2 amplification and/or overexpression...AD_________________ Award Number: W81XWH-06-1-0392 TITLE: Negative Suppressors of Oncogenic...CONTRACT NUMBER Negative Suppressors of Oncogenic Activation of the Met Receptor 5b. GRANT NUMBER W81XWH-06-1-0392 5c. PROGRAM ELEMENT NUMBER

  13. HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis

    PubMed Central

    Nawkar, Ganesh M.; Kang, Chang Ho; Maibam, Punyakishore; Park, Joung Hun; Jung, Young Jun; Chae, Ho Byoung; Chi, Yong Hun; Jung, In Jung; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2017-01-01

    Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box–like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression. PMID:28167764

  14. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    NASA Astrophysics Data System (ADS)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  15. Negative reciprocal regulation between Sirt1 and Per2 modulates the circadian clock and aging

    PubMed Central

    Wang, Rui-Hong; Zhao, Tingrui; Cui, Kairong; Hu, Gangqing; Chen, Qiang; Chen, Weiping; Wang, Xin-Wei; Soto-Gutierrez, Alejandro; Zhao, Keji; Deng, Chu-Xia

    2016-01-01

    Sirtuin 1 (SIRT1) is involved in both aging and circadian-clock regulation, yet the link between the two processes in relation to SIRT1 function is not clear. Using Sirt1-deficient mice, we found that Sirt1 and Period 2 (Per2) constitute a reciprocal negative regulation loop that plays important roles in modulating hepatic circadian rhythmicity and aging. Sirt1-deficient mice exhibited profound premature aging and enhanced acetylation of histone H4 on lysine16 (H4K16) in the promoter of Per2, the latter of which leads to its overexpression; in turn, Per2 suppresses Sirt1 transcription through binding to the Sirt1 promoter at the Clock/Bmal1 site. This negative reciprocal relationship between SIRT1 and PER2 was also observed in human hepatocytes. We further demonstrated that the absence of Sirt1 or the ectopic overexpression of Per2 in the liver resulted in a dysregulated pace of the circadian rhythm. The similar circadian rhythm was also observed in aged wild type mice. The interplay between Sirt1 and Per2 modulates aging gene expression and circadian-clock maintenance. PMID:27346580

  16. HY5, a positive regulator of light signaling, negatively controls the unfolded protein response in Arabidopsis.

    PubMed

    Nawkar, Ganesh M; Kang, Chang Ho; Maibam, Punyakishore; Park, Joung Hun; Jung, Young Jun; Chae, Ho Byoung; Chi, Yong Hun; Jung, In Jung; Kim, Woe Yeon; Yun, Dae-Jin; Lee, Sang Yeol

    2017-02-21

    Light influences essentially all aspects of plant growth and development. Integration of light signaling with different stress response results in improvement of plant survival rates in ever changing environmental conditions. Diverse environmental stresses affect the protein-folding capacity of the endoplasmic reticulum (ER), thus evoking ER stress in plants. Consequently, the unfolded protein response (UPR), in which a set of molecular chaperones is expressed, is initiated in the ER to alleviate this stress. Although its underlying molecular mechanism remains unknown, light is believed to be required for the ER stress response. In this study, we demonstrate that increasing light intensity elevates the ER stress sensitivity of plants. Moreover, mutation of the ELONGATED HYPOCOTYL 5 (HY5), a key component of light signaling, leads to tolerance to ER stress. This enhanced tolerance of hy5 plants can be attributed to higher expression of UPR genes. HY5 negatively regulates the UPR by competing with basic leucine zipper 28 (bZIP28) to bind to the G-box-like element present in the ER stress response element (ERSE). Furthermore, we found that HY5 undergoes 26S proteasome-mediated degradation under ER stress conditions. Conclusively, we propose a molecular mechanism of crosstalk between the UPR and light signaling, mediated by HY5, which positively mediates light signaling, but negatively regulates UPR gene expression.

  17. The Histidine Kinase BinK Is a Negative Regulator of Biofilm Formation and Squid Colonization

    PubMed Central

    Brooks, John F.

    2016-01-01

    ABSTRACT Bacterial colonization of animal epithelial tissue is a dynamic process that relies on precise molecular communication. Colonization of Euprymna scolopes bobtail squid by Vibrio fischeri bacteria requires bacterial aggregation in host mucus as the symbiont transitions from a planktonic lifestyle in seawater to a biofilm-associated state in the host. We have identified a gene, binK (biofilm inhibitor kinase; VF_A0360), which encodes an orphan hybrid histidine kinase that negatively regulates the V. fischeri symbiotic biofilm (Syp) in vivo and in vitro. We identified binK mutants as exhibiting a colonization advantage in a global genetic screen, a phenotype that we confirmed in controlled competition experiments. Bacterial biofilm aggregates in the host are larger in strains lacking BinK, whereas overexpression of BinK suppresses biofilm formation and squid colonization. Signaling through BinK is required for temperature modulation of biofilm formation at 28°C. Furthermore, we present evidence that BinK acts upstream of SypG, the σ54-dependent transcriptional regulator of the syp biofilm locus. The BinK effects are dependent on intact signaling in the RscS-Syp biofilm pathway. Therefore, we propose that BinK antagonizes the signal from RscS and serves as an integral component in V. fischeri biofilm regulation. IMPORTANCE Bacterial lifestyle transitions underlie the colonization of animal hosts from environmental reservoirs. Formation of matrix-enclosed, surface-associated aggregates (biofilms) is common in beneficial and pathogenic associations, but investigating the genetic basis of biofilm development in live animal hosts remains a significant challenge. Using the bobtail squid light organ as a model, we analyzed putative colonization factors and identified a histidine kinase that negatively regulates biofilm formation at the host interface. This work reveals a novel in vivo biofilm regulator that influences the transition of bacteria from their

  18. With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

    PubMed

    Mercado, Adriana; de Los Heros, Paola; Melo, Zesergio; Chávez-Canales, María; Murillo-de-Ozores, Adrián R; Moreno, Erika; Bazúa-Valenti, Silvana; Vázquez, Norma; Hadchouel, Juliette; Gamba, Gerardo

    2016-07-01

    The K(+)-Cl(-) cotransporters (KCC1-KCC4) encompass a branch of the SLC12 family of electroneutral cation-coupled chloride cotransporters that translocate ions out of the cell to regulate various factors, including cell volume and intracellular chloride concentration, among others. L-WNK1 is an ubiquitously expressed kinase that is activated in response to osmotic stress and intracellular chloride depletion, and it is implicated in two distinct hereditary syndromes: the renal disease pseudohypoaldosteronism type II (PHAII) and the neurological disease hereditary sensory neuropathy 2 (HSN2). The effect of L-WNK1 on KCC activity is unknown. Using Xenopus laevis oocytes and HEK-293 cells, we show that the activation of KCCs by cell swelling was prevented by L-WNK1 coexpression. In contrast, the activity of the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 was remarkably increased with L-WNK1 coexpression. The negative effect of L-WNK1 on the KCCs is kinase dependent. Elimination of the STE20 proline-alanine rich kinase (SPAK)/oxidative stress-responsive kinase (OSR1) binding site or the HQ motif required for the WNK-WNK interaction prevented the effect of L-WNK1 on KCCs, suggesting a required interaction between L-WNK1 molecules and SPAK. Together, our data support that NKCC1 and KCCs are coordinately regulated by L-WNK1 isoforms.

  19. Positive and negative regulation of type II TGF-beta receptor signal transduction by autophosphorylation on multiple serine residues.

    PubMed Central

    Luo, K; Lodish, H F

    1997-01-01

    The type II transforming growth factor-beta (TGF-beta) receptor Ser/Thr kinase (TbetaRII) is responsible for the initiation of multiple TGF-beta signaling pathways, and loss of its function is associated with many types of human cancer. Here we show that TbetaRII kinase is regulated intricately by autophosphorylation on at least three serine residues. Ser213, in the membrane-proximal segment outside the kinase domain, undergoes intra-molecular autophosphorylation which is essential for the activation of TbetaRII kinase activity, activation of TbetaRI and TGF-beta-induced growth inhibition. In contrast, phosphorylation of Ser409 and Ser416, located in a segment corresponding to the substrate recognition T-loop region in a three-dimensional structural model of protein kinases, is enhanced by receptor dimerization and can occur via an intermolecular mechanism. Phosphorylation of Ser409 is essential for TbetaRII kinase signaling, while phosphorylation of Ser416 inhibits receptor function. Mutation of Ser416 to alanine results in a hyperactive receptor that is better able than wild-type to induce TbetaRI activation and subsequent cell cycle arrest. Since on a single receptor either Ser409 or Ser416, but not both simultaneously, can become autophosphorylated, our results show that TbetaRII phosphorylation is regulated intricately and affects TGF-beta receptor signal transduction both positively and negatively. PMID:9155023

  20. Aurora A kinase activates YAP signaling in triple-negative breast cancer.

    PubMed

    Chang, S-S; Yamaguchi, H; Xia, W; Lim, S-O; Khotskaya, Y; Wu, Y; Chang, W-C; Liu, Q; Hung, M-C

    2017-03-02

    The Yes-associated protein (YAP) is an effector that transduces the output of the Hippo pathway to transcriptional modulation. Considering the role of YAP in cancers, this protein has emerged as a key node in malignancy development. In this study, we determined that Aurora A kinase acts as a positive regulator for YAP-mediated transcriptional machinery. Specifically, YAP associates with Aurora A predominantly in the nucleus. Activation of Aurora A can impinge on YAP activity through direct phosphorylation. Moreover, aberrant expression of YAP and Aurora A signaling is highly correlated with triple-negative breast cancer (TNBC). We herein provide evidence to establish the functional relevance of this newly discovered regulatory axis in TNBC.

  1. Negative regulation in correct tissue-specific expression of mouse mammary tumor virus in transgenic mice.

    PubMed Central

    Ross, S R; Hsu, C L; Choi, Y; Mok, E; Dudley, J P

    1990-01-01

    Mouse mammary tumor virus (MMTV) is an endogenous murine retrovirus that is expressed in the epithelial cells of the mammary and salivary glands, lungs, kidneys, and seminal vesicles and in the lymphoid cells of the spleen and thymus. Several studies have shown that the long terminal repeat (LTR) of this virus can direct the expression of reporter genes to the same tissues in transgenic mice. To determine whether multiple regulatory elements within the LTR are involved in this tissue-specific expression, we have established lines of transgenic mice containing transgenes that have deletions in the MMTV LTR. Deletions of all LTR sequences upstream of -364 or of LTR sequences from -165 to -665 both result in the expression of linked reporter genes such as the simian virus 40 early region or the bacterial enzyme chloramphenicol acetyltransferase in novel sites, such as the heart, brain, and skeletal muscle; expression of endogenous MMTV and transgenes containing the full-length LTR is not detected in these organs. Negative regulation appears to involve more than one region, since deletion of sequences between either -201 and -471 or -201 and -344, as well as sequences upstream of -364, results in inappropriate expression in heart, brain, and skeletal muscle. Therefore, a negative regulatory element(s) in the MMTV LTR can suppress transcription from the viral promoter in several different organs. This represents the first example of generalized negative regulatory elements that act in many different tissues in transgenic mice to prevent inappropriate expression of a gene. Images PMID:1700274

  2. Ligand Binding to WW Tandem Domains of YAP2 Transcriptional Regulator Is Under Negative Cooperativity

    PubMed Central

    Schuchardt, Brett J.; Mikles, David C.; Hoang, Lawrence M.; Bhat, Vikas; McDonald, Caleb B.; Sudol, Marius; Farooq, Amjad

    2014-01-01

    YAP2 transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well-documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules. PMID:25283809

  3. Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

    PubMed

    Schuchardt, Brett J; Mikles, David C; Hoang, Lawrence M; Bhat, Vikas; McDonald, Caleb B; Sudol, Marius; Farooq, Amjad

    2014-12-01

    YES-associated protein 2 (YAP2) transcriptional regulator drives a multitude of cellular processes, including the newly discovered Hippo tumor suppressor pathway, by virtue of the ability of its WW domains to bind and recruit PPXY-containing ligands to specific subcellular compartments. Herein, we employ an array of biophysical tools to investigate allosteric communication between the WW tandem domains of YAP2. Our data show that the WW tandem domains of YAP2 negatively cooperate when binding to their cognate ligands. Moreover, the molecular origin of such negative cooperativity lies in an unfavorable entropic contribution to the overall free energy relative to ligand binding to isolated WW domains. Consistent with this notion, the WW tandem domains adopt a fixed spatial orientation such that the WW1 domain curves outwards and stacks onto the binding groove of the WW2 domain, thereby sterically hindering ligand binding to both itself and its tandem partner. Although ligand binding to both WW domains disrupts such interdomain stacking interaction, they reorient themselves and adopt an alternative fixed spatial orientation in the liganded state by virtue of their ability to engage laterally so as to allow their binding grooves to point outwards and away from each other. In short, while the ability of WW tandem domains to aid ligand binding is well documented, our demonstration that they may also be subject to negative binding cooperativity represents a paradigm shift in our understanding of the molecular action of this ubiquitous family of protein modules.

  4. Functional heterogeneity of osteocytes in FGF23 production: the possible involvement of DMP1 as a direct negative regulator

    PubMed Central

    Lee, Ji-Won; Yamaguchi, Akira; Iimura, Tadahiro

    2014-01-01

    Fibroblast growth factor 23 (FGF23) and dentin matrix protein (DMP1) are hallmarks of osteocytes in bone. However, the mechanisms underlying the actions of DMP1 as a local factor regulating FGF23 and bone mineralization are not well understood. We first observed spatially distinct distributions of FGF23- and DMP1-positive osteocytic lacunae in rat femurs using immunohistochemistry. Three-dimensional immunofluorescence morphometry further demonstrated that the distribution and relative expression levels of these two proteins exhibited reciprocally reversed patterns especially in midshaft cortical bone. These in vivo findings suggest a direct role of DMP1 in FGF23 expression in osteocytes. We next observed that the inoculation of recombinant DMP1 in UMR-106 osteoblast/osteocyte-like cells and long-cultured MC3T3-E1 osteoblastic cells showed significant downregulation of FGF23 production. This effect was rescued by incubation with an focal adhesion kinase (FAK) inhibitor or MEK (mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)) inhibitor but not inhibitors of phosphoinositide 3-kinase or Rho kinase. Consistently, the levels of phosphorylated FAK, ERK and p38 were significantly elevated, indicating that exogenous DMP1 is capable of activating FAK-mediated MAPK signaling. These findings suggest that DMP1 is a local, direct and negative regulator of FGF23 production in osteocytes involved in the FAK-mediated MAPK pathway, proposing a relevant pathway that coordinates the extracellular environment of osteocytic lacunae and bone metabolism. PMID:24991406

  5. Lipoprotein electrostatic properties regulate hepatic lipase association and activity.

    PubMed

    Boucher, Jonathan G; Nguyen, Trang; Sparks, Daniel L

    2007-12-01

    The effect of lipoprotein electrostatic properties on the catalytic regulation of hepatic lipase (HL) was investigated. Enrichment of serum or very low density lipoprotein (VLDL) with oleic acid increased lipoprotein negative charge and stimulated lipid hydrolysis by HL. Similarly, enrichment of serum or isolated lipoproteins with the anionic phospholipids phosphatidylinositol (PI), phosphatidic acid, or phosphatidylserine also increased lipoprotein negative charge and stimulated hydrolysis by HL. Anionic lipids had a small effect on phospholipid hydrolysis, but significantly stimulated triacylglyceride (TG) hydrolysis. High density lipoprotein (HDL) charge appears to have a specific effect on lipolysis. Enrichment of HDL with PI significantly stimulated VLDL-TG hydrolysis by HL. To determine whether HDL charge affects the association of HL with HDL and VLDL, HL-lipoprotein interactions were probed immunochemically. Under normal circumstances, HL associates with HDL particles, and only small amounts bind to VLDL. PI enrichment of HDL blocked the binding of HL with HDL. These data indicate that increasing the negative charge of HDL stimulates VLDL-TG hydrolysis by reducing the association of HL with HDL. Therefore, HDL controls the hydrolysis of VLDL by affecting the interlipoprotein association of HL. Lipoprotein electrostatic properties regulate lipase association and are an important regulator of the binding and activity of lipolytic enzymes.

  6. Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells.

    PubMed

    Li, Yanxiang; Yang, Xiaofeng; He, Yanhao; Wang, Weirong; Zhang, Jiye; Zhang, Wei; Jing, Ting; Wang, Bo; Lin, Rong

    2017-03-01

    NLRP3 inflammasome not only functions as a critical effector in innate immunity, but also triggers the production of proinflammatory cytokines involved in inflammation-associated diseases. Sirtuin 1 (SIRT1) plays an important role in the regulation of cellular inflammation. However, whether the activation of NLRP3 inflammasome is regulated by SIRT1 remains unknown. In this study, we investigated the regulatory effect of SIRT1 on NLRP3 inflammasome and the underlying mechanisms. We found that lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced the activation of NLRP3 inflammasome in human umbilical vein endothelial cells (HUVECs). Activation of SIRT1 inhibited NLRP3 inflammasome activation and subsequent caspase-1 cleavage as well as interleukin (IL)-1β secretion, whereas SIRT1 knockdown obviously enhanced the activation of NLRP3 inflammasome in HUVECs. Importantly, gene silencing of SIRT1 abrogated the inhibitory effect of SIRT1 activator on NLRP3 inflammasome formation and IL-1β production in HUVECs stimulated with LPS plus ATP. Further study indicated that cluster of differentiation 40 (CD40) may be involved in the regulation of NLRP3 inflammasome by SIRT1. In vivo studies indicated that implantation of the periarterial carotid collar increased the arterial expression levels of CD40 and CD40 Ligand (CD40L), but inhibited arterial SIRT1 expression in the rabbits. Moreover, treatment with SIRT1 activator decreased CD40 and CD40L levels in collared arteries. Meanwhile, serum IL-1β level, the marker of inflammasome activation, was also inhibited by SIRT1 activation. Taken together, these findings revealed a novel regulatory mechanism of NLRP3 inflammasome by SIRT1, which may be related to suppression of CD40.

  7. Regulation of p53 and MDM2 activity by MTBP.

    PubMed

    Brady, Mark; Vlatkovic, Nikolina; Boyd, Mark T

    2005-01-01

    p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not gamma-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.

  8. [Regulation of myostatin promoter activity by myocyte enhancer factor 2].

    PubMed

    Li, Jia; Deng, Jie; Zhang, Junlin; Cheng, De; Wang, Huayan

    2012-08-01

    Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.

  9. Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility

    NASA Technical Reports Server (NTRS)

    Morfini, Gerardo; Szebenyi, Gyorgyi; Elluru, Ravindhra; Ratner, Nancy; Brady, Scott T.

    2002-01-01

    Membrane-bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin-based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule-stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

  10. P. brasiliensis Virulence Is Affected by SconC, the Negative Regulator of Inorganic Sulfur Assimilation

    PubMed Central

    Menino, João Filipe; Saraiva, Margarida; Gomes-Rezende, Jéssica; Sturme, Mark; Pedrosa, Jorge; Castro, António Gil; Ludovico, Paula; Goldman, Gustavo H.; Rodrigues, Fernando

    2013-01-01

    Conidia/mycelium-to-yeast transition of Paracoccidioidesbrasiliensis is a critical step for the establishment of paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Thus, knowledge of the factors that mediate this transition is of major importance for the design of intervention strategies. So far, the only known pre-requisites for the accomplishment of the morphological transition are the temperature shift to 37°C and the availability of organic sulfur compounds. In this study, we investigated the auxotrophic nature to organic sulfur of the yeast phase of Paracoccidioides, with special attention to P. brasiliensis species. For this, we addressed the role of SconCp, the negative regulator of the inorganic sulfur assimilation pathway, in the dimorphism and virulence of this pathogen. We show that down-regulation of SCONC allows initial steps of mycelium-to-yeast transition in the absence of organic sulfur compounds, contrarily to the wild-type fungus that cannot undergo mycelium-to-yeast transition under such conditions. However, SCONC down-regulated transformants were unable to sustain yeast growth using inorganic sulfur compounds only. Moreover, pulses with inorganic sulfur in SCONC down-regulated transformants triggered an increase of the inorganic sulfur metabolism, which culminated in a drastic reduction of the ATP and NADPH cellular levels and in higher oxidative stress. Importantly, the down-regulation of SCONC resulted in a decreased virulence of P. brasiliensis, as validated in an in vivo model of infection. Overall, our findings shed light on the inability of P. brasiliensis yeast to rely on inorganic sulfur compounds, correlating its metabolism with cellular energy and redox imbalances. Furthermore, the data herein presented reveal SconCp as a novel virulence determinant of P. brasiliensis. PMID:24066151

  11. NDRG2 phosphorylation provides negative feedback for SGK1-dependent regulation of a kainate receptor in astrocytes

    PubMed Central

    Matschke, Veronika; Theiss, Carsten; Hollmann, Michael; Schulze-Bahr, Eric; Lang, Florian; Seebohm, Guiscard; Strutz-Seebohm, Nathalie

    2015-01-01

    Glutamate receptors play an important role in the function of astrocytes. Among their tasks is the regulation of gliotransmission, gene expression and exocytosis of the tissue-type plasminogen activator (tPA), which has an enhancing effect on N-methyl-D-aspartate (NMDA) receptors and thus prevent over-excitation of neighboring neurons. The kainate receptor GluK2, which is expressed in neurons and astrocytes, is under tight regulation of the PI3-kinase SGK pathway as shown in neurons. SGK1 targets include N-myc downstream-regulated genes (NDRGs) 1 and 2 (NDRG1, NDRG2), proteins with elusive function. In the present study, we analyzed the effects of SGK1, NDRG1, and NDRG2 on GluK2 current amplitude and plasma membrane localization in astrocytes and heterologous expression. We demonstrate that NDRG1 and NDRG2 themselves have no effect on GluK2 current amplitudes in heterologous expressed ion channels. However, when NDRG2 is coexpressed with GluK2 and SGK1, the stimulating effect of SGK1 on GluK2 is suppressed both in heterologous expression and in astrocytes. Here, we reveal a new negative feedback mechanism, whereby GluK2 stimulation by SGK1 is regulated by parallel phosphorylation of NDRG2. This regulation of GluK2 by SGK1 and NDRG2 in astrocytes may play an important role in gliotransmission, modulation of gene expression and regulation of exocytosis of tPA. PMID:26500492

  12. 50 CFR 665.904 - Regulated activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Regulated activities. 665.904 Section 665.904 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Marianas Trench...

  13. 50 CFR 665.904 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Regulated activities. 665.904 Section 665.904 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Marianas Trench...

  14. 50 CFR 665.964 - Regulated activities.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 13 2013-10-01 2013-10-01 false Regulated activities. 665.964 Section 665.964 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Rose Atoll Marine...

  15. 50 CFR 665.964 - Regulated activities.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 13 2014-10-01 2014-10-01 false Regulated activities. 665.964 Section 665.964 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT, NATIONAL OCEANIC AND ATMOSPHERIC ADMINISTRATION, DEPARTMENT OF COMMERCE (CONTINUED) FISHERIES IN THE WESTERN PACIFIC Rose Atoll Marine...

  16. Parasympathetic cardio-regulation during social interactions in individuals with obesity-The influence of negative body image.

    PubMed

    Schrimpf, Anne; Kube, Jana; Neumann, Jane; Horstmann, Annette; Villringer, Arno; Gaebler, Michael

    2017-04-01

    Individuals with obesity in Western societies often face weight-related stigmatization and social exclusion. Recurrent exposure to prejudice and negative social feedback alters one's behavior in future social interactions. In this study, we aimed to investigate autonomic nervous system and affective responses to social interactions in individuals with obesity. Women and men with (n = 56) and without (n = 56) obesity participated in episodes of social inclusion and social exclusion using a virtual ball-tossing game. During the experiment, heart rate was measured and parasympathetic activity (overall high-frequency power and event-related cardiac slowing) was analyzed. Our results show that in novel social interactions, women with obesity, relative to the other groups, exhibited the strongest increase in parasympathetic activity. Furthermore, parasympathetic activity was related to a more negative body image in individuals with obesity, but not in lean individuals. Additionally, women with obesity reported a stronger decrease in mood after social exclusion than did the other participants. Our results demonstrate influences of objective and subjective bodily characteristics on parasympathetic cardio-regulation during social interactions. In particular, they show behavioral and physiological alterations during social interactions in women with obesity.

  17. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    SciTech Connect

    Yamagata, Kazutsune; Kitabayashi, Issay

    2009-12-25

    Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of {gamma}-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.

  18. Disabled-2 is a negative immune regulator of lipopolysaccharide-stimulated Toll-like receptor 4 internalization and signaling

    PubMed Central

    Hung, Wei-Shan; Ling, Pin; Cheng, Ju-Chien; Chang, Shy-Shin; Tseng, Ching-Ping

    2016-01-01

    Toll-like receptor 4 (TLR4) plays a pivotal role in the host response to lipopolysaccharide (LPS), a major cell wall component of Gram-negative bacteria. Here, we elucidated whether the endocytic adaptor protein Disabled-2 (Dab2), which is abundantly expressed in macrophages, plays a role in LPS-stimulated TLR4 signaling and trafficking. Molecular analysis and transcriptome profiling of RAW264.7 macrophage-like cells expressing short-hairpin RNA of Dab2 revealed that Dab2 regulated the TLR4/TRIF pathway upon LPS stimulation. Knockdown of Dab2 augmented TRIF-dependent interferon regulatory factor 3 activation and the expression of subsets of inflammatory cytokines and interferon-inducible genes. Dab2 acted as a clathrin sponge and sequestered clathrin from TLR4 in the resting stage of macrophages. Upon LPS stimulation, clathrin was released from Dab2 to facilitate endocytosis of TLR4 for triggering the TRIF-mediated pathway. Dab2 functions as a negative immune regulator of TLR4 endocytosis and signaling, supporting a novel role for a Dab2-associated regulatory circuit in controlling the inflammatory response of macrophages to endotoxin. PMID:27748405

  19. Phytochrome-interacting factors PIF4 and PIF5 negatively regulate anthocyanin biosynthesis under red light in Arabidopsis seedlings.

    PubMed

    Liu, Zhongjuan; Zhang, Yongqiang; Wang, Jianfeng; Li, Ping; Zhao, Chengzhou; Chen, Yadi; Bi, Yurong

    2015-09-01

    Light is an important environmental factor inducing anthocyanin accumulation in plants. Phytochrome-interacting factors (PIFs) have been shown to be a family of bHLH transcription factors involved in light signaling in Arabidopsis. Red light effectively increased anthocyanin accumulation in wild-type Col-0, whereas the effects were enhanced in pif4 and pif5 mutants but impaired in overexpression lines PIF4OX and PIF5OX, indicating that PIF4 and PIF5 are both negative regulators for red light-induced anthocyanin accumulation. Consistently, transcript levels of several genes involved in anthocyanin biosynthesis and regulatory pathway, including CHS, F3'H, DFR, LDOX, PAP1 and TT8, were significantly enhanced in mutants pif4 and pif5 but decreased in PIF4OX and PIF5OX compared to in Col-0, indicating that PIF4 and PIF5 are transcriptional repressor of these gene. Transient expression assays revealed that PIF4 and PIF5 could repress red light-induced promoter activities of F3'H and DFR in Arabidopsis protoplasts. Furthermore, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) test and electrophoretic mobility shift assay (EMSA) showed that PIF5 could directly bind to G-box motifs present in the promoter of DFR. Taken together, these results suggest that PIF4 and PIF5 negatively regulate red light-induced anthocyanin accumulation through transcriptional repression of the anthocyanin biosynthetic genes in Arabidopsis.

  20. The soybean R2R3 MYB transcription factor GmMYB100 negatively regulates plant flavonoid biosynthesis.

    PubMed

    Yan, Junhui; Wang, Biao; Zhong, Yunpeng; Yao, Luming; Cheng, Linjing; Wu, Tianlong

    2015-09-01

    Soybean flavonoids, a group of important signaling molecules in plant-environment interaction, ubiquitously exist in soybean and are tightly regulated by many genes. Here we reported that GmMYB100, a gene encoding a R2R3 MYB transcription factor, is involved in soybean flavonoid biosynthesis. GmMYB100 is mainly expressed in flowers, leaves and immature embryo, and its level is decreased after pod ripening. Subcellular localization assay indicates that GmMYB100 is a nuclear protein. GmMYB100 has transactivation ability revealed by a yeast functional assay; whereas bioinformatic analysis suggests that GmMYB100 has a negative function in flavonoid biosynthesis. GmMYB100-overexpression represses the transcript levels of flavonoid-related genes in transgenic soybean hairy roots and Arabidopsis, and inhibits isoflavonoid (soybean) and flavonol (Arabidopsis) production in transgenic plants. Furthermore, the transcript levels of six flavonoid-related genes and flavonoid (isoflavonoid and flavone aglycones) accumulation are elevated in the GmMYB100-RNAi transgenic hairy roots. We also demonstrate that GmMYB100 protein depresses the promoter activities of soybean chalcone synthase and chalcone isomerase. These findings indicate that GmMYB100 is a negative regulator in soybean flavonoid biosynthesis pathway.

  1. Increased anterior cingulate cortex and hippocampus activation in Complex PTSD during encoding of negative words

    PubMed Central

    Dorrepaal, Ethy; Draijer, Nel; de Ruiter, Michiel B.; Elzinga, Bernet M.; Sjoerds, Zsuzsika; van Balkom, Anton J.; Smit, Johannes H.; Veltman, Dick J.

    2013-01-01

    Post-traumatic stress disorder (PTSD) is associated with impaired memory performance coupled with functional changes in brain areas involved in declarative memory and emotion regulation. It is not yet clear how symptom severity and comorbidity affect neurocognitive functioning in PTSD. We performed a functional magnetic resonance imaging (fMRI) study with an emotional declarative memory task in 28 Complex PTSD patients with comorbid depressive and personality disorders, and 21 healthy non-trauma-exposed controls. In Complex PTSD patients—compared to controls—encoding of later remembered negative words vs baseline was associated with increased blood oxygenation level dependent (BOLD) response in the left ventral anterior cingulate cortex (ACC) and dorsal ACC extending to the dorsomedial prefrontal cortex (dmPFC) together with a trend for increased left hippocampus activation. Patients tended to commit more False Alarms to negative words compared to controls, which was associated with enhanced left ventrolateral prefrontal and orbitofrontal cortex (vlPFC/OFC) responses. Severity of child abuse was positively correlated with left ventral ACC activity and severity of depression with (para) hippocampal and ventral ACC activity. Presented results demonstrate functional abnormalities in Complex PTSD in the frontolimbic brain circuit also implicated in fear conditioning models, but generally in the opposite direction, which may be explained by severity of the trauma and severity of comorbid depression in Complex PTSD. PMID:22156722

  2. Regulation of myostatin activity and muscle growth.

    PubMed

    Lee, S J; McPherron, A C

    2001-07-31

    Myostatin is a transforming growth factor-beta family member that acts as a negative regulator of skeletal muscle mass. To identify possible myostatin inhibitors that may have applications for promoting muscle growth, we investigated the regulation of myostatin signaling. Myostatin protein purified from mammalian cells consisted of a noncovalently held complex of the N-terminal propeptide and a disulfide-linked dimer of C-terminal fragments. The purified C-terminal myostatin dimer was capable of binding the activin type II receptors, Act RIIB and, to a lesser extent, Act RIIA. Binding of myostatin to Act RIIB could be inhibited by the activin-binding protein follistatin and, at higher concentrations, by the myostatin propeptide. To determine the functional significance of these interactions in vivo, we generated transgenic mice expressing high levels of the propeptide, follistatin, or a dominant-negative form of Act RIIB by using a skeletal muscle-specific promoter. Independent transgenic mouse lines for each construct exhibited dramatic increases in muscle mass comparable to those seen in myostatin knockout mice. Our findings suggest that the propeptide, follistatin, or other molecules that block signaling through this pathway may be useful agents for enhancing muscle growth for both human therapeutic and agricultural applications.

  3. Chondroitin sulfate addition to CD44H negatively regulates hyaluronan binding

    SciTech Connect

    Ruffell, Brian; Johnson, Pauline . E-mail: pauline@interchange.ubc.ca

    2005-08-26

    CD44 is a widely expressed cell adhesion molecule that binds hyaluronan, an extracellular matrix glycosaminoglycan, in a tightly regulated manner. This regulated interaction has been implicated in inflammation and tumor metastasis. CD44 exists in the standard form, CD44H, or as higher molecular mass isoforms due to alternative splicing. Here, we identify serine 180 in human CD44H as the site of chondroitin sulfate addition and show that lack of chondroitin sulfate addition at this site enhances hyaluronan binding by CD44. A CD44H-immunoglobulin fusion protein expressed in HEK293 cells, and CD44H expressed in murine L fibroblast cells were modified by chondroitin sulfate, as determined by reduced sulfate incorporation after chondroitinase ABC treatment. Mutation of serine 180 or glycine 181 in CD44H reduced chondroitin sulfate addition and increased hyaluronan binding, indicating that serine 180 is the site for chondroitin sulfate addition in CD44H and that this negatively regulates hyaluronan binding.

  4. Maf1 is a negative regulator of transcription in Trypanosoma brucei.

    PubMed

    Romero-Meza, Gabriela; Vélez-Ramírez, Daniel E; Florencio-Martínez, Luis E; Román-Carraro, Fiordaliso C; Manning-Cela, Rebeca; Hernández-Rivas, Rosaura; Martínez-Calvillo, Santiago

    2017-02-01

    RNA polymerase III (Pol III) produces small RNA molecules that play essential roles in mRNA processing and translation. Maf1, originally described as a negative regulator of Pol III transcription, has been studied from yeast to human. Here we characterized Maf1 in the parasitic protozoa Trypanosoma brucei (TbMaf1), representing the first report to analyse Maf1 in an early-diverged eukaryote. While Maf1 is generally encoded by a single-copy gene, the T. brucei genome contains two almost identical TbMaf1 genes. The TbMaf1 protein has the three conserved sequences and is predicted to fold into a globular structure. Unlike in yeast, TbMaf1 localizes to the nucleus in procyclic forms of T. brucei under normal growth conditions. Cell lines that either downregulate or overexpress TbMaf1 were generated, and growth curve analysis with them suggested that TbMaf1 participates in the regulation of cell growth of T. brucei. Nuclear run-on and chromatin immunoprecipitation analyses demonstrated that TbMaf1 represses Pol III transcription of tRNA and U2 snRNA genes by associating with their promoters. Interestingly, 5S rRNA levels do not change after TbMaf1 ablation or overexpression. Notably, our data also revealed that TbMaf1 regulates Pol I transcription of procyclin gene and Pol II transcription of SL RNA genes.

  5. Arabidopsis t