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  1. TWEAK Negatively Regulates Human Dicer

    OpenAIRE

    2016-01-01

    The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like w...

  2. Triple Negative Breast Cancer and Metabolic Regulation

    Science.gov (United States)

    2015-08-01

    Lactate Dehydrogenase A is an isoform of lactate dehydrogenase, which catalyzes the conversion of pyruvate to lactate . LDHA is expressed in cancer ...AWARD NUMBER: W81XWH-13-1-0167 TITLE: Triple Negative Breast Cancer and Metabolic Regulation PRINCIPAL INVESTIGATOR: Amy S. Yee, Ph.D...Negative Breast Cancer and Metabolic Regulation 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-13-1-0167 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Amy S

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

    Science.gov (United States)

    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.

  4. Nitric oxide negatively regulates mammalian adult neurogenesis

    Science.gov (United States)

    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.

  5. LINGO-1 negatively regulates myelination by oligodendrocytes.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  7. Negative regulators of brown adipose tissue (BAT)-mediated thermogenesis.

    Science.gov (United States)

    Sharma, Bal Krishan; Patil, Mallikarjun; Satyanarayana, Ande

    2014-12-01

    Brown adipose tissue (BAT) is specialized for energy expenditure, a process called adaptive thermogenesis. PET-CT scans recently demonstrated the existence of metabolically active BAT in adult humans, which revitalized our interest in BAT. Increasing the amount and/or activity of BAT holds tremendous promise for the treatment of obesity and its associated diseases. PGC1α is the master regulator of UCP1-mediated thermogenesis in BAT. A number of proteins have been identified to influence thermogenesis either positively or negatively through regulating the expression or transcriptional activity of PGC1α. Therefore, BAT activation can be achieved by either inducing the expression of positive regulators of PGC1α or by inhibiting the repressors of the PGC1α/UCP1 pathway. Here, we review the most important negative regulators of PGC1α/UCP1 signaling and their mechanism of action in BAT-mediated thermogenesis.

  8. How Novice EFL Teachers Regulate Their Negative Emotions

    Science.gov (United States)

    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…

  9. The power of extraverts: testing positive and negative mood regulation

    Directory of Open Access Journals (Sweden)

    Gonzalo Hervas

    Full Text Available Extraversion is a personality trait which has been systematically related to positive affect and well-being. One of the mechanisms that may account for these positive outcomes is the ability to regulate the responses to positive, as well as negative, moods. Prior research has found that extraverts' higher positive mood maintenance could explain their higher levels of positive affect. However, research exploring differences between extraverts and introverts in negative mood regulation has yielded mixed results. The aim of the current study was explore the role of different facets of mood regulation displayed by extraverts, ambiverts, and introverts. After been exposed to a sad vs. happy mood induction, participants underwent a mood regulation task. Extraverts and ambiverts exhibited higher positive mood regulation than introverts, but similar mood repair. Thus, this research highlights the importance of positive mood regulation in the psychological functioning of extraverts, and opens new conceptualizations for developing interventions for introverts to improve their positive mood regulation and, hence, overall positive affect and well-being.

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

    Science.gov (United States)

    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.

  11. Susi, a negative regulator of Drosophila PI3-kinase.

    Science.gov (United States)

    Wittwer, Franz; Jaquenoud, Malika; Brogiolo, Walter; Zarske, Marcel; Wüstemann, Philipp; Fernandez, Rafael; Stocker, Hugo; Wymann, Matthias P; Hafen, Ernst

    2005-06-01

    The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.

  12. Akt is negatively regulated by the MULAN E3 ligase

    Institute of Scientific and Technical Information of China (English)

    Seunghee Bae; Jongdoo Kim; Hong-Duck Um; In-Chul Park; Su-Jae Lee; Seon Young Nam; Young-Woo Jin; Jae Ho Lee; Sungkwan An; Sun-Yong Kim; Jin Hyuk Jung; Yeongmin Yoon; Hwa Jun Cha; Hyunjin Lee; Karam Kim; Jongran Kim; In-Sook An

    2012-01-01

    The serine/threonine kinase Akt functions in multiple cellular processes,including cell survival and tumor development.Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation.In this study,we identified a negative regulator of Akt,MULAN,which possesses both a RING finger domain and E3 ubiquitin ligase activity.Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo.Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN.The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability.These data provide insight into the Akt ubiquitination signaling network.

  13. How Novice EFL Teachers Regulate Their Negative Emotions

    Directory of Open Access Journals (Sweden)

    Silvia Arizmendi Tejeda

    2016-04-01

    Full Text Available 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 observations, and analyzed by microanalysis and constant comparative analysis. The participants were five novice teachers who study English at the same university, and who were giving classes as part of their internship. The results from this research revealed that these particular novice English as a foreign language teachers use different emotional strategies to regulate their negative emotions.

  14. Regulation of positive and negative emotion: Effects of sociocultural context

    Directory of Open Access Journals (Sweden)

    Sara A. Snyder

    2013-07-01

    Full Text Available 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, Heller, John, & Gross, 2011. In this sociocultural context, as compared to home, participants reported less use of expressive suppression (a strategy generally associated with maladaptive outcomes, and greater use of cognitive reappraisal (a strategy 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 not only replicated our previous findings, but also found that the decreased use of suppression is primarily driven by reports of decreased suppression of positive emotion at Burning Man. By contrast, the reported 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 report 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 home. These findings have implications for understanding the ways in which certain sociocultural contexts may decrease suppression, and possibly minimize its associated maladaptive effects.

  15. Expression of Androgen Receptor Is Negatively Regulated By p53

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    Fatouma Alimirah

    2007-12-01

    Full Text Available Increased expression of androgen receptor (AR in prostate cancer (PC is associated with transition to androgen independence. Because the progression of PC to advanced stages is often associated with the loss of p53 function, we tested whether the p53 could regulate the expression of AR gene. Here we report that p53 negatively regulates the expression of AR in prostate epithelial cells (PrECs. We found that in LNCaP human prostate cancer cells that express the wild-type p53 and AR and in human normal PrECs, the activation of p53 by genotoxic stress or by inhibition of p53 nuclear export downregulated the expression of AR. Furthermore, forced expression of p53 in LNCaP cells decreased the expression of AR. Conversely, knockdown of p53 expression in LNCaP cells increased the AR expression. Consistent with the negative regulation of AR expression by p53, the p53-null HCT116 cells expressed higher levels of AR compared with the isogenic HCT116 cells that express the wildtype p53. Moreover, we noted that in etoposide treated LNCaP cells p53 bound to the promoter region of the AR gene, which contains a potential p53 DNA-binding consensus sequence, in chromatin immunoprecipitation assays. Together, our observations provide support for the idea that the loss of p53 function in prostate cancer cells contributes to increased expression of AR.

  16. RAGE, receptor of advanced glycation endoproducts, negatively regulates chondrocytes differentiation.

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    Tatsuya Kosaka

    Full Text Available RAGE, receptor for advanced glycation endoproducts (AGE, has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.

  17. Negative regulation of bacterial quorum sensing tunes public goods cooperation.

    Science.gov (United States)

    Gupta, Rashmi; Schuster, Martin

    2013-11-01

    Bacterial quorum sensing (QS) often coordinates the expression of other, generally more costly public goods involved in virulence and nutrient acquisition. In many Proteobacteria, the basic QS circuitry consists of a synthase that produces a diffusible acyl-homoserine lactone and a cognate receptor that activates public goods expression. In some species, the circuitry also contains negative regulators that have the potential to modulate the timing and magnitude of activation. In this study, we experimentally investigated the contribution of this regulatory function to the evolutionary stability of public goods cooperation in the opportunistic pathogen Pseudomonas aeruginosa. We compared fitness and public goods expression rates of strains lacking either qteE or qscR, each encoding a distinct negative regulator, with those of the wild-type parent and a signal-blind receptor mutant under defined growth conditions. We found that (1) qteE and qscR mutations behave virtually identically and have a stronger effect on the magnitude than on the timing of expression, (2) high expression in qteE and qscR mutants imposes a metabolic burden under nutrient conditions that advance induction and (3) high expression in qteE and qscR mutants increases population growth when QS is required, but also permits invasion by both wild-type and receptor mutant strains. Our data indicate that negative regulation of QS balances the costs and benefits of public goods by attenuating expression after transition to the induced state. As the cells cannot accurately assess the amount of cooperation needed, such bet-hedging would be advantageous in changing parasitic and nonparasitic environments.

  18. RAGE, Receptor of Advanced Glycation Endoproducts, Negatively Regulates Chondrocytes Differentiation

    Science.gov (United States)

    Kurosaka, Yuko; Nishimura, Haruka; Tanabe, Motoki; Takakura, Yuuki; Iwai, Keisuke; Waki, Takuya; Fujita, Takashi

    2014-01-01

    RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms. PMID:25275461

  19. Positive and Negative Regulation of Poly(A) Nuclease

    Science.gov (United States)

    Mangus, David A.; Evans, Matthew C.; Agrin, Nathan S.; Smith, Mandy; Gongidi, Preetam; Jacobson, Allan

    2004-01-01

    PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo. PMID:15169912

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

    Science.gov (United States)

    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.

  1. Negative regulation of DSS-induced experimental colitis by PILRα.

    Science.gov (United States)

    Kishida, Kazuki; Kohyama, Masako; Kurashima, Yosuke; Kogure, Yuta; Wang, Jing; Hirayasu, Kouyuki; Suenaga, Tadahiro; Kiyono, Hiroshi; Kunisawa, Jun; Arase, Hisashi

    2015-06-01

    Inflammatory bowel disease is thought to be a complex multifactorial disease, in which an increased inflammatory response plays an important role. Paired immunoglobulin-like type 2 receptor α (PILRα), well conserved in almost all mammals, is an inhibitory receptor containing immunoreceptor tyrosine-based inhibitory motifs in the cytoplasmic domain. PILRα is mainly expressed on myeloid cells and plays an important role in the regulation of inflammation. In the present study, we investigated the function of PILRα in inflammatory bowel disease using PILRα-deficient mice. When mice were orally administered dextran sulfate sodium (DSS), colonic mucosal injury and inflammation were significantly exacerbated in DSS-treated PILRα-deficient mice compared with wild-type (WT) mice. Flow cytometric analysis revealed that neutrophil and macrophage cell numbers were higher in the colons of DSS-treated PILRα-deficient mice than in those of WT mice. Blockade of CXCR2 expressed on neutrophils using a CXCR2 inhibitor decreased the severity of colitis observed in PILRα-deficient mice. These results suggest that PILRα negatively regulates inflammatory colitis by regulating the infiltration of inflammatory cells such as neutrophils and macrophages.

  2. Vitamin a is a negative regulator of osteoblast mineralization.

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    Thomas Lind

    Full Text Available An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1 with the active metabolite of vitamin A; retinoic acid (RA, a retinoic acid receptor (RAR antagonist (AGN194310, and a Cyp26 inhibitor (R115866 which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  3. Vitamin a is a negative regulator of osteoblast mineralization.

    Science.gov (United States)

    Lind, Thomas; Sundqvist, Anders; Hu, Lijuan; Pejler, Gunnar; Andersson, Göran; Jacobson, Annica; Melhus, Håkan

    2013-01-01

    An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

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

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    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.

  5. Annexin A3 as a negative regulator of adipocyte differentiation.

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    Watanabe, Takenori; Ito, Yoshimasa; Sato, Asuka; Hosono, Takashi; Niimi, Shingo; Ariga, Toyohiko; Seki, Taiichiro

    2012-10-01

    Annexin A3 is a protein belonging to the annexin family, and it is mainly present in cellular membranes as a phospholipid-binding protein that binds via the calcium ion. However, its physiological function remains to be clarified. We examined the expression of annexin A3 in mouse tissues and found for the first time that annexin A3 mRNA and its protein were expressed more strongly in adipose tissues than in other tissues. In adipose tissues, annexin A3-expressing cells were present in the stromal vascular fraction, and precisely identical to Pref-1-positive preadipocytes, Pref-1 being an epidermal growth factor repeat-containing transmembrane protein that inhibits adipogenesis. In 3T3-L1 cells, used as a model of adipogenesis, annexin A3 was down-regulated at an early phase of adipocyte differentiation, and this pattern paralleled that of Pref-1. Suppression of annexin A3 in these cells with siRNA caused elevation of the PPARγ2 mRNA level and lipid droplet accumulation. In conclusion, our data suggest that annexin A3 is a negative regulator of adipocyte differentiation.

  6. Arfaptin-1 negatively regulates Arl1-mediated retrograde transport.

    Directory of Open Access Journals (Sweden)

    Lien-Hung Huang

    Full Text Available The small GTPase Arf-like protein 1 (Arl1 is well known for its role in intracellular vesicular transport at the trans-Golgi network (TGN. In this study, we used differential affinity chromatography combined with mass spectrometry to identify Arf-interacting protein 1b (arfaptin-1b as an Arl1-interacting protein and characterized a novel function for arfaptin-1 (including the arfaptin-1a and 1b isoforms in Arl1-mediated retrograde transport. Using a Shiga-toxin subunit B (STxB transportation assay, we demonstrated that knockdown of arfaptin-1 accelerated the retrograde transport of STxB from the endosome to the Golgi apparatus, whereas Arl1 knockdown inhibited STxB transport compared with control cells. Arfaptin-1 overexpression, but not an Arl1 binding-defective mutant (arfaptin-1b-F317A, consistently inhibited STxB transport. Exogenous arfaptin-1 expression did not interfere with the localization of the Arl1-interacting proteins golgin-97 and golgin-245 to the TGN and vice versa. Moreover, we found that the N-terminal region of arfaptin-1 was involved in the regulation of retrograde transport. Our results show that arfaptin-1 acts as a negative regulator in Arl1-mediated retrograde transport and suggest that different functional complexes containing Arl1 form in distinct microdomains and are responsible for different functions.

  7. Negative regulation of lymphocyte activation by the adaptor protein LAX.

    Science.gov (United States)

    Zhu, Minghua; Granillo, Olivia; Wen, Renren; Yang, Kaiyong; Dai, Xuezhi; Wang, Demin; Zhang, Weiguo

    2005-05-01

    The membrane-associated adaptor protein LAX is a linker for activation of T cells (LAT)-like molecule that is expressed in lymphoid tissues. Upon stimulation of T or B cells, it is phosphorylated and interacts with Grb2 and the p85 subunit of PI3K. LAX, however, is not capable of replacing LAT in the TCR signaling pathway. In this study we report that upon T or B cell activation, the LAX protein was up-regulated dramatically. Although disruption of the LAX gene by homologous recombination had no major impact on lymphocyte development, it caused a significant reduction in CD23 expression on mature B cells. Interestingly, naive LAX(-/-) mice had spontaneous germinal center formation. Compared with normal T and B cells, LAX(-/-) T and B cells were hyperresponsive and had enhanced calcium flux, protein tyrosine phosphorylation, MAPK and Akt activation, and cell survival upon engagement of the T or B AgRs. Our data demonstrate that LAX functions as a negative regulator in lymphocyte signaling.

  8. DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

  9. DMPD: PI3K and negative regulation of TLR signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12860525 PI3K and negative regulation of TLR signaling. Fukao T, Koyasu S. Trends I...mmunol. 2003 Jul;24(7):358-63. (.png) (.svg) (.html) (.csml) Show PI3K and negative regulation of TLR signal...ing. PubmedID 12860525 Title PI3K and negative regulation of TLR signaling. Authors Fukao T, Koyasu S. Publi

  10. Drosophila RSK negatively regulates bouton number at the neuromuscular junction.

    Science.gov (United States)

    Fischer, Matthias; Raabe, Thomas; Heisenberg, Martin; Sendtner, Michael

    2009-03-01

    Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, we investigated the effect of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk-cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling.

  11. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    Science.gov (United States)

    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

  12. Ribosomal Protein S14 Negatively Regulates c-Myc Activity*

    Science.gov (United States)

    Zhou, Xiang; Hao, Qian; Liao, Jun-ming; Liao, Peng; Lu, Hua

    2013-01-01

    The ribosomal gene RPS14 is associated with the cancer-prone 5q-syndrome, which is caused by an interstitial deletion of the long arm of human chromosome 5. Previously, we found that ribosomal protein S14 (RPS14) binds to and inactivates MDM2, consequently leading to p53-dependent cell-cycle arrest and growth inhibition. However, it remains elusive whether RPS14 regulates cell proliferation in a p53-independent manner. Here, we show that RPS14 interacts with the Myc homology box II (MBII) and the C-terminal basic helix-loop-helix leucine zipper (bHLH-LZ) domains of the oncoprotein c-Myc. Further, RPS14 inhibited c-Myc transcriptional activity by preventing the recruitment of c-Myc and its cofactor, TRRAP, to the target gene promoters, as thus suppressing c-Myc-induced cell proliferation. Also, siRNA-mediated RPS14 depletion elevated c-Myc transcriptional activity determined by its target gene, Nucleolin, expression. Interestingly, RPS14 depletion also resulted in the induction of c-Myc mRNA and subsequent protein levels. Consistent with this, RPS14 promoted c-Myc mRNA turnover through an Argonaute 2 (Ago2)- and microRNA-mediated pathway. Taken together, our study demonstrates that RPS14 negates c-Myc functions by directly inhibiting its transcriptional activity and mediating its mRNA degradation via miRNA. PMID:23775087

  13. A Lexical Framework for Semantic Annotation of Positive and Negative Regulation Relations in Biomedical Pathways

    DEFF Research Database (Denmark)

    Zambach, Sine; Lassen, Tine

    presented here, we analyze 6 frequently used verbs denoting the regulation relations regulates, positively regulates and negatively regulates through corpus analysis, and propose a formal representation of the acquired knowledge as domain speci¯c semantic frames. The acquired knowledge patterns can thus...

  14. Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts.

    Science.gov (United States)

    Ono, Noriaki; Nakashima, Kazuhisa; Rittling, Susan R; Schipani, Ernestina; Hayata, Tadayoshi; Soma, Kunimichi; Denhardt, David T; Kronenberg, Henry M; Ezura, Yoichi; Noda, Masaki

    2008-07-11

    Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.

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

    Science.gov (United States)

    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…

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

    OpenAIRE

    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 nuclea...

  17. MEIS1 functions as a potential AR negative regulator

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Liang [Department of Urology, Chinese PLA Medical School/Chinese PLA General Hospital, Beijing 100853 (China); Department of Urology, Civil Aviation General Hospital/Civil Aviation Medical College of Peking University, Beijing 100123 (China); Li, Mingyang [Department of Gastroenterology, Nan Lou Division, Chinese PLA Medical School/Chinese PLA General Hospital, Beijing 100853 (China); Feng, Fan [Department of Pharmacy, General Hospital of Shenyang Military Command, Shenyang 110016 (China); Yang, Yutao [Beijing Institute for Neuroscience, Capital Medical University, Beijing 100069 (China); Hang, Xingyi [National Scientific Data Sharing Platform for Population and Health, Beijing 100730 (China); Cui, Jiajun, E-mail: cuijn@ucmail.uc.edu [Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267 (United States); Gao, Jiangping, E-mail: jpgao@163.com [Department of Urology, Chinese PLA Medical School/Chinese PLA General Hospital, Beijing 100853 (China)

    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.

  18. Cbl negatively regulates JNK activation and cell death

    Institute of Scientific and Technical Information of China (English)

    Andrew A Sproul; Zhiheng Xu; Michael Wilhelm; Stephen Gire; Lloyd A Greene

    2009-01-01

    Here, we explore the role of Cbl proteins in regulation of neuronal apoptosis. In two paradigms of neuron apopto-sis--nerve growth factor (NGF) deprivation and DNA damage--cellular levels of c-Cbl and Cbl-b fell well before the onset of cell death. NGF deprivation also induced rapid loss of tyrosine phosphorylation (and most likely, activa-tion) of c-Cbl. Targeting e-Cbl and Cbl-b with siRNAs to mimic their loss/inactivation sensitized neuronal cells to death promoted by NGF deprivation or DNA damage. One potential mechanism by which Cbl proteins might affect neuronal death is by regulation of apoptotic c-Jun N-terminal kinase (JNK) signaling. We demonstrate that Cbl pro-teins interact with the JNK pathway components mixed lineage kinase (MLK) 3 and POSH and that knockdown of Cbl proteins is sufficient to increase JNK pathway activity. Furthermore, expression of c-Cbl blocks the ability of MLKs to signal to downstream components of the kinase cascade leading to JNK activation and protects neuronal cells from death induced by MLKs, but not from downstream JNK activators. On the basis of these findings, we propose that Cbls suppress cell death in healthy neurons at least in part by inhibiting the ability of MLKs to activate JNK signaling. Apoptotic stimuli lead to loss of Cbl protein/activity, thereby removing a critical brake on JNK acti-vation and on cell death.

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

    Science.gov (United States)

    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.

  20. Negative auto-regulators trap p53 in their web.

    Science.gov (United States)

    Zhou, Xiang; Cao, Bo; Lu, Hua

    2017-01-09

    The transcriptional factor p53 activates the expression of a myriad of target genes involving a complicated signalling network, resulting in various cellular outcomes, such as growth arrest, senescence, apoptosis, and metabolic changes, and leading to consequent suppression of tumour growth and progression. Because of the profoundly adverse effect of p53 on growth and proliferation of cancer cells, several feedback mechanisms have been employed by the cells to constrain p53 activity. Two major antagonists MDM2 and MDMX (the long forms) are transcriptionally induced by p53, but in return block p53 activity, forming a negative feedback circuit and rendering chemoresistance of several cancer cells. However, they are not alone, as cancer cells also employ other proteins encoded by p53 target genes to inhibit p53 activity at transcriptional, translational, and posttranslational levels. This essay is thus composed to review a recent progress in understanding the mechanisms for how cancer cells hijack the p53 autoregulation by these proteins for their growth advantage and to discuss the clinical implications of these autoregulatory loops.

  1. TRIM65 negatively regulates p53 through ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yang [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China); Ma, Chengyuan [Department of Neurosurgery, The First Hospital of Jilin University, Changchun 130021 (China); Zhou, Tong [Department of Endocrinology, The First Hospital of Jilin University, Changchun 130021 (China); Liu, Ying [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China); Sun, Luyao [Department of Infectious Diseases, The First Hospital of Jilin University, Changchun 130021 (China); Yu, Zhenxiang, E-mail: zhenxiangyu2015@gmail.com [Department of Respiration, The First Hospital of Jilin University, Changchun 130021 (China)

    2016-04-22

    Tripartite-motif protein family member 65 (TRIM65) is an important protein involved in white matter lesion. However, the role of TRIM65 in human cancer remains less understood. Through the Cancer Genome Atlas (TCGA) gene alteration database, we found that TRIM65 is upregulated in a significant portion of non-small cell lung carcinoma (NSCLC) patients. Our cell growth assay revealed that TRIM65 overexpression promotes cell proliferation, while knockdown of TRIM65 displays opposite effect. Mechanistically, TRIM65 binds to p53, one of the most critical tumor suppressors, and serves as an E3 ligase toward p53. Consequently, TRIM65 inactivates p53 through facilitating p53 poly-ubiquitination and proteasome-mediated degradation. Notably, chemotherapeutic reagent cisplatin induction of p53 is markedly attenuated in response to ectopic expression of TRIM65. Cell growth inhibition by TRIM65 knockdown is more significant in p53 positive H460 than p53 negative H1299 cells, and knockdown of p53 in H460 cells also shows compromised cell growth inhibition by TRIM65 knockdown, indicating that p53 is required, at least in part, for TRIM65 function. Our findings demonstrate TRIM65 as a potential oncogenic protein, highly likely through p53 inactivation, and provide insight into development of novel approaches targeting TRIM65 for NSCLC treatment, and also overcoming chemotherapy resistance. - Highlights: • TRIM65 expression is elevated in NSCLC. • TRIM65 inactivates p53 through mediating p53 ubiquitination and degradation. • TRIM65 attenuates the response of NSCLC cells to cisplatin.

  2. Somatostatin Negatively Regulates Parasite Burden and Granulomatous Responses in Cysticercosis

    Directory of Open Access Journals (Sweden)

    Mitra Khumbatta

    2014-01-01

    Full Text Available Cysticercosis is an infection of tissues with the larval cysts of the cestode, Taenia  solium. While live parasites elicit little or no inflammation, dying parasites initiate a granulomatous reaction presenting as painful muscle nodules or seizures when cysts are located in the brain. We previously showed in the T. crassiceps murine model of cysticercosis that substance P (SP, a neuropeptide, was detected in early granulomas and was responsible for promoting granuloma formation, while somatostatin (SOM, another neuropeptide and immunomodulatory hormone, was detected in late granulomas; SOM’s contribution to granuloma formation was not examined. In the current studies, we used somatostatin knockout (SOM−/− mice to examine the hypothesis that SOM downmodulates granulomatous inflammation in cysticercosis, thereby promoting parasite growth. Our results demonstrated that parasite burden was reduced 5.9-fold in SOM−/− mice compared to WT mice (P<0.05. This reduction in parasite burden in SOM−/− mice was accompanied by a 95% increase in size of their granulomas (P<0.05, which contained a 1.5-fold increase in levels of IFN-γ and a 26-fold decrease in levels of IL-1β (P<0.05 for both compared to granulomas from WT mice. Thus, SOM regulates both parasite burden and granulomatous inflammation perhaps through modulating granuloma production of IFN-γ and IL-1β.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2014-01-01

    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. PMID:22343166

  5. Mutation analysis of the negative regulator cyclin G2 in gastric cancer

    African Journals Online (AJOL)

    Jane

    2011-10-24

    Oct 24, 2011 ... Key words: Cyclin G2, gastric cancer, negative regulator, mutation screen. ... has been reported in thyroid cancer, breast cancer, oral cancer and acute ..... transformation of papillary carcinoma of the thyroid. Anticancer. Res.

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

    Science.gov (United States)

    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.…

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

    Science.gov (United States)

    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…

  8. A Computational Model of the Relation Between Regulation of Negative Emotions and Mood

    NARCIS (Netherlands)

    Abro, A.H.; Klein, M.C.A.; Manzoor, A.R.; Tabatabaei, S.A.; Treur, J.

    2014-01-01

    In this paper a computational model is presented that describes the role of emotion regulation to reduce the influences of negative events on long term mood. The model incorporates an earlier model of mood dynamics and a model for the dynamics of emotion generation and regulation. Example model simu

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. Negative Regulation of Tumor Suppressor p53 by microRNA miR-504

    OpenAIRE

    2010-01-01

    Tumor suppressor p53 plays a central role in tumor prevention. p53 protein levels and activity are under a tight and complex regulation in cells to maintain the proper function of p53. microRNAs play a key role in the regulation of gene expression. Here we report the regulation of p53 through microRNA miR-504. miR-504 acts as a negative regulator of human p53 through its direct binding to two sites in p53 3′-UTR. Overexpression of miR-504 decreases p53 protein levels and functions in cells, i...

  12. Beyond CTLA-4 and PD-1, the Generation Z of Negative Checkpoint Regulators.

    Science.gov (United States)

    Le Mercier, Isabelle; Lines, J Louise; Noelle, Randolph J

    2015-01-01

    In the last two years, clinical trials with blocking antibodies to the negative checkpoint regulators CTLA-4 and PD-1 have rekindled the hope for cancer immunotherapy. Multiple negative checkpoint regulators protect the host against autoimmune reactions but also restrict the ability of T cells to effectively attack tumors. Releasing these brakes has emerged as an exciting strategy for cancer treatment. Conversely, these pathways can be manipulated to achieve durable tolerance for treatment of autoimmune diseases and transplantation. In the future, treatment may involve combination therapy to target multiple cell types and stages of the adaptive immune responses. In this review, we describe the current knowledge on the recently discovered negative checkpoint regulators, future targets for immunotherapy.

  13. Metacognitive beliefs and emotion regulation strategies: obese women with negative and positive body images

    Directory of Open Access Journals (Sweden)

    Somayeh Nejati

    2017-07-01

    Full Text Available Women have higher vulnerability regarding to increase prevalence of obesity and its effect on people’s body image and women’s health on the society and future generations’ health is unquestionable role , negative body image influence on women’s eating habits and mental health, so aim of present research is to compare metacognitive beliefs and emotional regulation strategies in obese women with positive and negative body image. This study was a causal-comparative. The statistical population of this study consisted of 100 obese women with a BMI>30 who had referred to five nutritional clinics in Tehran. The clinics and the participants were selected by using the convenience sampling method. The data collection tools were the Structured Clinical Interview for DSM (SCID-I/II, Body Mass Index (BMI, the Metacognitions Questionnaire (MCQ-30, the cognitive emotion regulation questionnaire, and fisher’s body image scale. The multivariate hoteling t-test was used to compare the difference between the two groups. Results indicated that obese women with negative body image had higher mean scores in inefficient emotion regulation strategies including self-blame or focus on thought, catastrophizing and other-blame compared with obese women with positive body image. Moreover, the mean scores of obese women with positive body images was higher in efficient emotional regulation strategies include acceptance, positive refocusing, refocusing on planning, perspective taking and positive reappraisal. Metacognitive beliefs and emotion regulation strategies are significant variables in obese woman with positive and negative body images.

  14. Emotion regulation in broadly defined anorexia nervosa: association with negative affective memory bias.

    Science.gov (United States)

    Manuel, Amy; Wade, Tracey D

    2013-08-01

    Theoretical models in anorexia nervosa (AN) implicate difficulties with emotion regulation as a maintaining factor. To date little is known about how different factors might maintain these difficulties. Forty eight women were recruited, 24 receiving treatment for AN (called broadly defined AN) and 24 healthy controls. Self-report measures of difficulties with emotion regulation and current depression were used in addition to computerized tasks which provided measures of social attentional bias and anger-threat bias, as well negative affective memory and recognition bias. Compared to controls, women with AN had significantly higher levels of difficulties with emotion regulation, depression, and negative affective memory bias, as well as lower bias for anger-threat. Simultaneous examination of the two variables that met pre-conditions for mediation of the relationship between group membership and difficulties with emotion regulation (anger-threat bias and negative affective memory) indicated negative affective memory bias to be a mediator, accounting for around one-third of the total effect a diagnosis of AN has on difficulties with emotion regulation. The association of these variables with AN may indicate shared risk factors with depression, and the variety of therapeutic approaches found to be effective with depression may be useful to further incorporate into treatments for AN.

  15. Gibberellins negatively regulate light-induced nitrate reductase activity in Arabidopsis seedlings.

    Science.gov (United States)

    Zhang, Yongqiang; Liu, Zhongjuan; Liu, Rongzhi; Wang, Liguang; Bi, Yurong

    2011-12-15

    In the present study, the role of phytohormone gibberellins (GAs) on regulating the nitrate reductase (NR) activity was tested in Arabidopsis seedlings. The NR activity in light-grown Col-0 seedlings was reduced by exogenous GA₃ (an active form of GAs), but enhanced by exogenous paclobutrazol (PAC, a gibberellin biosynthesis inhibitor), suggesting that GAs negatively regulate the NR activity in light-grown seedlings. Light is known to influence the NR activity through both photosynthesis and phytochromes. When etiolated seedlings were transferred to white or red light, both exogenously applied GA₃ and PAC were found to function on the NR activity only in the presence of sucrose, implying that GAs are not involved in light signaling-induced but negatively regulate photoproducts-induced NR activity. NR is regulated by light mainly at two levels: transcript level and post-translational level. Our reverse transcription (RT)-PCR assays showed that GAs did not affect the transcript levels of NIA1 and NIA2, two genes that encode NR proteins. But the divalent cations (especially Mg²⁺) were required for GAs negative regulation of NR activity, in view of the importance of divalent cations during the process of post-translational regulation of NR activity, which indicates that GAs very likely regulate the NR activity at the post-translational level. In the following dark-light shift analyses, GAs were found to accelerate dark-induced decrease, but retard light-induced increase of the NR activity. Furthermore, it was observed that application of G₃ or PAC could impair diurnal variation of the NR activity. These results collectively indicate that GAs play a negative role during light regulation of NR activity in nature.

  16. No fear, no panic: probing negation as a means for emotion regulation.

    Science.gov (United States)

    Herbert, Cornelia; Deutsch, Roland; Platte, Petra; Pauli, Paul

    2013-08-01

    This electroencephalographic study investigated if negating one's emotion results in paradoxical effects or leads to effective emotional downregulation. Healthy participants were asked to downregulate their emotions to happy and fearful faces by using negated emotional cue words (e.g., no fun, no fear). Cue words were congruent with the emotion depicted in the face and presented prior to each face. Stimuli were presented in blocks of happy and fearful faces. Blocks of passive stimulus viewing served as control condition. Active regulation reduced amplitudes of early event-related brain potentials (early posterior negativity, but not N170) and the late positive potential for fearful faces. A fronto-central negativity peaking at about 250 ms after target face onset showed larger amplitude modulations during downregulation of fearful and happy faces. Behaviorally, negating was more associated with reappraisal than with suppression. Our results suggest that in an emotional context, negation processing could be quite effective for emotional downregulation but that its effects depend on the type of the negated emotion (pleasant vs unpleasant). Results are discussed in the context of dual process models of cognition and emotion regulation.

  17. A Novel Approach to Revealing Positive and Negative Co-Regulated Genes

    Institute of Scientific and Technical Information of China (English)

    Yu-Hai Zhao; Guo-Ren Wang; Ying Yin; Guang-Yu Xu

    2007-01-01

    As explored by biologists, there is a real and emerging need to identify co-regulated gene clusters, which includeboth positive and negative regulated gene clusters. However, the existing pattern-based and tendency-based clusteringapproaches are only designed for finding positive regulated gene clusters. In this paper, a new subspace clustering modelcalled g-Cluster is proposed for gene expression data. The proposed model has the following advantages: 1) find both positiveand negative co-regulated genes in a shot, 2) get away from the restriction of magnitude transformation relationship amongco-regulated genes, and 3) guarantee quality of clusters and significance of regulations using a novel similarity measurementgCode and a user-specified regulation threshold 5, respectively. No previous work measures up to the task which has been set.Moreover, MDL technique is introduced to avoid insignificant g-Clusters generated. A tree structure, namely GS-tree, is alsodesigned, and two algorithms combined with efficient pruning and optimization strategies to identify all qualified g-Clusters.Extensive experiments are conducted on real and synthetic datasets. The experimental results show that 1) the algorithmis able to find an amount of co-regulated gene clusters missed by previous models, which are potentially of high biologicalsignificance, and 2) the algorithms are effective and efficient, and outperform the existing approaches.

  18. dRYBP contributes to the negative regulation of the Drosophila Imd pathway.

    Directory of Open Access Journals (Sweden)

    Ricardo Aparicio

    Full Text Available The Drosophila humoral innate immune response fights infection by producing antimicrobial peptides (AMPs through the microbe-specific activation of the Toll or the Imd signaling pathway. Upon systemic infection, the production of AMPs is both positively and negatively regulated to reach a balanced immune response required for survival. Here, we report the function of the dRYBP (drosophila Ring and YY1 Binding Protein protein, which contains a ubiquitin-binding domain, in the Imd pathway. We have found that dRYBP contributes to the negative regulation of AMP production: upon systemic infection with Gram-negative bacteria, Diptericin expression is up-regulated in the absence of dRYBP and down-regulated in the presence of high levels of dRYBP. Epistatic analyses using gain and loss of function alleles of imd, Relish, or skpA and dRYBP suggest that dRYBP functions upstream or together with SKPA, a member of the SCF-E3-ubiquitin ligase complex, to repress the Imd signaling cascade. We propose that the role of dRYBP in the regulation of the Imd signaling pathway is to function as a ubiquitin adaptor protein together with SKPA to promote SCF-dependent proteasomal degradation of Relish. Beyond the identification of dRYBP as a novel component of Imd pathway regulation, our results also suggest that the evolutionarily conserved RYBP protein may be involved in the human innate immune response.

  19. Strong negative self regulation of prokaryotic transcription factors increases the intrinsic noise of protein expression.

    Science.gov (United States)

    Stekel, Dov J; Jenkins, Dafyd J

    2008-01-18

    Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic models with strong repressors.

  20. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    Directory of Open Access Journals (Sweden)

    Jenkins Dafyd J

    2008-01-01

    Full Text Available Abstract Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic

  1. Emc, a negative HLH regulator with multiple functions in Drosophila development.

    Science.gov (United States)

    Campuzano, S

    2001-12-20

    Expression and functional analyses of Emc have demonstrated that it is a prototype for a protein required for multiple processes in development. Initially characterized as a negative regulator of sensory organ development, it was later found to regulate many other developmental processes and cell proliferation. Its ability to block the function of bHLH proteins by forming heterodimers, which are ineffective in DNA binding, accounts for the role of Emc in preventing the acquisition of several cell fates which are under the control of bHLH proteins. However, while maintaining this repressive molecular mechanism, emc also appears to act as a positive regulator of differentiation.

  2. MLK4β functions as a negative regulator of MAPK signaling and cell invasion

    OpenAIRE

    Abi Saab, W F; Brown, M S; Chadee, D N

    2012-01-01

    Mixed lineage kinase (MLK) 4, or MLK4, is a member of the MLK family of mitogen-activated protein kinase kinase kinases (MAP3Ks). Typically, MAP3Ks function to activate the mitogen-activated protein kinase (MAPK)-signaling pathways and regulate different cellular responses. However, here we report that MLK4β, unlike the other MLKs, negatively regulates the activities of the MAPKs, p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, and the MAP2Ks, MEK3 and 6. Our results s...

  3. Conflict Management with Friends and Romantic Partners: The Role of Attachment and Negative Mood Regulation Expectancies.

    Science.gov (United States)

    Creasey, Gary; Kershaw, Kathy; Boston, Ada

    1999-01-01

    Studied the degree to which attachment orientations were related to negative mood regulation expectancies and conflict management strategies with best friends and romantic partners in a sample of 140 female college students. Discusses results in relation to previous research on attachment theory and implications for interventions. (SLD)

  4. Relationships among Burnout, Social Support, and Negative Mood Regulation Expectancies of Elementary School Teachers in Korea

    Science.gov (United States)

    Kim, Mi Y.; Lee, Jee Y.; Kim, Jinsook

    2009-01-01

    The purposes of this study are as follows: (1) to determine whether burnout among elementary school teachers in Korea differs on selected demographic variables, (2) to investigate the relationship between burnout and negative mood regulation expectancies, as an internal variable, and social support, as an external variable, and (3) to examine the…

  5. Evidence that dendritic mitochondria negatively regulate dendritic branching in pyramidal neurons in the neocortex.

    Science.gov (United States)

    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.

  6. Impaired down-regulation of negative emotion in self-referent social situations in bipolar disorder

    DEFF Research Database (Denmark)

    Kjærstad, Hanne L; Vinberg, Maj; Goldin, Philippe R

    2016-01-01

    Emotion dysregulation is a core feature of bipolar disorder (BD) that persists into periods of remission. Neuroimaging studies show aberrant neural responses during emotion regulation (ER) in patients with BD relative to healthy controls, but behavioural evidence for ER deficits is sparse...... naturally or dampen their emotional response to positive and negative social scenarios and associated self-beliefs. They were also given an established experimental task for comparison, involving reappraisal of negative affective picture stimuli, as well as a questionnaire of habitual ER strategies. BD...... patients showed reduced ability to down-regulate emotional responses in negative, but not positive, social scenarios relative to healthy controls and UD patients. In contrast, there were no between-group differences in the established ER task or in self-reported habitual reappraisal strategies. Findings...

  7. Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bren Anat

    2011-07-01

    Full Text Available Abstract 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

  8. Negative auto-regulation increases the input dynamic-range of the arabinose system of Escherichia coli.

    Science.gov (United States)

    Madar, Daniel; Dekel, Erez; Bren, Anat; Alon, Uri

    2011-07-12

    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. 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. 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.

  9. 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

    Directory of Open Access Journals (Sweden)

    Moris Topaz

    2012-01-01

    Full Text Available 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.

  10. Cut! That’s a wrap: Regulating negative emotion by ending emotion-eliciting situations

    Directory of Open Access Journals (Sweden)

    Lara eVujovic

    2014-02-01

    Full Text Available 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 Emotion Regulation 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.

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

    Science.gov (United States)

    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.

  12. N-wasp is essential for the negative regulation of B cell receptor signaling.

    Directory of Open Access Journals (Sweden)

    Chaohong Liu

    2013-11-01

    Full Text Available 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.

  13. When death is not a problem: Regulating implicit negative affect under mortality salience.

    Science.gov (United States)

    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.

  14. [Regulation of Positive and Negative Emotions as Mediator between Maternal Emotion Socialization and Child Problem Behavior].

    Science.gov (United States)

    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.

  15. Instrumental Motives in Negative Emotion Regulation in Daily Life: Frequency, Consistency, and Predictors.

    Science.gov (United States)

    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

  16. DMPD: A novel negative regulator for IL-1 receptor and Toll-like receptor 4. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15585304 A novel negative regulator for IL-1 receptor and Toll-like receptor 4. Lie...w FY, Liu H, Xu D. Immunol Lett. 2005 Jan 15;96(1):27-31. (.png) (.svg) (.html) (.csml) Show A novel negative... regulator for IL-1 receptor and Toll-like receptor 4. PubmedID 15585304 Title A novel negative regulator f

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2012-07-01

    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.

  19. Optomotor-blind negatively regulates Drosophila eye development by blocking Jak/STAT signaling.

    Directory of Open Access Journals (Sweden)

    Yu-Chen Tsai

    Full Text Available Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired.

  20. Optomotor-blind negatively regulates Drosophila eye development by blocking Jak/STAT signaling.

    Science.gov (United States)

    Tsai, Yu-Chen; Grimm, Stefan; Chao, Ju-Lan; Wang, Shih-Chin; Hofmeyer, Kerstin; Shen, Jie; Eichinger, Fred; Michalopoulou, Theoni; Yao, Chi-Kuang; Chang, Chih-Hsuan; Lin, Shih-Han; Sun, Y Henry; Pflugfelder, Gert O

    2015-01-01

    Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  4. Phytophthora sojae TatD nuclease positively regulates sporulation and negatively regulates pathogenesis.

    Science.gov (United States)

    Chen, Linlin; Shen, Danyu; Sun, Nannan; Xu, Jing; Wang, Wen; Dou, Daolong

    2014-10-01

    During pathogenic interactions, both the host and pathogen are exposed to conditions that induce programmed cell death (PCD). Certain aspects of PCD have been recently examined in eukaryotic microbes but not in oomycetes. Here, we identified conserved TatD proteins in Phytophthora sojae; the proteins are key components of DNA degradation in apoptosis. We selected PsTatD4 for further investigation because the enzyme is unique to the oomycete branch of the phylogenetic tree. The purified protein exhibited DNase activity in vitro. Its expression was upregulated in sporangia and later infective stages but downregulated in cysts and during early infection. Functional analysis revealed that the gene was required for sporulation and zoospore production, and the expression levels were associated with the numbers of hydrogen-peroxide-induced terminal dUTP nick end-labeling-positive cells. Furthermore, overexpression of PsTatD4 gene reduced the virulence in a susceptible soybean cultivar. Together, these data suggest that apoptosis may play different roles in the early and late infective stages of P. sojae, and that PsTatD4 is a key regulator of infection. The association of PsTatD4 and apoptosis will lay a foundation to understanding the basic biology of apoptosis and its roles in P. sojae disease cycle.

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

    Science.gov (United States)

    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. PTEN, a widely known negative regulator of insulin/PI3K signaling, positively regulates neuronal insulin resistance

    Science.gov (United States)

    Gupta, Amit; Dey, Chinmoy Sankar

    2012-01-01

    Lipid and protein tyrosine phosphatase, phosphatase and tension homologue (PTEN), is a widely known negative regulator of insulin/phosphoinositide 3-kinase signaling. Down-regulation of PTEN is thus widely documented to ameliorate insulin resistance in peripheral tissues such as skeletal muscle and adipose. However, not much is known about its exact role in neuronal insulin signaling and insulin resistance. Moreover, alterations of PTEN in neuronal systems have led to discovery of several unexpected outcomes, including in the neurodegenerative disorder Alzheimer's disease (AD), which is increasingly being recognized as a brain-specific form of diabetes. In addition, contrary to expectations, its neuron-specific deletion in mice resulted in development of diet-sensitive obesity. The present study shows that PTEN, paradoxically, positively regulates neuronal insulin signaling and glucose uptake. Its down-regulation exacerbates neuronal insulin resistance. The positive role of PTEN in neuronal insulin signaling is likely due to its protein phosphatase actions, which prevents the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), the kinases critically involved in neuronal energy impairment and neurodegeneration. Results suggest that PTEN acting through FAK, the direct protein substrate of PTEN, prevents ERK activation. Our findings provide an explanation for unexpected outcomes reported earlier with PTEN alterations in neuronal systems and also suggest a novel molecular pathway linking neuronal insulin resistance and AD, the two pathophysiological states demonstrated to be closely linked. PMID:22875989

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

    OpenAIRE

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

    2006-01-01

    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 signalin...

  8. Modeling the role of negative cooperativity in metabolic regulation and homeostasis.

    Directory of Open Access Journals (Sweden)

    Eliot C Bush

    Full Text Available A significant proportion of enzymes display cooperativity in binding ligand molecules, and such effects have an important impact on metabolic regulation. This is easiest to understand in the case of positive cooperativity. Sharp responses to changes in metabolite concentrations can allow organisms to better respond to environmental changes and maintain metabolic homeostasis. However, despite the fact that negative cooperativity is almost as common as positive, it has been harder to imagine what advantages it provides. Here we use computational models to explore the utility of negative cooperativity in one particular context: that of an inhibitor binding to an enzyme. We identify several factors which may contribute, and show that acting together they can make negative cooperativity advantageous.

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

    Energy Technology Data Exchange (ETDEWEB)

    Chiarello, Carmelina; Bortoloso, Elena; Carpi, Andrea; Furlan, Sandra; Volpe, Pompeo, E-mail: pompeo.volpe@unipd.it

    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.

  10. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo, E-mail: innks@khu.ac.kr

    2013-07-19

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways.

  11. Neuronal pentraxin 1 negatively regulates excitatory synapse density and synaptic plasticity.

    Science.gov (United States)

    Figueiro-Silva, Joana; Gruart, Agnès; Clayton, Kevin Bernard; Podlesniy, Petar; Abad, Maria Alba; Gasull, Xavier; Delgado-García, José María; Trullas, Ramon

    2015-04-08

    In mature neurons, the number of synapses is determined by a neuronal activity-dependent dynamic equilibrium between positive and negative regulatory factors. We hypothesized that neuronal pentraxin (NP1), a proapoptotic protein induced by low neuronal activity, could be a negative regulator of synapse density because it is found in dystrophic neurites in Alzheimer's disease-affected brains. Here, we report that knockdown of NP1 increases the number of excitatory synapses and neuronal excitability in cultured rat cortical neurons and enhances excitatory drive and long-term potentiation in the hippocampus of behaving mice. Moreover, we found that NP1 regulates the surface expression of the Kv7.2 subunit of the Kv7 family of potassium channels that control neuronal excitability. Furthermore, pharmacological activation of Kv7 channels prevents, whereas inhibition mimics, the increase in synaptic proteins evoked by the knockdown of NP1. These results indicate that NP1 negatively regulates excitatory synapse number by modulating neuronal excitability and show that NP1 restricts excitatory synaptic plasticity. Copyright © 2015 the authors 0270-6474/15/355504-18$15.00/0.

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

    Science.gov (United States)

    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.

  13. HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Won Hee Jung

    Full Text Available The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

  14. HapX positively and negatively regulates the transcriptional response to iron deprivation in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Won Hee Jung

    2010-11-01

    Full Text Available The fungal pathogen Cryptococcus neoformans is a major cause of illness in immunocompromised individuals such as AIDS patients. The ability of the fungus to acquire nutrients during proliferation in host tissue and the ability to elaborate a polysaccharide capsule are critical determinants of disease outcome. We previously showed that the GATA factor, Cir1, is a major regulator both of the iron uptake functions needed for growth in host tissue and the key virulence factors such as capsule, melanin and growth at 37°C. We are interested in further defining the mechanisms of iron acquisition from inorganic and host-derived iron sources with the goal of understanding the nutritional adaptation of C. neoformans to the host environment. In this study, we investigated the roles of the HAP3 and HAPX genes in iron utilization and virulence. As in other fungi, the C. neoformans Hap proteins negatively influence the expression of genes encoding respiratory and TCA cycle functions under low-iron conditions. However, we also found that HapX plays both positive and negative roles in the regulation of gene expression, including a positive regulatory role in siderophore transporter expression. In addition, HapX also positively regulated the expression of the CIR1 transcript. This situation is in contrast to the negative regulation by HapX of genes encoding GATA iron regulatory factors in Aspergillus nidulans and Schizosaccharomyces pombe. Although both hapX and hap3 mutants were defective in heme utilization in culture, only HapX made a contribution to virulence, and loss of HapX in a strain lacking the high-affinity iron uptake system did not cause further attenuation of disease. Therefore, HapX appears to have a minimal role during infection of mammalian hosts and instead may be an important regulator of environmental iron uptake functions. Overall, these results indicated that C. neoformans employs multiple strategies for iron acquisition during infection.

  15. The Proteasome Activator PA28γ, a Negative Regulator of p53, Is Transcriptionally Up-Regulated by p53

    Directory of Open Access Journals (Sweden)

    Zhen-Xing Wan

    2014-02-01

    Full Text Available PA28γ (also called REGγ, 11Sγ or PSME3 negatively regulates p53 activity by promoting its nuclear export and/or degradation. Here, using the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE method, we identified the transcription start site of the PA28γ gene. Assessment with the luciferase assay demonstrated that the sequence −193 to +16 is the basal promoter. Three p53 binding sites were found within the PA28γ promoter utilizing a bioinformatics approach and were confirmed by chromatin immunoprecipitation and biotinylated DNA affinity precipitation experiments. The p53 protein promotes PA28γ transcription, and p53-stimulated transcription of PA28γ can be inhibited by PA28γ itself. Our results suggest that PA28γ and p53 form a negative feedback loop, which maintains the balance of p53 and PA28γ in cells.

  16. Negative feedback regulation of auxin signaling by ATHB8/ACL5-BUD2 transcription module.

    Science.gov (United States)

    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.

  17. Negative and positive auto-regulation of BMP expression in early eye development.

    Science.gov (United States)

    Huang, Jie; Liu, Ying; Filas, Benjamen; Gunhaga, Lena; Beebe, David C

    2015-11-15

    Previous results have shown that Bone Morphogenetic Protein (BMP) signaling is essential for lens specification and differentiation. How BMP signals are regulated in the prospective lens ectoderm is not well defined. To address this issue we have modulated BMP activity in a chicken embryo pre-lens ectoderm explant assay, and also studied transgenic mice, in which the type I BMP receptors, Bmpr1a and Acvr1, are deleted from the prospective lens ectoderm. Our results show that chicken embryo pre-lens ectoderm cells express BMPs and require BMP signaling for lens specification in vitro, and that in vivo inhibition of BMP signals in the mouse prospective lens ectoderm interrupts lens placode formation and prevents lens invagination. Furthermore, our results provide evidence that BMP expression is negatively auto-regulated in the lens-forming ectoderm, decreasing when the tissue is exposed to exogenous BMPs and increasing when BMP signaling is prevented. In addition, eyes lacking BMP receptors in the prospective lens placode develop coloboma in the adjacent wild type optic cup. In these eyes, Bmp7 expression increases in the ventral optic cup and the normal dorsal-ventral gradient of BMP signaling in the optic cup is disrupted. Pax2 becomes undetectable and expression of Sfrp2 increases in the ventral optic cup, suggesting that increased BMP signaling alter their expression, resulting in failure to close the optic fissure. In summary, our results suggest that negative and positive auto-regulation of BMP expression is important to regulate early eye development.

  18. HIP-55 negatively regulates myocardial contractility at the single-cell level.

    Science.gov (United States)

    Xing, Rui; Li, Shanshan; Liu, Kai; Yuan, Yuan; Li, Qing; Deng, Hao; Yang, Chengzhi; Huang, Jianyong; Zhang, Youyi; Fang, Jing; Xiong, Chunyang; Li, Zijian

    2014-08-22

    Myocardial contractility is crucial for cardiac output and heart function. But the detailed mechanisms of regulation remain unclear. In the present study, we found that HIP-55, an actin binding protein, negatively regulates myocardial contractility at the single-cell level. HIP-55 was overexpressed and knocked down in cardiomyocytes with an adenovirus infection. The traction forces exerted by single cardiomyocyte were measured using cell traction force microscopy. The results showed that HIP-55 knockdown significantly increased the contractility of the cardiomyocytes and HIP-55 overexpression could markedly reverse this process. Furthermore, HIP-55 was obviously co-localized with F-actin in cardiomyocytes, suggesting that HIP-55 regulated cardiac contractile function through the interaction between HIP-55 and F-actin. This study reveals the regulatory mechanisms of myocardial contractility and provides a new target for preventing and treating cardiovascular disease. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Arabidopsis RAV1 is down-regulated by brassinosteroid and may act as a negative regulator during plant development

    Institute of Scientific and Technical Information of China (English)

    Yu Xin HU; Yong Hong WANG; Xin Fang LIU; Jia Yang LI

    2004-01-01

    RAV1 is a novel DNA-binding protein with two distinct DNA-binding domains unique in higher plants,but its role in plant growth and development remains unknown. Using cDNA array,we found that transcription of RAV1 is downregulated by epibrassinolide (epiBL) in Arabidopsis suspension cells. RNA gel blot analysis revealed that epiBL-regulated RAV1 transcription involves neither protein phosphorylation/dephosphorylation nor newly synthesized protein,and does not require the functional BRI1,suggesting that this regulation might be through a new BR signaling pathway.Overexpressing RAV1 in Arabidopsis results in a retardation of lateral root and rosette leaf development,and the underexpression causes an earlier flowering phenotype,implying that RAV1 may function as a negative regulatory component of growth and development.

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

    Directory of Open Access Journals (Sweden)

    Rachel Haviland

    Full Text Available 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

  1. DMPD: When signaling pathways collide: positive and negative regulation of toll-likereceptor signal transduction. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18631453 When signaling pathways collide: positive and negative regulation of toll-...l) Show When signaling pathways collide: positive and negative regulation of toll-likereceptor signal transd...uction. PubmedID 18631453 Title When signaling pathways collide: positive and neg

  2. DMPD: The negative regulation of Toll-like receptor and associated pathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17621314 The negative regulation of Toll-like receptor and associated pathways. Lan...g T, Mansell A. Immunol Cell Biol. 2007 Aug-Sep;85(6):425-34. Epub 2007 Jul 10. (.png) (.svg) (.html) (.csml) Show The... negative regulation of Toll-like receptor and associated pathways. PubmedID 17621314 Title The ne

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

    Science.gov (United States)

    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.

  4. Dynamics of brassinosteroid response modulated by negative regulator LIC in rice.

    Directory of Open Access Journals (Sweden)

    Cui Zhang

    Full Text Available Brassinosteroids (BRs regulate rice plant architecture, including leaf bending, which affects grain yield. Although BR signaling has been investigated in Arabidopsis thaliana, the components negatively regulating this pathway are less well understood. Here, we demonstrate that Oryza sativa LEAF and TILLER ANGLE INCREASED CONTROLLER (LIC acts as an antagonistic transcription factor of BRASSINAZOLE-RESISTANT 1 (BZR1 to attenuate the BR signaling pathway. The gain-of-function mutant lic-1 and LIC-overexpressing lines showed erect leaves, similar to BZR1-depleted lines, which indicates the opposite roles of LIC and BZR1 in regulating leaf bending. Quantitative PCR revealed LIC transcription rapidly induced by BR treatment. Image analysis and immunoblotting showed that upon BR treatment LIC proteins translocate from the cytoplasm to the nucleus in a phosphorylation-dependent fashion. Phosphorylation assay in vitro revealed LIC phosphorylated by GSK3-like kinases. For negative feedback, LIC bound to the core element CTCGC in the BZR1 promoter on gel-shift and chromatin immunoprecipitation assay and repressed its transcription on transient transformation assay. LIC directly regulated target genes such as INCREASED LEAF INCLINATION 1 (ILI1 to oppose the action of BZR1. Repression of LIC in ILI1 transcription in protoplasts was partially rescued by BZR1. Phenotypic analysis of the crossed lines depleted in both LIC and BZR1 suggested that BZR1 functionally depends on LIC. Molecular and physiology assays revealed that LIC plays a dominant role at high BR levels, whereas BZR1 is dominant at low levels. Thus, LIC regulates rice leaf bending as an antagonistic transcription factor of BZR1. The phenotypes of lic-1 and LIC-overexpressing lines in erect leaves contribute to ideal plant architecture. Improving this phenotype may be a potential approach to molecular breeding for high yield in rice.

  5. Social anxiety and emotion regulation in daily life: spillover effects on positive and negative social events.

    Science.gov (United States)

    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.

  6. Suppressor of IKKɛ is an essential negative regulator of pathological cardiac hypertrophy

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  8. Evolution of gene network activity by tuning the strength of negative-feedback regulation.

    Science.gov (United States)

    Peng, Weilin; Liu, Ping; Xue, Yuan; Acar, Murat

    2015-02-11

    Despite the examples of protein evolution via mutations in coding sequences, we have very limited understanding on gene network evolution via changes in cis-regulatory elements. Using the galactose network as a model, here we show how the regulatory promoters of the network contribute to the evolved network activity between two yeast species. In Saccharomyces cerevisiae, we combinatorially replace all regulatory network promoters by their counterparts from Saccharomyces paradoxus, measure the resulting network inducibility profiles, and model the results. Lowering relative strength of GAL80-mediated negative feedback by replacing GAL80 promoter is necessary and sufficient to have high network inducibility levels as in S. paradoxus. This is achieved by increasing OFF-to-ON phenotypic switching rates. Competitions performed among strains with or without the GAL80 promoter replacement show strong relationships between network inducibility and fitness. Our results support the hypothesis that gene network activity can evolve by optimizing the strength of negative-feedback regulation.

  9. MEK kinase 1 is a negative regulator of virus-specific CD8(+) T cells

    DEFF Research Database (Denmark)

    Labuda, Tord; Christensen, Jan Pravsgaard; Rasmussen, Susanne;

    2006-01-01

    MEK kinase 1 (MEKK1) is a potent JNK-activating kinase, a regulator of T helper cell differentiation, cytokine production and proliferation in vitro. Using mice deficient for MEKK1 activity (Mekk1(DeltaKD)) exclusively in their hematopoietic system, we show that MEKK1 has a negative regulatory role...... in the generation of a virus-specific immune response. Mekk1(DeltaKD) mice challenged with vesicular stomatitis virus (VSV) showed a fourfold increase in splenic CD8(+) T cell numbers. In contrast, the number of splenic T cells in infected WT mice was only marginally increased. The CD8(+) T cell expansion in Mekk1...... suggest that MEKK1 plays a negative regulatory role in the expansion of virus-specific CD8(+) T cells in vivo....

  10. Regulation of negative affect in schizophrenia: the effectiveness of acceptance versus reappraisal and suppression.

    Science.gov (United States)

    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.

  11. MDM2/MDMX: Master negative regulators for p53 and RB.

    Science.gov (United States)

    Hu, Linshan; Zhang, Haibo; Bergholz, Johann; Sun, Shengnan; Xiao, Zhi-Xiong Jim

    2016-03-01

    MDM2 (mouse double minute 2 homolog) and MDMX (double minute X human homolog, also known as MDM4) are critical negative regulators of tumor protein p53. Our recent work shows that MDMX binds to and promotes degradation of retinoblastoma protein (RB) in an MDM2-dependent manner. In a xenograft tumor growth mouse model, silencing of MDMX results in inhibition of p53-deficient tumor growth, which can be effectively reversed by concomitant RB silencing. Thus, MDMX exerts its oncogenic activity via suppression of RB.

  12. High mobility group protein DSP1 negatively regulates HSP70 transcription in Crassostrea hongkongensis

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Zongyu; Xu, Delin; Cui, Miao; Zhang, Qizhong, E-mail: zhangqzdr@126.com

    2016-06-10

    HSP70 acts mostly as a molecular chaperone and plays important roles in facilitating the folding of nascent peptides as well as the refolding or degradation of the denatured proteins. Under stressed conditions, the expression level of HSP70 is upregulated significantly and rapidly, as is known to be achieved by various regulatory factors controlling the transcriptional level. In this study, a high mobility group protein DSP1 was identified by DNA-affinity purification from the nuclear extracts of Crassostrea hongkongensis using the ChHSP70 promoter as a bait. The specific interaction between the prokaryotically expressed ChDSP1 and the FITC-labeled ChHSP70 promoter was confirmed by EMSA analysis. ChDSP1 was shown to negatively regulate ChHSP70 promoter expression by Luciferase Reporter Assay in the heterologous HEK293T cells. Both ChHSP70 and ChDSP1 transcriptions were induced by either thermal or CdCl{sub 2} stress, while the accumulated expression peaks of ChDSP1 were always slightly delayed when compared with that of ChHSP70. This indicates that ChDSP1 is involved, very likely to exert its suppressive role, in the recovery of the ChHSP70 expression from the induced level to its original state. This study is the first to report negative regulator of HSP70 gene transcription, and provides novel insights into the mechanisms controlling heat shock protein expression. -- Highlights: •HMG protein ChDSP1 shows affinity to ChHSP70 promoter in Crassostrea hongkongensis. •ChDSP1 negatively regulates ChHSP70 transcription. •ChHSP70 and ChDSP1 transcriptions were coordinately induced by thermal/Cd stress. •ChDSP1 may contribute to the recovery of the induced ChHSP70 to its original state. •This is the first report regarding negative regulator of HSP70 transcription.

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

    Science.gov (United States)

    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.

  14. Anaplastic Lymphoma Kinase Acts in the Drosophila Mushroom Body to Negatively Regulate Sleep.

    Directory of Open Access Journals (Sweden)

    Lei Bai

    2015-11-01

    Full Text Available Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk, the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1 to regulate growth and associative learning in flies. We show that Alk mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes.

  15. HDAC3 Is a Critical Negative Regulator of Long-Term Memory Formation

    Science.gov (United States)

    McQuown, Susan C.; Barrett, Ruth M.; Matheos, Dina P.; Post, Rebecca J.; Rogge, George A.; Alenghat, Theresa; Mullican, Shannon E.; Jones, Steven; Rusche, James R.; Lazar, Mitchell A.; Wood, Marcelo A.

    2011-01-01

    Gene expression is dynamically regulated by chromatin modifications on histone tails, such as acetylation. In general, histone acetylation promotes transcription, whereas histone deacetylation negatively regulates transcription. The interplay between histone acetyl-transerases and histone deacetylases (HDACs) is pivotal for the regulation of gene expression required for long-term memory processes. Currently, very little is known about the role of individual HDACs in learning and memory. We examined the role of HDAC3 in long-term memory using a combined genetic and pharmacologic approach. We used HDAC3–FLOX genetically modified mice in combination with adeno-associated virus-expressing Cre recombinase to generate focal homozygous deletions of Hdac3 in area CA1 of the dorsal hippocampus. To complement this approach, we also used a selective inhibitor of HDAC3, RGFP136 [N-(6-(2-amino-4-fluorophenylamino)-6-oxohexyl)-4-methylbenzamide]. Immunohistochemistry showed that focal deletion or intrahippocampal delivery of RGFP136 resulted in increased histone acetylation. Both the focal deletion of HDAC3 as well as HDAC3 inhibition via RGFP136 significantly enhanced long-term memory in a persistent manner. Next we examined expression of genes implicated in long-term memory from dorsal hippocampal punches using quantitative reverse transcription-PCR. Expression of nuclear receptor subfamily 4 group A, member 2 (Nr4a2) and c-fos was significantly increased in the hippocampus of HDAC3–FLOX mice compared with wild-type controls. Memory enhancements observed in HDAC3–FLOX mice were abolished by intrahippocampal delivery of Nr4a2 small interfering RNA, suggesting a mechanism by which HDAC3 negatively regulates memory formation. Together, these findings demonstrate a critical role for HDAC3 in the molecular mechanisms underlying long-term memory formation. PMID:21228185

  16. Importin beta negatively regulates nuclear membrane fusion and nuclear pore complex assembly.

    Science.gov (United States)

    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.

  17. Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport.

    Directory of Open Access Journals (Sweden)

    Mariah Rayl

    Full Text Available 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.

  18. Osa-miR169 Negatively Regulates Rice Immunity against the Blast Fungus Magnaporthe oryzae

    Science.gov (United States)

    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

  19. Negative regulation of the innate antiviral immune response by TRIM62 from orange spotted grouper.

    Science.gov (United States)

    Yang, Ying; Huang, Youhua; Yu, Yepin; Zhou, Sheng; Wang, Shaowen; Yang, Min; Qin, Qiwei; Huang, Xiaohong

    2016-10-01

    Increased reports uncovered that mammalian tripartite motif-containing 62 (TRIM62) exerts crucial roles in cancer and innate immune response. However, the roles of fish TRIM62 in antiviral immune response remained uncertain. In this study, a TRIM62 gene was cloned from orange spotted grouper (EcTRIM62) and its roles in grouper RNA virus infection was elucidated in vitro. EcTRIM62 shared 99% and 83% identity to bicolor damselfish (Stegastes partitus) and human (Homo sapiens), respectively. Sequence alignment indicated that EcTRIM62 contained three domains, including a RING-finger domain, a B-box domain and a SPRY domain. In healthy grouper, the transcript of EcTRIM62 was predominantly detected in brain and liver, followed by heart, skin, spleen, fin, gill, intestine, and stomach. Subcellular localization analysis indicated that bright fluorescence spots were observed in the cytoplasm of EcTRIM62-transfected grouper spleen (GS) cells. During red-spotted grouper nervous necrosis (RGNNV) infection, overexpression of EcTRIM62 significantly enhanced the severity of CPE and increased viral gene transcriptions. Furthermore, the ectopic expression of EcTRIM62 significantly decreased the transcription level of interferon signaling molecules, including interferon regulatory factor 3 (IRF3), IRF7, interferon-stimulated gene 15 (ISG15), melanoma differentiation-associated protein 5 (MDA5), myxovirus resistance gene MXI, and MXII, suggesting that the negative regulation of interferon immune response by EcTRIM62 might directly contributed to its enhancing effect on RGNNV replication. Furthermore, our results also demonstrated that overexpression of EcTRIM62 was able to differently regulate the expression levels of pro-inflammation cytokines. In addition, we found the ectopic expression of EcTIRM62 negatively regulated MDA5-, but not mediator of IRF3 activation (MITA)-induced interferon immune response. Further studies showed that the deletion of RING domain and SPRY domain

  20. Endoglin negatively regulates transforming growth factor beta1-induced profibrotic responses in intestinal fibroblasts.

    LENUS (Irish Health Repository)

    Burke, J P

    2012-02-01

    BACKGROUND: Fibroblasts isolated from strictures in Crohn\\'s disease (CD) exhibit reduced responsiveness to stimulation with transforming growth factor (TGF) beta1. TGF-beta1, acting through the smad pathway, is critical to fibroblast-mediated intestinal fibrosis. The membrane glycoprotein, endoglin, is a negative regulator of TGF-beta1. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies of patients undergoing intestinal resection for CD strictures or from control patients. Endoglin expression was assessed using confocal microscopy, flow cytometry and western blot. The effect of small interfering (si) RNA-mediated knockdown and plasmid-mediated overexpression of endoglin on fibroblast responsiveness to TGF-beta1 was assessed by examining smad phosphorylation, smad binding element (SBE) promoter activity, connective tissue growth factor (CTGF) expression and ability to contract collagen. RESULTS: Crohn\\'s stricture fibroblasts expressed increased constitutive cell-surface and whole-cell endoglin relative to control cells. Endoglin co-localized with filamentous actin. Fibroblasts treated with siRNA directed against endoglin exhibited enhanced TGF-beta1-mediated smad-3 phosphorylation, and collagen contraction. Cells transfected with an endoglin plasmid did not respond to TGF-beta1 by exhibiting SBE promoter activity or producing CTGF. CONCLUSION: Fibroblasts from strictures in CD express increased constitutive endoglin. Endoglin is a negative regulator of TGF-beta1 signalling in the intestinal fibroblast, modulating smad-3 phosphorylation, SBE promoter activity, CTGF production and collagen contraction.

  1. The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing.

    Science.gov (United States)

    Lee, Soung-Hoon; Kim, Mi-Yeon; Kim, Hyun-Yi; Lee, Young-Mi; Kim, Heesu; Nam, Kyoung Ae; Roh, Mi Ryung; Min, Do Sik; Chung, Kee Yang; Choi, Kang-Yell

    2015-06-29

    Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5(-/-) mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)-Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing. © 2015 Lee et al.

  2. The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing

    Science.gov (United States)

    Lee, Soung-Hoon; Kim, Mi-Yeon; Kim, Hyun-Yi; Lee, Young-Mi; Kim, Heesu; Nam, Kyoung Ae; Roh, Mi Ryung; Min, Do Sik; Chung, Kee Yang

    2015-01-01

    Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5−/− mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)–Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing. PMID:26056233

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

    Directory of Open Access Journals (Sweden)

    Wang-Jian Zha

    2013-01-01

    Full Text Available 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.

  4. Negative regulation of receptor tyrosine kinases: unexpected links to c-Cbl and receptor ubiquitylation

    Institute of Scientific and Technical Information of China (English)

    Chanan RUBIN; Gal GUR; Yosef YARDEN

    2005-01-01

    Intracellular signals mediated by the family of receptor tyrosine kinases play pivotal roles in morphogenesis, cell fate determination and pathogenesis. Precise control of signal amplitude and duration is critical for the fidelity and robustness of these processes. Activation of receptor tyrosine kinases by their cognate growth factors not only leads to propagation of the signal through various biochemical cascades, but also sets in motion multiple attenuation mechanisms that ultimately terminate the active state. Early attenuators pre-exist prior to receptor activation and they act to limit signal propagation. Subsequently, late attenuators, such as Lrig and Sprouty, are transcriptionally induced and further act to dampen the signal. Central to the process of signaling attenuation is the role of the E3 ubiquitin ligase c-Cbl. While Cblmediated processes of receptor ubiquitylation and endocytosis are relatively well understood, the links of Cbl to other negative regulators are just now beginning to be appreciated. Here we review some emerging interfaces between Cbl and the transcriptionally induced negative regulators Lrig and Sprouty.

  5. Protein kinase Gin4 negatively regulates flippase function and controls plasma membrane asymmetry.

    Science.gov (United States)

    Roelants, Françoise M; Su, Brooke M; von Wulffen, Joachim; Ramachandran, Subramaniam; Sartorel, Elodie; Trott, Amy E; Thorner, Jeremy

    2015-02-02

    Plasma membrane function requires distinct leaflet lipid compositions. Two of the P-type ATPases (flippases) in yeast, Dnf1 and Dnf2, translocate aminoglycerophospholipids from the outer to the inner leaflet, stimulated via phosphorylation by cortically localized protein kinase Fpk1. By monitoring Fpk1 activity in vivo, we found that Fpk1 was hyperactive in cells lacking Gin4, a protein kinase previously implicated in septin collar assembly. Gin4 colocalized with Fpk1 at the cortical site of future bud emergence and phosphorylated Fpk1 at multiple sites, which we mapped. As judged by biochemical and phenotypic criteria, a mutant (Fpk1(11A)), in which 11 sites were mutated to Ala, was hyperactive, causing increased inward transport of phosphatidylethanolamine. Thus, Gin4 is a negative regulator of Fpk1 and therefore an indirect negative regulator of flippase function. Moreover, we found that decreasing flippase function rescued the growth deficiency of four different cytokinesis mutants, which suggests that the primary function of Gin4 is highly localized control of membrane lipid asymmetry and is necessary for optimal cytokinesis. © 2015 Roelants et al.

  6. BMX Negatively Regulates BAK Function, Thereby Increasing Apoptotic Resistance to Chemotherapeutic Drugs.

    Science.gov (United States)

    Fox, Joanna L; Storey, Alan

    2015-04-01

    The ability of chemotherapeutic agents to induce apoptosis, predominantly via the mitochondrial (intrinsic) apoptotic pathway, is thought to be a major determinant of the sensitivity of a given cancer to treatment. Intrinsic apoptosis, regulated by the BCL2 family, integrates diverse apoptotic signals to determine cell death commitment and then activates the nodal effector protein BAK to initiate the apoptotic cascade. In this study, we identified the tyrosine kinase BMX as a direct negative regulator of BAK function. BMX associates with BAK in viable cells and is the first kinase to phosphorylate the key tyrosine residue needed to maintain BAK in an inactive conformation. Importantly, elevated BMX expression prevents BAK activation in tumor cells treated with chemotherapeutic agents and is associated with increased resistance to apoptosis and decreased patient survival. Accordingly, BMX expression was elevated in prostate, breast, and colon cancers compared with normal tissue, including in aggressive triple-negative breast cancers where BMX overexpression may be a novel biomarker. Furthermore, BMX silencing potentiated BAK activation, rendering tumor cells hypersensitive to otherwise sublethal doses of clinically relevant chemotherapeutic agents. Our finding that BMX directly inhibits a core component of the intrinsic apoptosis machinery opens opportunities to improve the efficacy of existing chemotherapy by potentiating BAK-driven cell death in cancer cells.

  7. Zac1 functions through TGFβII to negatively regulate cell number in the developing retina

    Directory of Open Access Journals (Sweden)

    Götz Magdalena

    2007-06-01

    Full Text Available Abstract Background Organs are programmed to acquire a particular size during development, but the regulatory mechanisms that dictate when dividing progenitor cells should permanently exit the cell cycle and stop producing additional daughter cells are poorly understood. In differentiated tissues, tumor suppressor genes maintain a constant cell number and intact tissue architecture by controlling proliferation, apoptosis and cell dispersal. Here we report a similar role for two tumor suppressor genes, the Zac1 zinc finger transcription factor and that encoding the cytokine TGFβII, in the developing retina. Results Using loss and gain-of-function approaches, we show that Zac1 is an essential negative regulator of retinal size. Zac1 mutants develop hypercellular retinae due to increased progenitor cell proliferation and reduced apoptosis at late developmental stages. Consequently, supernumerary rod photoreceptors and amacrine cells are generated, the latter of which form an ectopic cellular layer, while other retinal cells are present in their normal number and location. Strikingly, Zac1 functions as a direct negative regulator of a rod fate, while acting cell non-autonomously to modulate amacrine cell number. We implicate TGFβII, another tumor suppressor and cytokine, as a Zac1-dependent amacrine cell negative feedback signal. TGFβII and phospho-Smad2/3, its downstream effector, are expressed at reduced levels in Zac1 mutant retinae, and exogenous TGFβII relieves the mutant amacrine cell phenotype. Moreover, treatment of wild-type retinae with a soluble TGFβ inhibitor and TGFβ receptor II (TGFβRII conditional mutants generate excess amacrine cells, phenocopying the Zac1 mutant phenotype. Conclusion We show here that Zac1 has an essential role in cell number control during retinal development, akin to its role in tumor surveillance in mature tissues. Furthermore, we demonstrate that Zac1 employs a novel cell non-autonomous strategy to

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

    Energy Technology Data Exchange (ETDEWEB)

    Sung, Nak-Yun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Yang, Mi-So [Department of Microbiology, Infection Signaling Network Research Center, College of Medicine, Chungnam National University, Daejeon (Korea, Republic of); Song, Du-Sub [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); School of life sciences and Biotechnology, Korea University 5-ka, Anam-Dong, Sungbuk-ku, Seoul 136-701 (Korea, Republic of); Kim, Jae-Kyung; Park, Jong-Heum; Song, Beom-Seok; Park, Sang-Hyun; Lee, Ju-Woon [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Park, Hyun-Jin [School of life sciences and Biotechnology, Korea University 5-ka, Anam-Dong, Sungbuk-ku, Seoul 136-701 (Korea, Republic of); Kim, Jae-Hun [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Baek, E-mail: ebbyun80@kaeri.re.kr [Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185 (Korea, Republic of); Byun, Eui-Hong, E-mail: ehbyun80@kongju.ac.k [Department of Food Science and Technology, Kongju National University, Yesan 340-800 (Korea, Republic of)

    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

  9. Negative regulation of TGFβ-induced lens epithelial to mesenchymal transition (EMT) by RTK antagonists.

    Science.gov (United States)

    Zhao, Guannan; Wojciechowski, Magdalena C; Jee, Seonah; Boros, Jessica; McAvoy, John W; Lovicu, Frank J

    2015-03-01

    An eclectic range of ocular growth factors with differing actions are present within the aqueous and vitreous humors that bathe the lens. Growth factors that exert their actions via receptor tyrosine kinases (RTKs), such as FGF, play a normal regulatory role in lens; whereas other factors, such as TGFβ, can lead to an epithelial to mesenchymal transition (EMT) that underlies several forms of cataract. The respective downstream intracellular signaling pathways of these factors are in turn tightly regulated. One level of negative regulation is thought to be through RTK-antagonists, namely, Sprouty (Spry), Sef and Spred that are all expressed in the lens. In this study, we tested these different negative regulators and compared their ability to block TGFβ-induced EMT in rat lens epithelial cells. Spred expression within the rodent eye was confirmed using RT-PCR, western blotting and immunofluorescence. Rat lens epithelial explants were used to examine the morphological changes associated with TGFβ-induced EMT over 3 days of culture, as well as α-smooth muscle actin (α-sma) immunolabeling. Cells in lens epithelial explants were transfected with either a reporter (EGFP) vector (pLXSG), or with plasmids also coding for different RTK-antagonists (i.e. pLSXG-Spry1, pLSXG-Spry2, pLXSG-Sef, pLSXG-Spred1, pLSXG-Spred2, pLSXG-Spred3), before treating with TGFβ for up to 3 days. The percentages of transfected cells that underwent TGFβ-induced morphological changes consistent with an EMT were determined using cell counts and validated with a paired two-tailed t-test. Explants transfected with pLXSG demonstrated a distinct transition in cell morphology after TGFβ treatment, with ∼60% of the cells undergoing fibrotic-like cell elongation. This percentage was significantly reduced in cells overexpressing the different antagonists, indicative of a block in lens EMT. Of the antagonists tested under these in vitro conditions, Spred1 was the most potent demonstrating the

  10. Ligand binding to WW tandem domains of YAP2 transcriptional regulator is under negative cooperativity.

    Science.gov (United States)

    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.

  11. Ligand Binding to WW Tandem Domains of YAP2 Transcriptional Regulator Is Under Negative Cooperativity

    Science.gov (United States)

    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

  12. Unkempt is negatively regulated by mTOR and uncouples neuronal differentiation from growth control.

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    Amélie Avet-Rochex

    2014-09-01

    Full Text Available Neuronal differentiation is exquisitely controlled both spatially and temporally during nervous system development. Defects in the spatiotemporal control of neurogenesis cause incorrect formation of neural networks and lead to neurological disorders such as epilepsy and autism. The mTOR kinase integrates signals from mitogens, nutrients and energy levels to regulate growth, autophagy and metabolism. We previously identified the insulin receptor (InR/mTOR pathway as a critical regulator of the timing of neuronal differentiation in the Drosophila melanogaster eye. Subsequently, this pathway has been shown to play a conserved role in regulating neurogenesis in vertebrates. However, the factors that mediate the neurogenic role of this pathway are completely unknown. To identify downstream effectors of the InR/mTOR pathway we screened transcriptional targets of mTOR for neuronal differentiation phenotypes in photoreceptor neurons. We identified the conserved gene unkempt (unk, which encodes a zinc finger/RING domain containing protein, as a negative regulator of the timing of photoreceptor differentiation. Loss of unk phenocopies InR/mTOR pathway activation and unk acts downstream of this pathway to regulate neurogenesis. In contrast to InR/mTOR signalling, unk does not regulate growth. unk therefore uncouples the role of the InR/mTOR pathway in neurogenesis from its role in growth control. We also identified the gene headcase (hdc as a second downstream regulator of the InR/mTOR pathway controlling the timing of neurogenesis. Unk forms a complex with Hdc, and Hdc expression is regulated by unk and InR/mTOR signalling. Co-overexpression of unk and hdc completely suppresses the precocious neuronal differentiation phenotype caused by loss of Tsc1. Thus, Unk and Hdc are the first neurogenic components of the InR/mTOR pathway to be identified. Finally, we show that Unkempt-like is expressed in the developing mouse retina and in neural stem

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

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    Shahaf, Galit; Rotem-Dai, Noa; Koifman, Gabriela; Raveh-Amit, Hadas; Frost, Sigal A.; Livneh, Etta, E-mail: etta@bgu.ac.il

    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.

  14. Negative feedback regulation of Wnt4 signaling by EAF1 and EAF2/U19.

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    Xiaoyang Wan

    Full Text Available Previous studies indicated that EAF (ELL-associated factor family members, EAF1 and EAF2/U19, play a role in cancer and embryogenesis. For example, EAF2/U19 may serve as a tumor suppressor in prostate cancer. At the same time, EAF2/U19 is a downstream factor in the non-canonical Wnt 4 signaling pathway required for eye development in Xenopus laevis, and along with EAF1, contributes to convergence and extension movements in zebrafish embryos through Wnt maintenance. Here, we used zebrafish embryos and mammalian cells to show that both EAF1 and EAF2/U19 were up-regulated by Wnt4 (Wnt4a. Furthermore, we found that EAF1 and EAF2/U19 suppressed Wnt4 expression by directly binding to the Wnt4 promoter as seen in chromatin immunoprecipitation assays. These findings indicate that an auto-regulatory negative feedback loop occurs between Wnt4 and the EAF family, which is conserved between zebrafish and mammalian. The rescue experiments in zebrafish embryos showed that early embryonic development required the maintenance of the appropriate levels of Wnt4a through the feedback loop. Others have demonstrated that the tumor suppressors p63, p73 and WT1 positively regulate Wnt4 expression while p21 has the opposite effect, suggesting that maintenance of appropriate Wnt4 expression may also be critical for adult tissue homeostasis and prevention against tumor initiation. Thus, the auto-regulatory negative feedback loop that controls expression of Wnt4 and EAF proteins may play an important role in both embryonic development and tumor suppression. Our findings provide the first convincing line of evidence that EAF and Wnt4 form an auto-regulatory negative feedback loop in vivo.

  15. P. brasiliensis Virulence Is Affected by SconC, the Negative Regulator of Inorganic Sulfur Assimilation

    Science.gov (United States)

    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

  16. P. brasiliensis virulence is affected by SconC, the negative regulator of inorganic sulfur assimilation.

    Directory of Open Access Journals (Sweden)

    João Filipe Menino

    Full Text Available 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.

  17. MicroRNA-1299 is a negative regulator of STAT3 in colon cancer.

    Science.gov (United States)

    Wang, Yong; Lu, Zhi; Wang, Ningning; Zhang, Man; Zeng, Xiandong; Zhao, Wei

    2017-06-01

    Signal transducers and activators of transcription (STAT) is a family of transcription factors which regulate cell proliferation, differentiation, apoptosis, metastasis, immune and inflammatory responses, and angiogenesis. STAT3 is a latent cytoplasmic transcription factor that belongs to STATs. STAT3 has been reported be regulates genes involved with cellular growth, proliferation and metastasis. Worldwide, colon cancer is one of the leading causes of cancer-related deaths. Cumulative evidence has established that STAT3 is essential for colon cancer progression to advanced malignancy. In our study, we showed that microRNA-1299 (miR-1299) was closely related to the TNM stage of colon cancer, and that the expression of miR-1299 was negatively correlated with the expression of STAT3 in colon cancer which means that miR-1299 can be a negative regulator of STAT3 in colon cancer. A total of 60 cases of different grades of colon samples were used to detect the expression of miR-1299. Results showed that miR-1299 was significantly lower in high-grade colons both in mRNA and protein levels. Furthermore, Overall survival (OS) in patients with low miR-1299 is shorter than 25.6 months, as compared with an OS of 28.4 months in patients with high level of miR-1299. We also confirmed that the overexpression of miR-1299 can not only downregulate the STAT3 pathway, but also inhibited colon cancer cell growth. Our findings could provide new insights into the molecular therapeutic of colon cancer.

  18. Plexin-B2 negatively regulates macrophage motility, Rac, and Cdc42 activation.

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    Kelly E Roney

    Full Text Available Plexins are cell surface receptors widely studied in the nervous system, where they mediate migration and morphogenesis though the Rho family of small GTPases. More recently, plexins have been implicated in immune processes including cell-cell interaction, immune activation, migration, and cytokine production. Plexin-B2 facilitates ligand induced cell guidance and migration in the nervous system, and induces cytoskeletal changes in overexpression assays through RhoGTPase. The function of Plexin-B2 in the immune system is unknown. This report shows that Plexin-B2 is highly expressed on cells of the innate immune system in the mouse, including macrophages, conventional dendritic cells, and plasmacytoid dendritic cells. However, Plexin-B2 does not appear to regulate the production of proinflammatory cytokines, phagocytosis of a variety of targets, or directional migration towards chemoattractants or extracellular matrix in mouse macrophages. Instead, Plxnb2(-/- macrophages have greater cellular motility than wild type in the unstimulated state that is accompanied by more active, GTP-bound Rac and Cdc42. Additionally, Plxnb2(-/- macrophages demonstrate faster in vitro wound closure activity. Studies have shown that a closely related family member, Plexin-B1, binds to active Rac and sequesters it from downstream signaling. The interaction of Plexin-B2 with Rac has only been previously confirmed in yeast and bacterial overexpression assays. The data presented here show that Plexin-B2 functions in mouse macrophages as a negative regulator of the GTPases Rac and Cdc42 and as a negative regulator of basal cell motility and wound healing.

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

    Science.gov (United States)

    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. Zebrafish foxo3b negatively regulates canonical Wnt signaling to affect early embryogenesis.

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    Xun-wei Xie

    Full Text Available FOXO genes are involved in many aspects of development and vascular homeostasis by regulating cell apoptosis, proliferation, and the control of oxidative stress. In addition, FOXO genes have been showed to inhibit Wnt/β-catenin signaling by competing with T cell factor to bind to β-catenin. However, how important of this inhibition in vivo, particularly in embryogenesis is still unknown. To demonstrate the roles of FOXO genes in embryogenesis will help us to further understand their relevant physiological functions. Zebrafish foxo3b gene, an orthologue of mammalian FOXO3, was expressed maternally and distributed ubiquitously during early embryogenesis and later restricted to brain. After morpholino-mediated knockdown of foxo3b, the zebrafish embryos exhibited defects in axis and neuroectoderm formation, suggesting its critical role in early embryogenesis. The embryo-developmental marker gene staining at different stages, phenotype analysis and rescue assays revealed that foxo3b acted its role through negatively regulating both maternal and zygotic Wnt/β-catenin signaling. Moreover, we found that foxo3b could interact with zebrafish β-catenin1 and β-catenin2 to suppress their transactivation in vitro and in vivo, further confirming its role relevant to the inhibition of Wnt/β-catenin signaling. Taken together, we revealed that foxo3b played a very important role in embryogenesis and negatively regulated maternal and zygotic Wnt/β-catenin signaling by directly interacting with both β-catenin1 and β-catenin2. Our studies provide an in vivo model for illustrating function of FOXO transcription factors in embryogenesis.

  1. Polypyrimidine tract binding protein functions as a negative regulator of feline calicivirus translation.

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    Ioannis Karakasiliotis

    Full Text Available BACKGROUND: Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5' or 3' extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB interacts with the 5'end of the feline calicivirus (FCV genomic and subgenomic RNAs, playing a role in the FCV life cycle. PRINCIPAL FINDINGS: We have demonstrated that PTB interacts with at least two binding sites within the 5'end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication. CONCLUSIONS: Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but

  2. Interferon regulatory factor 7 functions as a novel negative regulator of pathological cardiac hypertrophy.

    Science.gov (United States)

    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

    2014-04-01

    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 κBα(S32A/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-β(S177E/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.

  3. Arabidopsis type B cytokinin response regulators ARR1, ARR10, and ARR12 negatively regulate plant responses to drought.

    Science.gov (United States)

    Nguyen, Kien Huu; Ha, Chien Van; Nishiyama, Rie; Watanabe, Yasuko; Leyva-González, Marco Antonio; Fujita, Yasunari; Tran, Uven Thi; Li, Weiqiang; Tanaka, Maho; Seki, Motoaki; Schaller, G Eric; Herrera-Estrella, Luis; Tran, L S

    2016-03-15

    In this study, we used a loss-of-function approach to elucidate the functions of three Arabidopsis type B response regulators (ARRs)--namely ARR1, ARR10, and ARR12--in regulating the Arabidopsis plant responses to drought. The arr1,10,12 triple mutant showed a significant increase in drought tolerance versus WT plants, as indicated by its higher relative water content and survival rate on drying soil. This enhanced drought tolerance of arr1,10,12 plants can be attributed to enhanced cell membrane integrity, increased anthocyanin biosynthesis, abscisic acid (ABA) hypersensitivity, and reduced stomatal aperture, but not to altered stomatal density. Further drought-tolerance tests of lower-order double and single mutants indicated that ARR1, ARR10, and ARR12 negatively and redundantly control plant responses to drought, with ARR1 appearing to bear the most critical function among the three proteins. In agreement with these findings, a comparative genome-wide analysis of the leaves of arr1,10,12 and WT plants under both normal and dehydration conditions suggested a cytokinin (CK) signaling-mediated network controlling plant adaptation to drought via many dehydration/drought- and/or ABA-responsive genes that can provide osmotic adjustment and protection to cellular and membrane structures. Expression of all three ARR genes was repressed by dehydration and ABA treatments, inferring that plants down-regulate these genes as an adaptive mechanism to survive drought. Collectively, our results demonstrate that repression of CK response, and thus CK signaling, is one of the strategies plants use to cope with water deficit, providing novel insight for the design of drought-tolerant plants by genetic engineering.

  4. Grouper TRIM13 exerts negative regulation of antiviral immune response against nodavirus.

    Science.gov (United States)

    Huang, Youhua; Yang, Min; Yu, Yepin; Yang, Ying; Zhou, Linli; Huang, Xiaohong; Qin, Qiwei

    2016-08-01

    The tripartite motif (TRIM)-containing proteins have attracted particular attention to their multiple functions in different biological processes. TRIM13, a member of the TRIM family, is a RING domain-containing E3 ubiquitin ligase which plays critical roles in diverse cellular processes including cell death, cancer and antiviral immunity. In this study, a TRIM13 homolog from orange spotted grouper, Epinephelus coioides (EcTRIM13) was cloned and characterized. The full-length of EcTRIM13 cDNA encoded a polypeptide of 399 amino acids which shared 81% identity with TRIM13 homolog from large yellow croaker (Larimichthys crocea). Amino acid alignment analysis showed that EcTRIM13 contained conserved RING finger and B-box domain. Expression patterns analysis indicated that EcTRIM13 was abundant in liver, spleen, kidney, intestine and gill. Moreover, the transcript of EcTRIM13 in grouper spleen was differently regulated after injection with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C). Under fluorescence microscopy, we observed the tubular structure in wild type EcTRIM13 transfected cells, but the RING domain mutant resulted in the fluorescence distribution was changed and the bright punctate fluorescence was evenly situated throughout the cytoplasm, suggesting that the RING domain was essential for its accurate localization. Overexpression of EcTRIM13 in vitro obviously increased the replication of red spotted grouper nervous necrosis virus (RGNNV), and the enhancing effect of EcTRIM13 on virus replication was affected by the RING domain. Furthermore, the ectopic expression of EcTRIM13 not only negatively regulated the interferon promoter activity induced by interferon regulator factor (IRF) 3, IRF7, and melanoma differentiation-associated protein 5 (MDA5), but also decreased the expression of several interferon related factors. In addition, the overexpression of EcTRIM13 also differently regulated the transcription of pro

  5. Negative regulation of NF-κB by the ING4 tumor suppressor in breast cancer.

    Directory of Open Access Journals (Sweden)

    Sara A Byron

    Full Text Available Nuclear Factor kappa B (NF-κB is a key mediator of normal immune response but contributes to aggressive cancer cell phenotypes when aberrantly activated. Here we present evidence that the Inhibitor of Growth 4 (ING4 tumor suppressor negatively regulates NF-κB in breast cancer. We surveyed primary breast tumor samples for ING4 protein expression using tissue microarrays and a newly generated antibody. We found that 34% of tumors expressed undetectable to low levels of the ING4 protein (n = 227. Tumors with low ING4 expression were frequently large in size, high grade, and lymph node positive, suggesting that down-regulation of ING4 may contribute to breast cancer progression. In the same tumor set, we found that low ING4 expression correlated with high levels of nuclear phosphorylated p65/RelA (p-p65, an activated form of NF-κB (p = 0.018. Fifty seven percent of ING4-low/p-p65-high tumors were lymph node-positive, indicating a high metastatic tendency of these tumors. Conversely, ectopic expression of ING4 inhibited p65/RelA phosphorylation in T47D and MCF7 breast cancer cells. In addition, ING4 suppressed PMA-induced cell invasion and NF-κB-target gene expression in T47D cells, indicating that ING4 inhibited NF-κB activity in breast cancer cells. Supportive of the ING4 function in the regulation of NF-κB-target gene expression, we found that ING4 expression levels inversely correlated with the expression of NF-κB-target genes in primary breast tumors by analyzing public gene expression datasets. Moreover, low ING4 expression or high expression of the gene signature composed of a subset of ING4-repressed NF-κB-target genes was associated with reduced disease-free survival in breast cancer patients. Taken together, we conclude that ING4 negatively regulates NF-κB in breast cancer. Consequently, down-regulation of ING4 leads to activation of NF-κB, contributing to tumor progression and reduced disease-free patient survival in

  6. Characterization of a negative regulator AveI for avermectin biosynthesis in Streptomyces avermitilis NRRL8165.

    Science.gov (United States)

    Chen, Lei; Lu, Yinhua; Chen, Jun; Zhang, Weiwen; Shu, Dan; Qin, Zhongjun; Yang, Sheng; Jiang, Weihong

    2008-08-01

    A transcriptional activator for actinorhodin biosynthesis, AtrA, was previously characterized in Streptomyces coelicolor A3(2), and an orthologue of atrA, named aveI, is identified in the Streptomyces avermitilis NRRL8165 genome (Uguru et al., Mol Microbiol, 58:131-150, 2005). In this study, genetic and functional characterization of aveI gene was reported. Deletion of aveI gene led to increased biosynthesis of avermectin B1a by about 16-fold. The increased synthesis of avermectin B1a was suppressed by complementation with either aveI gene or its orthologue gene atrA from S. coelicolor, suggesting AveI and AtrA shared the similar functionality and were negative regulators for avermectin biosynthesis in S. avermitilis. However, when aveI was introduced into S. coelicolor on a multi-copy plasmid, the production of actinorhodin was significantly increased, indicating that aveI had a positive effect on actinorhodin biosynthesis in S. coelicolor, the same as its orthologue atrA. Electrophoretic mobility shift assays revealed AveI can bind specifically to the promoter region of actII-ORF4 in vitro but not that of aveR. Although its mechanism still needs to be defined, the species-differential regulation by the same regulator may represent an example of the evolutional strategy that enables bacteria to adapt the existing molecular machinery to a variety of functionalities for growth and survival.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha; Jeong, Jae Hoon; Pak, Youngmi Kim, E-mail: ykpak@khu.ac.kr

    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.

  8. An Arabidopsis SUMO E3 Ligase, SIZ1, Negatively Regulates Photomorphogenesis by Promoting COP1 Activity

    KAUST Repository

    Lin, Xiao-Li

    2016-04-29

    COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1), a ubiquitin E3 ligase, is a central negative regulator of photomorphogenesis. However, how COP1 activity is regulated by post-translational modifications remains largely unknown. Here we show that SUMO (small ubiquitin-like modifier) modification enhances COP1 activity. Loss-of-function siz1 mutant seedlings exhibit a weak constitutive photomorphogenic phenotype. SIZ1 physically interacts with COP1 and mediates the sumoylation of COP1. A K193R substitution in COP1 blocks its SUMO modification and reduces COP1 activity in vitro and in planta. Consistently, COP1 activity is reduced in siz1 and the level of HY5, a COP1 target protein, is increased in siz1. Sumoylated COP1 may exhibits higher transubiquitination activity than does non-sumoylated COP1, but SIZ1-mediated SUMO modification does not affect COP1 dimerization, COP1-HY5 interaction, and nuclear accumulation of COP1. Interestingly, prolonged light exposure reduces the sumoylation level of COP1, and COP1 mediates the ubiquitination and degradation of SIZ1. These regulatory mechanisms may maintain the homeostasis of COP1 activity, ensuing proper photomorphogenic development in changing light environment. Our genetic and biochemical studies identify a function for SIZ1 in photomorphogenesis and reveal a novel SUMO-regulated ubiquitin ligase, COP1, in plants.

  9. SMARCAL1 Negatively Regulates C-Myc Transcription By Altering The Conformation Of The Promoter Region.

    Science.gov (United States)

    Sharma, Tapan; Bansal, Ritu; Haokip, Dominic Thangminlen; Goel, Isha; Muthuswami, Rohini

    2015-12-09

    SMARCAL1, a member of the SWI2/SNF2 protein family, stabilizes replication forks during DNA damage. In this manuscript, we provide the first evidence that SMARCAL1 is also a transcriptional co-regulator modulating the expression of c-Myc, a transcription factor that regulates 10-15% genes in the human genome. BRG1, SMARCAL1 and RNAPII were found localized onto the c-myc promoter. When HeLa cells were serum starved, the occupancy of SMARCAL1 on the c-myc promoter increased while that of BRG1 and RNAPII decreased correlating with repression of c-myc transcription. Using Active DNA-dependent ATPase A Domain (ADAAD), the bovine homolog of SMARCAL1, we show that the protein can hydrolyze ATP using a specific region upstream of the CT element of the c-myc promoter as a DNA effector. The energy, thereby, released is harnessed to alter the conformation of the promoter DNA. We propose that SMARCAL1 negatively regulates c-myc transcription by altering the conformation of its promoter region during differentiation.

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

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    Full Text Available 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.

  11. MLK4β functions as a negative regulator of MAPK signaling and cell invasion.

    Science.gov (United States)

    Abi Saab, W F; Brown, M S; Chadee, D N

    2012-03-26

    Mixed lineage kinase (MLK) 4, or MLK4, is a member of the MLK family of mitogen-activated protein kinase kinase kinases (MAP3Ks). Typically, MAP3Ks function to activate the mitogen-activated protein kinase (MAPK)-signaling pathways and regulate different cellular responses. However, here we report that MLK4β, unlike the other MLKs, negatively regulates the activities of the MAPKs, p38, c-Jun N-terminal kinase and extracellular signal-regulated kinase, and the MAP2Ks, MEK3 and 6. Our results show that MLK4β inhibits sorbitol- and tumor necrosis factor-induced activation of p38. Furthermore, MLK4β interacts with another MLK family member, MLK3, in HCT116 cells. Exogenous expression of MLK4β inhibits activation of MLK3 and also blocks matrix metalloproteinase-9 gelatinase activity and invasion in SKOV3 ovarian cancer cells. Collectively, our data establish MLK4β as a novel suppressor of MLK3 activation, MAPK signaling and cell invasion.

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

    Science.gov (United States)

    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.

  13. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell

    Directory of Open Access Journals (Sweden)

    Yumei Luo

    2015-01-01

    Full Text Available The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C; knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

  14. Voltage gated calcium channels negatively regulate protective immunity to Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Shashank Gupta

    Full Text Available Mycobacterium tuberculosis modulates levels and activity of key intracellular second messengers to evade protective immune responses. Calcium release from voltage gated calcium channels (VGCC regulates immune responses to pathogens. In this study, we investigated the roles of VGCC in regulating protective immunity to mycobacteria in vitro and in vivo. Inhibiting L-type or R-type VGCC in dendritic cells (DCs either using antibodies or by siRNA increased calcium influx in an inositol 1,4,5-phosphate and calcium release calcium activated channel dependent mechanism that resulted in increased expression of genes favoring pro-inflammatory responses. Further, VGCC-blocked DCs activated T cells that in turn mediated killing of M. tuberculosis inside macrophages. Likewise, inhibiting VGCC in infected macrophages and PBMCs induced calcium influx, upregulated the expression of pro-inflammatory genes and resulted in enhanced killing of intracellular M. tuberculosis. Importantly, compared to healthy controls, PBMCs of tuberculosis patients expressed higher levels of both VGCC, which were significantly reduced following chemotherapy. Finally, blocking VGCC in vivo in M. tuberculosis infected mice using specific antibodies increased intracellular calcium and significantly reduced bacterial loads. These results indicate that L-type and R-type VGCC play a negative role in M. tuberculosis infection by regulating calcium mobilization in cells that determine protective immunity.

  15. Negative regulators of insulin signaling revealed in a genome-wide functional screen.

    Directory of Open Access Journals (Sweden)

    Shih-Min A Huang

    Full Text Available BACKGROUND: Type 2 diabetes develops due to a combination of insulin resistance and beta-cell failure and current therapeutics aim at both of these underlying causes. Several negative regulators of insulin signaling are known and are the subject of drug discovery efforts. We sought to identify novel contributors to insulin resistance and hence potentially novel targets for therapeutic intervention. METHODOLOGY: An arrayed cDNA library encoding 18,441 human transcripts was screened for inhibitors of insulin signaling and revealed known inhibitors and numerous potential novel regulators. The novel hits included proteins of various functional classes such as kinases, phosphatases, transcription factors, and GTPase associated proteins. A series of secondary assays confirmed the relevance of the primary screen hits to insulin signaling and provided further insight into their modes of action. CONCLUSION/SIGNIFICANCE: Among the novel hits was PALD (KIAA1274, paladin, a previously uncharacterized protein that when overexpressed led to inhibition of insulin's ability to down regulate a FOXO1A-driven reporter gene, reduced upstream insulin-stimulated AKT phosphorylation, and decreased insulin receptor (IR abundance. Conversely, knockdown of PALD gene expression resulted in increased IR abundance, enhanced insulin-stimulated AKT phosphorylation, and an improvement in insulin's ability to suppress FOXO1A-driven reporter gene activity. The present data demonstrate that the application of arrayed genome-wide screening technologies to insulin signaling is fruitful and is likely to reveal novel drug targets for insulin resistance and the metabolic syndrome.

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

    Science.gov (United States)

    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

  17. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell.

    Science.gov (United States)

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells.

  18. Selective androgen receptor modulators (SARMs negatively regulate triple-negative breast cancer growth and epithelial:mesenchymal stem cell signaling.

    Directory of Open Access Journals (Sweden)

    Ramesh Narayanan

    Full Text Available The androgen receptor (AR is the most highly expressed steroid receptor in breast cancer with 75-95% of estrogen receptor (ER-positive and 40-70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer.Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC co-culture signaling studies were performed to understand the mechanisms of action.Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures.1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.

  19. Selective androgen receptor modulators (SARMs) negatively regulate triple-negative breast cancer growth and epithelial:mesenchymal stem cell signaling.

    Science.gov (United States)

    Narayanan, Ramesh; Ahn, Sunjoo; Cheney, Misty D; Yepuru, Muralimohan; Miller, Duane D; Steiner, Mitchell S; Dalton, James T

    2014-01-01

    The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75-95% of estrogen receptor (ER)-positive and 40-70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer. Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR) were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC) co-culture signaling studies were performed to understand the mechanisms of action. Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures. 1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.

  20. How Is Emotional Awareness Related to Emotion Regulation Strategies and Self-Reported Negative Affect in the General Population?

    OpenAIRE

    Claudia Subic-Wrana; Beutel, Manfred E.; Elmar Brähler; Yve Stöbel-Richter; Achim Knebel; Lane, Richard D.; Jörg Wiltink

    2014-01-01

    OBJECTIVE: The Levels of Emotional Awareness Scale (LEAS) as a performance task discriminates between implicit or subconscious and explicit or conscious levels of emotional awareness. An impaired awareness of one's feeling states may influence emotion regulation strategies and self-reports of negative emotions. To determine this influence, we applied the LEAS and self-report measures for emotion regulation strategies and negative affect in a representative sample of the German general populat...

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

    Science.gov (United States)

    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.

  2. Snail transcription factor negatively regulates maspin tumor suppressor in human prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Neal Corey L

    2012-08-01

    Full Text Available Abstract Background Maspin, a putative tumor suppressor that is down-regulated in breast and prostate cancer, has been associated with decreased cell motility. Snail transcription factor is a zinc finger protein that is increased in breast cancer and is associated with increased tumor motility and invasion by induction of epithelial-mesenchymal transition (EMT. We investigated the molecular mechanisms by which Snail increases tumor motility and invasion utilizing prostate cancer cells. Methods Expression levels were analyzed by RT-PCR and western blot analyses. Cell motility and invasion assays were performed, while Snail regulation and binding to maspin promoter was analyzed by luciferase reporter and chromatin immunoprecipitation (ChIP assays. Results Snail protein expression was higher in different prostate cancer cells lines as compared to normal prostate epithelial cells, which correlated inversely with maspin expression. Snail overexpression in 22Rv1 prostate cancer cells inhibited maspin expression and led to increased migration and invasion. Knockdown of Snail in DU145 and C4-2 cancer cells resulted in up-regulation of maspin expression, concomitant with decreased migration. Transfection of Snail into 22Rv1 or LNCaP cells inhibited maspin promoter activity, while stable knockdown of Snail in C4-2 cells increased promoter activity. ChIP analysis showed that Snail is recruited to the maspin promoter in 22Rv1 cells. Conclusions Overall, this is the first report showing that Snail can negatively regulate maspin expression by directly repressing maspin promoter activity, leading to increased cell migration and invasion. Therefore, therapeutic targeting of Snail may be useful to re-induce expression of maspin tumor suppressor and prevent prostate cancer tumor progression.

  3. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

    Science.gov (United States)

    Chen, Nai-Yu; Nagarajan, Govindarajulu; Chiou, Pinwen Peter

    2015-01-01

    Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish. PMID:25955250

  4. Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish.

    Directory of Open Access Journals (Sweden)

    Frank Fang-Yao Lee

    Full Text Available Toll-like receptor 9 (TLR9 recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length and gTLR9B (with a truncated C'-terminal signal transducing domain, whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides, whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN, gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.

  5. Abscisic acid is a negative regulator of root gravitropism in Arabidopsis thaliana.

    Science.gov (United States)

    Han, Woong; Rong, Honglin; Zhang, Hanma; Wang, Myeong-Hyeon

    2009-01-23

    The plant hormone abscisic acid (ABA) plays a role in root gravitropism and has led to an intense debate over whether ABA acts similar to auxin by translating the gravitational signal into directional root growth. While tremendous advances have been made in the past two decades in establishing the role of auxin in root gravitropism, little progress has been made in characterizing the role of ABA in this response. In fact, roots of plants that have undetectable levels of ABA and that display a normal gravitropic response have raised some serious doubts about whether ABA plays any role in root gravitropism. Here, we show strong evidence that ABA plays a role opposite to that of auxin and that it is a negative regulator of the gravitropic response of Arabidopsis roots.

  6. Negative regulation of parathyroid hormone-related protein expression by steroid hormones.

    Science.gov (United States)

    Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko; Chikamori, Minoru; Iizuka, Masayoshi; Okazaki, Tomoki

    2011-04-15

    Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression. Copyright © 2011 Elsevier Inc. All rights reserved.

  7. amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis

    Directory of Open Access Journals (Sweden)

    Turner Jane

    2001-08-01

    Full Text Available Abstract Background The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation. Results We constructed a deletion mutant in one of the upstream ORFs (amiA. This mutant (Mad1 showed a constitutively high level of acetamidase expression. We identified four promoters in the upstream region using a β-galactosidase reporter gene. One of these (P2 was inducible in the wild-type, but was constitutively active in Mad1. Conclusions These results demonstrate that amiA encodes a negative regulatory protein which interacts with P2. Since amiA has homology to DNA-binding proteins, it is likely that it exerts the regulatory effect by binding to the promoter to prevent transcription.

  8. Positive and negative regulation of the human heme oxygenase-1 gene expression in cultured cells.

    Science.gov (United States)

    Takahashi, S; Takahashi, Y; Ito, K; Nagano, T; Shibahara, S; Miura, T

    1999-10-28

    To elucidate the regulation of the human heme oxygenase-1 (hHO-1) gene expression, we assessed approximately 4 kb of the 5'-flanking region of the hHO-1 gene for basal promoter activity and sequenced approximately 2 kb of the 5'-flanking region. A series of deletion mutants of the 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in HepG2 human hepatoma cells and HeLa cervical cancer cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found a positive regulatory region at position -1976 to -1655 bp. This region functions in HepG2 cells but not in HeLa cells. A negative regulatory region was also found at position -981 to -412 bp that functions in both HepG2 cells and HeLa cells.

  9. Models of Aire-dependent gene regulation for thymic negative selection

    Directory of Open Access Journals (Sweden)

    Dina eDanso-Abeam

    2011-05-01

    Full Text Available Mutations in the Autoimmune Regulator (AIRE gene lead to Autoimmune Polyendocrinopathy Syndrome type 1 (APS1, characterized by the development of multi-organ autoimmune damage. The mechanism by which defects in AIRE result in autoimmunity has been the subject of intense scrutiny. At the cellular level, the working model explains most of the clinical and immunological characteristics of APS1, with AIRE driving the expression of tissue restricted antigens (TRAs in the epithelial cells of the thymic medulla. This TRA expression results in effective negative selection of TRA-reactive thymocytes, preventing autoimmune disease. At the molecular level, the mechanism by which AIRE initiates TRA expression in the thymic medulla remains unclear. Multiple different models for the molecular mechanism have been proposed, ranging from classical transcriptional activity, to random induction of gene expression, to epigenetic tag recognition effect, to altered cell biology. In this review, we evaluate each of these models and discuss their relative strengths and weaknesses.

  10. SarA is a negative regulator of Staphylococcus epidermidis biofilm formation

    DEFF Research Database (Denmark)

    Martin, Christer; Heinze, C.; Busch, M.

    2012-01-01

    Biofilm formation is essential for Staphylococcus epidermidis pathogenicity in implant-associated infections. Nonetheless, large proportions of invasive S. epidermidis isolates fail to show accumulative biofilm growth in vitro. We here tested the hypothesis that this apparent paradox is related...... to the existence of superimposed regulatory systems suppressing a multi-cellular biofilm life style in vitro. Transposon mutagenesis of clinical significant but biofilm-negative S. epidermidis 1585 was used to isolate a biofilm positive mutant carrying a Tn917 insertion in sarA,chief regulator of staphylococcal...... virulence. Genetic analysis revealed that inactivation of sarA induced biofilm formation via over-expression of the giant 1 MDa extracellular matrix binding protein (Embp), serving as an intercellular adhesin. In addition to Embp, increased extracellular DNA (eDNA) release significantly contributed...

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

    Science.gov (United States)

    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

  12. F-spondin negatively regulates dental follicle differentiation through the inhibition of TGF-β activity.

    Science.gov (United States)

    Orimoto, Ai; Kurokawa, Misaki; Handa, Keisuke; Ishikawa, Masaki; Nishida, Eisaku; Aino, Makoto; Mitani, Akio; Ogawa, Miho; Tsuji, Takashi; Saito, Masahiro

    2017-07-01

    F-spondin is an extracellular matrix (ECM) protein that belongs to the thrombospondin type I repeat superfamily and is a negative regulator of bone mass. We have previously shown that f-spondin is specifically expressed in the dental follicle (DF), which gives rise to the periodontal ligament (PDL) during the tooth root formation stage. To investigate the molecular mechanism of PDL formation, we investigated the function of f-spondin in DF differentiation. The expression patterning of f-spondin in the developing tooth germ was compared with that of periodontal ligament-related genes, including runx2, type I collagen and periostin, by in situ hybridization analysis. To investigate the function of f-spondin during periodontal ligament formation, an f-spondin adenovirus was infected into the bell stage of the developing tooth germ, and the effect on dental differentiation was analyzed. F-spondin was specifically expressed in the DF of the developing tooth germ; by contrast, type I collagen, runx2 and periostin were expressed in the DF and in the alveolar bone. F-spondin-overexpresssing tooth germ exhibited a reduction in gene expression of periostin and type I collagen in the DF. By contrast, the knockdown of f-spondin in primary DF cells increased the expression of these genes. Treatment with recombinant f-spondin protein functionally inhibited periostin expression induced by transforming growth factor-β (TGF-β). Our data indicated that f-spondin inhibits the differentiation of DF cells into periodontal ligament cells by inhibiting TGF-β. These data suggested that f-spondin negatively regulates PDL differentiation which may play an important role in the immature phenotype of DF. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. HMGA1: a master regulator of tumor progression in triple-negative breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Sandeep N Shah

    Full Text Available Emerging evidence suggests that tumor cells metastasize by co-opting stem cell transcriptional networks, although the molecular underpinnings of this process are poorly understood. Here, we show for the first time that the high mobility group A1 (HMGA1 gene drives metastatic progression in triple negative breast cancer cells (MDA-MB-231, Hs578T by reprogramming cancer cells to a stem-like state. Silencing HMGA1 expression in invasive, aggressive breast cancer cells dramatically halts cell growth and results in striking morphologic changes from mesenchymal-like, spindle-shaped cells to cuboidal, epithelial-like cells. Mesenchymal genes (Vimentin, Snail are repressed, while E-cadherin is induced in the knock-down cells. Silencing HMGA1 also blocks oncogenic properties, including proliferation, migration, invasion, and orthotopic tumorigenesis. Metastatic progression following mammary implantation is almost completely abrogated in the HMGA1 knock-down cells. Moreover, silencing HMGA1 inhibits the stem cell property of three-dimensional mammosphere formation, including primary, secondary, and tertiary spheres. In addition, knock-down of HMGA1 depletes cancer initiator/cancer stem cells and prevents tumorigenesis at limiting dilutions. We also discovered an HMGA1 signature in triple negative breast cancer cells that is highly enriched in embryonic stem cells. Together, these findings indicate that HMGA1 is a master regulator of tumor progression in breast cancer by reprogramming cancer cells through stem cell transcriptional networks. Future studies are needed to determine how to target HMGA1 in therapy.

  14. Interleukin-19 acts as a negative autocrine regulator of activated microglia.

    Directory of Open Access Journals (Sweden)

    Hiroshi Horiuchi

    Full Text Available Activated microglia can exert either neurotoxic or neuroprotective effects, and they play pivotal roles in the pathogenesis and progression of various neurological diseases. In this study, we used cDNA microarrays to show that interleukin-19 (IL-19, an IL-10 family cytokine, is markedly upregulated in activated microglia. Furthermore, we found that microglia are the only cells in the nervous system that express the IL-19 receptor, a heterodimer of the IL-20Rα and IL-20Rβ subunits. IL-19 deficiency increased the production of such pro-inflammatory cytokines as IL-6 and tumor necrosis factor-α in activated microglia, and IL-19 treatment suppressed this effect. Moreover, in a mouse model of Alzheimer's disease, we observed upregulation of IL-19 in affected areas in association with disease progression. Our findings demonstrate that IL-19 is an anti-inflammatory cytokine, produced by activated microglia, that acts negatively on microglia in an autocrine manner. Thus, microglia may self-limit their inflammatory response by producing the negative regulator IL-19.

  15. N-cadherin negatively regulates collective Drosophila glial migration through actin cytoskeleton remodeling.

    Science.gov (United States)

    Kumar, Arun; Gupta, Tripti; Berzsenyi, Sara; Giangrande, Angela

    2015-03-01

    Cell migration is an essential and highly regulated process. During development, glia cells and neurons migrate over long distances - in most cases collectively - to reach their final destination and build the sophisticated architecture of the nervous system, the most complex tissue of the body. Collective migration is highly stereotyped and efficient, defects in the process leading to severe human diseases that include mental retardation. This dynamic process entails extensive cell communication and coordination, hence, the real challenge is to analyze it in the entire organism and at cellular resolution. We here investigate the impact of the N-cadherin adhesion molecule on collective glial migration, by using the Drosophila developing wing and cell-type specific manipulation of gene expression. We show that N-cadherin timely accumulates in glial cells and that its levels affect migration efficiency. N-cadherin works as a molecular brake in a dosage-dependent manner, by negatively controlling actin nucleation and cytoskeleton remodeling through α/β catenins. This is the first in vivo evidence for N-cadherin negatively and cell autonomously controlling collective migration.

  16. 5-Lipoxygenase negatively regulates Th1 response during Brucella abortus infection in mice.

    Science.gov (United States)

    Fahel, Júlia Silveira; de Souza, Mariana Bueno; Gomes, Marco Túlio Ribeiro; Corsetti, Patricia P; Carvalho, Natalia B; Marinho, Fabio A V; de Almeida, Leonardo A; Caliari, Marcelo V; Machado, Fabiana Simão; Oliveira, Sergio Costa

    2015-03-01

    Brucella abortus is a Gram-negative bacterium that infects humans and cattle, causing a chronic inflammatory disease known as brucellosis. A Th1-mediated immune response plays a critical role in host control of this pathogen. Recent findings indicate contrasting roles for lipid mediators in host responses against infections. 5-Lipoxygenase (5-LO) is an enzyme required for the production of the lipid mediators leukotrienes and lipoxins. To determine the involvement of 5-LO in host responses to B. abortus infection, we intraperitoneally infected wild-type and 5-LO-deficient mice and evaluated the progression of infection and concomitant expression of immune mediators. Here, we demonstrate that B. abortus induced the upregulation of 5-LO mRNA in wild-type mice. Moreover, this pathogen upregulated the production of the lipid mediators leukotriene B4 and lipoxin A4 in a 5-LO-dependent manner. 5-LO-deficient mice displayed lower bacterial burdens in the spleen and liver and less severe liver pathology, demonstrating an enhanced resistance to infection. Host resistance paralleled an increased expression of the proinflammatory mediators interleukin-12 (IL-12), gamma interferon (IFN-γ), and inducible nitric oxide synthase (iNOS) during the course of infection. Moreover, we demonstrated that 5-LO downregulated the expression of IL-12 in macrophages during B. abortus infection. Our results suggest that 5-LO has a major involvement in B. abortus infection, by functioning as a negative regulator of the protective Th1 immune responses against this pathogen.

  17. The nuclear protein-coding gene ANKRD23 negatively regulates myoblast differentiation.

    Science.gov (United States)

    Wang, Xiaojing; Zeng, Rui; Xu, Haiyang; Xu, Zaiyan; Zuo, Bo

    2017-09-20

    Muscle fiber formation is a complex process and subject to fine regulation of a variety of protein-coding genes and non-coding RNA. In this study, we identified a nuclear protein-coding gene ANKRD23 which was highly expressed in muscle. Quantitative real-time PCR, western blotting and immunofluorescence were used to detect the expression change of myoblast differentiation marker genes after knockdown and overexpression of ANKRD23. The results showed that the expression of myoblast differentiation marker genes were increased by interference and reduced by ANKRD23 overexpression, indicating that ANKRD23 played a negative role in the myoblast differentiation. Interestingly, we discovered a long non-coding RNA-AK004293 which was overlapped with the 3'UTR of ANKRD23 gene. Then we detected the effect of AK004293 on the expression of ANKRD23 and myoblast differentiation marker genes in C2C12 myoblasts. The results showed that AK004293 had no significant effect on the expression of myoblast differentiation maker genes and ANKRD23. In conclusion, our results established the foundation for further studies about the regulation mechanism of ANKRD23 in muscle development. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. JMJD6 promotes colon carcinogenesis through negative regulation of p53 by hydroxylation.

    Directory of Open Access Journals (Sweden)

    Feng Wang

    2014-03-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  20. miR-27 negatively regulates pluripotency-associated genes in human embryonal carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Heiko Fuchs

    Full Text Available Human embryonic stem cells and human embryonal carcinoma cells have been studied extensively with respect to the transcription factors (OCT4, SOX2 and NANOG, epigenetic modulators and associated signalling pathways that either promote self-renewal or induce differentiation in these cells. The ACTIVIN/NODAL axis (SMAD2/3 of the TGFß signalling pathway coupled with FGF signalling maintains self-renewal in these cells, whilst the BMP (SMAD1,5,8 axis promotes differentiation. Here we show that miR-27, a somatic-enriched miRNA, is activated upon RNAi-mediated suppression of OCT4 function in human embryonic stem cells. We further demonstrate that miR-27 negatively regulates the expression of the pluripotency-associated ACTIVIN/NODAL axis (SMAD2/3 of the TGFß signalling pathway by targeting ACVR2A, TGFßR1 and SMAD2. Additionally, we have identified a number of pluripotency-associated genes such as NANOG, LIN28, POLR3G and NR5A2 as novel miR-27 targets. Transcriptome analysis revealed that miR-27 over-expression in human embryonal carcinoma cells leads indeed to a significant up-regulation of genes involved in developmental pathways such as TGFß- and WNT-signalling.

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

    Science.gov (United States)

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

    2014-03-11

    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.

  2. AMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation.

    Science.gov (United States)

    Bandow, Kenjiro; Kusuyama, Joji; Kakimoto, Kyoko; Ohnishi, Tomokazu; Matsuguchi, Tetsuya

    2015-05-01

    Chondrocytes are derived from mesenchymal stem cells, and play an important role in cartilage formation. Sex determining region Y box (Sox) family transcription factors are essential for chondrogenic differentiation, whereas the intracellular signal pathways of Sox activation have not been clearly elucidated. AMP-activated protein kinase (AMPK) is a serine-threonine kinase generally regarded as a key regulator of cellular energy homeostasis. It is known that the catalytic alpha subunit of AMPK is activated by upstream AMPK kinases (AMPKKs) including liver kinase B1 (LKB1). We have previously reported that AMPK is a negative regulator of osteoblastic differentiation. Here, we have explored the role of AMPK in chondrogenic differentiation using in vitro culture models. The phosphorylation level of the catalytic AMPK alpha subunit significantly decreased during chondrogenic differentiation of primary chondrocyte precursors as well as ATDC-5, a well-characterized chondrogenic cell line. Treatment with metformin, an activator of AMPK, significantly reduced cartilage matrix formation and inhibited gene expression of sox6, sox9, col2a1 and aggrecan core protein (acp). Thus, chondrocyte differentiation is functionally associated with decreased AMPK activity. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Cif is negatively regulated by the TetR family repressor CifR.

    Science.gov (United States)

    MacEachran, Daniel P; Stanton, Bruce A; O'Toole, George A

    2008-07-01

    We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene.

  4. Cif Is Negatively Regulated by the TetR Family Repressor CifR▿

    Science.gov (United States)

    MacEachran, Daniel P.; Stanton, Bruce A.; O'Toole, George A.

    2008-01-01

    We previously reported that the novel Pseudomonas aeruginosa toxin Cif is capable of decreasing apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR). We further demonstrated that Cif is capable of degrading the synthetic epoxide hydrolase (EH) substrate S-NEPC [(2S,3S)-trans-3-phenyl-2-oxiranylmethyl 4-nitrophenol carbonate], suggesting that Cif may be reducing apical membrane expression of CFTR via its EH activity. Here we report that Cif is capable of degrading the xenobiotic epoxide epibromohydrin (EBH) to its vicinal diol 3-bromo-1,2-propanediol. We also demonstrate that this epoxide is a potent inducer of cif gene expression. We show that the predicted TetR family transcriptional repressor encoded by the PA2931 gene, which is immediately adjacent to and divergently transcribed from the cif-containing, three-gene operon, negatively regulates cif gene expression by binding to the promoter region immediately upstream of the cif-containing operon. Furthermore, this protein-DNA interaction is disrupted by the epoxide EBH in vitro, suggesting that the binding of EBH by the PA2931 protein product drives the disassociation from its DNA-binding site. Given its role as a repressor of cif gene expression, we have renamed PA2931 as CifR. Finally, we demonstrate that P. aeruginosa strains isolated from cystic fibrosis patient sputum with increased cif gene expression are impaired for the expression of the cifR gene. PMID:18458065

  5. Marburgvirus Hijacks Nrf2-Dependent Pathway by Targeting Nrf2-Negative Regulator Keap1

    Directory of Open Access Journals (Sweden)

    Audrey Page

    2014-03-01

    Full Text Available Marburg virus (MARV has a high fatality rate in humans, causing hemorrhagic fever characterized by massive viral replication and dysregulated inflammation. Here, we demonstrate that VP24 of MARV binds Kelch-like ECH-associated protein 1 (Keap1, a negative regulator of nuclear transcription factor erythroid-derived 2 (Nrf2. Binding of VP24 to Keap1 Kelch domain releases Nrf2 from Keap1-mediated inhibition promoting persistent activation of a panoply of cytoprotective genes implicated in cellular responses to oxidative stress and regulation of inflammatory responses. Increased expression of Nrf2-dependent genes was demonstrated both during MARV infection and upon ectopic expression of MARV VP24. We also show that Nrf2-deficient mice can control MARV infection when compared to lethal infection in wild-type animals, indicating that Nrf2 is critical for MARV infection. We conclude that VP24-driven activation of the Nrf2-dependent pathway is likely to contribute to dysregulation of host antiviral inflammatory responses and that it ensures survival of MARV-infected cells despite these responses.

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

    Science.gov (United States)

    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. P90 Ribosomal s6 kinase 2 negatively regulates axon growth in motoneurons.

    Science.gov (United States)

    Fischer, Matthias; Pereira, Patricia Marques; Holtmann, Bettina; Simon, Christian M; Hanauer, Andre; Heisenberg, Martin; Sendtner, Michael

    2009-10-01

    Mutations in Ribosomal s6 kinase 2 (Rsk2) are associated with severe neuronal dysfunction in Coffin-Lowry syndrome (CLS) patients, flies and mice. So far, the mechanisms of how Rsk2 regulates development, maintenance and activity of neurons are not understood. We have investigated the consequences of Rsk2 deficiency in mouse spinal motoneurons. Survival of isolated Rsk2 deficient motoneurons is not reduced, but these cells grow significantly longer neurites. Conversely, overexpression of a constitutively active form of Rsk2 leads to reduced axon growth. Increased axon growth in Rsk2 deficient neurons was accompanied by higher Erk 1/2 phosphorylation, and the knockout phenotype could be rescued by pharmacological inhibition of MAPK/Erk kinase (Mek). These data indicate that Rsk2 negatively regulates axon elongation via the MAPK pathway. Thus, the functional defects observed in the nervous system of CLS patients and animal models with Rsk2 deficiency might be caused by dysregulated neurite growth rather than primary neurodegeneration.

  8. Regnase-1 in microglia negatively regulates high mobility group box 1-mediated inflammation and neuronal injury.

    Science.gov (United States)

    Liu, Xiao-Xi; Wang, Chen; Huang, Shao-Fei; Chen, Qiong; Hu, Ya-Fang; Zhou, Liang; Gu, Yong

    2016-04-05

    Extracellular high mobility group box 1 (HMGB1) has been demonstrated to function as a proinflammatory cytokine and induces neuronal injury in response to various pathological stimuli in central nervous system (CNS). However, the regulatory factor involved in HMGB1-mediated inflammatory signaling is largely unclear. Regulatory RNase 1 (Regnase-1) is a potent anti-inflammation enzyme that can degrade a set of mRNAs encoding proinflammatory cytokines. The present study aims to determine the role of Regnase-1 in the regulation of HMGB1-mediated inflammatory injury in CNS. Cultured microglia and rat brain were treated with recombinant HMGB1 to examine the induction of Regnase-1 expression. Moreover, the role of Regnase-1 in modulating the expression of inflammatory cytokines and neuronal injury was then investigated in microglia by specific siRNA knockdown upon HMGB1 treatment. Results showed that HMGB1 could significantly induce the de novo synthesis of Regnase-1 in cultured microglia. Consistently, Regnase-1 was elevated and found to be co-localized with microglia marker in the brain of rat treated with HMGB1. Silencing Regnase-1 in microglia enhanced HMGB1-induced expression of proinflammatory cytokines and exacerbated neuronal toxicity. Collectively, these results suggest that Regnase-1 can be induced by HMGB1 in microglia and negatively regulates HMGB1-mediated neuroinflammation and neuronal toxicity.

  9. proBDNF negatively regulates neuronal remodeling, synaptic transmission, and synaptic plasticity in hippocampus.

    Science.gov (United States)

    Yang, Jianmin; Harte-Hargrove, Lauren C; Siao, Chia-Jen; Marinic, Tina; Clarke, Roshelle; Ma, Qian; Jing, Deqiang; Lafrancois, John J; Bath, Kevin G; Mark, Willie; Ballon, Douglas; Lee, Francis S; Scharfman, Helen E; Hempstead, Barbara L

    2014-05-08

    Experience-dependent plasticity shapes postnatal development of neural circuits, but the mechanisms that refine dendritic arbors, remodel spines, and impair synaptic activity are poorly understood. Mature brain-derived neurotrophic factor (BDNF) modulates neuronal morphology and synaptic plasticity, including long-term potentiation (LTP) via TrkB activation. BDNF is initially translated as proBDNF, which binds p75(NTR). In vitro, recombinant proBDNF modulates neuronal structure and alters hippocampal long-term plasticity, but the actions of endogenously expressed proBDNF are unclear. Therefore, we generated a cleavage-resistant probdnf knockin mouse. Our results demonstrate that proBDNF negatively regulates hippocampal dendritic complexity and spine density through p75(NTR). Hippocampal slices from probdnf mice exhibit depressed synaptic transmission, impaired LTP, and enhanced long-term depression (LTD) in area CA1. These results suggest that proBDNF acts in vivo as a biologically active factor that regulates hippocampal structure, synaptic transmission, and plasticity, effects that are distinct from those of mature BDNF. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  10. Akt2 negatively regulates assembly of the POSH-MLK-JNK signaling complex.

    Science.gov (United States)

    Figueroa, Claudia; Tarras, Samantha; Taylor, Jennifer; Vojtek, Anne B

    2003-11-28

    We demonstrate that POSH, a scaffold for the JNK signaling pathway, binds to Akt2. A POSH mutant that is unable to bind Akt2 (POSH W489A) exhibits enhanced-binding to MLK3, and this increase in binding is accompanied by increased activation of the JNK signaling pathway. In addition, we show that the association of MLK3 with POSH is increased upon inhibition of the endogenous phosphatidylinositol 3-kinase/Akt signaling pathway. Thus, the assembly of an active JNK signaling complex by POSH is negatively regulated by Akt2. Further, the level of Akt-phosphorylated MLK3 is reduced in cells expressing the Akt2 binding domain of POSH, which acts as a dominant interfering protein. Taken together, our results support a model in which Akt2 binds to a POSH-MLK-MKK-JNK complex and phosphorylates MLK3; phosphorylation of MLK3 by Akt2 results in the disassembly of the JNK complex bound to POSH and down-regulation of the JNK signaling pathway.

  11. The PhoP transcription factor negatively regulates avermectin biosynthesis in Streptomyces avermitilis.

    Science.gov (United States)

    Yang, Renjun; Liu, Xingchao; Wen, Ying; Song, Yuan; Chen, Zhi; Li, Jilun

    2015-12-01

    Bacteria sense and respond to the stress of phosphate limitation, anticipating Pi deletion/starvation via the two-component PhoR-PhoP system. The role of the response regulator PhoP in primary metabolism and avermectin biosynthesis in Streptomyces avermitilis was investigated. In response to phosphate starvation, S. avermitilis PhoP, like Streptomyces coelicolor and Streptomyces lividans PhoP, activates the expression of phoRP, phoU, and pstS by binding to the PHO boxes in their promoter regions. Avermectin biosynthesis was significantly increased in ΔphoP deletion mutants. Electrophoretic mobility gel shift assay (EMSA) and DNase I footprinting assays showed that PhoP can bind to a PHO box formed by two direct repeat units of 11 nucleotides located downstream of the transcriptional start site of aveR. By negatively regulating the transcription of aveR, PhoP directly affects avermectin biosynthesis in S. avermitilis. PhoP indirectly affects melanogenesis on Casaminoacids Minimal Medium (MMC) lacking supplemental phosphate. Nitrogen metabolism and some key genes involved in morphological differentiation and antibiotic production in S. avermitilis are also under the control of PhoP.

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

    Science.gov (United States)

    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.

  13. APUM5, encoding a Pumilio RNA binding protein, negatively regulates abiotic stress responsive gene expression

    Science.gov (United States)

    2014-01-01

    Background A mutant screening was carried out previously to look for new genes related to the Cucumber mosaic virus infection response in Arabidopsis. A Pumilio RNA binding protein-coding gene, Arabidopsis Pumilio RNA binding protein 5 (APUM5), was obtained from this screening. Results APUM5 transcriptional profiling was carried out using a bioinformatics tool. We found that APUM5 was associated with both biotic and abiotic stress responses. However, bacterial and fungal pathogen infection susceptibility was not changed in APUM5 transgenic plants compared to that in wild type plants although APUM5 expression was induced upon pathogen infection. In contrast, APUM5 was involved in the abiotic stress response. 35S-APUM5 transgenic plants showed hypersensitive phenotypes under salt and drought stresses during germination, primary root elongation at the seedling stage, and at the vegetative stage in soil. We also showed that some abiotic stress-responsive genes were negatively regulated in 35S-APUM5 transgenic plants. The APUM5-Pumilio homology domain (PHD) protein bound to the 3′ untranslated region (UTR) of the abiotic stress-responsive genes which contained putative Pumilio RNA binding motifs at the 3′ UTR. Conclusions These results suggest that APUM5 may be a new post-transcriptional regulator of the abiotic stress response by direct binding of target genes 3′ UTRs. PMID:24666827

  14. SOCS1 mimetics and antagonists: a complementary approach to positive and negative regulation of immune function

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    Chulbul M. Ahmed

    2015-04-01

    Full Text Available 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.

  15. Arabidopsis MSI1 Is Required for Negative Regulation of the Response to Drought Stress

    Institute of Scientific and Technical Information of China (English)

    Cristina Alexandre; Yvonne M(o)ller-Steinbach; Nicole Sch(o)nrock; Wilhelm Gruissem; Lars Hennig

    2009-01-01

    Arabidopsis MSI1 has fundamental functions in plant development.MSI1 is a subunit of Polycomb group protein complexes and Chromatin assembly factor 1,and it interacts with the Retinoblastoma-related protein 1.Altered levels of MSI1 result in pleiotropic phenotypes,reflecting the complexity of MSI1 protein functions.In order to uncover additional functions of MSI1,we performed transcriptional profiling of wild-type and plants with highly reduced MSI1 levels (msil-cs).Surprisingly,the known functions of MSI1 could only account for a minor part of the transcriptional changes in msi1-cs plants.One of the most striking unexpected observations was the up-regulation of a subset of ABA-responsive genes eliciting the response to drought and salt stress.We report that MSI1 can bind to the chromatin of the drought-inducible downstream target RD20 and suggest a new role for MSI1 in the negative regulation of the Arabidopsis drought-stress response.

  16. p27(Kip1) negatively regulates the activation of murine primordial oocytes.

    Science.gov (United States)

    Hirashima, Yumiko; Moniruzzaman, Mohammad; Miyano, Takashi

    2011-04-01

    In mice, small oocytes (primordial oocytes) are enclosed within flattened granulosa cells to form primordial follicles around birth. A small number of primordial oocytes enter the growth phase, whereas others are quiescent. The mechanism regulating this selection of primordial oocytes is not well understood. The objective of the present study was to understand the role of p27(Kip1), which regulates cell cycle progression in somatic cells, in the growth initiation of primordial oocytes in neonatal mice. We studied the localization of p27(Kip1) in 0-, 3-, 5-, 7- and 21-day-old mouse ovaries by immunohistochemistry. Ovaries from 3-day-old mice were treated with p27(Kip1) siRNAs (small interfering RNAs), and knockdown of p27(Kip1) was determined by immunohistochemistry and Western blotting. Ovaries treated with siRNAs were organ-cultured for 6 days, and oocyte growth was estimated histologically. Expression of p27(Kip1) was undetectable in the primordial oocytes of newborn mice. In the 3-day-old ovaries (n=3), p27(Kip1) was demonstrated in the nucleus of 36 ± 6% primordial oocytes. The percentage of p27(Kip1)-positive primordial oocytes increased to 72 ± 8 (n=3), 85 ± 7 (n=3) and 93 ± 5 (n=3) in the 5-, 7- and 21-day-old mouse ovaries, respectively. After knockdown of the p27(Kip1) protein by siRNAs, a higher proportion of oocytes entered the growth phase in cultured ovaries than those in the control. These results suggest that p27(Kip1) negatively regulates primordial oocyte growth and that knockdown of p27(Kip1) leads primordial oocytes to enter the growth phase in vitro.

  17. Negative regulation of the endocytic adaptor disabled-2 (Dab2) in mitosis.

    Science.gov (United States)

    Chetrit, David; Barzilay, Lior; Horn, Galit; Bielik, Tom; Smorodinsky, Nechama I; Ehrlich, Marcelo

    2011-02-18

    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.

  18. ABSCISIC ACID-INSENSITIVE 4 negatively regulates flowering through directly promoting Arabidopsis FLOWERING LOCUS C transcription

    Science.gov (United States)

    Shu, Kai; Chen, Qian; Wu, Yaorong; Liu, Ruijun; Zhang, Huawei; Wang, Shengfu; Tang, Sanyuan; Yang, Wenyu; Xie, Qi

    2016-01-01

    During the life cycle of a plant, one of the major biological processes is the transition from the vegetative to the reproductive stage. In Arabidopsis, flowering time is precisely controlled by extensive environmental and internal cues. Gibberellins (GAs) promote flowering, while abscisic acid (ABA) is considered as a flowering suppressor. However, the detailed mechanism through which ABA inhibits the floral transition is poorly understood. Here, we report that ABSCISIC ACID-INSENSITIVE 4 (ABI4), a key component in the ABA signalling pathway, negatively regulates floral transition by directly promoting FLOWERING LOCUS C (FLC) transcription. The abi4 mutant showed the early flowering phenotype whereas ABI4-overexpressing (OE-ABI4) plants had delayed floral transition. Consistently, quantitative reverse transcription–PCR (qRT–PCR) assay revealed that the FLC transcription level was down-regulated in abi4, but up-regulated in OE-ABI4. The change in FT level was consistent with the pattern of FLC expression. Chromatin immunoprecipitation-qPCR (ChIP-qPCR), electrophoretic mobility shift assay (EMSA), and tobacco transient expression analysis showed that ABI4 promotes FLC expression by directly binding to its promoter. Genetic analysis demonstrated that OE-ABI4::flc-3 could not alter the flc-3 phenotype. OE-FLC::abi4 showed a markedly delayed flowering phenotype, which mimicked OE-FLC::WT, and suggested that ABI4 acts upstream of FLC in the same genetic pathway. Taken together, these findings suggest that ABA inhibits the floral transition by activating FLC transcription through ABI4. PMID:26507894

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

    Science.gov (United States)

    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.

  20. Bone morphogenetic protein-2 is a negative regulator of hepatocyte proliferation downregulated in the regenerating liver

    Institute of Scientific and Technical Information of China (English)

    Cui-Ping Xu; Wen-Min Ji; Gijs R van den Brink; Maikel P Peppelenbosch

    2006-01-01

    AIM: To characterize the expression and dynamic changes of bone morphogenetic protein (BMP)-2 in hepatocytes in the regenerating liver in rats after partial hepatectomy (PH), and examine the effects of BMP-2 on proliferation of human Huh7 hepatoma cells.METHODS: Fifty-four adult male Wistar rats were randomly divided into three groups: A normal control (NC) group, a partial hepatectomized (PH) group and a sham operated (SO) group. To study the effect of liver regeneration on BMP-2 expression, rats were sacrificed before and at different time points after PH or the sham intervention (6, 12, 24 and 48 h). For each time point, six rats were used in parallel. Expression and distribution of BMP-2 protein were determined in regenerating liver tissue by Western blot analysis and immunohistochemistry. Effects of BMP-2 on cell proliferation of human Huh7 hepatoma cell line were assessed using an MTT assay.RESULTS: In the normal liver strong BMP-2 expression was observed around the central and portal veins. The expression of BMP-2 decreased rapidly as measured by both immunohistochemistry and Western blot analysis.This decrease was at a maximum of 3.22 fold after 12 h and returned to normal levels at 48 h after PH. No significant changes in BMP-2 immunoreactivity were observed in the SO group. BMP-2 inhibited serum induced Huh7 cell proliferation.CONCLUSION: BMP-2 is expressed in normal adult rat liver and negatively regulates hepatocyte proliferation.The observed down regulation of BMP-2 following partial hepatectomy suggests that such down regulation may be necessary for hepatocyte proliferation.

  1. Polypyrimidine Tract Binding Protein Functions as a Negative Regulator of Feline Calicivirus Translation

    Science.gov (United States)

    Karakasiliotis, Ioannis; Vashist, Surender; Bailey, Dalan; Abente, Eugenio J.; Green, Kim Y.; Roberts, Lisa O.; Sosnovtsev, Stanislav V.; Goodfellow, Ian G.

    2010-01-01

    Background Positive strand RNA viruses rely heavily on host cell RNA binding proteins for various aspects of their life cycle. Such proteins interact with sequences usually present at the 5′ or 3′ extremities of the viral RNA genome, to regulate viral translation and/or replication. We have previously reported that the well characterized host RNA binding protein polypyrimidine tract binding protein (PTB) interacts with the 5′end of the feline calicivirus (FCV) genomic and subgenomic RNAs, playing a role in the FCV life cycle. Principal Findings We have demonstrated that PTB interacts with at least two binding sites within the 5′end of the FCV genome. In vitro translation indicated that PTB may function as a negative regulator of FCV translation and this was subsequently confirmed as the translation of the viral subgenomic RNA in PTB siRNA treated cells was stimulated under conditions in which RNA replication could not occur. We also observed that PTB redistributes from the nucleus to the cytoplasm during FCV infection, partially localizing to viral replication complexes, suggesting that PTB binding may be involved in the switch from translation to replication. Reverse genetics studies demonstrated that synonymous mutations in the PTB binding sites result in a cell-type specific defect in FCV replication. Conclusions Our data indicates that PTB may function to negatively regulate FCV translation initiation. To reconcile this with efficient virus replication in cells, we propose a putative model for the function of PTB in the FCV life cycle. It is possible that during the early stages of infection, viral RNA is translated in the absence of PTB, however, as the levels of viral proteins increase, the nuclear-cytoplasmic shuttling of PTB is altered, increasing the cytoplasmic levels of PTB, inhibiting viral translation. Whether PTB acts directly to repress translation initiation or via the recruitment of other factors remains to be determined but this may

  2. Arabidopsis ETO1 specifically interacts with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases

    Directory of Open Access Journals (Sweden)

    Saito Koji

    2005-08-01

    Full Text Available Abstract Background In Arabidopsis, ETO1 (ETHYLENE-OVERPRODUCER1 is a negative regulator of ethylene evolution by interacting with AtACS5, an isoform of the rate-limiting enzyme, 1-aminocyclopropane-1-carboxylate synthases (ACC synthase or ACS, in ethylene biosynthetic pathway. ETO1 directly inhibits the enzymatic activity of AtACS5. In addition, a specific interaction between ETO1 and AtCUL3, a constituent of a new type of E3 ubiquitin ligase complex, suggests the molecular mechanism in promoting AtACS5 degradation by the proteasome-dependent pathway. Because orthologous sequences to ETO1 are found in many plant species including tomato, we transformed tomato with Arabidopsis ETO1 to evaluate its ability to suppress ethylene production in tomato fruits. Results Transgenic tomato lines that overexpress Arabidopsis ETO1 (ETO1-OE did not show a significant delay of fruit ripening. So, we performed yeast two-hybrid assays to investigate potential heterologous interaction between ETO1 and three isozymes of ACC synthases from tomato. In the yeast two-hybrid system, ETO1 interacts with LE-ACS3 as well as AtACS5 but not with LE-ACS2 or LE-ACS4, two major isozymes whose gene expression is induced markedly in ripening fruits. According to the classification of ACC synthases, which is based on the C-terminal amino acid sequences, both LE-ACS3 and AtACS5 are categorized as type 2 isozymes and possess a consensus C-terminal sequence. In contrast, LE-ACS2 and LE-ACS4 are type 1 and type 3 isozymes, respectively, both of which do not possess this specific C-terminal sequence. Yeast two-hybrid analysis using chimeric constructs between LE-ACS2 and LE-ACS3 revealed that the type-2-ACS-specific C-terminal tail is required for interaction with ETO1. When treated with auxin to induce LE-ACS3, seedlings of ETO1-OE produced less ethylene than the wild type, despite comparable expression of the LE-ACS3 gene in the wild type. Conclusion These results suggest that ETO1

  3. Perfectionism, emotion regulation and their relationship to negative affect in patients with social phobia

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    Systla Rukmini

    2014-01-01

    Full Text Available Context: Research on the perfectionism and emotion regulation strategies in anxiety disorders has gained increased attention. These have an important implication for formulation of therapies. Aims: We examined perfectionism, emotion regulation were examined in 30 patients with social phobia (SP and 30 community participants. Settings and Design: A cross-sectional design using a clinical and a community control sample was adopted in this exploratory study. Materials and Methods: Participants were assessed on The Mini-International Neuropsychiatric Interview, Frost′s-Multidimensional Perfectionism Scale, Ruminative Response Scale of the response style questionnaire, cognitive emotion regulation questionnaire, Social Interaction Anxiety Scale and the Beck′s Depression Inventory. Statistical Analysis: Data was analyzed using independents samples t-test and Pearson′s Product moment correlations and step-wise linear regression. Results: Individuals with SP had higher perfectionism (mean = 100.30, SD = ±17.73, t = 7.29, P < 0.001, rumination (mean = 61.47, SD = ±11.96, t = 6.71, P < 0.001 and lower levels of positive reappraisal (mean = 11.53, SD = ±3.85, t = 4.90, P < 0.001. Perfectionism was correlated with social anxiety (r = 0.44, P < 0.05 and rumination (r = 0.43, P < 0.05, but not with depression. Rumination was positively correlated with both social anxiety (r = 0.513, P < 0.01 and depression (r = 0.485, P < 0.01.Positive reappraisal was negatively correlated with depression (r = -0.396, P < 0.05 and anxiety (r = -0.335, P < 0.05. Acceptance was found to be significantly correlated only to the reflective pondering subscale of rumination. Parental criticism was a significant predictor of social anxiety (F = 11.11, P < 0.01 and brooding predicted depression (F = 10.49, P < 0.01. Conclusions: This study highlights the role of perfectionism as a maintaining factor in SP and the importance of adaptive forms of emotion regulation that need to

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

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    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}.

  5. Sli-1, a Negative Regulator of Let-23-Mediated Signaling in C. Elegans

    Science.gov (United States)

    Jongeward, G. D.; Clandinin, T. R.; Sternberg, P. W.

    1995-01-01

    By screening for suppressors of hypomorphic mutations of let-23, a receptor tyrosine kinase necessary for vulval induction in Caenorhabditis elegans, we recovered >/=12 mutations defining the sli-1 (suppressor of lineage defect) locus. sli-1 mutations suppress four of five phenotypes associated with hypomorphic alleles of let-23 but do not suppress let-23 null alleles. Thus, a sli-1 mutation does not bypass the requirement for functional let-23 but rather allows more potent LET-23-dependent signaling. Mutations at the sli-1 locus are otherwise silent with respect to vulval differentiation and cause only a low-penetrance abnormal head phenotype. Mutations at sli-1 also suppress the vulval defects but not other defects associated with mutations of sem-5, whose product likely interacts with LET-23 protein during vulval induction. Mutations at sli-1 suppress lin-2, lin-7 and lin-10 mutations but only partially suppress lin-3 and let-60 mutations and do not suppress a lin-45 mutation. The sli-1 locus displays dosage sensitivity: severe reduction of function alleles of sli-1 are semidominant suppressors; a duplication of the sli-1 (+) region enhances the vulvaless phenotype of hypomorphic mutations of let-23. We propose that sli-1 is a negative regulator that acts at or near the LET-23-mediated step of the vulval induction pathway. Our analysis suggests that let-23 can activate distinct signaling pathways in different tissues: one pathway is required for vulval induction; another pathway is involved in hermaphrodite fertilty and is not regulated by sli-1. PMID:7789760

  6. EphrinA5 suppresses colon cancer development by negatively regulating epidermal growth factor receptor stability.

    Science.gov (United States)

    Wang, Tong-Hong; Chang, Junn-Liang; Ho, Jar-Yi; Wu, Hsiao-Chun; Chen, Tse-Ching

    2012-01-01

    Colon cancer is one of the most common human cancers worldwide. Owing to its aggressiveness and lethality, it is necessary to determine the mechanisms regulating the carcinogenesis of colon cancer. EphrinA5 has been reported to act as a putative tumor suppressor in glioma; however, little is known concerning the role of this protein in the context of colon cancer. To elucidate the biological significance of ephrinA5 in colon cancer, we examined ephrinA5 and epidermal growth factor receptor (EGFR) expression profiles in both colon cancer and normal tissues, using immunohistochemistry on a 96-spot tissue microarray. Gain-of-function and loss-of-function experiments were performed on the human colon cancer cell lines SW480 and WiDr to determine the biological effects of ephrinA5 in relation to cell proliferation, survival, and migration. It was found that ephrinA5 mRNA and protein levels were significantly reduced in colon cancer as compared with normal colon tissue specimens. EphrinA5 expression was also negatively associated with tumor differentiation and clinical stage. In colon cancer cell line models, ephrinA5 exerted an inhibitory effect on EGFR by promoting c-Cbl-mediated EGFR ubiquitination and degradation. EphrinA5 did not affect the transcriptional regulation of EGFR mRNA expression in colon cancer cells. Expression of ephrinA5 suppressed colon cancer cell proliferation, migration, and chemotherapeutic resistance. In conclusion, ephrinA5 inhibited colon cancer progression by promoting c-Cbl-mediated EGFR degradation. Our findings identify a novel mechanism that could be utilized to improve the therapeutic efficiency of EGFR-targeting strategies.

  7. Cereblon negatively regulates TLR4 signaling through the attenuation of ubiquitination of TRAF6.

    Science.gov (United States)

    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.

  8. TRIM11 negatively regulates IFNβ production and antiviral activity by targeting TBK1.

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    Younglang Lee

    Full Text Available The innate immune response is a host defense mechanism against infection by viruses and bacteria. Type I interferons (IFNα/β play a crucial role in innate immunity. If not tightly regulated under normal conditions and during immune responses, IFN production can become aberrant, leading to inflammatory and autoimmune diseases. In this study, we identified TRIM11 (tripartite motif containing 11 as a novel negative regulator of IFNβ production. Ectopic expression of TRIM11 decreased IFNβ promoter activity induced by poly (I:C stimulation or overexpression of RIG-I (retinoic acid-inducible gene-I signaling cascade components RIG-IN (constitutively active form of RIG-I, MAVS (mitochondrial antiviral signaling protein, or TBK1 (TANK-binding kinase-1. Conversely, TRIM11 knockdown enhanced IFNβ promoter activity induced by these stimuli. Moreover, TRIM11 overexpression inhibited the phosphorylation and dimerization of IRF3 and expression of IFNβ mRNA. By contrast, TRIM11 knockdown increased the IRF3 phosphorylation and IFNβ mRNA expression. We also found that TRIM11 and TBK1, a key kinase that phosphorylates IRF3 in the RIG-I pathway, interacted with each other through CC and CC2 domain, respectively. This interaction was enhanced in the presence of the TBK1 adaptor proteins, NAP1 (NF-κB activating kinase-associated protein-1, SINTBAD (similar to NAP1 TBK1 adaptor or TANK (TRAF family member-associated NF-κB activator. Consistent with its inhibitory role in RIG-I-mediated IFNβ signaling, TRIM11 overexpression enhanced viral infectivity, whereas TRIM11 knockdown produced the opposite effect. Collectively, our results suggest that TRIM11 inhibits RIG-I-mediated IFNβ production by targeting the TBK1 signaling complex.

  9. Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

    Science.gov (United States)

    Liang, Jingjing; Sagum, Cari A.; Bedford, Mark T.; Sudol, Marius; Han, Ziying

    2017-01-01

    Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles. PMID:28076420

  10. PUF-8 negatively regulates RAS/MAPK signalling to promote differentiation of C. elegans germ cells.

    Science.gov (United States)

    Vaid, Samir; Ariz, Mohd; Chaturbedi, Amaresh; Kumar, Ganga Anil; Subramaniam, Kuppuswamy

    2013-04-01

    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.

  11. The negative regulation of red cell mass by neocytolysis: physiologic and pathophysiologic manifestations.

    Science.gov (United States)

    Rice, Lawrence; Alfrey, Clarence P

    2005-01-01

    We have uncovered a physiologic process which negatively regulates the red cell mass by selectively hemolyzing young circulating red blood cells. This allows fine control of the number of circulating red blood cells under steady-state conditions and relatively rapid adaptation to new environments. Neocytolysis is initiated by a fall in erythropoietin levels, so this hormone remains the major regulator of red cell mass both with anemia and with red cell excess. Physiologic situations in which there is increased neocytolysis include the emergence of newborns from the hypoxic uterine environment and the descent of polycythemic high-altitude dwellers to sea level. The process first became apparent while investigating the mechanism of the anemia that invariably occurs after spaceflight. Astronauts experience acute central plethora on entering microgravity resulting in erythropoietin suppression and neocytolysis, but the reduced blood volume and red cell mass become suddenly maladaptive on re-entry to earth's gravity. The pathologic erythropoietin deficiency of renal disease precipitates neocytolysis, which explains the prolongation of red cell survival consistently resulting from erythropoietin therapy and points to optimally efficient erythropoietin dosing schedules. Implications should extend to a number of other physiologic and pathologic situations including polycythemias, hemolytic anemias, 'blood-doping' by elite athletes, and oxygen therapy. It is likely that erythropoietin influences endothelial cells which in turn signal reticuloendothelial phagocytes to destroy or permit the survival of young red cells marked by surface molecules. Ongoing studies to identify the molecular targets and cytokine intermediaries should facilitate detection, dissection and eventual therapeutic manipulation of the process. Copyright (c) 2005 S. Karger AG, Basel.

  12. Infant negative reactivity defines the effects of parent-child synchrony on physiological and behavioral regulation of social stress.

    Science.gov (United States)

    Pratt, Maayan; Singer, Magi; Kanat-Maymon, Yaniv; Feldman, Ruth

    2015-11-01

    How infants shape their own development has puzzled developmentalists for decades. Recent models suggest that infant dispositions, particularly negative reactivity and regulation, affect outcome by determining the extent of parental effects. Here, we used a microanalytic experimental approach and proposed that infants with varying levels of negative reactivity will be differentially impacted by parent-infant synchrony in predicting physiological and behavioral regulation of increasing social stress during an experimental paradigm. One hundred and twenty-two mother-infant dyads (4-6 months) were observed in the face-to-face still face (SF) paradigm and randomly assigned to three experimental conditions: SF with touch, standard SF, and SF with arms' restraint. Mother-infant synchrony and infant negative reactivity were observed at baseline, and three mechanisms of behavior regulation were microcoded; distress, disengagement, and social regulation. Respiratory sinus arrhythmia baseline, reactivity, and recovery were quantified. Structural equation modeling provided support for our hypothesis. For physiological regulation, infants high in negative reactivity receiving high mother-infant synchrony showed greater vagal withdrawal, which in turn predicted comparable levels of vagal recovery to that of nonreactive infants. In behavioral regulation, only infants low in negative reactivity who received high synchrony were able to regulate stress by employing social engagement cues during the SF phase. Distress was reduced only among calm infants to highly synchronous mothers, and disengagement was lowest among highly reactive infants experiencing high mother-infant synchrony. Findings chart two pathways by which synchrony may bolster regulation in infants of high and low reactivity. Among low reactive infants, synchrony builds a social repertoire for handling interpersonal stress, whereas in highly reactive infants, it constructs a platform for repeated reparation of

  13. Negative regulation of parathyroid hormone-related protein expression by steroid hormones

    Energy Technology Data Exchange (ETDEWEB)

    Kajitani, Takashi; Tamamori-Adachi, Mimi [Department of Biochemistry, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605 (Japan); Okinaga, Hiroko [Department of Internal Medicine, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605 (Japan); Chikamori, Minoru; Iizuka, Masayoshi [Department of Biochemistry, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605 (Japan); Okazaki, Tomoki, E-mail: okbgeni@med.teikyo-u.ac.jp [Department of Biochemistry, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605 (Japan)

    2011-04-15

    Highlights: {yields} Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. {yields} Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. {yields} Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor {alpha}, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.

  14. Negative regulation of TLR-signaling pathways by activating transcription factor-3.

    Science.gov (United States)

    Whitmore, Mark M; Iparraguirre, Amaya; Kubelka, Lindsey; Weninger, Wolfgang; Hai, Tsonwin; Williams, Bryan R G

    2007-09-15

    Activating transcription factor-3 (ATF3) is rapidly induced by LPS in mouse macrophages and regulates TLR4 responses. We show that ATF3 is rapidly induced by various TLRs in mouse macrophages and plasmacytoid dendritic cells (DCs), as well as plasmacytoid and myeloid subsets of human DCs. In primary macrophages from mice with a targeted deletion of the atf3 gene (ATF3-knockout (KO)), TLR-stimulated levels of IL-12 and IL-6 were elevated relative to responses in wild-type macrophages. Similarly, targeted deletion of atf3 correlated with enhanced responsiveness of myeloid DCs to TLR activation as measured by IL-12 secretion. Ectopic expression of ATF3 antagonized TLR-stimulated IL-12p40 activation in a reporter assay. In vivo, CpG-oligodeoxynucleotide, a TLR9 agonist, given i.p. to ATF3-KO mice resulted in enhanced cytokine production from splenocytes. Furthermore, while ATF3-KO mice challenged with a sublethal dose of PR8 influenza virus were delayed in body weight recovery in comparison to wild type, the ATF3-KO mice showed higher titers of serum neutralizing Ab against PR8 5 mo postinfection. Thus, ATF3 behaves as a negative regulatory transcription factor in TLR pathways and, accordingly, deficiency in atf3 alters responses to immunological challenges in vivo. ATF3 dysregulation merits further exploration in diseases such as type I diabetes and cancer, where altered innate immunity has been implicated in their pathogenesis.

  15. The lin-15 locus encodes two negative regulators of Caenorhabditis elegans vulval development.

    Science.gov (United States)

    Huang, L S; Tzou, P; Sternberg, P W

    1994-01-01

    During Caenorhabditis elegans vulval development, an inductive signal from the anchor cell stimulates three of the six vulval precursor cells (VPCs) to adopt vulval rather than nonvulval epidermal fates. Genes necessary for this induction include the lin-3 growth factor, the let-23 receptor tyrosine kinase, and let-60 ras. lin-15 is a negative regulator of this inductive pathway. In lin-15 mutant animals, all six VPCs adopt vulval fates, even in the absence of inductive signal. Previous genetic studies suggested that lin-15 is a complex locus with two independently mutable activities, A and B. We have cloned the lin-15 locus by germline transformation and find that it encodes two nonoverlapping transcripts that are transcribed in the same direction. The downstream transcript encodes the lin-15A function; the upstream transcript encodes the lin-15B function. The predicted lin-15A and lin-15B proteins are novel and hydrophilic. We have identified a molecular null allele of lin-15 and have used it to analyze the role of lin-15 in the signaling pathway. We find that lin-15 acts upstream of let-23 and in parallel to the inductive signal. Images PMID:8054684

  16. Cyclooxygenase-2 regulates TGFβ-induced cancer stemness in triple-negative breast cancer

    Science.gov (United States)

    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

  17. Mitochondrial transcription termination factor 2 binds to entire mitochondrial DNA and negatively regulates mitochondrial gene expression

    Institute of Scientific and Technical Information of China (English)

    Weiwei Huang; Min Yu; Yang Jiao; Jie Ma; Mingxing Ma; Zehua Wang; Hong Wu; Deyong Tan

    2011-01-01

    Mitochondrial transcription termination factor 2 (mTERF2) is a mitochondriai matrix protein that binds to the mitochondriai DNA.Previous studies have shown that overexpression of mTERF2 can inhibit cell proliferation, but the mechanism has not been well defined so far.This study aimed to present the binding pattern of mTERF2 to the mitochondrial DNA (mtDNA) in vivo, and investigated the biological function of mTERF2 on the replication of mtDNA, mRNA transcription, and protein translation.The mTERF2 binding to entire mtDNA was identified via the chromatin immunoprecipitation analysis.The mtDNA replication efficiency and expression levels of mitochondria genes were significantly inhibited when the mTERF2 was overexpressed in HeLa cells.The inhibition level of mtDNA content was the same with the decreased levels of mRNA and mitochondrial protein expression.Overall, the mTERF2 might be a cell growth inhibitor based on its negative effect on mtDNA replication, which eventually own-regulated all of the oxidative phosphorylation components in the mitochondria that were essential for the cell's energy metabolism.

  18. MEK-dependent IL-8 induction regulates the invasiveness of triple-negative breast cancer cells.

    Science.gov (United States)

    Kim, Sangmin; Lee, Jeongmin; Jeon, Myeongjin; Lee, Jeong Eon; Nam, Seok Jin

    2016-04-01

    Interleukin-8 (IL-8) serves as a prognostic marker for breast cancer, and its expression level correlates with metastatic breast cancer and poor prognosis. Here, we investigated the levels of IL-8 expression in a variety of breast cancer cells and the regulatory mechanism of IL-8 in triple-negative breast cancer (TNBC) cells. Our results showed that IL-8 expression correlated positively with overall survival in basal-type breast cancer patients. The levels of IL-8 mRNA expression and protein secretion were significantly increased in TNBC cells compared with non-TNBC cells. In addition, the invasiveness of the TNBC cells was dramatically increased by IL-8 treatment and then augmented invasion-related proteins such as matrix metalloproteinase (MMP)-2 or MMP-9. We observed that elevated IL-8 mRNA expression and protein secretion were suppressed by a specific MEK1/2 inhibitor, UO126. In contrast, the overexpression of constitutively active MEK significantly increased the level of IL-8 mRNA expression in BT474 non-TNBC cells. Finally, we investigated the effect of UO126 on the tumorigenecity of TNBC cells. Our results showed that anchorage-independent growth, cell invasion, and cell migration were also decreased by UO126 in TNBC cells. As such, we demonstrated that IL-8 expression is regulated through MEK/ERK-dependent pathways in TNBC cells. A diversity of MEK blockers, including UO126, may be promising for treating TNBC patients.

  19. SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling.

    Science.gov (United States)

    Honorat, Jean-François; Ragab, Ashraf; Lamant, Laurence; Delsol, Georges; Ragab-Thomas, Jeannie

    2006-05-15

    Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

  20. TIPE2 negatively regulates inflammation by switching arginine metabolism from nitric oxide synthase to arginase.

    Directory of Open Access Journals (Sweden)

    Yunwei Lou

    Full Text Available TIPE2, the tumor necrosis factor (TNF-alpha-induced protein 8-like 2 (TNFAIP8L2, plays an essential role in maintaining immune homeostasis. It is highly expressed in macrophages and negatively regulates inflammation through inhibiting Toll-like receptor signaling. In this paper, we utilized RAW264.7 cells stably transfected with a TIPE2 expression plasmid, as well as TIPE2-deficient macrophages to study the roles of TIPE2 in LPS-induced nitric oxide (NO and urea production. The results showed that TIPE2-deficiency significantly upregulated the levels of iNOS expression and NO production in LPS-stimulated macrophages, but decreased mRNA levels of arginase I and urea production. However, TIPE2 overexpression in macrophages was capable of downregulating protein levels of LPS-induced iNOS and NO, but generated greater levels of arginase I and urea production. Furthermore, TIPE2-/- mice had higher iNOS protein levels in lung and liver and higher plasma NO concentrations, but lower levels of liver arginase I compared to LPS-treated WT controls. Interestingly, significant increases in IκB degradation and phosphorylation of JNK, p38, and IκB were observed in TIPE2-deficient macrophages following LPS challenge. These results strongly suggest that TIPE2 plays an important role in shifting L-arginase metabolism from production of NO to urea, during host inflammatory response.

  1. TRIF is a critical negative regulator of TLR agonist mediated activation of dendritic cells in vivo.

    Directory of Open Access Journals (Sweden)

    Sergey S Seregin

    Full Text Available 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.

  2. PRKCI negatively regulates autophagy via PIK3CA/AKT–MTOR signaling

    Energy Technology Data Exchange (ETDEWEB)

    Qu, Liujing; Li, Ge; Xia, Dan; Hongdu, Beiqi; Xu, Chentong; Lin, Xin [Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Sciences Center, Beijing (China); Peking University Center for Human Disease Genomics, Peking University, Beijing (China); Chen, Yingyu, E-mail: yingyu_chen@bjmu.edu.cn [Key Laboratory of Medical Immunology, Ministry of Health, Peking University Health Sciences Center, Beijing (China); Peking University Center for Human Disease Genomics, Peking University, Beijing (China)

    2016-02-05

    The atypical protein kinase C isoform PRKC iota (PRKCI) plays a key role in cell proliferation, differentiation, and carcinogenesis, and it has been shown to be a human oncogene. Here, we show that PRKCI overexpression in U2OS cells impaired functional autophagy in normal or cell stress conditions, as characterized by decreased levels of light chain 3B-II protein (LC3B-II) and weakened degradation of endogenous and exogenous autophagic substrates. Conversely, PRKCI knockdown by small interference RNA resulted in opposite effects. Additionally, we identified two novel PRKCI mutants, PRKCI{sup L485M} and PRKCI{sup P560R}, which induced autophagy and exhibited dominant negative effects. Further studies indicated that PRKCI knockdown–mediated autophagy was associated with the inactivation of phosphatidylinositol 3-kinase alpha/AKT–mammalian target of rapamycin (PIK3CA/AKT–MTOR) signaling. These data underscore the importance of PRKCI in the regulation of autophagy. Moreover, the finding may be useful in treating PRKCI-overexpressing carcinomas that are characterized by increased levels of autophagy. - Highlights: • The atypical protein kinase C iota isoform (PRKCI) is a human oncogene. • PRKCI overexpression impairs functional autophagy in U2OS cells. • It reduces LC3B-II levels and weakens SQSTM1 and polyQ80 aggregate degradation. • PRKCI knockdown has the opposite effect. • The effect of PRKCI knockdown is related to PIK3CA/AKT–MTOR signaling inactivation.

  3. Negative regulation of NaF-induced apoptosis by Bad-CAII complex.

    Science.gov (United States)

    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.

  4. Positive and negative signaling through SLAM receptors regulate synapse organization and thresholds of cytolysis.

    Science.gov (United States)

    Zhao, Fang; Cannons, Jennifer L; Dutta, Mala; Griffiths, Gillian M; Schwartzberg, Pamela L

    2012-06-29

    X-linked lymphoproliferative syndrome, characterized by fatal responses to Epstein-Barr virus infection, is caused by mutations affecting the adaptor SAP, which links SLAM family receptors to downstream signaling. Although cytotoxic defects in SAP-deficient T cells are documented, the mechanism remains unclear. We show that SAP-deficient murine CD8(+) T cells exhibited normal cytotoxicity against fibrosarcoma targets, yet had impaired adhesion to and killing of B cell and low-avidity T cell targets. SAP-deficient cytotoxic lymphocytes showed specific defects in immunological synapse organization with these targets, resulting in inefficient actin clearance. In the absence of SAP, signaling through the SLAM family members Ly108 and 2B4 resulted in increased recruitment of the SHP-1 phosphatase, associated with altered SHP-1 localization and decreased activation of Src kinases at the synapse. Hence, SAP and SLAM receptors regulate positive and negative signals required for organizing the T cell:B cell synapse and setting thresholds for cytotoxicity against distinct cellular targets.

  5. Negative density dependence regulates two tree species at later life stage in a temperate forest.

    Directory of Open Access Journals (Sweden)

    Tiefeng Piao

    Full Text Available Numerous studies have demonstrated that tree survival is influenced by negative density dependence (NDD and differences among species in shade tolerance could enhance coexistence via resource partitioning, but it is still unclear how NDD affects tree species with different shade-tolerance guilds at later life stages. In this study, we analyzed the spatial patterns for trees with dbh (diameter at breast height ≥2 cm using the pair-correlation g(r function to test for NDD in a temperate forest in South Korea after removing the effects of habitat heterogeneity. The analyses were implemented for the most abundant shade-tolerant (Chamaecyparis obtusa and shade-intolerant (Quercus serrata species. We found NDD existed for both species at later life stages. We also found Quercus serrata experienced greater NDD compared with Chamaecyparis obtusa. This study indicates that NDD regulates the two abundant tree species at later life stages and it is important to consider variation in species' shade tolerance in NDD study.

  6. Negative density dependence regulates two tree species at later life stage in a temperate forest.

    Science.gov (United States)

    Piao, Tiefeng; Chun, Jung Hwa; Yang, Hee Moon; Cheon, Kwangil

    2014-01-01

    Numerous studies have demonstrated that tree survival is influenced by negative density dependence (NDD) and differences among species in shade tolerance could enhance coexistence via resource partitioning, but it is still unclear how NDD affects tree species with different shade-tolerance guilds at later life stages. In this study, we analyzed the spatial patterns for trees with dbh (diameter at breast height) ≥2 cm using the pair-correlation g(r) function to test for NDD in a temperate forest in South Korea after removing the effects of habitat heterogeneity. The analyses were implemented for the most abundant shade-tolerant (Chamaecyparis obtusa) and shade-intolerant (Quercus serrata) species. We found NDD existed for both species at later life stages. We also found Quercus serrata experienced greater NDD compared with Chamaecyparis obtusa. This study indicates that NDD regulates the two abundant tree species at later life stages and it is important to consider variation in species' shade tolerance in NDD study.

  7. Novel function of perforin in negatively regulating CD4+T cell activation by affecting calcium signaling

    Institute of Scientific and Technical Information of China (English)

    Enguang Bi; Kairui Mao; Jia Zou; Yuhan Zheng; Bing Sun; Chunjian Huang; Yu Hu; Xiaodong Wu; Weiwen Deng; Guomei Lin; Zhiduo Liu; Lin Tian; Shuhui Sun

    2009-01-01

    Perforin is a pore-forming protein engaged mainly in mediating target T cell death and is employed by cytotoxic Tlymphocytes (CTLs) and natural killer cells. However, whether it also plays a role in conventional CD4+ T cell func-tion remains unclear. Here we report that in perforin-deficient (PKO) mice, CD4+ T cells are hyperproliferative in response to T cell receptor (TCR) stimulation. This feature of hyperproliferation is accompanied by the enhancement both in cell division and in IL-2 secretion. It seems that the perforin deficiency does not influence T cell development in thymus spleen and lymph node. In vivo, perforin deficiency results in increased antigen-specific T cell prolifera-tion and antibody production. Furthermore, PKO mice are more susceptible to experimental autoimmune uveitis. To address the molecular mechanism, we found that after TCR stimulation, CD44 T cells from PKO mice display an increased intracellular calcium flux and subsequently enhance activation of transcription factor NFATI. Our results indicate that perforin plays a negative role in regulating CD4+ T cell activation and immune response by affecting TCR-dependent Ca2+ signaling.

  8. Identification and functional characterization of Bet protein as a negative regulator of BFV3026 replication.

    Science.gov (United States)

    Bing, Tiejun; Wu, Kai; Cui, Xiaoxu; Shao, Peng; Zhang, Qicheng; Bai, Xiaobo; Tan, Juan; Qiao, Wentao

    2014-06-01

    Foamy virus (FV) establishes persistent infection in the host without causing apparent disease. Besides the transactivator Tas protein, another auxiliary protein--Bet--has been reported in prototype foamy virus, equine foamy virus, and feline foamy virus. Here, we found the putative bbet gene in clone C74 from a cDNA library of bovine foamy virus strain 3026 (BFV3026) by comparison of gene localization, composition, and splicing features with other known bet genes. Subsequently, BBet protein was detected in BFV3026-infected cells by Western blot and immunofluorescence analyses. Analysis of the BBet mutant infectious clone (pBS-BFVdelBBet) revealed that BBet could inhibit BFV3026 replication. Consistent with this result, overexpression of BBet in Cf2Th cells reduced BFV replication by approximately threefold. Furthermore, virus replication levels similarly were reduced by approximately threefold in pBS-BFV-transfected and BFV3026-infected Cf2Th cells stably expressing BBet compared with control cells. After three passages, BFV3026 replicated more slowly in BBet-expressing cells. This study implicates BBet as a negative regulator of BFV replication and provides a resource for future studies on the function of this protein in the virus lifecycle.

  9. PERK–KIPK–KCBP signalling negatively regulates root growth in Arabidopsis thaliana

    Science.gov (United States)

    Humphrey, Tania V.; Haasen, Katrina E.; Aldea-Brydges, May Grace; Sun, He; Zayed, Yara; Indriolo, Emily; Goring, Daphne R.

    2015-01-01

    The Arabidopsis proline-rich, extensin-like receptor-like kinases (PERKs) are a small group of receptor-like kinases that are thought to act as sensors at the cell wall through their predicted proline-rich extracellular domains. In this study, we focused on the characterization of a subclade of three Arabidopsis predicted PERK genes, PERK8, -9, and -10, for which no functions were known. Yeast two-hybrid interaction studies were conducted with the PERK8,- 9, and -10 cytosolic kinase domains, and two members of the Arabidopsis AGC VIII kinase family were identified as interacting proteins: AGC1-9 and the closely related kinesin-like calmodulin-binding protein (KCBP)-interacting protein kinase (KIPK). As KIPK has been identified previously as an interactor of KCBP, these interactions were also examined further and confirmed in this study. Finally, T-DNA mutants for each gene were screened for altered phenotypes under different conditions, and from these screens, a role for the PERK, KIPK, and KCBP genes in negatively regulating root growth was uncovered. PMID:25262228

  10. How is emotional awareness related to emotion regulation strategies and self-reported negative affect in the general population?

    Directory of Open Access Journals (Sweden)

    Claudia Subic-Wrana

    Full Text Available OBJECTIVE: The Levels of Emotional Awareness Scale (LEAS as a performance task discriminates between implicit or subconscious and explicit or conscious levels of emotional awareness. An impaired awareness of one's feeling states may influence emotion regulation strategies and self-reports of negative emotions. To determine this influence, we applied the LEAS and self-report measures for emotion regulation strategies and negative affect in a representative sample of the German general population. SAMPLE AND METHODS: A short version of the LEAS, the Hospital Anxiety and Depression Scale (HADS and the Emotion Regulation Questionnaire (ERQ, assessing reappraisal and suppression as emotion regulation strategies, were presented to N = 2524 participants of a representative German community study. The questionnaire data were analyzed with regard to the level of emotional awareness. RESULTS: LEAS scores were independent from depression, but related to self-reported anxiety. Although of small or medium effect size, different correlational patters between emotion regulation strategies and negative affectivity were related to implict and explict levels of emotional awareness. In participants with implicit emotional awareness, suppression was related to higher anxiety and depression, whereas in participants with explicit emotional awareness, in addition to a positive relationship of suppression and depression, we found a negative relationship of reappraisal to depression. These findings were independent of age. In women high use of suppression and little use of reappraisal were more strongly related to negative affect than in men. DISCUSSION: Our first findings suggest that conscious awareness of emotions may be a precondition for the use of reappraisal as an adaptive emotion regulation strategy. They encourage further research in the relation between subconsious and conscious emotional awareness and the prefarance of adaptive or maladaptive emotion

  11. How is emotional awareness related to emotion regulation strategies and self-reported negative affect in the general population?

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    Subic-Wrana, Claudia; Beutel, Manfred E; Brähler, Elmar; Stöbel-Richter, Yve; Knebel, Achim; Lane, Richard D; Wiltink, Jörg

    2014-01-01

    The Levels of Emotional Awareness Scale (LEAS) as a performance task discriminates between implicit or subconscious and explicit or conscious levels of emotional awareness. An impaired awareness of one's feeling states may influence emotion regulation strategies and self-reports of negative emotions. To determine this influence, we applied the LEAS and self-report measures for emotion regulation strategies and negative affect in a representative sample of the German general population. A short version of the LEAS, the Hospital Anxiety and Depression Scale (HADS) and the Emotion Regulation Questionnaire (ERQ), assessing reappraisal and suppression as emotion regulation strategies, were presented to N = 2524 participants of a representative German community study. The questionnaire data were analyzed with regard to the level of emotional awareness. LEAS scores were independent from depression, but related to self-reported anxiety. Although of small or medium effect size, different correlational patters between emotion regulation strategies and negative affectivity were related to implict and explict levels of emotional awareness. In participants with implicit emotional awareness, suppression was related to higher anxiety and depression, whereas in participants with explicit emotional awareness, in addition to a positive relationship of suppression and depression, we found a negative relationship of reappraisal to depression. These findings were independent of age. In women high use of suppression and little use of reappraisal were more strongly related to negative affect than in men. Our first findings suggest that conscious awareness of emotions may be a precondition for the use of reappraisal as an adaptive emotion regulation strategy. They encourage further research in the relation between subconsious and conscious emotional awareness and the prefarance of adaptive or maladaptive emotion regulation strategies The correlational trends found in a representative

  12. Mel-18 negatively regulates stem cell-like properties through downregulation of miR-21 in gastric cancer.

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    Wang, Xiao-Feng; Zhang, Xiao-Wei; Hua, Rui-Xi; Du, Yi-Qun; Huang, Ming-Zhu; Liu, Yong; Cheng, Yu Fang; Guo, Wei-Jian

    2016-09-27

    Mel-18, a polycomb group protein, has been reported to act as a tumor suppressor and be down-regulated in several human cancers including gastric cancer. It was also found that Mel-18 negatively regulates self-renewal of hematopoietic stem cells and breast cancer stem cells (CSCs). This study aimed to clarify its role in gastric CSCs and explore the mechanisms. We found that low-expression of Mel-18 was correlated with poor prognosis and negatively correlated with overexpression of stem cell markers Oct4, Sox2, and Gli1 in 101 gastric cancer tissues. Mel-18 was down-regulated in cultured spheroid cells, which possess CSCs, and overexpression of Mel-18 inhibits cells sphere-forming ability and tumor growth in vivo. Besides, Mel-18 was lower-expressed in ovary metastatic lesions compared with that in primary lesions of gastric cancer, and Mel-18 overexpression inhibited the migration ability of gastric cancer cells. Interestingly, overexpression of Mel-18 resulted in down-regulation of miR-21 in gastric cancer cells and the expression of Mel-18 was negatively correlated with the expression of miR-21 in gastric cancer tissues. Furthermore, miR-21 overexpression partially restored sphere-forming ability, migration potential and chemo-resistance in Mel-18 overexpressing gastric cancer cells. These results suggests Mel-18 negatively regulates stem cell-like properties through downregulation of miR-21 in gastric cancer cells.

  13. Relationships between negative affectivity, emotion regulation, anxiety, and depressive symptoms in adolescents as examined through structural equation modeling.

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    Tortella-Feliu, Miquel; Balle, Maria; Sesé, Albert

    2010-10-01

    The relationship between negative affectivity (NA) and emotion regulation (ER) in determining anxiety and depressive symptomatology was examined in a large (n=1441) sample of adolescents (12-17 years old). Two models, diverging only as to inclusion or exclusion of a path from NA to negative ER, were analyzed through structural equation modeling; the goal was to explore the mediational or non-mediational role of ER in determining anxiety symptoms. The models yielded similar adequate fit to data, indicating that both NA and negative ER contribute to anxiety symptoms which, in turn, significantly determine depressive symptomatology. The mediational model better captures the relationships revealed in the data, with NA determining negative ER to a great extent. Additionally, most individuals scoring highly in NA also tend to score highly in negative ER, indicating that adolescents with heightened NA are prone to a dysfunctional style of ER.

  14. miR-103 regulates triple negative breast cancer cells migration and invasion through targeting olfactomedin 4.

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    Xiong, Bin; Lei, Xuefeng; Zhang, Lei; Fu, Jia

    2017-03-18

    Our previous study showed olfactomedin 4 (OLFM4) suppressed triple-negative breast cancer cells migration, invasion and metastasis-associated protein MMP 9 expression. OLFM4 was identified as a potential target of miR-103 according to microRNA target databases and published studies. The aim of this study is to validate the relationship between miR-103 and OLFM4, and explore the function and clinical significance of miR-103 in triple-negative breast cancer patients. In our results, miR-103 negatively regulated OLFM4 expression by directly targeting its 3'-UTR. OLFM4 was a functional target of miR-103 to regulate triple-negative breast cancer cells migration, invasion and MMP 9 expression. Moreover, miR-103 overexpression was observed in triple-negative breast cancer tissues and cell lines, and associated with lymph node metastasis, distant metastasis and clinical stage. Univariate and multivariate analyses suggested that miR-103 overexpression was a poor independent prognostic factor for triple-negative breast cancer patients. In conclusion, miR-103 acts as an oncogene miRNA to promote triple-negative breast cancer cells migration and invasion through targeting OLFM4.

  15. Efflux Transporters Regulate Arsenite Induced Genotoxicity in Double Negative and Double Positive T Cells.

    Science.gov (United States)

    Xu, Huan; Medina, Sebastian; Lauer, Fredine T; Douillet, Christelle; Jian Liu, Ke; Hudson, Laurie G; Stýblo, Miroslav; Aleksunes, Lauren M; Burchiel, Scott W

    2017-04-29

    Arsenite (As+3) exposure is known to cause immunotoxicity in human and animal models. Our previous studies demonstrated that As+3 at 50 to 500 nM concentrations induced both genotoxicity and non-genotoxicity in mouse thymus cells. Developing T cells at CD4-CD8- double negative (DN) stage, the first stage after early T cells are transported from bone marrow to thymus, were found to be more sensitive to As+3 toxicity than the T cells at CD4+CD8+ double positive (DP) stage in vitro. Induction of Mdr1 (Abcb1) and Mrp1 (Abcc1), two multidrug resistance transporters and exporters of As+3, was associated with the reversal of As+3-induced double strand breaks and DNA damage. In order to confirm that the thymus cell populations have different sensitivity to As+3in vivo, male C57BL/6J mice were exposed to 0, 100 and 500 ppb As+3 in drinking water for 30 d. A significant decrease in DN cell percentage was observed with exposure to 500 ppb As+3. Low to moderate concentrations of As+3 were shown to induce higher genotoxicity in sorted DN cells than DP cells in vitro. Calcein AM uptake and Mdr1/Mrp1 mRNA quantification results revealed that DN cells not only had limited As+3 exporter activity, but also lacked the ability to activate these exporters with As+3 treatments, resulting in a higher accumulation of intracellular As+3. Knockdown study of As+3 exporters in the DN thymic cell line, D1 using siRNA, demonstrated that Mdr1 and Mrp1 regulate intracellular As+3 accumulation and genotoxicity. Taken together, the results indicate that transporter regulation is an important mechanism for differential genotoxicity induced by As+3 in thymocytes at different developmental stages. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  16. The pif1 helicase, a negative regulator of telomerase, acts preferentially at long telomeres.

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    Jane A Phillips

    2015-04-01

    Full Text Available Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB. Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX, which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80 -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.

  17. A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Iimori, Makoto; Ozaki, Kanako [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Chikashige, Yuji [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Habu, Toshiyuki [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan); Hiraoka, Yasushi [Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, 651-2492 (Japan); Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, 565-0871 (Japan); Maki, Takahisa; Hayashi, Ikuko [Graduate School of Nanobioscience, Yokohama City University, Tsurumi, Yokohama, 230-0045 (Japan); Obuse, Chikashi [Graduate School of Life Science, Hokkaido University, Sapporo 001-0021 (Japan); Matsumoto, Tomohiro, E-mail: tmatsumo@house.rbc.kyoto-u.ac.jp [Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwake cho, Sakyo ku, Kyoto, 606-8502 (Japan); Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo ku, Kyoto, 606-8501 (Japan)

    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.

  18. BP1, an Isoform of DLX4 Homeoprotein, Negatively Regulates BRCA1 in Sporadic Breast Cancer

    Directory of Open Access Journals (Sweden)

    Brian J. Kluk, Yebo Fu, Trina A. Formolo, Lei Zhang, Anne K. Hindle, Yan-gao Man, Robert S. Siegel, Patricia E. Berg, Chuxia Deng, Timothy A. McCaffrey, Sidney W. Fu

    2010-01-01

    Full Text Available Introduction: Several lines of evidence point to an important role for BP1, an isoform of DLX4 homeobox gene, in breast carcinogenesis and progression. BRCA1 is a well-known player in the etiology of breast cancer. While familial breast cancer is often marked by BRCA1 mutation and subsequent loss of heterozygosity, sporadic breast cancers exhibit reduced expression of wild type BRCA1, and loss of BRCA1 expression may result in tumor development and progression.Methods: The Cister algorithm and Genomatix program were used to identify potential BP1 binding sites in BRCA1 gene. Real-time PCR, Western blot and immunohistochemistry analysis were performed to verify the expression of BRCA1 and BP1 in cell lines and breast cancer tissues. Double-stranded siRNA transfection was carried out for silencing BP1 expression. ChIP and EMSA were used to confirm that BP1 specifically binds to BRCA1.Results: A putative BP1 binding site was identified in the first intron of BRCA1, which was confirmed by chromatin immunoprecipiation and electrophoresis mobility shift assay. BP1 and BRCA1 expression were inversely correlated in breast cancer cell lines and tissues, suggesting that BP1 may suppress BRCA1 transcription through consensus sequence binding.Conclusions: BP1 homeoprotein represses BRCA1 expression through direct binding to its first intron, which is consistent with a previous study which identified a novel transcriptional repressor element located more than 500 base pairs into the first intron of BRCA1, suggesting that the first intron plays an important role in the negative regulation of BRCA1. Although further functional studies are necessary to confirm its repressor activity towards BRCA1, the elucidation of the role of BP1 in breast tumorigenesis holds great promise in establishing BP1 as a novel target for drug therapy.

  19. RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA

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    Howe Grant A

    2012-02-01

    Full Text Available Abstract Recent studies have suggested a role for the small GTPase RhoB in the control of processes required for angiogenesis. However, the mechanisms whereby RhoB exerts control over these processes are not well understood. Given the role of vascular endothelial growth factor (VEGF in pathological angiogenesis, we were interested in examining whether RhoB contributed to VEGF-induced angiogenic processes. To assess this, RhoB was specifically depleted in human umbilical vein endothelial cells (HUVEC, using siRNA-targeted strategies. The effects of RhoB depletion on VEGF-induced angiogenic activities were assessed using a variety of standard in vitro angiogenesis assays to assess endothelial cell viability, migration and capillary morphogenesis. Effects of RhoB depletion on signaling from other Rho family member proteins was also assessed using specific activity assays for RhoA and RhoC. We observed that although RhoB appeared dispensable for HUVEC viability, RhoB was required for endothelial cell migration, sprouting, and capillary morphogenesis. We also observed that siRNA-mediated depletion of RhoB in HUVEC resulted in increased RhoA activation in response to VEGF stimulation. This increased RhoA activation contributed to the cellular morphogenesis defects observed in RhoB-depleted cells, as inhibition of RhoA activity using C3 transferase, or inhibition of the activity of the downstream RhoA effectors Rho-dependent kinases I and II (ROCK I and II led to a partial restoration of capillary morphogenesis in the absence of RhoB. Thus our data indicate that RhoB plays a significant role in VEGF-induced endothelial cell morphogenesis in part by negatively regulating the activity of RhoA and the RhoA/ROCK pathway.

  20. Negative regulation of active zone assembly by a newly identified SR protein kinase.

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    Ervin L Johnson

    2009-09-01

    Full Text Available Presynaptic, electron-dense, cytoplasmic protrusions such as the T-bar (Drosophila or ribbon (vertebrates are believed to facilitate vesicle movement to the active zone (AZ of synapses throughout the nervous system. The molecular composition of these structures including the T-bar and ribbon are largely unknown, as are the mechanisms that specify their synapse-specific assembly and distribution. In a large-scale, forward genetic screen, we have identified a mutation termed air traffic controller (atc that causes T-bar-like protein aggregates to form abnormally in motoneuron axons. This mutation disrupts a gene that encodes for a serine-arginine protein kinase (SRPK79D. This mutant phenotype is specific to SRPK79D and is not secondary to impaired kinesin-dependent axonal transport. The srpk79D gene is neuronally expressed, and transgenic rescue experiments are consistent with SRPK79D kinase activity being necessary in neurons. The SRPK79D protein colocalizes with the T-bar-associated protein Bruchpilot (Brp in both the axon and synapse. We propose that SRPK79D is a novel T-bar-associated protein kinase that represses T-bar assembly in peripheral axons, and that SRPK79D-dependent repression must be relieved to facilitate site-specific AZ assembly. Consistent with this model, overexpression of SRPK79D disrupts AZ-specific Brp organization and significantly impairs presynaptic neurotransmitter release. These data identify a novel AZ-associated protein kinase and reveal a new mechanism of negative regulation involved in AZ assembly. This mechanism could contribute to the speed and specificity with which AZs are assembled throughout the nervous system.

  1. Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin

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    Hadas Smadar

    2012-07-01

    Full Text Available Abstract Background Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3 is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin remodeling (i.e., disassembly and reassembly by shifting between active unphosphorylated and inactive phosphorylated states. Results Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia, which, as we also revealed, are instrumental in myelin phagocytosis. Conclusions CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive

  2. Protein tyrosine phosphatase receptor type z negatively regulates oligodendrocyte differentiation and myelination.

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    Kazuya Kuboyama

    Full Text Available BACKGROUND: Fyn tyrosine kinase-mediated down-regulation of Rho activity through activation of p190RhoGAP is crucial for oligodendrocyte differentiation and myelination. Therefore, the loss of function of its counterpart protein tyrosine phosphatase (PTP may enhance myelination during development and remyelination in demyelinating diseases. To test this hypothesis, we investigated whether Ptprz, a receptor-like PTP (RPTP expressed abuntantly in oligodendrocyte lineage cells, is involved in this process, because we recently revealed that p190RhoGAP is a physiological substrate for Ptprz. METHODOLOGY/PRINCIPAL FINDINGS: We found an early onset of the expression of myelin basic protein (MBP, a major protein of the myelin sheath, and early initiation of myelination in vivo during development of the Ptprz-deficient mouse, as compared with the wild-type. In addition, oligodendrocytes appeared earlier in primary cultures from Ptprz-deficient mice than wild-type mice. Furthermore, adult Ptprz-deficient mice were less susceptible to experimental autoimmune encephalomyelitis (EAE induced by active immunization with myelin/oligodendrocyte glycoprotein (MOG peptide than were wild-type mice. After EAE was induced, the tyrosine phosphorylation of p190RhoGAP increased significantly, and the EAE-induced loss of MBP was markedly suppressed in the white matter of the spinal cord in Ptprz-deficient mice. Here, the number of T-cells and macrophages/microglia infiltrating into the spinal cord did not differ between the two genotypes after MOG immunization. All these findings strongly support the validity of our hypothesis. CONCLUSIONS/SIGNIFICANCE: Ptprz plays a negative role in oligodendrocyte differentiation in early central nervous system (CNS development and remyelination in demyelinating CNS diseases, through the dephosphorylation of substrates such as p190RhoGAP.

  3. Tctex1d2 Is a Negative Regulator of GLUT4 Translocation and Glucose Uptake.

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    Shimoda, Yoko; Okada, Shuichi; Yamada, Eijiro; Pessin, Jeffrey E; Yamada, Masanobu

    2015-10-01

    Tctex1d2 (Tctex1 domain containing 2) is an open reading frame that encodes for a functionally unknown protein that contains a Tctex1 domain found in dynein light chain family members. Examination of gene expression during adipogenesis demonstrated a marked increase in Tctex1d2 protein expression that was essentially undetectable in preadipocytes and markedly induced during 3T3-L1 adipocyte differentiation. Tctex1d2 overexpression significantly inhibited insulin-stimulated glucose transporter 4 (GLUT4) translocation and 2-deoxyglucose uptake. In contrast, Tctex1d2 knockdown significantly increased insulin-stimulated GLUT4 translocation and 2-deoxyglucose uptake. However, acute insulin stimulation (up to 30 min) in 3T3-L1 adipocytes with overexpression or knockdown of Tctex1d2 had no effect on Akt phosphorylation, a critical signal transduction target required for GLUT4 translocation. Although overexpression of Tctex1d2 had no significant effect on GLUT4 internalization, Tctex1d2 was found to associate with syntaxin 4 in an insulin-dependent manner and inhibit Doc2b binding to syntaxin 4. In addition, glucose-dependent insulinotropic polypeptide rescued the Tctex1d2 inhibition of insulin-stimulated GLUT4 translocation by suppressing the Tctex1d2-syntaxin 4 interaction and increasing Doc2b-Synatxin4 interactions. Taking these results together, we hypothesized that Tctex1d2 is a novel syntaxin 4 binding protein that functions as a negative regulator of GLUT4 plasma membrane translocation through inhibition of the Doc2b-syntaxin 4 interaction.

  4. Different Fear-Regulation Behaviors in Toddlerhood: Relations to Preceding Infant Negative Emotionality, Maternal Depression, and Sensitivity

    Science.gov (United States)

    Gloggler, Bettina; Pauli-Pott, Ursula

    2008-01-01

    In the study presented, the development of different fear regulation behaviors and their associations with preceding maternal sensitivity and depression is addressed. A sample of 64 mother-child pairs was examined at the children's ages of 4, 12, and 30 months. Four-month negative reactivity and 12- and 30- month behavioral inhibition and fear…

  5. Adolescent depression and negative life events, the mediating role of cognitive emotion regulation

    NARCIS (Netherlands)

    Stikkelbroek, Y.A.J.; Bodden, Denise; Kleinjan, Marloes; Reijnders, Mirjam; van Baar, Anneloes

    2016-01-01

    Background: Depression during adolescence is a serious mental health problem. Difficulties in regulating evoked emotions after stressful life events are considered to lead to depression. This study examined if depressive symptoms were mediated by various cognitive emotion regulation strategies after

  6. Limitation of immune tolerance-inducing thymic epithelial cell development by Spi-B-mediated negative feedback regulation.

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    Akiyama, Nobuko; Shinzawa, Miho; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shimo, Yusuke; Ohshima, Daisuke; Matsuo, Koichi; Sasaki, Izumi; Hoshino, Katsuaki; Wu, Guoying; Yagi, Shintaro; Inoue, Jun-ichiro; Kaisho, Tsuneyasu; Akiyama, Taishin

    2014-11-17

    Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B-mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage-specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL-Spi-B-OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.

  7. Negative regulation of the antiviral response by grouper LGP2 against fish viruses.

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    Yu, Yepin; Huang, Youhua; Yang, Ying; Wang, Shaowen; Yang, Min; Huang, Xiaohong; Qin, Qiwei

    2016-09-01

    Laboratory of genetics and physiology 2 (LGP2), a member of RIG-I like receptor (RLR) family, plays crucial roles in modulating cellular antiviral response during viral infection. However, the detailed roles of LGP2 in different virus infection were controversial up to now. Here, we cloned a LGP2 gene from orange-spotted grouper (EcLGP2) and investigated its roles in response to grouper virus infection. EcLGP2 encoded a 678-aa protein which shared 83% identity to sea perch (Lateolabrax japonicas). Amino acid alignment showed that EcLGP2 contained three conserved domains, including a DEAD/DEAH box helicase domain, a helicase superfamily C-terminal domain and a C-terminal domain of RIG-I. In healthy grouper, the transcript of EcLGP2 could be predominantly detected in kidney, gill, fin, spleen and skin. Subcellular localization analysis showed that EcLGP2 distributed throughout the cytoplasm in grouper cells. Notably, the intracellular distribution of EcLGP2 was altered at the late stage of Singapore grouper iridovirus (SGIV) infection, but remained unchanged during red-spotted grouper nervous necrosis virus (RGNNV) infection. Moreover, overexpression of EcLGP2 in vitro significantly enhanced the viral replication of SGIV and RGNNV, evidenced by the acceleration of CPE occurrence and the up-regulation of the viral gene transcription or protein synthesis. Further studies indicated that overexpression of EcLGP2 decreased the expression level of interferon related molecules or effectors, including IRF3, IRF7, ISG15, IFP35, MXI, MXII, and MDA5, suggesting that the negative feedback of interferon immune response by EcLGP2 might contribute to the enhancement of RGNNV infection. Moreover, the expression levels of pro-inflammation cytokines, including IL-8 and TNFα were significantly decreased, but that of IL-6 was increased by the ectopic expression of EcLGP2. Thus, our results will contribute greatly to understanding the roles of fish LGP2 in innate immune response during

  8. The receptor tyrosine kinase FGFR4 negatively regulates NF-kappaB signaling.

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    Kristine A Drafahl

    Full Text Available BACKGROUND: NFκB signaling is of paramount importance in the regulation of apoptosis, proliferation, and inflammatory responses during human development and homeostasis, as well as in many human cancers. Receptor Tyrosine Kinases (RTKs, including the Fibroblast Growth Factor Receptors (FGFRs are also important in development and disease. However, a direct relationship between growth factor signaling pathways and NFκB activation has not been previously described, although FGFs have been known to antagonize TNFα-induced apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we demonstrate an interaction between FGFR4 and IKKβ (Inhibitor of NFκB Kinase β subunit, an essential component in the NFκB pathway. This novel interaction was identified utilizing a yeast two-hybrid screen [1] and confirmed by coimmunoprecipitation and mass spectrometry analysis. We demonstrate tyrosine phosphorylation of IKKβ in the presence of activated FGFR4, but not kinase-dead FGFR4. Following stimulation by TNFα (Tumor Necrosis Factor α to activate NFκB pathways, FGFR4 activation results in significant inhibition of NFκB signaling as measured by decreased nuclear NFκB localization, by reduced NFκB transcriptional activation in electophoretic mobility shift assays, and by inhibition of IKKβ kinase activity towards the substrate GST-IκBα in in vitro assays. FGF19 stimulation of endogenous FGFR4 in TNFα-treated DU145 prostate cancer cells also leads to a decrease in IKKβ activity, concomitant reduction in NFκB nuclear localization, and reduced apoptosis. Microarray analysis demonstrates that FGF19 + TNFα treatment of DU145 cells, in comparison with TNFα alone, favors proliferative genes while downregulating genes involved in apoptotic responses and NFκB signaling. CONCLUSIONS/SIGNIFICANCE: These results identify a compelling link between FGFR4 signaling and the NFκB pathway, and reveal that FGFR4 activation leads to a negative effect on NFκB signaling

  9. Orphan Nuclear Receptor ERRα Controls Macrophage Metabolic Signaling and A20 Expression to Negatively Regulate TLR-Induced Inflammation.

    Science.gov (United States)

    Yuk, Jae-Min; Kim, Tae Sung; Kim, Soo Yeon; Lee, Hye-Mi; Han, Jeongsu; Dufour, Catherine Rosa; Kim, Jin Kyung; Jin, Hyo Sun; Yang, Chul-Su; Park, Ki-Sun; Lee, Chul-Ho; Kim, Jin-Man; Kweon, Gi Ryang; Choi, Hueng-Sik; Vanacker, Jean-Marc; Moore, David D; Giguère, Vincent; Jo, Eun-Kyeong

    2015-07-21

    The orphan nuclear receptor estrogen-related receptor α (ERRα; NR3B1) is a key metabolic regulator, but its function in regulating inflammation remains largely unknown. Here, we demonstrate that ERRα negatively regulates Toll-like receptor (TLR)-induced inflammation by promoting Tnfaip3 transcription and fine-tuning of metabolic reprogramming in macrophages. ERRα-deficient (Esrra(-/-)) mice showed increased susceptibility to endotoxin-induced septic shock, leading to more severe pro-inflammatory responses than control mice. ERRα regulated macrophage inflammatory responses by directly binding the promoter region of Tnfaip3, a deubiquitinating enzyme in TLR signaling. In addition, Esrra(-/-) macrophages showed an increased glycolysis, but impaired mitochondrial respiratory function and biogenesis. Further, ERRα was required for the regulation of NF-κB signaling by controlling p65 acetylation via maintenance of NAD(+) levels and sirtuin 1 activation. These findings unravel a previously unappreciated role for ERRα as a negative regulator of TLR-induced inflammatory responses through inducing Tnfaip3 transcription and controlling the metabolic reprogramming.

  10. ZAT11, a zinc finger transcription factor, is a negative regulator of nickel ion tolerance in Arabidopsis.

    Science.gov (United States)

    Liu, Xiao-Min; An, Jonguk; Han, Hay Ju; Kim, Sun Ho; Lim, Chae Oh; Yun, Dae-Jin; Chung, Woo Sik

    2014-12-01

    ZAT11, a Zinc Finger of Arabidopsis Thaliana 11, is a dual-function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni (2+) tolerance. Zinc Finger of Arabidopsis Thaliana 11 (ZAT11) is a C2H2-type zinc finger protein that has been reported to function as an active transcriptional repressor. However, the biological function of ZAT11 remains unknown. Here we show that GFP-tagged ZAT11 is targeted to the nucleus. Analysis of plants expressing ZAT11 promoter-GUS showed that ZAT11 is highly expressed in roots and particularly in root tips. To identify the biological function of ZAT11, we constructed three independent lines of ZAT11 overexpressing transgenic plant (ZAT11 OE). ZAT11 OE enhanced the elongation of primary root but reduced the metal tolerance against nickel ion (Ni(2+)). The reduced Ni(2+) tolerance of ZAT11 OE was correlated with decreased accumulation of Ni(2+) in plants. The decreased accumulation of Ni(2+) in ZAT11 OE was caused by the reduced transcription of a vacuolar Ni(2+) transporter gene. Taken together, our results suggest that ZAT11 is a dual function transcriptional regulator that positively regulates primary root growth but negatively regulates Ni(2+) tolerance.

  11. Affect regulation training (ART) for alcohol use disorders: development of a novel intervention for negative affect drinkers.

    Science.gov (United States)

    Stasiewicz, Paul R; Bradizza, Clara M; Schlauch, Robert C; Coffey, Scott F; Gulliver, Suzy B; Gudleski, Gregory D; Bole, Christopher W

    2013-01-01

    Although negative affect is a common precipitant of alcohol relapse, there are few interventions for alcohol dependence that specifically target negative affect. In this stage 1a/1b treatment development study, several affect regulation strategies (e.g., mindfulness, prolonged exposure, distress tolerance) were combined to create a new treatment supplement called affect regulation training (ART), which could be added to enhance cognitive-behavioral therapy (CBT) for alcohol dependence. A draft therapy manual was given to therapists and treatment experts before being administered to several patients who also provided input. After two rounds of manual development (stage 1a), a pilot randomized clinical trial (N=77) of alcohol-dependent outpatients who reported drinking often in negative affect situations was conducted (stage 1b). Participants received 12-weekly, 90-minute sessions of either CBT for alcohol dependence plus ART (CBT+ART) or CBT plus a healthy lifestyles control condition (CBT+HLS). Baseline, end-of-treatment, and 3- and 6-month posttreatment interviews were conducted. For both treatment conditions, participant ratings of treatment satisfaction were high, with CBT+ART rated significantly higher. Drinking outcome results indicated greater reductions in alcohol use for CBT+ART when compared to CBT+HLS, with moderate effect sizes for percent days abstinent, drinks per day, drinks per drinking day, and percent heavy drinking days. Overall, findings support further research on affect regulation interventions for negative affect drinkers.

  12. sli-3 negatively regulates the LET-23/epidermal growth factor receptor-mediated vulval induction pathway in Caenorhabditis elegans.

    Science.gov (United States)

    Gupta, Bhagwati P; Liu, Jing; Hwang, Byung J; Moghal, Nadeem; Sternberg, Paul W

    2006-11-01

    The LIN-3-LET-23-mediated inductive signaling pathway plays a major role during vulval development in C. elegans. Studies on the components of this pathway have revealed positive as well as negative regulators that function to modulate the strength and specificity of the signal transduction cascade. We have carried out genetic screens to identify new regulators of this pathway by screening for suppressors of lin-3 vulvaless phenotype. The screens recovered three loci including alleles of gap-1 and a new gene represented by sli-3. Our genetic epistasis experiments suggest that sli-3 functions either downstream or in parallel to nuclear factors lin-1 and sur-2. sli-3 synergistically interacts with the previously identified negative regulators of the let-23 signaling pathway and causes excessive cell proliferation. However, in the absence of any other mutation sli-3 mutant animals display wild-type vulval induction and morphology. We propose that sli-3 functions as a negative regulator of vulval induction and defines a branch of the inductive signaling pathway. We provide evidence that sli-3 interacts with the EGF signaling pathway components during vulval induction but not during viability and ovulation processes. Thus, sli-3 helps define specificity of the EGF signaling to induce the vulva.

  13. Marital conflict and parental responses to infant negative emotions: Relations with toddler emotional regulation.

    Science.gov (United States)

    Frankel, Leslie A; Umemura, Tomo; Jacobvitz, Deborah; Hazen, Nancy

    2015-08-01

    According to family systems theory, children's emotional development is likely to be influenced by family interactions at multiple levels, including marital, mother-child, and father-child interactions, as well as by interrelations between these levels. The purpose of the present study was to examine parents' marital conflict and mothers' and fathers' distressed responses to their infant's negative emotions, assessed when their child was 8 and 24 months old, in addition to interactions between parents' marital conflict and their distressed responses, as predictors of their toddler's negative and flat/withdrawn affect at 24 months. Higher marital conflict during infancy and toddlerhood predicted both increased negative and increased flat/withdrawn affect during toddlerhood. In addition, toddlers' negative (but not flat) affect was related to mothers' distressed responses, but was only related to father's distressed responses when martial conflict was high. Implications of this study for parent education and family intervention were discussed.

  14. An shRNA-Based Screen of Splicing Regulators Identifies SFRS3 as a Negative Regulator of IL-1β Secretion

    Science.gov (United States)

    Pacheco, Teresa Raquel; D'Almeida, Bruno; Rodrigues, Raquel; Cadima-Couto, Iris; Chora, Ângelo; Oliveira, Mariana; Gama-Carvalho, Margarida; Hacohen, Nir; Moita, Luis F.

    2011-01-01

    The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1β secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1β secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1β secretion due to elevation of IL-1β and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases. PMID:21611201

  15. Applying a dual process model of self-regulation: The association between executive working memory capacity, negative urgency, and negative mood induction on pre-potent response inhibition.

    Science.gov (United States)

    Gunn, Rachel L; Finn, Peter R

    2015-03-01

    This study tested a dual-process model of self-control where the combination of high impulsivity (negative urgency - NU), weak reflective / control processes (low executive working memory capacity - E-WMC), and a cognitive load is associated with increased failures to inhibit pre-potent responses on a cued go/no-go task. Using a within-subjects design, a cognitive load with and without negative emotional load was implemented to consider situational factors. Results suggested that: (1) high NU was associated with low E-WMC; (2) low E-WMC significantly predicted more inhibitory control failures across tasks; and (3) there was a significant interaction of E-WMC and NU, revealing those with low E-WMC and high NU had the highest rates of inhibitory control failures on all conditions of the task. In conclusion, results suggest that while E-WMC is a strong independent predictor of inhibitory control, NU provides additional information for vulnerability to problems associated with self-regulation.

  16. Fat mass and obesity associated gene (FTO expression is regulated negatively by the transcription factor Foxa2.

    Directory of Open Access Journals (Sweden)

    Jianjin Guo

    Full Text Available Fat mass and obesity associated gene (FTO is the first gene associated with body mass index (BMI and risk for diabetes. FTO is highly expressed in the brain and pancreas, and is involved in regulating dietary intake and energy expenditure. To investigate the transcriptional regulation of FTO expression, we created 5'-deletion constructs of the FTO promoter to determine which transcription factors are most relevant to FTO expression. The presence of an activation region at -201/+34 was confirmed by luciferase activity analysis. A potential Foxa2 (called HNF-3β binding site and an upstream stimulatory factor (USF-binding site was identified in the -100 bp fragment upstream of the transcription start site (TSS. Furthermore, using mutagenesis, we identified the Foxa2 binding sequence (-26/-14 as a negative regulatory element to the activity of the human FTO promoter. The USF binding site did not affect the FTO promoter activity. Chromatin immunoprecipitation (ChIP assays were performed to confirm Foxa2 binding to the FTO promoter. Overexpression of Foxa2 in HEK 293 cells significantly down-regulated FTO promoter activity and expression. Conversely, knockdown of Foxa2 by siRNA significantly up-regulated FTO expression. These findings suggest that Foxa2 negatively regulates the basal transcription and expression of the human FTO gene.

  17. Negative Effects of Antimonopoly Regulation on the Russian Electric Power Industry

    Directory of Open Access Journals (Sweden)

    Elena NEPRINTSEVA

    2017-07-01

    Full Text Available With the antimonopoly regulation in the domestic economy getting more stringent an analysis of the current measures of antimonopoly regulation in terms of their efficiency is now becoming ever more relevant. The aim of the study - analyze how the measures of antimonopoly regulation affect competitive relationships in the electric power industry. The following methods have been used in this work: empirical method, cause-effect method and scientific abstraction method. The article sets out an analysis of the antimonopoly regulation measures that the antimonopoly authority applies. It also provides an assessment of consequences that follow from such methods being applied for the promotion of competitive relationships on the market of electric power and capacity. A conclusion has been reached that the antimonopoly regulation measures being applied impede the progress of competitive relationships on the market of electric power and capacity. The continuing process of reformation in electric power industry aims to liberalize relationships in the area of electric power production. Yet, as a result of this process, generating capacities are becoming increasingly more concentrated mainly around state companies. This is mainly caused by the lack of certainty regarding the results of the industry reformation and a more stringent state regulation over the last years of the reforms. At the same time, for the purposes of limiting the market force, measures of antimonopoly regulation are being applied to generating companies. Such measures have an adverse effect on competitive relationships and stimulate further concentration.

  18. A Loss-of-Function Screen for Phosphatases that Regulate Neurite Outgrowth Identifies PTPN12 as a Negative Regulator of TrkB Tyrosine Phosphorylation

    DEFF Research Database (Denmark)

    Ambjørn, Malene; Dubreuil, Véronique; Miozzo, Federico

    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...... 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...

  19. Positive and Negative Associations between Adolescents’ Religiousness and Health Behaviors via Self-Regulation

    Science.gov (United States)

    Holmes, Christopher J.; Kim-Spoon, Jungmeen

    2015-01-01

    It has been proposed that self-regulation may be the explanatory mechanism for the relation between religiousness and positive health behaviors. However, different religious motivations have differential effects on a variety of health related outcomes, which may explain the adverse effects of religiousness found in some studies. The current study hypothesized that higher identification as religious motivation would be linked to higher health-promoting behavior and lower health-risk behavior through higher self-regulation, whereas higher introjection would be linked to lower health-promoting behavior and higher health-risk behavior through lower self-regulation. The sample included 220 adolescents (mean age = 15 years, 55% male) and their primary caregivers. Structural equation modeling results supported the hypotheses and indicated that adolescent self-regulation mediated the relations between their religious motivation and health behavior. The findings suggest that different types of religious motivation may be promotive or hindering for adolescents’ health. PMID:27595048

  20. Epigenetic Regulation of microRNA Expression: Targeting the Triple-Negative Breast Cancer Phenotype

    Science.gov (United States)

    2011-10-01

    CTCE-9908 inhibits breast cancer metastasis to lung and bone, Oncol. Rep. 21 (2009) 761–767. [36] N.T. Holm, F. Abreo, L.W. Johnson, B.D. Li, Q.D. Chu...Kawai, T. Inoue, H. Ito, M. Oshimura, T. Ochiya, MicroRNA-143 regulates human osteosarcoma metastasis by regulating matrix metalloprotease-13...cancers with increased potential for metastasis and recurrence (2). Basal-like breast carcinomas express genes associated with an EMT phenotype and

  1. Perfectionism, Emotion Regulation and Their Relationship to Negative Affect in Patients with Social Phobia

    OpenAIRE

    Systla Rukmini; Sudhir, Paulomi M.; Suresh Bada Math

    2014-01-01

    Context: Research on the perfectionism and emotion regulation strategies in anxiety disorders has gained increased attention. These have an important implication for formulation of therapies. Aims: We examined perfectionism, emotion regulation were examined in 30 patients with social phobia (SP) and 30 community participants. Settings and Design: A cross-sectional design using a clinical and a community control sample was adopted in this exploratory study. Materials and Methods: Participants ...

  2. A negative-feedback loop regulating ERK1/2 activation and mediated by RasGPR2 phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Jinqi [Departments of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Cook, Aaron A.; Bergmeier, Wolfgang [Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Sondek, John, E-mail: sondek@med.unc.edu [Departments of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States); Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599 (United States)

    2016-05-20

    The dynamic regulation of ERK1 and -2 (ERK1/2) is required for precise signal transduction controlling cell proliferation, differentiation, and survival. However, the underlying mechanisms regulating the activation of ERK1/2 are not completely understood. In this study, we show that phosphorylation of RasGRP2, a guanine nucleotide exchange factor (GEF), inhibits its ability to activate the small GTPase Rap1 that ultimately leads to decreased activation of ERK1/2 in cells. ERK2 phosphorylates RasGRP2 at Ser394 located in the linker region implicated in its autoinhibition. These studies identify RasGRP2 as a novel substrate of ERK1/2 and define a negative-feedback loop that regulates the BRaf–MEK–ERK signaling cascade. This negative-feedback loop determines the amplitude and duration of active ERK1/2. -- Highlights: •ERK2 phosphorylates the guanine nucleotide exchange factor RasGRP2 at Ser394. •Phosphorylated RasGRP2 has decreased capacity to active Rap1b in vitro and in cells. •Phosphorylation of RasGRP2 by ERK1/2 introduces a negative-feedback loop into the BRaf-MEK-ERK pathway.

  3. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Science.gov (United States)

    Ikebe, Tadayoshi; Ato, Manabu; Matsumura, Takayuki; Hasegawa, Hideki; Sata, Tetsutaro; Kobayashi, Kazuo; Watanabe, Haruo

    2010-04-01

    Streptococcal toxic shock syndrome (STSS) is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates) and non-invasive infections (59 isolates), 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%). The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

  4. MicroRNA-221/222 negatively regulates estrogen receptor alpha and is associated with tamoxifen resistance in breast cancer.

    Science.gov (United States)

    Zhao, Jian-Jun; Lin, Jianhong; Yang, Hua; Kong, William; He, Lili; Ma, Xu; Coppola, Domenico; Cheng, Jin Q

    2008-11-07

    A search for regulators of estrogen receptor alpha (ERalpha) expression has yielded a set of microRNAs (miRNAs) for which expression is specifically elevated in ERalpha-negative breast cancer. Here we show distinct expression of a panel of miRNAs between ERalpha-positive and ERalpha-negative breast cancer cell lines and primary tumors. Of the elevated miRNAs in ERalpha-negative cells, miR-221 and miR-222 directly interact with the 3'-untranslated region of ERalpha. Ectopic expression of miR-221 and miR-222 in MCF-7 and T47D cells resulted in a decrease in expression of ERalpha protein but not mRNA, whereas knockdown of miR-221 and miR-222 partially restored ERalpha in ERalpha protein-negative/mRNA-positive cells. Notably, miR-221- and/or miR-222-transfected MCF-7 and T47D cells became resistant to tamoxifen compared with vector-treated cells. Furthermore, knockdown of miR-221 and/or miR-222 sensitized MDA-MB-468 cells to tamoxifen-induced cell growth arrest and apoptosis. These findings indicate that miR-221 and miR-222 play a significant role in the regulation of ERalpha expression at the protein level and could be potential targets for restoring ERalpha expression and responding to antiestrogen therapy in a subset of breast cancers.

  5. Dlk1 is a negative regulator of emerging hematopoietic stem and progenitor cells

    NARCIS (Netherlands)

    B. Mirshekar-Syahkal (Bahar); E. Haak (Esther); G.M. Kimber (Gillian); K. van Leusden (Kevin); K. Harvey (Kirsten); R.A. O'Rourke; J. Laborda (Jorge); S.R. Bauer (Steven); M.F.T.R. de Bruijn (Marella F.T.R); A. Ferguson-Smith (Anne); E.A. Dzierzak (Elaine); K. Ottersbach (Katrin)

    2013-01-01

    textabstractThe first mouse adult-repopulating hematopoietic stem cells emerge in the aorta-gonad-mesonephros region at embryonic day (E) 10.5. Their numbers in this region increase thereafter and begin to decline at E12.5, thus pointing to the possible existence of both positive and negative regula

  6. CML20, an Arabidopsis Calmodulin-like Protein, Negatively Regulates Guard Cell ABA Signaling and Drought Stress Tolerance

    Directory of Open Access Journals (Sweden)

    Xiaomeng Wu

    2017-05-01

    Full Text Available Guard cells shrink in response to drought and abscisic acid (ABA, which is caused by efflux of ions that in turn reduces stomatal aperture and improves the plant’s ability to retain moisture. Cytosolic free calcium is an essential secondary messenger in guard cell ABA signaling, but the details of this regulatory pathway remain sketchy. Here, the calmodulin-like protein CML20, which has four EF-hand domains and calcium-binding activity in vitro, was found to be a negative regulator of ABA-induced stomatal movement in Arabidopsis. The guard cells of cml20 loss-of-function mutant plants were hypersensitive to both ABA-activated S-type anion currents, and ABA inhibited inward K+ currents than those of wild type. Additional, due to smaller stomatal aperture, cml20 showed less water loss from the leaves than wild type. These phenotypes of CML20 overexpressing plants contrasted with wild type in the opposite direction. In the cml20 mutant, the transcripts of stress responsive genes, such as MYB2, RAB18, ERD10, COR47, and RD29A were up-regulated in response to drought and ABA, while down-regulated of APX2 transcription and higher reactive oxygen species (ROS accumulation. These observations support the CML20, a functional Ca2+ sensor, is a negative regulator in guard cell ABA signaling.

  7. MicroRNA-370 suppresses proliferation and promotes endometrioid ovarian cancer chemosensitivity to cDDP by negatively regulating ENG.

    Science.gov (United States)

    Chen, Xiao-Ping; Chen, You-Guo; Lan, Jian-Yun; Shen, Zong-Ji

    2014-10-28

    MicroRNAs (miRNAs) are a class of non-coding RNAs that post-transcriptionally inhibit gene expression. In this study, we discovered that microRNA-370 (miR-370) was down-regulated in endometrioid ovarian cancer cells. In IGROV1 and TOV112D endometrioid ovarian cancer cells, miR-370 suppressed cellular viability and colony formation. miR-370 also enhanced endometrioid ovarian cancer cell chemosensitivity to cDDP. Endoglin (ENG) was directly and negatively regulated by miR-370. In addition, hypermethylation was a potential mechanism of miR-370 epigenetic silencing. We conclude that miR-370 acts as a tumor suppressor in endometrioid ovarian cancer via ENG regulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  9. SIRT1 is regulated by a PPARγ–SIRT1 negative feedback loop associated with senescence

    Science.gov (United States)

    Zhou, Rui; Niu, Jing; McNutt, Michael A.; Wang, Pan; Tong, Tanjun

    2010-01-01

    Human Silent Information Regulator Type 1 (SIRT1) is an NAD+-dependent deacetylase protein which is an intermediary of cellular metabolism in gene silencing and aging. SIRT1 has been extensively investigated and shown to delay senescence; however, less is known about the regulation of SIRT1 during aging. In this study, we show that the peroxisome proliferator-activated receptor-γ (PPARγ), which is a ligand-regulated modular nuclear receptor that governs adipocyte differentiation and inhibits cellular proliferation, inhibits SIRT1 expression at the transcriptional level. Moreover, both PPARγ and SIRT1 can bind the SIRT1 promoter. PPARγ directly interacts with SIRT1 and inhibits SIRT1 activity, forming a negative feedback and self-regulation loop. In addition, our data show that acetylation of PPARγ increased with increasing cell passage number. We propose that PPARγ is subject to regulation by acetylation and deacetylation via p300 and SIRT1 in cellular senescence. These results demonstrate a mutual regulation between PPARγ and SIRT1 and identify a new posttranslational modification that affects cellular senescence. PMID:20660480

  10. Characterization of Adapter Protein NRBP as a Negative Regulator of T Cell Activation

    Institute of Scientific and Technical Information of China (English)

    WANG Hui; LIN Zhi-xin; WU Jun

    2008-01-01

    Adapter proteins can regulate the gene transcriptions in disparate signaling pathway by interacting with multiple signaling molecules, including T cell activation signaling. Nuclear receptor binding protein (NRBP), a novel adapter protein, represents a small family of evolutionarily conserved proteins with homologs in Caenorhabditis elegans (C. elegans), Drosophila melanogaster (D.melanogaster), mouse and human. Here, we demonstrated that overexpression of NRBP in Jurkat TAg cells specifically impairs T cell receptor (TCR) or phorbol myristate acetate (PMA)/ionomycin-mediated signaling leading to nuclear factor of activated T cells (NFAT) promoter activation. Furthermore, the N-terminal of NRBP is necessary for its regulation of NFAT activation. Finally, we showed that NRBP has minimal effect on both TCR- and PMA-induced CD69 up-regulation in Jurkat TAg cells, which suggests that NRBP may function downstream of protein kinase C (PKC)/Ras pathway.

  11. SENP2 negatively regulates cellular antiviral response by deSUMOylating IRF3 and conditioning it for ubiquitination and degradation

    Institute of Scientific and Technical Information of China (English)

    Yong Ran; Tian-Tian Liu; Qian Zhou; Shu Li; Ai-Ping Mao; Ying Li; Li-Juan Liu; Jin-Ke Cheng; Hong-Bing Shu

    2011-01-01

    Transcription factor IRF3-mediated type I interferon induction is essential for antiviral innate immunity.We identified the deSUMOylating enzyme Sentrin/SUMO-specific protease (SENP) 2 as a negative regulator of virus-triggered IFN-β induction.Overexpression of SENP2 caused IRF3 deSUMOylation,K48-linked ubiquitination,and degradation,whereas depletion of SENP2 had opposite effects.Both the SUMOylation and K48-linked ubiquitination of IRF3 occurred at iysines 70 and 87,and these processes are competitive.The level of virus-triggered IFN-β was markedly up-regulated and viral replication was reduced in SENP2-deficient cells comparing with wild-type controls.Our findings suggest that SENP2 regulates antiviral innate immunity by deSUMOylating IRF3 and conditioning it for ubiquitination and degradation,and provide an example of cross-talk between the ubiquitin and SUMO pathways in innate immunity.%Transcription factor IRF3-mediated type I interferon induction is essential for antiviral innate immunity. We identified the deSUMOylating enzyme Sentrin/SUMO-specific protease (SENP) 2 as a negative regulator of virus-triggered IFN-p induction. Overexpression of SENP2 caused IRF3 deSUMOylation, K48-linked ubiquitination, and degradation, whereas depletion of SENP2 had opposite effects. Both the SUMOylation and K48-linked ubiquitination of IRF3 occurred at lysines 70 and 87, and these processes are competitive. The level of virus-triggered IFN-β was markedly up-regulated and viral replication was reduced in SENP2-deficient cells comparing with wild-type controls. Our findings suggest that SENP2 regulates antiviral innate immunity by deSUMOylating IRF3 and conditioning it for ubiquitination and degradation, and provide an example of cross-talk between the ubiquitin and SUMO pathways in innate immunity.

  12. Functional analysis of Arabidopsis immune-related MAPKs uncovers a role for MPK3 as negative regulator of inducible defences

    KAUST Repository

    Frei dit Frey, Nicolas

    2014-06-30

    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.

  13. ST2 negatively regulates TLR2 signaling, but is not required for bacterial lipoprotein-induced tolerance.

    LENUS (Irish Health Repository)

    Liu, Jinghua

    2010-05-15

    Activation of TLR signaling is critical for host innate immunity against bacterial infection. Previous studies reported that the ST2 receptor, a member of the Toll\\/IL-1 receptor superfamily, functions as a negative regulator of TLR4 signaling and maintains LPS tolerance. However, it is undetermined whether ST2 negatively regulates TLR2 signaling and furthermore, whether a TLR2 agonist, bacterial lipoprotein (BLP)-induced tolerance is dependent on ST2. In this study, we show that BLP stimulation-induced production of proinflammatory cytokines and immunocomplex formation of TLR2-MyD88 and MyD88-IL-1R-associated kinase (IRAK) were significantly enhanced in ST2-deficient macrophages compared with those in wild-type controls. Furthermore, overexpression of ST2 dose-dependently attenuated BLP-induced NF-kappaB activation, suggesting a negative regulatory role of ST2 in TLR2 signaling. A moderate but significantly attenuated production of TNF-alpha and IL-6 on a second BLP stimulation was observed in BLP-pretreated, ST2-deficient macrophages, which is associated with substantially reduced IRAK-1 protein expression and downregulated TLR2-MyD88 and MyD88-IRAK immunocomplex formation. ST2-deficient mice, when pretreated with a nonlethal dose of BLP, benefitted from an improved survival against a subsequent lethal BLP challenge, indicating BLP tolerance develops in the absence of the ST2 receptor. Taken together, our results demonstrate that ST2 acts as a negative regulator of TLR2 signaling, but is not required for BLP-induced tolerance.

  14. Negative Regulation of DsbA-L Gene Expression by the Transcription Factor Sp1

    OpenAIRE

    Fang, Qichen; Yang, Wenjing; Li, Huating; Hu, Wenxiu; Chen, Lihui; Jiang, Shan; Dong, Kun; Song, Qianqian; Wang, Chen; Chen, Shuo; LIU, FENG; Jia, Weiping

    2014-01-01

    Disulfide-bond A oxidoreductase-like protein (DsbA-L) possesses beneficial effects such as promoting adiponectin multimerization and stability, increasing insulin sensitivity, and enhancing energy metabolism. The expression level of DsbA-L is negatively correlated with obesity in mice and humans, but the underlying mechanisms remain unknown. To address this question, we generated reporter gene constructs containing the promoter sequence of the mouse DsbA-L gene. Deletion analysis showed that ...

  15. Ubiquitin-Specific Peptidase USP22 Negatively Regulates the STAT Signaling Pathway by Deubiquitinating SIRT1

    Directory of Open Access Journals (Sweden)

    Ning Ao

    2014-06-01

    Full Text Available Background/Aims: The ubiquitin-specific peptidase USP22 mediates various cellular and organismal processes, such as cell growth, apoptosis, and tumor malignancy. However, the molecular mechanisms that regulate USP22 activity remain poorly understood. Here we identify STAT3 as a new USP22 interactor. Methods:· We used western blotting and RT-PCR to measure key protein, acetylated STAT3, and mRNA levels in HEK293 and colorectal cancer cell lines transfected with expression plasmids or specific siRNAs. Co-immunoprecipitation was used to demonstrate protein-protein interaction and protein complex composition. Results: USP22 overexpression down-regulated STAT3 acetylation by deubiquitinating SIRT1. The three proteins were found to be present in a single protein complex. SiRNA-mediated depletion of endogenous USP22 resulted in SIRT1 destabilization and elevated STAT3 acetylation. Consistent with this finding, USP22 also down-regulated the expression of two known STAT3 target genes, MMP9 and TWIST. Conclusion: We show that USP22 is a new regulator of the SIRT1-STAT3 signaling pathway and report a new mechanistic explanation for cross talk between USP22 and the SIRT1-STAT pathways.

  16. Growth Hormone Receptor Signaling Pathways and its Negative Regulation by SOCS2

    DEFF Research Database (Denmark)

    Fernández Pérez, Leandro; Flores-Morales, Amilcar; Guerra, Borja

    2016-01-01

    Growth hormone (GH) is a critical regulator of linear body growth during childhood but continues to have important metabolic actions throughout life. The GH receptor (GHR) is ubiquitously expressed, and deficiency of GHR signaling causes a dramatic impact on normal physiology during somatic devel...

  17. The Potato ERF Transcription Factor StERF3 Negatively Regulates Resistance to Phytophthora infestans and Salt Tolerance in Potato.

    Science.gov (United States)

    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.

  18. Burkholderia mallei and Burkholderia pseudomallei cluster 1 type VI secretion system gene expression is negatively regulated by iron and zinc.

    Directory of Open Access Journals (Sweden)

    Mary N Burtnick

    Full Text Available Burkholderia mallei is a facultative intracellular pathogen that causes glanders in humans and animals. Previous studies have demonstrated that the cluster 1 type VI secretion system (T6SS-1 expressed by this organism is essential for virulence in hamsters and is positively regulated by the VirAG two-component system. Recently, we have shown that T6SS-1 gene expression is up-regulated following internalization of this pathogen into phagocytic cells and that this system promotes multinucleated giant cell formation in infected tissue culture monolayers. In the present study, we further investigated the complex regulation of this important virulence factor. To assess T6SS-1 expression, B. mallei strains were cultured in various media conditions and Hcp1 production was analyzed by Western immunoblotting. Transcript levels of several VirAG-regulated genes (bimA, tssA, hcp1 and tssM were also determined using quantitative real time PCR. Consistent with previous observations, T6SS-1 was not expressed during growth of B. mallei in rich media. Curiously, growth of the organism in minimal media (M9G or minimal media plus casamino acids (M9CG facilitated robust expression of T6SS-1 genes whereas growth in minimal media plus tryptone (M9TG did not. Investigation of this phenomenon confirmed a regulatory role for VirAG in this process. Additionally, T6SS-1 gene expression was significantly down-regulated by the addition of iron and zinc to M9CG. Other genes under the control of VirAG did not appear to be as tightly regulated by these divalent metals. Similar results were observed for B. pseudomallei, but not for B. thailandensis. Collectively, our findings indicate that in addition to being positively regulated by VirAG, B. mallei and B. pseudomallei T6SS-1 gene expression is negatively regulated by iron and zinc.

  19. A loss-of-function screen for phosphatases that regulate neurite outgrowth identifies PTPN12 as a negative regulator of TrkB tyrosine phosphorylation.

    Directory of Open Access Journals (Sweden)

    Malene Ambjørn

    Full Text Available 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

  20. Regulator of G protein signaling 20 correlates with clinicopathological features and prognosis in triple-negative breast cancer.

    Science.gov (United States)

    Li, Quan; Jin, Wenxu; Cai, Yefeng; Yang, Fang; Chen, Endong; Ye, Danrong; Wang, Qingxuan; Guan, Xiaoxiang

    2017-04-08

    Triple-negative breast cancer (TNBC) is a highly aggressive tumor subtype lacking effective prognostic indicators or therapeutic targets. Therefore, finding a novel molecular biomarker for TNBC to achieve target therapy and predict its prognosis is crucial in preventing inappropriate treatment. Regulator of G-protein signaling (RGS) families of protein can negatively regulate signaling of heterotrimeric G proteins and are known to be upregulated in various tumors. In this study, we demonstrated that RGS20 was more highly expressed in TNBC tumor tissue than in adjacent normal tissue by analyzing the cancer genome atlas (TCGA) database. However, RGS20 expression was low in all breast cancer and luminal breast cancer patients. Validated by the TCGA cohort, RGS20 was upregulated in lymph node-positive TNBC compared with that in lymph node-negative breast cancer. High expression of RGS20 had a risk of lymph node metastasis, ki-67 > 14%, poor N stage, and poor clinical stage in the immunohistochemistry of tissue microarrays. Moreover, K-M plot analysis showed that TNBC patients with high RGS20 expression had poor relapse-free survival. In summary, the findings revealed that RGS20 was a special TNBC oncogene that promoted tumor progression and influenced TNBC prognosis. This study is the first to show that RGS20 was a special oncogene, and its high expression was significantly associated with the progression and prognosis of TNBC. RGS20 may be a novel molecular biomarker for the targeted therapy and prognosis of TNBC.

  1. GSK3beta is a negative regulator of the transcriptional coactivator MAML1.

    Science.gov (United States)

    Saint Just Ribeiro, Mariana; Hansson, Magnus L; Lindberg, Mikael J; Popko-Scibor, Anita E; Wallberg, Annika E

    2009-11-01

    Glycogen synthase kinase 3beta (GSK3beta) is involved in several cellular signaling systems through regulation of the activity of diverse transcription factors such as Notch, p53 and beta-catenin. Mastermind-like 1 (MAML1) was originally identified as a Notch coactivator, but has also been reported to function as a transcriptional coregulator of p53, beta-catenin and MEF2C. In this report, we show that active GSK3beta directly interacts with the MAML1 N-terminus and decreases MAML1 transcriptional activity, suggesting that GSK3beta might target a coactivator in its regulation of gene expression. We have previously shown that MAML1 increases global acetylation of histones, and here we show that the GSK3 inhibitor SB41, further enhances MAML1-dependent histone acetylation in cells. Finally, MAML1 translocates GSK3beta to nuclear bodies; this function requires full-length MAML1 protein.

  2. Ski and SnoN,potent negative regulators of TGF-β signaling

    Institute of Scientific and Technical Information of China (English)

    Julien Deheuninck; Kunxin Luo

    2009-01-01

    SKi and the closely related SnoN were discovered as oncogenes by their ability to transform chicken embryo fi-broblasts upon overexpression.While elevated expressions of Ski and SnoN have also been reported in many human cancer cells and tissues,consistent with their pro-oncogenic activity,emerging evidence also suggests a potential anti-oncogenic activity for both.In addition,Ski and SnoN have been implicated in regulation of cell differentiation,especially in the muscle and neuronal lineages.Multiple cellular partners of Ski and SnoN have been identified in an effort to understand the molecular mechanisms underlying the complex roles of Ski and SnoN.In this review,we summarize recent findings on the biological functions of Ski and SnoN.their mechanisms of action and how theirlevels of expression are regulated.

  3. Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein

    DEFF Research Database (Denmark)

    Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro;

    2011-01-01

    In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear....... In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased...... significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO...

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

    Science.gov (United States)

    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.

  5. PTPN14 interacts with and negatively regulates the oncogenic function of YAP

    OpenAIRE

    Liu, X; Yang, N; Figel, SA; Wilson, KE; Morrison, CD; Gelman, IH; Zhang, J

    2012-01-01

    The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition and malignant transformation. Here, we report a novel regulatory mechanism for the YAP oncogenic function via direct interaction with non-receptor tyrosine phosphatase 14 (PTPN14) thro...

  6. Eos negatively regulates human γ-globin gene transcription during erythroid differentiation.

    Directory of Open Access Journals (Sweden)

    Hai-Chuan Yu

    Full Text Available BACKGROUND: Human globin gene expression is precisely regulated by a complicated network of transcription factors and chromatin modifying activities during development and erythropoiesis. Eos (Ikaros family zinc finger 4, IKZF4, a member of the zinc finger transcription factor Ikaros family, plays a pivotal role as a repressor of gene expression. The aim of this study was to examine the role of Eos in globin gene regulation. METHODOLOGY/PRINCIPAL FINDINGS: Western blot and quantitative real-time PCR detected a gradual decrease in Eos expression during erythroid differentiation of hemin-induced K562 cells and Epo-induced CD34+ hematopoietic stem/progenitor cells (HPCs. DNA transfection and lentivirus-mediated gene transfer demonstrated that the enforced expression of Eos significantly represses the expression of γ-globin, but not other globin genes, in K562 cells and CD34+ HPCs. Consistent with a direct role of Eos in globin gene regulation, chromatin immunoprecipitaion and dual-luciferase reporter assays identified three discrete sites located in the DNase I hypersensitivity site 3 (HS3 of the β-globin locus control region (LCR, the promoter regions of the Gγ- and Aγ- globin genes, as functional binding sites of Eos protein. A chromosome conformation capture (3C assay indicated that Eos may repress the interaction between the LCR and the γ-globin gene promoter. In addition, erythroid differentiation was inhibited by enforced expression of Eos in K562 cells and CD34+ HPCs. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that Eos plays an important role in the transcriptional regulation of the γ-globin gene during erythroid differentiation.

  7. Negative regulation of hepatic fat mass and obesity associated (Fto) gene expression by insulin.

    Science.gov (United States)

    Mizuno, Tooru M; Lew, Pei San; Luo, Yanming; Leckstrom, Arnold

    2017-02-01

    To investigate the role of glucose and insulin in the regulation of hepatic fat mass and obesity associated (Fto) gene expression and the role of hepatic Fto in the regulation of gluconeogenic gene expression. To determine the effect of hyperglycemia on hepatic Fto expression, levels of Fto mRNA in liver were compared between normoglycemic/normoinsulinemic, hypereglycemic/hyperinsulinemic, and hyperglycemic/hypoinsulinemic mice. To determine the direct effect of insulin on Fto expression, levels of Fto, glucose-6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (Pepck) mRNA levels were compared between control and insulin-treated mouse liver tissues cultured ex vivo and immortalized mouse hepatocytes AML12. To determine the role of Fto in the regulation of gluconeogenic gene expression, we examined the effect of enhanced Fto expression on G6pase and Pepck mRNA levels in AML12 cells. Fto mRNA levels were significantly reduced in hyperglycemic/hyperinsulinemic mice compared to normoglycemic/normoinsulinemic mice, while they were indistinguishable between hyperglycemic/hypoinsulinemic mice and normoglycemic/normoinsulinemic mice. Insulin treatment reduced Fto, G6pase, and Pepck mRNA levels compared to control vehicle treatment in both ex vivo cultured mouse liver tissues and AML12 cells. Enhanced Fto expression significantly increased G6pase and Pepck mRNA level in AML12 cells. Our findings support the hypothesis that hepatic Fto participates in the maintenance of glucose homeostasis possibly by mediating the inhibitory effect of glucose and insulin on gluconeogenic gene expression in liver. It is further suggested that impairments in nutritional and hormonal regulation of hepatic Fto expression may lead to impairments in glycemic control in diabetes. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. miR-27a is a negative regulator of adipocyte differentiation via suppressing PPARgamma expression.

    Science.gov (United States)

    Kim, Sang Yun; Kim, A Young; Lee, Hyun Woo; Son, You Hwa; Lee, Gha Young; Lee, Joo-Won; Lee, Yun Sok; Kim, Jae Bum

    2010-02-12

    microRNAs (miRNAs) are non-coding small RNAs regulating gene expression, cell growth, and differentiation. Although several miRNAs have been implicated in cell growth and differentiation, it is barely understood their roles in adipocyte differentiation. In the present study, we reveal that miR-27a is involved in adipocyte differentiation by binding to the PPARgamma 3'-UTR whose sequence motifs are highly conserved in mammals. During adipogenesis, the expression level of miR-27a was inversely correlated with that of adipogenic marker genes such as PPARgamma and adiponectin. In white adipose tissue, miR-27a was more abundantly expressed in stromal vascular cell fraction than in mature adipocyte fraction. Ectopic expression of miR-27a in 3T3-L1 pre-adipocytes repressed adipocyte differentiation by reducing PPARgamma expression. Interestingly, the level of miR-27a in mature adipocyte fraction of obese mice was down-regulated than that of lean mice. Together, these results suggest that miR-27a would suppress adipocyte differentiation through targeting PPARgamma and thereby down-regulation of miR-27a might be associated with adipose tissue dysregulation in obesity.

  9. Negative feedback regulation of Wnt signaling via N-linked fucosylation in zebrafish.

    Science.gov (United States)

    Feng, Lei; Jiang, Hao; Wu, Peng; Marlow, Florence L

    2014-11-15

    L-fucose, a monosaccharide widely distributed in eukaryotes and certain bacteria, is a determinant of many functional glycans that play central roles in numerous biological processes. The molecular mechanism, however, by which fucosylation mediates these processes remains largely elusive. To study how changes in fucosylation impact embryonic development, we up-regulated N-linked fucosylation via over-expression of a key GDP-Fucose transporter, Slc35c1, in zebrafish. We show that Slc35c1 overexpression causes elevated N-linked fucosylation and disrupts embryonic patterning in a transporter activity dependent manner. We demonstrate that patterning defects associated with enhanced N-linked fucosylation are due to diminished canonical Wnt signaling. Chimeric analyses demonstrate that elevated Slc35c1 expression in receiving cells decreases the signaling range of Wnt8a during zebrafish embryogenesis. Moreover, we provide biochemical evidence that this decrease is associated with reduced Wnt8 ligand and elevated Lrp6 coreceptor, which we show are both substrates for N-linked fucosylation in zebrafish embryos. Strikingly, slc35c1 expression is regulated by canonical Wnt signaling. These results suggest that Wnt limits its own signaling activity in part via up-regulation of a transporter, slc35c1 that promotes terminal fucosylation and thereby limits Wnt activity.

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

    Science.gov (United States)

    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.

  11. MicroRNA-142-3p Negatively Regulates Canonical Wnt Signaling Pathway.

    Directory of Open Access Journals (Sweden)

    Tanyu Hu

    Full Text Available Wnt/β-catenin signaling pathway plays essential roles in mammalian development and tissue homeostasis. MicroRNAs (miRNAs are a class of regulators involved in modulating this pathway. In this study, we screened miRNAs regulating Wnt/β-catenin signaling by using a TopFlash based luciferase reporter. Surprisingly, we found that miR-142 inhibited Wnt/β-catenin signaling, which was inconsistent with a recent study showing that miR-142-3p targeted Adenomatous Polyposis Coli (APC to upregulate Wnt/β-catenin signaling. Due to the discordance, we elaborated experiments by using extensive mutagenesis, which demonstrated that the stem-loop structure was important for miR-142 to efficiently suppress Wnt/β-catenin signaling. Moreover, the inhibitory effect of miR-142 relies on miR-142-3p rather than miR-142-5p. Further, we found that miR-142-3p directly modulated translation of Ctnnb1 mRNA (encoding β-catenin through binding to its 3' untranslated region (3' UTR. Finally, miR-142 was able to repress cell cycle progression by inhibiting active Wnt/β-catenin signaling. Thus, our findings highlight the inhibitory role of miR-142-3p in Wnt/β-catenin signaling, which help to understand the complex regulation of Wnt/β-catenin signaling.

  12. PAK1 negatively regulates the activity of the Rho exchange factor NET1.

    Science.gov (United States)

    Alberts, Arthur S; Qin, Huajun; Carr, Heather S; Frost, Jeffrey A

    2005-04-01

    Rho family small G-protein activity is controlled by guanine nucleotide exchange factors that stimulate the release of GDP, thus allowing GTP binding. Once activated, Rho proteins control cell signaling through interactions with downstream effector proteins, leading to changes in cytoskeletal organization and gene expression. The ability of Rho family members to modulate the activity of other Rho proteins is also intrinsic to these processes. In this work we show that the Rac/Cdc42hs-regulated protein kinase PAK1 down-regulates the activity of the RhoA-specific guanine nucleotide exchange factor NET1. Specifically, PAK1 phosphorylates NET1 on three sites in vitro: serines 152, 153, and 538. Replacement of serines 152 and 153 with glutamate residues down-regulates the activity of NET1 as an exchange factor in vitro and its ability to stimulate actin stress fiber formation in cells. Using a phospho-specific antibody that recognizes NET1 phosphorylated on serine 152, we show that PAK1 phosphorylates NET1 on this site in cells and that Rac1 stimulates serine 152 phosphorylation in a PAK1-dependent manner. Furthermore, coexpression of constitutively active PAK1 inhibits the ability of NET1 to stimulate actin polymerization only when serines 152 and 153 are present. These data provide a novel mechanism for the control of RhoA activity by Rac1 through the PAK-dependent phosphorylation of NET1 to reduce its activity as a guanine nucleotide exchange factor.

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

    Science.gov (United States)

    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. Germ-Cell-Specific Inflammasome Component NLRP14 Negatively Regulates Cytosolic Nucleic Acid Sensing to Promote Fertilization.

    Science.gov (United States)

    Abe, Takayuki; Lee, Albert; Sitharam, Ramaswami; Kesner, Jordan; Rabadan, Raul; Shapira, Sagi D

    2017-04-18

    Cytosolic sensing of nucleic acids initiates tightly regulated programs to limit infection. Oocyte fertilization represents a scenario wherein inappropriate responses to exogenous yet non-pathogen-derived nucleic acids would have negative consequences. We hypothesized that germ cells express negative regulators of nucleic acid sensing (NAS) in steady state and applied an integrated data-mining and functional genomics approach to identify a rheostat of DNA and RNA sensing-the inflammasome component NLRP14. We demonstrated that NLRP14 interacted physically with the nucleic acid sensing pathway and targeted TBK1 (TANK binding kinase 1) for ubiquitination and degradation. We further mapped domains in NLRP14 and TBK1 that mediated the inhibitory function. Finally, we identified a human nonsense germline variant associated with male sterility that results in loss of NLRP14 function and hyper-responsiveness to nucleic acids. The discovery points to a mechanism of nucleic acid sensing regulation that may be of particular importance in fertilization. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex.

    Science.gov (United States)

    Zhang, Wenjing; Gao, Yijun; Li, Peixue; Shi, Zhubing; Guo, Tong; Li, Fei; Han, Xiangkun; Feng, Yan; Zheng, Chao; Wang, Zuoyun; Li, Fuming; Chen, Haiquan; Zhou, Zhaocai; Zhang, Lei; Ji, Hongbin

    2014-03-01

    Lung cancer is one of the most devastating diseases worldwide with high incidence and mortality. Hippo (Hpo) pathway is a conserved regulator of organ size in both Drosophila and mammals. Emerging evidence has suggested the significance of Hpo pathway in cancer development. In this study, we identify VGLL4 as a novel tumor suppressor in lung carcinogenesis through negatively regulating the formation of YAP-TEAD complex, the core component of Hpo pathway. Our data show that VGLL4 is frequently observed to be lowly expressed in both mouse and human lung cancer specimens. Ectopic expression of VGLL4 significantly suppresses the growth of lung cancer cells in vitro. More importantly, VGLL4 significantly inhibits lung cancer progression in de novo mouse model. We further find that VGLL4 inhibits the activity of the YAP-TEAD transcriptional complex. Our data show that VGLL4 directly competes with YAP in binding to TEADs and executes its growth-inhibitory function through two TDU domains. Collectively, our study demonstrates that VGLL4 is a novel tumor suppressor for lung cancer through negatively regulating the YAP-TEAD complex formation and thus the Hpo pathway.

  16. A G-protein β subunit, AGB1, negatively regulates the ABA response and drought tolerance by down-regulating AtMPK6-related pathway in Arabidopsis.

    Science.gov (United States)

    Xu, Dong-bei; Chen, Ming; Ma, Ya-nan; Xu, Zhao-shi; Li, Lian-cheng; Chen, Yao-feng; Ma, You-zhi

    2015-01-01

    Heterotrimeric G-proteins are versatile regulators involved in diverse cellular processes in eukaryotes. In plants, the function of G-proteins is primarily associated with ABA signaling. However, the downstream effectors and the molecular mechanisms in the ABA pathway remain largely unknown. In this study, an AGB1 mutant (agb1-2) was found to show enhanced drought tolerance, indicating that AGB1 might negatively regulate drought tolerance in Arabidopsis. Data showed that AGB1 interacted with protein kinase AtMPK6 that was previously shown to phosphorylate AtVIP1, a transcription factor responding to ABA signaling. Our study found that transcript levels of three ABA responsive genes, AtMPK6, AtVIP1 and AtMYB44 (downstream gene of AtVIP1), were significantly up-regulated in agb1-2 lines after ABA or drought treatments. Other ABA-responsive and drought-inducible genes, such as RD29A (downstream gene of AtMYB44), were also up-regulated in agb1-2 lines. Furthermore, overexpression of AtVIP1 resulted in hypersensitivity to ABA at seed germination and seedling stages, and significantly enhanced drought tolerance in transgenic plants. These results suggest that AGB1 was involved in the ABA signaling pathway and drought tolerance in Arabidopsis through down-regulating the AtMPK6, AtVIP1 and AtMYB44 cascade.

  17. A G-protein β subunit, AGB1, negatively regulates the ABA response and drought tolerance by down-regulating AtMPK6-related pathway in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Dong-bei Xu

    Full Text Available Heterotrimeric G-proteins are versatile regulators involved in diverse cellular processes in eukaryotes. In plants, the function of G-proteins is primarily associated with ABA signaling. However, the downstream effectors and the molecular mechanisms in the ABA pathway remain largely unknown. In this study, an AGB1 mutant (agb1-2 was found to show enhanced drought tolerance, indicating that AGB1 might negatively regulate drought tolerance in Arabidopsis. Data showed that AGB1 interacted with protein kinase AtMPK6 that was previously shown to phosphorylate AtVIP1, a transcription factor responding to ABA signaling. Our study found that transcript levels of three ABA responsive genes, AtMPK6, AtVIP1 and AtMYB44 (downstream gene of AtVIP1, were significantly up-regulated in agb1-2 lines after ABA or drought treatments. Other ABA-responsive and drought-inducible genes, such as RD29A (downstream gene of AtMYB44, were also up-regulated in agb1-2 lines. Furthermore, overexpression of AtVIP1 resulted in hypersensitivity to ABA at seed germination and seedling stages, and significantly enhanced drought tolerance in transgenic plants. These results suggest that AGB1 was involved in the ABA signaling pathway and drought tolerance in Arabidopsis through down-regulating the AtMPK6, AtVIP1 and AtMYB44 cascade.

  18. Some methods to regulate low-bias negative differential resistance in σ barrier separating nanoscale molecular transport systems

    Science.gov (United States)

    Shen, Ji-Mei; Liu, Jing; Min, Yi; Zhou, Li-Ping

    2016-12-01

    Using the first-principles method which combines the nonequilibrium Green’s function (NEGF) with density functional theory (DFT), the role of defect, dopant, barrier length and geometric deformation for low-bias negative differential resistance (NDR) in two capped armchair carbon nanotubes (CNTs) sandwiching σ barrier are systematically analyzed. We found that this method can regulate the negative differential resistance (NDR) effects such as current peak and peak position. The adjusting mechanism may originate from orbital interaction and orbital reconstruction. Our calculations try to manipulate the transport characteristics in energy space by simply manipulating the structure in real space, which may promise the potential applications in nanomolecular-electronics in the future.

  19. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  20. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  1. NF-κB (p65) negatively regulates myocardin-induced cardiomyocyte hypertrophy through multiple mechanisms.

    Science.gov (United States)

    Liao, Xing-Hua; Wang, Nan; Zhao, Dong-Wei; Zheng, De-Liang; Zheng, Li; Xing, Wen-Jing; Zhou, Hao; Cao, Dong-Sun; Zhang, Tong-Cun

    2014-12-01

    Myocardin is well known to play a key role in the development of cardiomyocyte hypertrophy. But the exact molecular mechanism regulating myocardin stability and transactivity to affect cardiomyocyte hypertrophy has not been studied clearly. We now report that NF-κB (p65) can inhibit myocardin-induced cardiomyocyte hypertrophy. Then we explore the molecular mechanism of this response. First, we show that p65 can functionally repress myocardin transcriptional activity and also reduce the protein expression of myocardin. Second, the function of myocardin can be regulated by epigenetic modifications. Myocardin sumoylation is known to transactivate cardiac genes, but whether p65 can inhibit SUMO modification of myocardin is still not clear. Our data show that p65 weakens myocardin transcriptional activity through attenuating SUMO modification of myocardin by SUMO1/PIAS1, thereby impairing myocardin-mediated cardiomyocyte hypertrophy. Furthermore, the expression of myocardin can be regulated by several microRNAs, which play important roles in the development and function of the heart and muscle. We next investigated potential role of miR-1 in cardiac hypotrophy. Our results show that p65 can upregulate the level of miR-1 and miR-1 can decrease protein expression of myocardin in cardiac myocytes. Notably, miR-1 expression is also controlled by myocardin, leading to a feedback loop. These data thus provide important and novel insights into the function that p65 inhibits myocardin-mediated cardiomyocyte hypertrophy by downregulating the expression and SUMO modification of myocardin and enhancing the expression of miR-1. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. CHIP promotes human telomerase reverse transcriptase degradation and negatively regulates telomerase activity.

    Science.gov (United States)

    Lee, Ji Hoon; Khadka, Prabhat; Baek, Seung Han; Chung, In Kwon

    2010-12-31

    The maintenance of eukaryotic telomeres requires telomerase, which is minimally composed of a telomerase reverse transcriptase (TERT) and an associated RNA component. Telomerase activity is tightly regulated by expression of human (h) TERT at both the transcriptional and post-translational levels. The Hsp90 and p23 molecular chaperones have been shown to associate with hTERT for the assembly of active telomerase. Here, we show that CHIP (C terminus of Hsc70-interacting protein) physically associates with hTERT in the cytoplasm and regulates the cellular abundance of hTERT through a ubiquitin-mediated degradation. Overexpression of CHIP prevents nuclear translocation of hTERT and promotes hTERT degradation in the cytoplasm, thereby inhibiting telomerase activity. In contrast, knockdown of endogenous CHIP results in the stabilization of cytoplasmic hTERT. However, it does not affect the level of nuclear hTERT and has no effect on telomerase activity and telomere length. We further show that the binding of CHIP and Hsp70 to hTERT inhibits nuclear translocation of hTERT by dissociating p23. However, Hsp90 binding to hTERT was not affected by CHIP overexpression. These results suggest that CHIP can remodel the hTERT-chaperone complexes. Finally, the amount of hTERT associated with CHIP peaks in G(2)/M phases but decreases during S phase, suggesting a cell cycle-dependent regulation of hTERT. Our data suggest that CHIP represents a new pathway for modulating telomerase activity in cancer.

  3. AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora.

    Directory of Open Access Journals (Sweden)

    Dongping Wang

    Full Text Available In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.

  4. AmyR is a novel negative regulator of amylovoran production in Erwinia amylovora.

    Science.gov (United States)

    Wang, Dongping; Korban, Schuyler S; Pusey, P Lawrence; Zhao, Youfu

    2012-01-01

    In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.

  5. Negative regulation of NLRP3 inflammasome by SIRT1 in vascular endothelial cells.

    Science.gov (United States)

    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. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates.

    Directory of Open Access Journals (Sweden)

    Tadayoshi Ikebe

    2010-04-01

    Full Text Available Streptococcal toxic shock syndrome (STSS is a severe invasive infection characterized by the sudden onset of shock and multiorgan failure; it has a high mortality rate. Although a number of studies have attempted to determine the crucial factors behind the onset of STSS, the responsible genes in group A Streptococcus have not been clarified. We previously reported that mutations of csrS/csrR genes, a two-component negative regulator system for multiple virulence genes of Streptococcus pyogenes, are found among the isolates from STSS patients. In the present study, mutations of another negative regulator, rgg, were also found in clinical isolates of STSS patients. The rgg mutants from STSS clinical isolates enhanced lethality and impaired various organs in the mouse models, similar to the csrS mutants, and precluded their being killed by human neutrophils, mainly due to an overproduction of SLO. When we assessed the mutation frequency of csrS, csrR, and rgg genes among S. pyogenes isolates from STSS (164 isolates and non-invasive infections (59 isolates, 57.3% of the STSS isolates had mutations of one or more genes among three genes, while isolates from patients with non-invasive disease had significantly fewer mutations in these genes (1.7%. The results of the present study suggest that mutations in the negative regulators csrS/csrR and rgg of S. pyogenes are crucial factors in the pathogenesis of STSS, as they lead to the overproduction of multiple virulence factors.

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

    Science.gov (United States)

    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.

  8. CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling

    DEFF Research Database (Denmark)

    Abdallah, Basem M.; Figeac, Florence; Larsen, Kenneth H.

    2017-01-01

    We identified the neuroprotein collapsing response mediator protein-4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1-CRMP......5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain-of-function and loss-of-function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4-deficient mice (Crmp4(-/-) ) displayed a 40...

  9. E2F-HDAC complexes negatively regulate the tumor suppressor gene ARHI in breast cancer

    DEFF Research Database (Denmark)

    Lu, Z; Luo, R Z; Peng, H;

    2006-01-01

    to the P2 region of the ARHI promoter and regulate its activity. Sequence analysis and oligonucleotide competition in electrophoretic mobility shift assays identified an A2 fragment containing an E2F-binding site. Using specific antibodies in supershift assays, we have shown that anti-E2F1 and 4 antibodies...... and increased E2F DNA-binding activity. Moreover, chromatin immunoprecipitation experiments revealed that both E2F1 and 4 bind to the ARHI promoter in breast cancer cells in vivo. This binding was reduced when the cells were treated with the histone deacetylase (HDAC) inhibitor--trichostatin A (TSA). When SKBr3...

  10. Distributed Self-regulation Induced by Negative Feedbacks in Ecological and Economic Systems

    CERN Document Server

    Gafiychuk, V V; Ulanowicz, R E; Ulanowicz, Robert E.

    1998-01-01

    We consider an ecological system governed by Lotka-Volterra dynamics and an example of an economic system as a mesomarket with perfect competition. We propose a mechanism for cooperative self-regulation that enables the system under consideration to respond properly to changes in the environment. This mechanism is based on (1) active individual behavior of the system elements at each hierarchical level and (2) self-processing of information caused by the hierarchical organization. It is shown how the proposed mechanism suppresses nonlocal interaction of elements belonging to a particular level as mediated by higher levels.

  11. Caenorhabditis elegans inositol 5-phosphatase homolog negatively regulates inositol 1,4,5-triphosphate signaling in ovulation.

    Science.gov (United States)

    Bui, Yen Kim; Sternberg, Paul W

    2002-05-01

    Ovulation in Caenorhabditis elegans requires inositol 1,4,5-triphosphate (IP(3)) signaling activated by the epidermal growth factor (EGF)-receptor homolog LET-23. We generated a deletion mutant of a type I 5-phosphatase, ipp-5, and found a novel ovulation phenotype whereby the spermatheca hyperextends to engulf two oocytes per ovulation cycle. The temporal and spatial expression of IPP-5 is consistent with its proposed inhibition of IP(3) signaling in the adult spermatheca. ipp-5 acts downstream of let-23, and interacts with let-23-mediated IP(3) signaling pathway genes. We infer that IPP-5 negatively regulates IP(3) signaling to ensure proper spermathecal contraction.

  12. Estrogen Negatively Regulates the Pro-apoptotic Function of Mixed Lineage Kinase 3 in Estrogen Receptor Positive Breast Cancer

    OpenAIRE

    Rangasamy, Velusamy; Mishra, Rajakishore; Mehrotra, Suneet; Sondarva, Gautam; Ray, Rajarshi S.; Rao, Arundhati; Chatterjee,Malay; Rana, Basabi; Rana, Ajay

    2010-01-01

    Estrogen stimulates growth and inhibits apoptosis of breast cancer cells via genomic and non-genomic actions. However, the detailed mechanism by which estrogen inhibits the pro-apoptotic pathways that might impede the normal homeostasis and action of chemotherapeutic drugs in breast cancer cells is not well understood. Here, we report a negative regulation of a pro-apoptotic kinase, Mixed Lineage Kinase 3 (MLK3) by 17β-estradiol (E2) that hinders cytotoxic drug-induced cell death in estrogen ...

  13. TGIF1 is a negative regulator of MLL-rearranged acute myeloid leukemia

    DEFF Research Database (Denmark)

    Willer, Anton; Jakobsen, Janus Schou; Ohlsson, E

    2015-01-01

    Members of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing transcription factors are important downstream effectors of oncogenic fusion proteins involving the mixed lineage leukemia (MLL) gene. A well-characterized member of this protein family is MEIS1, which...... orchestrates a transcriptional program required for the maintenance of MLL-rearranged acute myeloid leukemia (AML). TGIF1/TGIF2 are relatively uncharacterized TALE transcription factors, which, in contrast to the remaining family, have been shown to act as transcriptional repressors. Given the general...... influence the clinical outcome. Collectively, these findings demonstrate that TALE family members can act both positively and negatively on transcriptional programs responsible for leukemic maintenance and provide novel insights into the regulatory gene expression circuitries in MLL-rearranged AML.Leukemia...

  14. BMP signaling negatively regulates bone mass through sclerostin by inhibiting the canonical Wnt pathway.

    Science.gov (United States)

    Kamiya, Nobuhiro; Ye, Ling; Kobayashi, Tatsuya; Mochida, Yoshiyuki; Yamauchi, Mitsuo; Kronenberg, Henry M; Feng, Jian Q; Mishina, Yuji

    2008-11-01

    Bone morphogenetic proteins (BMPs) are known to induce ectopic bone. However, it is largely unknown how BMP signaling in osteoblasts directly regulates endogenous bone. This study investigated the mechanism by which BMP signaling through the type IA receptor (BMPR1A) regulates endogenous bone mass using an inducible Cre-loxP system. When BMPR1A in osteoblasts was conditionally disrupted during embryonic bone development, bone mass surprisingly was increased with upregulation of canonical Wnt signaling. Although levels of bone formation markers were modestly reduced, levels of resorption markers representing osteoclastogenesis were severely reduced, resulting in a net increase in bone mass. The reduction of osteoclastogenesis was primarily caused by Bmpr1a-deficiency in osteoblasts, at least through the RANKL-OPG pathway. Sclerostin (Sost) expression was downregulated by about 90% and SOST protein was undetectable in osteoblasts and osteocytes, whereas the Wnt signaling was upregulated. Treatment of Bmpr1a-deficient calvariae with sclerostin repressed the Wnt signaling and restored normal bone morphology. By gain of Smad-dependent BMPR1A signaling in mice, Sost expression was upregulated and osteoclastogenesis was increased. Finally, the Bmpr1a-deficient bone phenotype was rescued by enhancing BMPR1A signaling, with restoration of osteoclastogenesis. These findings demonstrate that BMPR1A signaling in osteoblasts restrain endogenous bone mass directly by upregulating osteoclastogenesis through the RANKL-OPG pathway, or indirectly by downregulating canonical Wnt signaling through sclerostin, a Wnt inhibitor and a bone mass mediator.

  15. Chondroitin sulfate proteoglycans negatively regulate the positioning of mitochondria and endoplasmic reticulum to distal axons.

    Science.gov (United States)

    Sainath, Rajiv; Armijo-Weingart, Lorena; Ketscheck, Andrea; Xu, Zhuxuan; Li, Shuxin; Gallo, Gianluca

    2017-09-13

    Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit the extension and regeneration of axons. However, the underlying mechanism of action remains poorly understood. Mitochondria and endoplasmic reticulum (ER) are functionally inter-linked organelles important to axon development and maintenance. We report that CSPGs impair the targeting of mitochondria and ER to the growth cones of chicken embryonic sensory axons. The effect of CSPGs on the targeting of mitochondria is blocked by inhibition of the LAR receptor for CSPGs. The regulation of the targeting of mitochondria and ER to the growth cone by CSPGs is due to attenuation of PI3K signaling, which is known to be downstream of LAR receptor activation. Dynactin is a required component of the dynein motor complex that drives the normally occurring retrograde evacuation of mitochondria from growth cones. CSPGs elevate the levels of p150(Glu) dynactin found in distal axons, and inhibition of the interaction of dynactin with dynein increased axon lengths on CSPGs. CSPGs decreased the membrane potential of mitochondria, and pharmacological inhibition of mitochondria respiration at the growth cone independent of manipulation of mitochondria positioning impaired axon extension. Combined inhibition of dynactin and potentiation of mitochondria respiration further increased axon lengths on CSPGs relative to inhibition of dynactin alone. These data reveal that the regulation of the localization of mitochondria and ER to growth cones is a previously unappreciated aspect of the effects of CSPGs on embryonic axons. © 2017 Wiley Periodicals, Inc. Develop Neurobiol, 2017. © 2017 Wiley Periodicals, Inc.

  16. Inhibition of IRF8 Negatively Regulates Macrophage Function and Impairs Cutaneous Wound Healing.

    Science.gov (United States)

    Guo, Yuanyuan; Yang, Zhiyin; Wu, Shan; Xu, Peng; Peng, Yinbo; Yao, Min

    2017-02-01

    The inflammatory response is essential for normal cutaneous wound healing. Macrophages, as critical inflammatory cells, coordinate inflammation and angiogenesis phases during wound healing. It has been reported that the transcription factor interferon regulatory factor 8 (IRF8), a member of the IRF family, plays a critical role in the development and function of macrophages and is associated with inflammation. However, the role of IRF8 in cutaneous wound healing and its underlying mechanism remain elusive. Through immunohistochemical (IHC) staining, we showed that IRF8 is involved in the wound repair process in mice and patients. Furthermore, we ascertain that the repression of IRF8 by small interfering RNA (siRNA) leads to delayed wound healing. To explore the mechanism by which IRF8 impacts wound healing, we observed its effect on macrophage-related mediators by IHC or real-time PCR. The results demonstrated that the inhibition of IRF8 decreases the mRNA expression of inflammatory mediators associated with M1 macrophage (il-1b, il-6, inos, and tnf-a) but no impact on M2 macrophage-related mediators (arg-1, mrc-1, and il-10) and the number of macrophages in the wounds. Furthermore, the inhibition of IRF8 induced apoptosis in the wounds. In summary, this study demonstrates that the down-regulation of IRF8 in the wound leads to impaired wound healing possibly through the regulation of macrophage function and apoptosis in skin wound.

  17. The CKH2/PKL chromatin remodeling factor negatively regulates cytokinin responses in Arabidopsis calli.

    Science.gov (United States)

    Furuta, Kaori; Kubo, Minoru; Sano, Kiyomi; Demura, Taku; Fukuda, Hiroo; Liu, Yao-Guang; Shibata, Daisuke; Kakimoto, Tatsuo

    2011-04-01

    Cytokinins promote cell division and chloroplast development in tissue culture. We previously isolated two mutants of Arabidopsis thaliana, ckh1 (cytokinin-hypersensitive 1) and ckh2, which produce rapidly growing green calli in response to lower levels of cytokinins than those found in the wild type. Here we report that the product of the CKH2 gene is PICKLE, a protein resembling the CHD3 class of SWI/SNF chromatin remodeling factors. We also show that inhibition of histone deacetylase by trichostatin A (TSA) partially substituted for cytokinins, but not for auxin, in the promotion of callus growth, indicating that chromatin remodeling and histone deacetylation are intimately related to cytokinin-induced callus growth. A microarray experiment revealed that either the ckh1 mutation or the ckh2 mutation caused hypersensitivity to cytokinins in terms of gene expression, especially of photosynthesis-related genes. The ckh1 and ckh2 mutations up-regulated nuclear-encoded genes, but not plastid-encoded genes, whereas TSA deregulated both nuclear- and plastid-encoded genes. The ckh1 ckh2 double mutant showed synergistic phenotypes: the callus grew with a green color independently of exogenous cytokinins. A yeast two-hybrid experiment showed protein interaction between CKH1/EER4/AtTAF12b and CKH2/PKL. These results suggest that CKH1/EER4/AtTAF12b and CKH2/PKL may act together on cytokinin-regulated genes.

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

    Energy Technology Data Exchange (ETDEWEB)

    Mayi, Therese Hervee; Rigamonti, Elena [Univ Lille Nord de France, F-59000 Lille (France); INSERM UR1011, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Pattou, Francois [Univ Lille Nord de France, F-59000 Lille (France); Department of Endocrine Surgery, University Hospital, Lille (France); U859 Biotherapies for Diabetes, INSERM, Lille (France); Staels, Bart, E-mail: bart.staels@pasteur-lille.fr [Univ Lille Nord de France, F-59000 Lille (France); INSERM UR1011, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France); Chinetti-Gbaguidi, Giulia [Univ Lille Nord de France, F-59000 Lille (France); INSERM UR1011, F-59000 Lille (France); UDSL, F-59000 Lille (France); Institut Pasteur de Lille, F-59019 Lille (France)

    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.

  19. MicroRNA-146a: A dominant, negative regulator of the innate immune response

    Directory of Open Access Journals (Sweden)

    Stephanie eBooth

    2014-11-01

    Full Text Available MicroRNAs (miRNAs are a class of small non-coding RNA molecules that can play critical roles as regulators of numerous pathways and biological processes including the immune response. Emerging as one of the most important miRNAs to orchestrate immune and inflammatory signaling, often through its recognized target genes, IRAK1 and TRAF6, is microRNA-146a (miR-146a. MiR-146a is one, of a small number of miRNAs, whose expression is strongly induced following challenge of cells with bacterial endotoxin, and prolonged expression has been linked to immune tolerance, implying that it acts as a fine tuning mechanism to prevent an overstimulation of the inflammatory response. In other cells, miR-146a has been shown to play a role in the control of the differentiation of megakaryocytic and monocytic lineages, adaptive immunity and cancer. In this review, we discuss the central role prescribed to miR-146a in innate immunity. We particularly focus on the role played by miR-146a in the regulation and signaling mediated by one of the main pattern recognition receptors, Toll/IL-1 receptors (TLRs. Additionally, we also discuss the role of miR-146a in several classes of autoimmune pathologies where this miRNA has been shown to be dysregulated, as well as its potential role in the pathobiology of neurodegenerative diseases.

  20. FGF-23 Is a Negative Regulator of Prenatal and Postnatal Erythropoiesis*

    Science.gov (United States)

    Coe, Lindsay M.; Madathil, Sangeetha Vadakke; Casu, Carla; Lanske, Beate; Rivella, Stefano; Sitara, Despina

    2014-01-01

    Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23−/−) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD. PMID:24509850

  1. Negative regulation of bone formation by the transmembrane Wnt antagonist Kremen-2.

    Directory of Open Access Journals (Sweden)

    Jochen Schulze

    Full Text Available Wnt signalling is a key pathway controlling bone formation in mice and humans. One of the regulators of this pathway is Dkk1, which antagonizes Wnt signalling through the formation of a ternary complex with the transmembrane receptors Krm1/2 and Lrp5/6, thereby blocking the induction of Wnt signalling by the latter ones. Here we show that Kremen-2 (Krm2 is predominantly expressed in bone, and that its osteoblast-specific over-expression in transgenic mice (Col1a1-Krm2 results in severe osteoporosis. Histomorphometric analysis revealed that osteoblast maturation and bone formation are disturbed in Col1a1-Krm2 mice, whereas bone resorption is increased. In line with these findings, primary osteoblasts derived from Col1a1-Krm2 mice display a cell-autonomous differentiation defect, impaired canonical Wnt signalling and decreased production of the osteoclast inhibitory factor Opg. To determine whether the observed effects of Krm2 on bone remodeling are physiologically relevant, we analyzed the skeletal phenotype of 24 weeks old Krm2-deficient mice and observed high bone mass caused by a more than three-fold increase in bone formation. Taken together, these data identify Krm2 as a regulator of bone remodeling and raise the possibility that antagonizing KRM2 might prove beneficial in patients with bone loss disorders.

  2. FGF-23 is a negative regulator of prenatal and postnatal erythropoiesis.

    Science.gov (United States)

    Coe, Lindsay M; Madathil, Sangeetha Vadakke; Casu, Carla; Lanske, Beate; Rivella, Stefano; Sitara, Despina

    2014-04-04

    Abnormal blood cell production is associated with chronic kidney disease (CKD) and cardiovascular disease (CVD). Bone-derived FGF-23 (fibroblast growth factor-23) regulates phosphate homeostasis and bone mineralization. Genetic deletion of Fgf-23 in mice (Fgf-23(-/-)) results in hypervitaminosis D, abnormal mineral metabolism, and reduced lymphatic organ size. Elevated FGF-23 levels are linked to CKD and greater risk of CVD, left ventricular hypertrophy, and mortality in dialysis patients. However, whether FGF-23 is involved in the regulation of erythropoiesis is unknown. Here we report that loss of FGF-23 results in increased hematopoietic stem cell frequency associated with increased erythropoiesis in peripheral blood and bone marrow in young adult mice. In particular, these hematopoietic changes are also detected in fetal livers, suggesting that they are not the result of altered bone marrow niche alone. Most importantly, administration of FGF-23 in wild-type mice results in a rapid decrease in erythropoiesis. Finally, we show that the effect of FGF-23 on erythropoiesis is independent of the high vitamin D levels in these mice. Our studies suggest a novel role for FGF-23 in erythrocyte production and differentiation and suggest that elevated FGF-23 levels contribute to the pathogenesis of anemia in patients with CKD and CVD.

  3. PTPN14 interacts with and negatively regulates the oncogenic function of YAP.

    Science.gov (United States)

    Liu, X; Yang, N; Figel, S A; Wilson, K E; Morrison, C D; Gelman, I H; Zhang, J

    2013-03-07

    The Hippo signaling pathway regulates cellular proliferation and survival, thus exerting profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP, a transcriptional co-activator amplified in mouse and human cancers where it promotes epithelial-to-mesenchymal transition and malignant transformation. Here, we report a novel regulatory mechanism for the YAP oncogenic function via direct interaction with non-receptor tyrosine phosphatase 14 (PTPN14) through the WW domain of YAP and the PPxY domain of PTPN14. We also found that YAP is a direct substrate of PTPN14. In addition, luciferase reporter assay showed that the inhibition of the YAP transcriptional co-activator function by PTPN14 is mediated through their protein interactions and may result from an increase in the inactive cytoplasmic form of YAP. Last, knockdown of PTPN14 induces the nuclear retention of YAP and increases the YAP-dependent cell migration. In summary, our results indicate a potential regulatory role of PTPN14 on YAP and demonstrate a novel mechanism in YAP regulation.

  4. Tolloid-like 1 is negatively regulated by stress and glucocorticoids.

    Science.gov (United States)

    Tamura, Goichiro; Olson, Dawne; Miron, Joel; Clark, Timothy G

    2005-12-14

    Glucocorticoids affect a variety of tissues to enable the organism to adapt to the stress. Hippocampal neurons contain glucocorticoid receptors and respond to elevated glucocorticoid levels by down-regulating the HPA axis. Chronically, however, stress is deleterious to hippocampal neurons. Chronically elevated levels of glucocorticoids result in a decrease in the number of dendritic spines, reduced axonal growth and synaptogenesis, and decreased neurogenesis in the hippocampus. Tolloid-like 1 (Tll-1) is a metalloprotease that potentiates the activity of the bone morphogenetic proteins (BMPs). Neurogenesis in the hippocampus of both developing and adult mammals requires BMPs. In this study, we demonstrate that Tll-1 expression is increased in mice that have increased neurogenesis. The Tll-1 promoter contains glucocorticoid response elements which are capable of binding to purified glucocorticoid receptor. Glucocorticoids decrease Tll-1 expression in vitro. Finally, prenatal stress leads to a decrease in Tll-1 mRNA expression in the hippocampus of adult female mice that is not observed in adult male mice indicating that Tll-1 expression is differentially regulated in males and females. The results of this study indicate that Tll-1 is responsive to glucocorticoids and this mechanism might influence neurogenesis in the hippocampus.

  5. VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.

    Science.gov (United States)

    Yu, Xiao; Chen, Shuliang; Hou, Panpan; Wang, Min; Chen, Yu; Guo, Deyin

    2015-04-03

    Eukaryotic cellular and most viral RNAs carry a 5'-terminal cap structure, a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2'-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda

    2007-01-01

    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  7. miR-340 and ZEB1 negative feedback loop regulates TGF-β- mediated breast cancer progression

    Science.gov (United States)

    Xie, Ye-Gong; Wang, Jie; Mao, Jie-Fei; Zhang, Bin; Wang, Xin; Cao, Xu-Chen

    2016-01-01

    MicroRNAs act as key regulators in carcinogenesis and progression in various cancers. In present study, we explored the role of miR-340 in the breast cancer progression. Our results showed that overexpression of miR-340 inhibits breast cancer cell proliferation and invasion, whereas depletion of miR-340 promotes breast cancer progression. Molecularly, ZEB1 was identified as a target gene of miR-340 and miR-340 suppressed the expression of ZEB1 by directly binding to the 3′-UTR of ZEB1. Furthermore, ZEB1 transcriptionally suppresses miR-340 expression. The negative feedback loop regulated TGF-β-mediated breast cancer progression. In conclusion, our data suggested that miR-340 acted as a tumor suppressor in breast cancer progression. PMID:27036021

  8. CPC,a Single-Repeat R3 MYB,Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Hui-Fen Zhu; Karen Fitzsimmons; Abha Khandelwal; Robert G.Kranz

    2009-01-01

    Single-repeat R3 MYB transcription factors like CPC (CAPRICE) are known to play roles in developmental processes such as root hair differentiation and trichome initiation.However,none of the six Arabidopsis single-repeat R3 MYB members has been reported to regulate flavonoid biosynthesis.We show here that CPC is a negative regulator of anthocyanin biosynthesis.In the process of using CPC to test GAL4-dependent driver lines,we observed a repression of anthocyanin synthesis upon GAL4-mediated CPC overexpression,We demonstrated that this is not due to an increase in nutrient uptake because of more root hairs.Rather,CPC expression level tightly controls anthocyanin accumulation.Microarray analysis on the whole genome showed that,of 37 000 features tested,85 genes are repressed greater than three-fold by CPC overexpression.Of these 85,seven are late anthocyanin biosynthesis genes.Also,anthocyanin synthesis genes were shown to be down-regulated in 35S::CPC overexpression plants.Transient expression results suggest that CPC competes with the R2R3-MYB transcription factor PAP1/2,which is an activator of anthocyanin biosynthesis genes.This report adds anthocyanin biosynthesis to the set of programs that are under CPC control,indicating that this regulator is not only for developmental programs (e.g.root hairs,trichomes),but can influence anthocyanin pigment synthesis.

  9. The TAM family receptor tyrosine kinase TYRO3 is a negative regulator of type 2 immunity

    Science.gov (United States)

    Chan, Pamela Y.; Carrera Silva, Eugenio A.; De Kouchkovsky, Dimitri; Joannas, Leonel D.; Hao, Liming; Hu, Donglei; Huntsman, Scott; Eng, Celeste; Licona-Limón, Paula; Weinstein, Jason S.; Herbert, De’Broski R.; Craft, Joseph E.; Flavell, Richard A.; Repetto, Silvia; Correale, Jorge; Burchard, Esteban G.; Torgerson, Dara G.; Ghosh, Sourav; Rothlin, Carla V.

    2016-01-01

    Host responses against metazoan parasites or an array of environmental substances elicit type 2 immunity. Despite its protective function, type 2 immunity also drives allergic diseases. The mechanisms that regulate the magnitude of the type 2 response remain largely unknown. Here, we show that genetic ablation of a receptor tyrosine kinase encoded by Tyro3 in mice or the functional neutralization of its ortholog in human dendritic cells resulted in enhanced type 2 immunity. Furthermore, the TYRO3 agonist PROS1 was induced in T cells by the quintessential type 2 cytokine, interleukin-4. T cell–specific Pros1 knockouts phenocopied the loss of Tyro3. Thus, a PROS1-mediated feedback from adaptive immunity engages a rheostat, TYRO3, on innate immune cells to limit the intensity of type 2 responses. PMID:27034374

  10. The Slx5-Slx8 complex affects sumoylation of DNA repair proteins and negatively regulates recombination

    DEFF Research Database (Denmark)

    Burgess, Rebecca C; Rahman, Sadia; Lisby, Michael

    2007-01-01

    Recombination is important for repairing DNA lesions, yet it can also lead to genomic rearrangements. This process must be regulated, and recently, sumoylation-mediated mechanisms were found to inhibit Rad51-dependent recombination. Here, we report that the absence of the Slx5-Slx8 complex, a newly...... identified player in the SUMO (small ubiquitin-like modifier) pathway, led to increased Rad51-dependent and Rad51-independent recombination. The increases were most striking during S phase, suggesting an accumulation of DNA lesions during replication. Consistent with this view, Slx8 protein localized...... propose that, during replication, the Slx5-Slx8 complex helps prevent DNA lesions that are acted upon by recombination. In addition, the complex inhibits Rad51-independent recombination via modulating the sumoylation of DNA repair proteins....

  11. Cullin4B/E3-ubiquitin ligase negatively regulates -catenin

    Indian Academy of Sciences (India)

    Rachana Tripathi; Satya Keerthi Kota; Usha K Srinivas

    2007-09-01

    -catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, -catenin is targeted to ubiquitin–proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in -catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased -catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and -catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of -catenin levels.

  12. Amygdalin Regulates Apoptosis and Adhesion in Hs578T Triple-Negative Breast Cancer Cells.

    Science.gov (United States)

    Lee, Hye Min; Moon, Aree

    2016-01-01

    Amygdalin, D-mandelonitrile-β-D-glucoside-6-β-glucoside, belongs to aromatic cyanogenic glycoside group derived from rosaceous plant seed. Mounting evidence has supported the anti-cancer effects of amygdalin. However, whether amygdalin indeed acts as an anti-tumor agent against breast cancer cells is not clear. The present study aimed to investigate the effect of amygdalin on the proliferation of human breast cancer cells. Here, we show that amygdalin exerted cytotoxic activities on estrogen receptors (ER)-positive MCF7 cells, and MDA-MB-231 and Hs578T triple-negative breast cancer (TNBC) cells. Amygdalin induced apoptosis of Hs578T TNBC cells. Amygdalin downregulated B-cell lymphoma 2 (Bcl-2), upregulated Bcl-2-associated X protein (Bax), activated of caspase-3 and cleaved poly ADP-ribose polymerase (PARP). Amygdalin activated a pro-apoptotic signaling molecule p38 mitogen-activated protein kinases (p38 MAPK) in Hs578T cells. Treatment of amygdalin significantly inhibited the adhesion of Hs578T cells, in which integrin α5 may be involved. Taken together, this study demonstrates that amygdalin induces apoptosis and inhibits adhesion of breast cancer cells. The results suggest a potential application of amygdalin as a chemopreventive agent to prevent or alleviate progression of breast cancer, especially TNBC.

  13. Ubiquinol (QH(2)) functions as a negative regulator of purine nucleotide inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein.

    Science.gov (United States)

    Woyda-Ploszczyca, Andrzej; Jarmuszkiewicz, Wieslawa

    2011-01-01

    We compared the influence of different adenine and guanine nucleotides on the free fatty acid-induced uncoupling protein (UCP) activity in non-phosphorylating Acanthamoeba castellanii mitochondria when the membranous ubiquinone (Q) redox state was varied. The purine nucleotides exhibit an inhibitory effect in the following descending order: GTP>ATP>GDP>ADP≫GMP>AMP. The efficiency of guanine and adenine nucleotides to inhibit UCP-sustained uncoupling in A. castellanii mitochondria depends on the Q redox state. Inhibition by purine nucleotides can be increased with decreasing Q reduction level (thereby ubiquinol, QH₂ concentration) even with nucleoside monophosphates that are very weak inhibitors at the initial respiration. On the other hand, the inhibition can be alleviated with increasing Q reduction level (thereby QH₂ concentration). The most important finding was that ubiquinol (QH₂) but not oxidised Q functions as a negative regulator of UCP inhibition by purine nucleotides. For a given concentration of QH₂, the linoleic acid-induced GTP-inhibited H(+) leak was the same for two types of A. castellanii mitochondria that differ in the endogenous Q content. When availability of the inhibitor (GTP) or the negative inhibition modulator (QH₂) was changed, a competitive influence on the UCP activity was observed. QH₂ decreases the affinity of UCP for GTP and, vice versa, GTP decreases the affinity of UCP for QH₂. These results describe the kinetic mechanism of regulation of UCP affinity for purine nucleotides by endogenous QH₂ in the mitochondria of a unicellular eukaryote.

  14. Cbl participates in shikonin-induced apoptosis by negatively regulating phosphoinositide 3-kinase/protein kinase B signaling.

    Science.gov (United States)

    Qu, Dan; Xu, Xiao-Man; Zhang, Meng; Jiang, Ting-Shu; Zhang, Yi; Li, Sheng-Qi

    2015-07-01

    Shikonin, a naturally occurring naphthoquinone, exhibits anti-tumorigenic activity. However, its precise mechanisms of action have remained elusive. In the present study, the involvement in the action of shikonin of the ubiquitin ligases Cbl-b and c-Cbl, which are negative regulators of phosphoinositide 3-kinase (PI3K) activation, was investigated. Shikonin was observed to reduce cell viability and induce apoptosis and G2/M phase arrest in lung cancer cells. In addition, shikonin increased the protein levels of B-cell lymphoma 2 (Bcl-2)-associated X and p53 and reduced those of Bcl-2. Additionally, shikonin inhibited PI3k/Akt activity and upregulated Cbl protein expression. In addition, a specific inhibitor of PI3K, LY294002, was observed to have a synergistic effect on the proliferation inhibition and apoptotic induction of A549 cells with shikonin. In conclusion, the results of the present study suggested that Cbl proteins promote shikonin-induced apoptosis by negatively regulating PI3K/Akt signaling in lung cancer cells.

  15. Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX

    Energy Technology Data Exchange (ETDEWEB)

    Yamagata, Kazutsune, E-mail: kyamagat@ncc.go.jp [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Kitabayashi, Issay [Department of Molecular Oncology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan)

    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.

  16. MiR-21 promoted proliferation and migration in hepatocellular carcinoma through negative regulation of Navigator-3

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Zhipeng, E-mail: dr_zpwang@163.com [The Digestive and Vascural Surgery Center, the First Affiliated Hospital of Xinjiang Medical University, Urumqi 830054, Xinjiang Uygur Autonomous Region (China); Yang, Huan [The Department of Liver and Biliary Pancreatic Surgery, Affiliated Tumor Hospital of Xinjiang Medical University, Urumqi 830000, Xinjiang Uygur Autonomous Region (China); Ren, Lei [The Department of General Surgery, Branching Hospital of the First People' s Hospital of Urumqi, 830000, Xinjiang Uygur Autonomous Region (China)

    2015-09-04

    MicroRNA-21 (miR-21) has been well-established and found to be over-expressed in various human cancers and has been associated with hepatocellular carcinoma (HCC) progression. However, the underlying mechanism of miR-21 involvement in the development and progression of HCC remains to be understood. In the present study, we firstly identified that the Navigator-3 (NAV-3) gene as a novel direct target of miR-21. Knock-down of NAV-3 using shRNA can rescue the effects of anti-miR-21 inhibitor in HCC cell lines, whereas re-expression of miR-21 using transfection with miR-21 mimics phenocopied the NAV-3 knock-down model. Additionally, miR-21 levels inversely correlated with NAV-3 both in HCC cells and tissues. Knock-down of NAV-3 promoted both the proliferation and migration in HCC cells. Together, our findings suggest an important role for miR-21 in the progression of HCC, which negatively regulated Navigator-3 in the migration of HCC. - Highlights: • Navigator-3 (NAV-3) suppresses proliferation, migration and tumorigenesis of HCC cells. • NAV-3 was a novel target of miR-21. • MiR-21 negatively regulates NAV-3 in HCC.

  17. Dihydropyrimidine dehydrogenase (DPD) expression is negatively regulated by certain microRNAs in human lung tissues.

    Science.gov (United States)

    Hirota, Takeshi; Date, Yuko; Nishibatake, Yu; Takane, Hiroshi; Fukuoka, Yasushi; Taniguchi, Yuuji; Burioka, Naoto; Shimizu, Eiji; Nakamura, Hiroshige; Otsubo, Kenji; Ieiri, Ichiro

    2012-07-01

    Dihydropyrimidine dehydrogenase (DPD) is important to the antitumor effect of 5-fluorouracil (5-FU). DPD gene (DPYD) expression in tumors is correlated with sensitivity to 5-FU. Because the 5-FU accumulated in cancer cells is also rapidly converted into inactivated metabolites through catabolic pathways mediated by DPD, high DPD activity in cancer cells is an important determinant of the response to 5-FU. DPD activity is highly variable and reduced activity causes a high risk of 5-FU toxicity. Genetic variation in DPYD has been proposed as the main factor responsible for the variation in DPD activity. However, only a small proportion of the activity of DPD can be explained by DPYD mutations. In this study, we found that DPYD is a target of the following microRNAs (miRNA): miR-27a, miR-27b, miR-134, and miR-582-5p. In luciferase assays with HepG2 cells, the overexpression of these miRNAs was associated with significantly decreased reporter activity in a plasmid containing the 3'-UTR of DYPD mRNA. The level of DPD protein in MIAPaca-2 cells was also significantly decreased by the overexpression of these four miRNAs. The results suggest that miR-27a, miR-27b, miR-134, and miR-582-5p post-transcriptionally regulate DPD protein expression. The levels of miRNAs in normal lung tissue and lung tumors were compared; miR-27b and miR-134 levels were significantly lower in the tumors than normal tissue (3.64 ± 4.02 versus 9.75 ± 6.58 and 0.64 ± 0.75 versus 1.48 ± 1.39). DPD protein levels were significantly higher in the tumors. Thus, the decreased expression of miR-27b would be responsible for the high levels of DPD protein. This study is the first to show that miRNAs regulate the DPD protein, and provides new insight into 5-FU-based chemotherapy.

  18. Induction of DKK1 by ox-LDL negatively regulates intracellular lipid accumulation in macrophages.

    Science.gov (United States)

    Zhang, Yu; Ge, Cheng; Wang, Lin; Liu, Xinxin; Chen, Yifei; Li, Mengmeng; Zhang, Mei

    2015-01-01

    Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux.

  19. MAGED1 is a negative regulator of bone remodeling in mice.

    Science.gov (United States)

    Liu, Mei; Xu, Lijuan; Ma, Xiao; Xu, Jiake; Wang, Jing; Xian, Mengmeng; Zhou, Xiaotian; Wang, Min; Wang, Fang; Qin, An; Pan, Qiuhui; Wen, Chuanjun

    2015-10-01

    Melanoma antigen family D1 (MAGED1), an important adaptor protein, has been shown to ubiquitously express and play critical roles in many aspects of cellular events and physiological functions. However, its role in bone remodeling remains unknown. We, therefore, analyzed the bone phenotype of Maged1-deficient mice. Maged1-deficient mice displayed a significant osteoporotic phenotype with a marked decrease in bone density and deterioration of trabecular architecture. Histomorphometric analysis demonstrated an increased mineral apposition rate as well as increased osteoclast number and surface in Maged1 knockout mice. At the cellular level, Maged1-deficient osteoblasts exhibited an increased proliferation rate and accelerated differentiation. MAGED1 deficiency also caused a promotion in osteoclastogenesis, and that was attributed to the cell autonomous acceleration of differentiation in osteoclasts and an increased receptor activator of NF-κB ligand/osteoprotegerin ratio, a major index of osteoclastogenesis, in osteoblasts. Thus, we identified MAGED1 as a novel regulator of osteoblastogenesis, osteoclastogenesis, and bone remodeling in a mouse model.

  20. Primary Cilia Negatively Regulate Melanogenesis in Melanocytes and Pigmentation in a Human Skin Model

    Science.gov (United States)

    Kim, Eun Sung; Park, So Jung; Bae, Il-Hong; Jo, Yoon Kyung; Jeong, In Young; Kim, Hyoung-June; Lee, Youngjin; Park, Hea Chul; Jeon, Hong Bae; Kim, Ki Woo; Lee, Tae Ryong; Cho, Dong-Hyung

    2016-01-01

    The primary cilium is an organelle protruding from the cell body that senses external stimuli including chemical, mechanical, light, osmotic, fluid flow, and gravitational signals. Skin is always exposed to the external environment and responds to external stimuli. Therefore, it is possible that primary cilia have an important role in skin. Ciliogenesis was reported to be involved in developmental processes in skin, such as keratinocyte differentiation and hair formation. However, the relation between skin pigmentation and primary cilia is largely unknown. Here, we observed that increased melanogenesis in melanocytes treated with a melanogenic inducer was inhibited by a ciliogenesis inducer, cytochalasin D, and serum-free culture. However, these inhibitory effects disappeared in GLI2 knockdown cells. In addition, activation of sonic hedgehog (SHH)-smoothened (Smo) signaling pathway by a Smo agonist, SAG inhibited melanin synthesis in melanocytes and pigmentation in a human skin model. On the contrary, an inhibitor of primary cilium formation, ciliobrevin A1, activated melanogenesis in melanocytes. These results suggest that skin pigmentation may be regulated partly by the induction of ciliogenesis through Smo-GLI2 signaling. PMID:27941997

  1. Hid, Rpr and Grim negatively regulate DIAP1 levels through distinct mechanisms.

    Science.gov (United States)

    Yoo, Soon Ji; Huh, Jun R; Muro, Israel; Yu, Hong; Wang, Lijuan; Wang, Susan L; Feldman, R M Renny; Clem, Rollie J; Müller, H-Arno J; Hay, Bruce A

    2002-06-01

    Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Here we show that Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis.

  2. Lrrc75b is a novel negative regulator of C2C12 myogenic differentiation

    Science.gov (United States)

    Zhong, Yuechun; Zou, Liyi; Wang, Zonggui; Pan, Yaqiong; Dai, Zhong; Liu, Xinguang; Cui, Liao; Zuo, Changqing

    2016-01-01

    Many transcription factors and signaling molecules involved in the guidance of myogenic differentiation have been investigated in previous studies. However, the precise molecular mechanisms of myogenic differentiation remain largely unknown. In the present study, by performing a meta-analysis of C2C12 myogenic differentiation microarray data, we found that leucine-rich repeat-containing 75B (Lrrc75b), also known as AI646023, a molecule of unknown biological function, was downregulated during C2C12 myogenic differentiation. The knockdown of Lrrc75b using specific siRNA in C2C12 myoblasts markedly enhanced the expression of muscle-specific myogenin and increased myoblast fusion and the myotube diameter. By contrast, the adenovirus-mediated overexpression of Lrrc75b in C2C12 cells markedly inhibited myoblast differentiation accompanied by a decrease in myogenin expression. In addition, the phosphorylation of extracellular signal-regulated kinase 1/2 (Erk1/2) was suppressed in the cells in which Lrrc75b was silenced. Taken together, our results demonstrate that Lrrc75b is a novel suppressor of C2C12 myogenic differentiation by modulating myogenin and Erk1/2 signaling. PMID:27633041

  3. Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion.

    Science.gov (United States)

    Liu, Kai; Jian, Youli; Sun, Xiaojuan; Yang, Chengkui; Gao, Zhiyang; Zhang, Zhili; Liu, Xuezhao; Li, Yang; Xu, Jing; Jing, Yudong; Mitani, Shohei; He, Sudan; Yang, Chonglin

    2016-01-18

    Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.

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

    Science.gov (United States)

    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. Positive and Negative Regulation of Cellular Immune Responses in Physiologic Conditions and Diseases

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    S. Viganò

    2012-01-01

    Full Text Available The immune system has evolved to allow robust responses against pathogens while avoiding autoimmunity. This is notably enabled by stimulatory and inhibitory signals which contribute to the regulation of immune responses. In the presence of a pathogen, a specific and effective immune response must be induced and this leads to antigen-specific T-cell proliferation, cytokines production, and induction of T-cell differentiation toward an effector phenotype. After clearance or control of the pathogen, the effector immune response must be terminated in order to avoid tissue damage and chronic inflammation and this process involves coinhibitory molecules. When the immune system fails to eliminate or control the pathogen, continuous stimulation of T cells prevents the full contraction and leads to the functional exhaustion of effector T cells. Several evidences both in vitro and in vivo suggest that this anergic state can be reverted by blocking the interactions between coinhibitory molecules and their ligands. The potential to revert exhausted or inactivated T-cell responses following selective blocking of their function made these markers interesting targets for therapeutic interventions in patients with persistent viral infections or cancer.

  6. A negative regulation loop of long noncoding RNA HOTAIR and p53 in non-small-cell lung cancer

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    Zhai N

    2016-09-01

    Full Text Available Nailiang Zhai,1 Yongfu Xia,1 Rui Yin,2 Jinping Liu,3 Fuquan Gao1 1Department of Respiratory Medicine, Affiliated Hospital of Binzhou Medical University, 2Department of Respiratory Medicine, People’s Hospital of Binzhou City, 3Department of Pharmacology, Binzhou Medical University, Binzhou, Shandong, People’s Republic of China Abstract: Non-small-cell lung cancer (NSCLC is one of the leading causes of cancer-related death worldwide, and the 5-year survival rate is still low despite advances in diagnosis and therapeutics. A long noncoding RNA (lncRNA HOX antisense intergenic RNA (HOTAIR has been revealed to play important roles in NSCLC carcinogenesis but the detailed mechanisms are still unclear. In the current study, we aimed to investigate the regulation between the lncRNA HOTAIR and p53 in the NSCLC patient samples and cell lines. Our results showed that HOTAIR expression was significantly higher in the cancer tissues than that in the adjacent normal tissue, and was negatively correlated with p53 functionality rather than expression. When p53 was overexpressed in A549 cells, the lncRNA HOTAIR expression was downregulated, and the cell proliferation rate and cell invasion capacity decreased as a consequence. We identified two binding sites of p53 on the promoter region of HOTAIR, where the p53 protein would bind to and suppress the HOTAIR mRNA transcription. Inversely, overexpression of lncRNA HOTAIR inhibited the expression of p53 in A549 cells. Mechanistic studies revealed that HOTAIR modified the promoter of p53 and enhanced histone H3 lysine 27 trimethylation (H3K27me3. These studies identified a specific negative regulation loop of lncRNA HOTAIR and p53 in NSCLC cells, which revealed a new understanding of tumorigenesis in p53 dysfunction NSCLC cells. Keywords: NSCLC, LncRNA HOTAIR, p53, negative loop

  7. Negative Regulation of Ectoine Uptake and Catabolism in Sinorhizobium meliloti: Characterization of the EhuR Gene.

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    Yu, Qinli; Cai, Hanlin; Zhang, Yanfeng; He, Yongzhi; Chen, Lincai; Merritt, Justin; Zhang, Shan; Dong, Zhiyang

    2017-01-01

    Ectoine has osmoprotective effects on Sinorhizobium meliloti that differ from its effects in other bacteria. Ectoine does not accumulate in S. meliloti cells; instead, it is degraded. The products of the ehuABCD-eutABCDE operon were previously discovered to be responsible for the uptake and catabolism of ectoine in S. meliloti However, the mechanism by which ectoine is involved in the regulation of the ehuABCD-eutABCDE operon remains unclear. The ehuR gene, which is upstream of and oriented in the same direction as the ehuABCD-eutABCDE operon, encodes a member of the MocR/GntR family of transcriptional regulators. Quantitative reverse transcription-PCR and promoter-lacZ reporter fusion experiments revealed that EhuR represses transcription of the ehuABCD-eutABCDE operon, but this repression is inhibited in the presence of ectoine. Electrophoretic mobility shift assays and DNase I footprinting assays revealed that EhuR bound specifically to the DNA regions overlapping the -35 region of the ehuA promoter and the +1 region of the ehuR promoter. Surface plasmon resonance assays further demonstrated direct interactions between EhuR and the two promoters, although EhuR was found to have higher affinity for the ehuA promoter than for the ehuR promoter. In vitro, DNA binding by EhuR could be directly inhibited by a degradation product of ectoine. Our work demonstrates that EhuR is an important negative transcriptional regulator involved in the regulation of ectoine uptake and catabolism and is likely regulated by one or more end products of ectoine catabolism.

  8. Tumor microenvironment tenascin-C promotes glioblastoma invasion and negatively regulates tumor proliferation.

    Science.gov (United States)

    Xia, Shuli; Lal, Bachchu; Tung, Brian; Wang, Shervin; Goodwin, C Rory; Laterra, John

    2016-04-01

    Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults. Recent research on cancer stroma indicates that the brain microenvironment plays a substantial role in tumor malignancy and treatment responses to current antitumor therapy. In this work, we have investigated the effect of alterations in brain tumor extracellular matrix tenascin-C (TNC) on brain tumor growth patterns including proliferation and invasion. Since intracranial xenografts from patient-derived GBM neurospheres form highly invasive tumors that recapitulate the invasive features demonstrated in human patients diagnosed with GBM, we studied TNC gain-of-function and loss-of function in these GBM neurospheres in vitro and in vivo. TNC loss-of-function promoted GBM neurosphere cell adhesion and actin cytoskeleton organization. Yet, TNC loss-of-function or exogenous TNC had no effect on GBM neurosphere cell growth in vitro. In animal models, decreased TNC in the tumor microenvironment was accompanied by decreased tumor invasion and increased tumor proliferation, suggesting that TNC regulates the "go-or-grow" phenotypic switch of glioma in vivo. We demonstrated that decreased TNC in the tumor microenvironment modulated behaviors of stromal cells including endothelial cells and microglia, resulting in enlarged tumor blood vessels and activated microglia in tumors. We further demonstrated that tumor cells with decreased TNC expression are sensitive to anti-proliferative treatment in vitro. Our findings suggest that detailed understanding of how TNC in the tumor microenvironment influences tumor behavior and the interactions between tumor cells and surrounding nontumor cells will benefit novel combinatory antitumor strategies to treat malignant brain tumors. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Positive and negative regulation of Gli activity by Kif7 in the zebrafish embryo.

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    Ashish Kumar Maurya

    Full Text Available Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles.

  10. Positive and negative regulation of Gli activity by Kif7 in the zebrafish embryo.

    Science.gov (United States)

    Maurya, Ashish Kumar; Ben, Jin; Zhao, Zhonghua; Lee, Raymond Teck Ho; Niah, Weixin; Ng, Ashley Shu Mei; Iyu, Audrey; Yu, Weimiao; Elworthy, Stone; van Eeden, Fredericus J M; Ingham, Philip William

    2013-01-01

    Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles.

  11. PGRP negatively regulates NOD-mediated cytokine production in rainbow trout liver cells.

    Science.gov (United States)

    Jang, Ju Hye; Kim, Hyun; Jang, Mi Jung; Cho, Ju Hyun

    2016-12-19

    Pattern-recognition receptors (PRRs) initiate innate immunity via pathogen recognition. Recent studies suggest that signalling pathways downstream of different PRRs and their crosstalk effectively control immune responses. However, the cross-regulation among PRRs and its effects have yet to be fully described in fish. Here, we examined the crosstalk between OmPGRP-L1, a long form of PGRP in rainbow trout, and other PRRs during pathogenic infections. OmPGRP-L1 expression was increased in RTH-149 cells by iE-DAP and MDP, which are agonists of NOD1 and NOD2, respectively. The silencing of NOD1 and NOD2 specifically inhibited the upregulation of OmPGRP-L1 expression induced by their cognate ligands. Suppression of RIP2 and NF-κB activation prevented the induction of OmPGRP-L1 expression. An in silico analysis and electrophoretic mobility shift assay revealed that the promoter of OmPGRP-L1 has NF-κB binding sites, suggesting that OmPGRP-L1 is produced through the NOD-RIP2-NF-κB signalling pathway. Loss-of-function and gain-of-function experiments indicated that OmPGRP-L1 downregulates the induction of NOD-mediated pro-inflammatory cytokine expression. Mechanistically, secreted OmPGRP-L1 inhibited the activation of the NOD-induced NF-κB pathway via downregulation of TAK1 and IκBα phosphorylation through A20 expression. Our data demonstrate that OmPGRP-L1 and NODs might play interdependent roles in the inflammatory response to bacterial infections in rainbow trout.

  12. Aryl hydrocarbon receptors in osteoclast lineage cells are a negative regulator of bone mass.

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    Tai-yong Yu

    Full Text Available Aryl hydrocarbon receptors (AhRs play a critical role in various pathological and physiological processes. Although recent research has identified AhRs as a key contributor to bone metabolism following studies in systemic AhR knockout (KO or transgenic mice, the cellular and molecular mechanism(s in this process remain unclear. In this study, we explored the function of AhR in bone metabolism using AhR(RANKΔOc/ΔOc (RANK(Cre/+;AhR(flox/flox mice. We observed enhanced bone mass together with decreased resorption in both male and female 12 and 24-week-old AhR(RANKΔOc/ΔOc mice. Control mice treated with 3-methylcholanthrene (3MC, an AhR agonist, exhibited decreased bone mass and increased bone resorption, whereas AhR(CtskΔOc/ΔOc (Ctsk(Cre/+;AhR(flox/flox mice injected with 3MC appeared to have a normal bone phenotype. In vitro, bone marrow-derived macrophages (BMDMs from AhR(RANKΔOc/ΔOc mice exhibited impaired osteoclastogenesis and repressed differentiation with downregulated expression of B lymphocyte-induced maturation protein 1 (Blimp1, and cytochrome P450 genes Cyp1b1 and Cyp1a2. Collectively, our results not only demonstrated that AhR in osteoclast lineage cells is a physiologically relevant regulator of bone resorption, but also highlighted the need for further studies on the skeletal actions of AhR inhibitors in osteoclast lineage cells commonly associated with bone diseases, especially diseases linked to environmental pollutants known to induce bone loss.

  13. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

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    Sayaka Aizawa

    Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

  14. A negative regulator encoded by a rice WRKY gene represses both abscisic acid and gibberellins signaling in aleurone cells.

    Science.gov (United States)

    Zhang, Zhong-Lin; Shin, Margaret; Zou, Xiaolu; Huang, Jianzhi; Ho, Tun-hua David; Shen, Qingxi J

    2009-05-01

    Abscisic acid (ABA) and gibberellins (GAs) control several developmental processes including seed maturation, dormancy, and germination. The antagonism of these two hormones is well-documented. However, recent data from transcription profiling studies indicate that they can function as agonists in regulating the expression of many genes although the underlying mechanism is unclear. Here we report a rice WRKY gene, OsWRKY24, which encodes a protein that functions as a negative regulator of both GA and ABA signaling. Overexpression of OsWRKY24 via particle bombardment-mediated transient expression in aleurone cells represses the expression of two reporter constructs: the beta-glucuronidase gene driven by the GA-inducible Amy32b alpha-amylase promoter (Amy32b-GUS) and the ABA-inducible HVA22 promoter (HVA22-GUS). OsWRKY24 is unlikely a general repressor because it has little effect on the expression of the luciferase reporter gene driven by a constitutive ubiquitin promoter (UBI-Luciferase). As to the GA signaling, OsWRKY24 differs from OsWRKY51 and -71, two negative regulators specifically function in the GA signaling pathway, in several ways. First, OsWRKY24 contains two WRKY domains while OsWRKY51 and -71 have only one; both WRKY domains are essential for the full repressing activity of OsWRKY24. Second, binding of OsWRKY24 to the Amy32b promoter appears to involve sequences in addition to the TGAC cores of the W-boxes. Third, unlike OsWRKY71, OsWRKY24 is stable upon GA treatment. Together, these data demonstrate that OsWRKY24 is a novel type of transcriptional repressor that inhibits both GA and ABA signaling.

  15. Sonic hedgehog acts as a negative regulator of {beta}-catenin signaling in the adult tongue epithelium.

    Science.gov (United States)

    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.

  16. Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons.

    Science.gov (United States)

    Palomba, Letizia; Silvestri, Cristoforo; Imperatore, Roberta; Morello, Giovanna; Piscitelli, Fabiana; Martella, Andrea; Cristino, Luigia; Di Marzo, Vincenzo

    2015-05-29

    The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL(-/-) mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.

  17. Histone Deacetylase 1 (HDAC1) Negatively Regulates Thermogenic Program in Brown Adipocytes via Coordinated Regulation of Histone H3 Lysine 27 (H3K27) Deacetylation and Methylation.

    Science.gov (United States)

    Li, Fenfen; Wu, Rui; Cui, Xin; Zha, Lin; Yu, Liqing; Shi, Hang; Xue, Bingzhong

    2016-02-26

    Inhibiting class I histone deacetylases (HDACs) increases energy expenditure, reduces adiposity, and improves insulin sensitivity in obese mice. However, the precise mechanism is poorly understood. Here, we demonstrate that HDAC1 is a negative regulator of the brown adipocyte thermogenic program. The Hdac1 level is lower in mouse brown fat (BAT) than white fat, is suppressed in mouse BAT during cold exposure or β3-adrenergic stimulation, and is down-regulated during brown adipocyte differentiation. Remarkably, overexpressing Hdac1 profoundly blocks, whereas deleting Hdac1 significantly enhances, β-adrenergic activation-induced BAT-specific gene expression in brown adipocytes. β-Adrenergic activation in brown adipocytes results in a dissociation of HDAC1 from promoters of BAT-specific genes, including uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor γ co-activator 1α (Pgc1α), leading to increased acetylation of histone H3 lysine 27 (H3K27), an epigenetic mark of gene activation. This is followed by dissociation of the polycomb repressive complexes, including the H3K27 methyltransferase enhancer of zeste homologue (EZH2), suppressor of zeste 12 (SUZ12), and ring finger protein 2 (RNF2) from (and concomitant recruitment of H3K27 demethylase ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) to) Ucp1 and Pgc1α promoters, leading to decreased H3K27 trimethylation, a histone transcriptional repression mark. Thus, HDAC1 negatively regulates the brown adipocyte thermogenic program, and inhibiting Hdac1 promotes BAT-specific gene expression through a coordinated control of increased acetylation and decreased methylation of H3K27, thereby switching the transcriptional repressive state to the active state at the promoters of Ucp1 and Pgc1α. Targeting HDAC1 may be beneficial in prevention and treatment of obesity by enhancing BAT thermogenesis.

  18. Up-Regulation of RFC3 Promotes Triple Negative Breast Cancer Metastasis and is Associated With Poor Prognosis Via EMT

    Directory of Open Access Journals (Sweden)

    Zhen-Yu He

    2017-02-01

    Full Text Available Triple-negative breast cancer (TNBC was regarded as the most aggressive and mortal subtype of breast cancer (BC since the molecular subtype system has been established. Abundant studies have revealed that epithelial-mesenchymal transition (EMT played a pivotal role during breast cancer metastasis and progression, especially in TNBC. Herein, we showed that inhibition the expression of replication factor C subunit 3 (RFC3 significantly attenuated TNBC metastasis and progression, which was associated with EMT signal pathway. In TNBC cells, knockdown of RFC3 can down-regulate mesenchymal markers and up-regulate epithelial markers, significantly attenuated cell proliferation, migration and invasion. Additionally, silencing RFC3 expression can decrease nude mice tumor volume, weight and relieve lung metastasis in vivo. Furthermore, we also demonstrated that overexpression of RFC3 in TNBC showed increased metastasis, progression and poor prognosis. We confirmed all of these results by immunohistochemistry analysis in 127 human TNBC tissues and found that RFC3 expression was significantly associated with poor prognosis in TNBC. Taken all these findings into consideration, we can conclude that up-regulation of RFC3 promotes TNBC progression through EMT signal pathway. Therefore, RFC3 could be an independent prognostic factor and therapeutic target for TNBC.

  19. The role of emotion regulation in situational empathy-related responding and prosocial behaviour in the presence of negative affect.

    Science.gov (United States)

    Hein, Sascha; Röder, Mandy; Fingerle, Michael

    2016-12-15

    Empathy and prosocial behaviour are crucial factors for children's positive social adjustment. Contemporary models of empathy highlight the capacity to regulate vicariously experienced emotions as a precursor to empathy-related responses (e.g., prosocial behaviour). The goal of this study was to examine the role of emotion regulation (ER) in situational empathy-related responding and prosocial behaviour. A sample of 157 children (76 boys and 81 girls; Mage = 9.94 years) participated in a two-tiered interview procedure that utilised vignettes to assess empathy and prosocial behaviour. Between both phases of the interview, a negative affect was induced to investigate the influence of ER on the change between the two phases. Results from a latent change model showed that ER strategies positively predicted change scores, that is, children with higher abilities to regulate emotions showed a higher increase in empathy and prosocial behaviour. Implications for the promotion of social-emotional learning in school are discussed. © 2016 International Union of Psychological Science.

  20. Sarco(endo)plasmic reticulum ATPase is a molecular partner of Wolfram syndrome 1 protein, which negatively regulates its expression.

    Science.gov (United States)

    Zatyka, Malgorzata; Da Silva Xavier, Gabriela; Bellomo, Elisa A; Leadbeater, Wendy; Astuti, Dewi; Smith, Joel; Michelangeli, Frank; Rutter, Guy A; Barrett, Timothy G

    2015-02-01

    Wolfram syndrome is an autosomal recessive disorder characterized by neurodegeneration and diabetes mellitus. The gene responsible for the syndrome (WFS1) encodes an endoplasmic reticulum (ER)-resident transmembrane protein that is involved in the regulation of the unfolded protein response (UPR), intracellular ion homeostasis, cyclic adenosine monophosphate production and regulation of insulin biosynthesis and secretion. In this study, single cell Ca(2+) imaging with fura-2 and direct measurements of free cytosolic ATP concentration ([ATP]CYT) with adenovirally expressed luciferase confirmed a reduced and delayed rise in cytosolic free Ca(2+) concentration ([Ca(2+)]CYT), and additionally, diminished [ATP]CYT rises in response to elevated glucose concentrations in WFS1-depleted MIN6 cells. We also observed that sarco(endo)plasmic reticulum ATPase (SERCA) expression was elevated in several WFS1-depleted cell models and primary islets. We demonstrated a novel interaction between WFS1 and SERCA by co-immunoprecipitation in Cos7 cells and with endogenous proteins in human neuroblastoma cells. This interaction was reduced when cells were treated with the ER stress inducer dithiothreitol. Treatment of WFS1-depleted neuroblastoma cells with the proteasome inhibitor MG132 resulted in reduced accumulation of SERCA levels compared with wild-type cells. Together these results reveal a role for WFS1 in the negative regulation of SERCA and provide further insights into the function of WFS1 in calcium homeostasis.

  1. Negative Regulation of Anthocynanin Biosynthesis in Arabidopsis by a miR156-Targeted SPL Transcription Factor

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    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.

  2. Root-expressed maize lipoxygenase 3 negatively regulates induced systemic resistance to Colletotrichum graminicola in shoots.

    Science.gov (United States)

    Constantino, Nasie N; Mastouri, Fatemeh; Damarwinasis, Ramadhika; Borrego, Eli J; Moran-Diez, Maria E; Kenerley, Charley M; Gao, Xiquan; Kolomiets, Michael V

    2013-01-01

    evidence that SM1 function in ISR, at least in part, by suppressing host ZmLOX3 gene. This study and the genetic tools generated herein will allow the identification of the signals regulating the induction of resistance to aboveground attackers by beneficial soil microorganisms in the future.

  3. Brassica napus DS-3, encoding a DELLA protein, negatively regulates stem elongation through gibberellin signaling pathway.

    Science.gov (United States)

    Zhao, Bo; Li, Haitao; Li, Juanjuan; Wang, Bo; Dai, Cheng; Wang, Jing; Liu, Kede

    2017-04-01

    Identification and characterization of a semi-dwarfing gene ds-3 encoding a mutant DELLA protein regulating plant height through gibberellin signaling pathway. Lodging is one of the most important factors causing severe yield loss in oilseed rape. Utilization of semi-dwarf varieties has been proved the most effective way to increase lodging resistance and yield in many crops. To develop semi-dwarf germplasm in oilseed rape, we identified a semi-dwarf mutant ds-3 which showed a reduced response to phytohormones gibberellins (GAs). Genetic analysis indicated the dwarfism was controlled by a single semi-dominant gene, ds-3. The DS-3 gene was mapped to a genomic region on chromosome C07, which is syntenic to the region of a previously identified semi-dwarf gene ds-1 (BnaA06.RGA). In this region, DS-3 (BnaC07.RGA) gene was identified to encode a DELLA protein that functions as a repressor in GA signaling pathway. A substitution of proline to leucine was identified in ds-3 in the conserved VHYNP motif, which is essential for GA-dependent interaction between gibberellin receptor GID1 and DELLA proteins. Segregation analysis in the F2 population derived from the cross between ds-1 and ds-3 demonstrated that BnaA06.RGA displayed a stronger effect on plant height than BnaC07.RGA, indicating that different RGA genes may play different roles in stem elongation. In addition to BnaA06.RGA and BnaC07.RGA, two more RGA genes (BnaA09.RGA and BnaC09.RGA) were identified in the Brassica napus (B. napus) genome. Reverse-transcription polymerase chain reaction (RT-PCR) and yeast two-hybrid (Y2H) assays suggest that both BnaA09.RGA and BnaC09.RGA are transcribed in leaves and stems and can mediate GA signaling in vivo. These genes represent potential targets for screening ideal semi-dwarfing alleles for oilseed rape breeding.

  4. Os2 MAP kinase-mediated osmostress tolerance in Penicillium digitatum is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

    Science.gov (United States)

    Wang, Mingshuang; Chen, Changsheng; Zhu, Congyi; Sun, Xuepeng; Ruan, Ruoxin; Li, Hongye

    2014-01-01

    High osmolarity glycerol (HOG) pathway is ubiquitously distributed among eukaryotic organisms and plays an important role in adaptation to changes in the environment. In this study, the Hog1 ortholog in Penicillium digitatum, designated Pdos2, was identified and characterized using a gene knock-out strategy. The ΔPdos2 mutant showed a considerably increased sensitivity to salt stress and cell wall-disturbing agents and a slightly increased resistance to fungicides iprodione and fludioxonil, indicating that Pdos2 is involved in response to hyperosmotic stress, regulation of cell wall integrity and sensitivity to fungicides iprodione and fludioxonil. Surprisingly, the mutant was not affected in response to oxidative stress caused by H2O2. The average lesion size in citrus fruits caused by ΔPdos2 mutant was smaller (approximately 25.0% reduction) than that caused by the wild-type strain of P. digitatum at 4 days post inoculation, which suggests that Pdos2 is needed for full virulence of P. digitatum. Interestingly, in the presence of 0.7 M NaCl, the glycerol content was remarkably increased and the ergosterol was decreased in mycelia of the wide-type P. digitatum, whereas the glycerol content was only slightly increased and the ergosterol content remained stable in the ΔPdos2 mutant, suggesting that Pdos2-mediated osmotic adaption is associated with its positive regulation on glycerol synthesis and negative regulation on ergosterol synthesis.

  5. Orphan nuclear receptor NR4A1 is a negative regulator of DHT-induced rat preantral follicular growth.

    Science.gov (United States)

    Xue, Kai; Liu, Jia-yin; Murphy, Bruce D; Tsang, Benjamin K

    2012-12-01

    Nuclear receptor subfamily 4 group A member1 (NR4A1), an orphan nuclear receptor, is involved in the transcriptional regulation of thecal cell androgen biosynthesis and paracrine factor insulin-like 3 (INSL3) expression. Androgens are known to play an important regulatory role in ovarian follicle growth. Using a chronically androgenized rat model, a preantral follicle culture model and virus-mediated gene delivery, we examined the role and regulation of NR4A1 in the androgenic control of preantral follicular growth. In the present study, Ki67 staining was increased in preantral follicles on ovarian sections from 5α-dihydrotestosterone (DHT)-treated rats. Preantral follicles from DHT-treated rats cultured for 4 d exhibited increased growth and up-regulation of mRNA abundance of G(1)/S-specific cyclin-D2 (Ccnd2) and FSH receptor (Fshr). Similarly, DHT (1 μm) increased preantral follicular growth and Ccnd2 and Fshr mRNA abundance in vitro. The NR4A1 expression was high in theca cells and was down-regulated by DHT in vivo and in vitro. Forced expression of NR4A1 augmented preantral follicular growth, androstenedione production, and Insl3 expression in vitro. Inhibiting the action of androgen (with androgen receptor antagonist flutamide) or INSL3 (with INSL3 receptor antagonist INSL3 B-chain) reduced NR4A1-induced preantral follicular growth. Furthermore, NR4A1 overexpression enhanced DHT-induced preantral follicular growth, a response attenuated by inhibiting INSL3. In conclusion, DHT promotes preantral follicular growth and attenuates thecal NR4A1 expression in vivo and in vitro. Our findings are consistent with the notion that NR4A1 serves as an important point of negative feedback to minimize the excessive preantral follicle growth in hyperandrogenism.

  6. ZFP36L1 negatively regulates plasmacytoid differentiation of BCL1 cells by targeting BLIMP1 mRNA.

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    Asghar Nasir

    Full Text Available The ZFP36/Tis11 family of zinc-finger proteins regulate cellular processes by binding to adenine uridine rich elements in the 3' untranslated regions of various mRNAs and promoting their degradation. We show here that ZFP36L1 expression is largely extinguished during the transition from B cells to plasma cells, in a reciprocal pattern to that of ZFP36 and the plasma cell transcription factor, BLIMP1. Enforced expression of ZFP36L1 in the mouse BCL1 cell line blocked cytokine-induced differentiation while shRNA-mediated knock-down enhanced differentiation. Reconstruction of regulatory networks from microarray gene expression data using the ARACNe algorithm identified candidate mRNA targets for ZFP36L1 including BLIMP1. Genes that displayed down-regulation in plasma cells were significantly over-represented (P = <0.0001 in a set of previously validated ZFP36 targets suggesting that ZFP36L1 and ZFP36 target distinct sets of mRNAs during plasmacytoid differentiation. ShRNA-mediated knock-down of ZFP36L1 in BCL1 cells led to an increase in levels of BLIMP1 mRNA and protein, but not for mRNAs of other transcription factors that regulate plasmacytoid differentiation (xbp1, irf4, bcl6. Finally, ZFP36L1 significantly reduced the activity of a BLIMP1 3' untranslated region-driven luciferase reporter. Taken together, these findings suggest that ZFP36L1 negatively regulates plasmacytoid differentiation, at least in part, by targeting the expression of BLIMP1.

  7. Caenorhabditis elegans Inositol 5-Phosphatase Homolog Negatively Regulates Inositol 1,4,5-Triphosphate Signaling in Ovulation V⃞

    Science.gov (United States)

    Bui, Yen Kim; Sternberg, Paul W.

    2002-01-01

    Ovulation in Caenorhabditis elegans requires inositol 1,4,5-triphosphate (IP3) signaling activated by the epidermal growth factor (EGF)-receptor homolog LET-23. We generated a deletion mutant of a type I 5-phosphatase, ipp-5, and found a novel ovulation phenotype whereby the spermatheca hyperextends to engulf two oocytes per ovulation cycle. The temporal and spatial expression of IPP-5 is consistent with its proposed inhibition of IP3 signaling in the adult spermatheca. ipp-5 acts downstream of let-23, and interacts with let-23–mediated IP3 signaling pathway genes. We infer that IPP-5 negatively regulates IP3 signaling to ensure proper spermathecal contraction. PMID:12006659

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

    Science.gov (United States)

    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.

  9. Agreeableness and the Self-Regulation of Negative Affect: Findings Involving the Neuroticism/Somatic Distress Relationship.

    Science.gov (United States)

    Ode, Scott; Robinson, Michael D

    2007-12-01

    In the temperament literature, agreeableness has been theoretically linked to effortful control. Further, research in this area has suggested that effortful control may play a broad role in moderating temperament-based tendencies toward negative affect. The present three studies, involving a total of 300 undergraduate participants, sought to extend this perspective to the adult literature by examining potential interactions between agreeableness and neuroticism in predicting reported somatic symptoms. Although such symptoms have been linked to neuroticism, they are not characteristic of the interpersonal concerns linked to agreeableness. Nevertheless, all studies found that agreeableness and neuroticism interacted to predict somatic symptoms such that high levels of agreeableness decoupled the relationship between neuroticism and somatic distress. These findings indicate a broad role for agreeableness in the self-regulation of neuroticism-linked distress.

  10. TRAF1 is a negative regulator of TNF signaling. enhanced TNF signaling in TRAF1-deficient mice.

    Science.gov (United States)

    Tsitsikov, E N; Laouini, D; Dunn, I F; Sannikova, T Y; Davidson, L; Alt, F W; Geha, R S

    2001-10-01

    TNF receptor-associated factor 1 (TRAF1) is a unique TRAF protein because it lacks a RING finger domain and is predominantly expressed in activated lymphocytes. To elucidate the function of TRAF1, we generated TRAF1-deficient mice. TRAF1(-/-) mice are viable and have normal lymphocyte development. TRAF1(-/-) T cells exhibit stronger than wild-type (WT) T cell proliferation to anti-CD3 mAb, which persisted in the presence of IL-2 or anti-CD28 antibodies. Activated TRAF1(-/-) T cells, but not TRAF1(+/+) T cells, responded to TNF by proliferation and activation of the NF-kappa B and AP-1 signaling pathways. This TNF effect was mediated by TNFR2 (p75) but not by TNFR1 (p55). Furthermore, skin from TRAF1(-/-) mice was hypersensitive to TNF-induced necrosis. These findings suggest that TRAF1 is a negative regulator of TNF signaling.

  11. Deleted in liver cancer 1 (DLC1 negatively regulates Rho/ROCK/MLC pathway in hepatocellular carcinoma.

    Directory of Open Access Journals (Sweden)

    Carmen Chak-Lui Wong

    Full Text Available AIMS: Deleted in liver cancer 1 (DLC1, a member of RhoGTPase activating protein (GAP family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immunofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2. These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1 phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage. CONCLUSION: Our data suggest that DLC1 negatively regulates Rho/ROCK/MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma.

  12. Coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of STING-mediated signaling.

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    Li Sun

    Full Text Available Viruses have evolved elaborate mechanisms to evade or inactivate the complex system of sensors and signaling molecules that make up the host innate immune response. Here we show that human coronavirus (HCoV NL63 and severe acute respiratory syndrome (SARS CoV papain-like proteases (PLP antagonize innate immune signaling mediated by STING (stimulator of interferon genes, also known as MITA/ERIS/MYPS. STING resides in the endoplasmic reticulum and upon activation, forms dimers which assemble with MAVS, TBK-1 and IKKε, leading to IRF-3 activation and subsequent induction of interferon (IFN. We found that expression of the membrane anchored PLP domain from human HCoV-NL63 (PLP2-TM or SARS-CoV (PLpro-TM inhibits STING-mediated activation of IRF-3 nuclear translocation and induction of IRF-3 dependent promoters. Both catalytically active and inactive forms of CoV PLPs co-immunoprecipitated with STING, and viral replicase proteins co-localize with STING in HCoV-NL63-infected cells. Ectopic expression of catalytically active PLP2-TM blocks STING dimer formation and negatively regulates assembly of STING-MAVS-TBK1/IKKε complexes required for activation of IRF-3. STING dimerization was also substantially reduced in cells infected with SARS-CoV. Furthermore, the level of ubiquitinated forms of STING, RIG-I, TBK1 and IRF-3 are reduced in cells expressing wild type or catalytic mutants of PLP2-TM, likely contributing to disruption of signaling required for IFN induction. These results describe a new mechanism used by CoVs in which CoV PLPs negatively regulate antiviral defenses by disrupting the STING-mediated IFN induction.

  13. CKIP-1 is an intrinsic negative regulator of T-cell activation through an interaction with CARMA1.

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    Takashi Sakamoto

    Full Text Available The transcription factor NF-κB plays a key regulatory role in lymphocyte activation and generation of immune response. Stimulation of T cell receptor (TCR induces phosphorylation of CARMA1 by PKCθ, resulting in formation of CARMA1-Bcl10-MALT1 (CBM complex at lipid rafts and subsequently leading to NF-κB activation. While many molecular events leading to NF-κB activation have been reported, it is less understood how this activation is negatively regulated. We performed a cell-based screening for negative regulators of TCR-mediated NF-κB activation, using mutagenesis and complementation cloning strategies. Here we show that casein kinase-2 interacting protein-1 (CKIP-1 suppresses PKCθ-CBM-NF-κB signaling. We found that CKIP-1 interacts with CARMA1 and competes with PKCθ for association. We further confirmed that a PH domain of CKIP-1 is required for association with CARMA1 and its inhibitory effect. CKIP-1 represses NF-κB activity in unstimulated cells, and inhibits NF-κB activation induced by stimulation with PMA or constitutively active PKCθ, but not by stimulation with TNFα. Interestingly, CKIP-1 does not inhibit NF-κB activation induced by CD3/CD28 costimulation, which caused dissociation of CKIP-1 from lipid rafts. These data suggest that CKIP-1 contributes maintenance of a resting state on NF-κB activity or prevents T cells from being activated by inadequate signaling. In conclusion, we demonstrate that CKIP-1 interacts with CARMA1 and has an inhibitory effect on PKCθ-CBM-NF-κB signaling.

  14. Negative and positive mRNA splicing elements act competitively to regulate human immunodeficiency virus type 1 vif gene expression.

    Science.gov (United States)

    Exline, C M; Feng, Z; Stoltzfus, C M

    2008-04-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5' splice site (5'ss) downstream of exon 2 (5'ss D2). Here we show that the mutations within 5'ss D2 that are predicted to lower or increase the affinity of the 5'ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5'ss D2 was not necessary for the effect of 5'ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5'ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5'-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5'ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication.

  15. Parasympathetic cardio-regulation during social interactions in individuals with obesity-The influence of negative body image.

    Science.gov (United States)

    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.

  16. Negative regulation of gamma-aminobutyric acid type A receptor on free calcium ion levels following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Fugao Zhu; Dawei Sun; Yanqing Wang; Rui Zhou; Junfeng Wen; Xiuming Wan; Yanjun Wang; Banghua Liu

    2010-01-01

    Previous studies have demonstrated that muscarinic, and nicotinic receptors increase free Ca2+ levels in the facial nerve nucleus via various channels following facial nerve injury. However, intracellular Ca2+ overload can trigger either necrotic or apoptotic cell death. Gamma-aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, exists in the facial nerve nucleus. It is assumed that GABA negatively regulates free Ca2+ levels in the facial nerve nucleus. The present study investigated GABA type A (GABAA) receptor expression in the facial nerve nucleus in a rat model of facial nerve injury using immunohistochemistry and laser confocal microscopy, as well as the regulatory effects of GABAA receptor on nicotinic receptor response following facial nerve injury. Subunits α1, α3, α5, β1, β2, δ, and γ3 of GABAA receptors were expressed in the facial nerve nucleus following facial nerve injury. In addition, GABAA receptor expression significantly inhibited the increase in nicotinic receptor-mediated free Ca2+ levels in the facial nerve nucleus following facial nerve injury in a concentration-dependent fashion. These results suggest that GABAA receptors exhibit negative effects on nicotinic receptor responses following facial nerve injury.

  17. Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100.

    Science.gov (United States)

    Liu, Shouan; Kracher, Barbara; Ziegler, Jörg; Birkenbihl, Rainer P; Somssich, Imre E

    2015-06-15

    The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity.

  18. Differential alterations of positive and negative regulators of beta catenin enhance endogenous expression and activity of beta catenin in A549 non small cell lung cancer (NSCLC cells

    Directory of Open Access Journals (Sweden)

    Supratim Ghatak

    2016-12-01

    Full Text Available Beta catenin has been well documented in previous studies to be involved in non small cell lung cancer (NSCLC. Beta catenin abundance and transcriptional activity are significantly regulated by several factors. Though it is well known that Akt and Gsk3 beta are respective positive and negative regulators of beta catenin, however, no single study has so far documented how the expression and activity of both positive as well as negative regulators play favorable role on beta catenin expression and activity in NSCLC. In this study, we compared expression and activity of beta catenin and its regulators in normal lung cell WI38 and NSCLC cell A549 by western blot, qRT-PCR and luciferase assay. We observed that beta catenin positive regulators (Akt and Hsp90 and negative regulators (Gsk3 beta and microRNA-214 have differential expression and/or activity in NSCLC cell A549. However the differentially altered statuses of both the positive and negative regulators rendered cumulative positive effect on beta catenin expression and activity in A549. Our study thus suggests that chemotherapeutic modulations of regulating factors are crucial when abrogation and/or inhibition of key oncogenic proteins are necessary for cancer chemotherapy.

  19. Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation.

    Science.gov (United States)

    Miles, Wayne O; Lembo, Antonio; Volorio, Angela; Brachtel, Elena; Tian, Bin; Sgroi, Dennis; Provero, Paolo; Dyson, Nicholas

    2016-12-15

    Alternative polyadenylation (APA) is a process that changes the posttranscriptional regulation and translation potential of mRNAs via addition or deletion of 3' untranslated region (3' UTR) sequences. To identify posttranscriptional-regulatory events affected by APA in breast tumors, tumor datasets were analyzed for recurrent APA events. Motif mapping of the changed 3' UTR regions found that APA-mediated removal of Pumilio regulatory elements (PRE) was unusually common. Breast tumor subtype-specific APA profiling identified triple-negative breast tumors as having the highest levels of APA. To determine the frequency of these events, an independent cohort of triple-negative breast tumors and normal breast tissue was analyzed for APA. APA-mediated shortening of NRAS and c-JUN was seen frequently, and this correlated with changes in the expression of downstream targets. mRNA stability and luciferase assays demonstrated APA-dependent alterations in RNA and protein levels of affected candidate genes. Examination of clinical parameters of these tumors found those with APA of NRAS and c-JUN to be smaller and less proliferative, but more invasive than non-APA tumors. RT-PCR profiling identified elevated levels of polyadenylation factor CSTF3 in tumors with APA. Overexpression of CSTF3 was common in triple-negative breast cancer cell lines, and elevated CSTF3 levels were sufficient to induce APA of NRAS and c-JUN. Our results support the hypothesis that PRE-containing mRNAs are disproportionately affected by APA, primarily due to high sequence similarity in the motifs utilized by polyadenylation machinery and the PUM complex. Cancer Res; 76(24); 7231-41. ©2016 AACR.

  20. Alternative Polyadenylation in Triple-Negative Breast Tumors Allows NRAS and c-JUN to Bypass PUMILIO Posttranscriptional Regulation

    Science.gov (United States)

    Miles, Wayne O.; Lembo, Antonio; Volorio, Angela; Brachtel, Elena; Tian, Bin; Sgroi, Dennis; Provero, Paolo; Dyson, Nicholas

    2017-01-01

    Alternative polyadenylation (APA) is a process that changes the posttranscriptional regulation and translation potential of mRNAs via addition or deletion of 3′ untranslated region (3′ UTR) sequences. To identify posttranscriptional-regulatory events affected by APA in breast tumors, tumor datasets were analyzed for recurrent APA events. Motif mapping of the changed 3′ UTR regions found that APA-mediated removal of Pumilio regulatory elements (PRE) was unusually common. Breast tumor subtype–specific APA profiling identified triple-negative breast tumors as having the highest levels of APA. To determine the frequency of these events, an independent cohort of triple-negative breast tumors and normal breast tissue was analyzed for APA. APA-mediated shortening of NRAS and c-JUN was seen frequently, and this correlated with changes in the expression of downstream targets. mRNA stability and luciferase assays demonstrated APA-dependent alterations in RNA and protein levels of affected candidate genes. Examination of clinical parameters of these tumors found those with APA of NRAS and c-JUN to be smaller and less proliferative, but more invasive than non-APA tumors. RT-PCR profiling identified elevated levels of polyadenylation factor CSTF3 in tumors with APA. Overexpression of CSTF3 was common in triple-negative breast cancer cell lines, and elevated CSTF3 levels were sufficient to induce APA of NRAS and c-JUN. Our results support the hypothesis that PRE-containing mRNAs are disproportionately affected by APA, primarily due to high sequence similarity in the motifs utilized by polyadenylation machinery and the PUM complex. PMID:27758885

  1. Galectin-3 Negatively Regulates Hippocampus-Dependent Memory Formation through Inhibition of Integrin Signaling and Galectin-3 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Yan-Chu Chen

    2017-07-01

    Full Text Available Galectin-3, a member of the galectin protein family, has been found to regulate cell proliferation, inhibit apoptosis and promote inflammatory responses. Galectin-3 is also expressed in the adult rat hippocampus, but its role in learning and memory function is not known. Here, we found that contextual fear-conditioning training, spatial training or injection of NMDA into the rat CA1 area each dramatically decreased the level of endogenous galectin-3 expression. Overexpression of galectin-3 impaired fear memory, whereas galectin-3 knockout (KO enhanced fear retention, spatial memory and hippocampal long-term potentiation. Galectin-3 was further found to associate with integrin α3, an association that was decreased after fear-conditioning training. Transfection of the rat CA1 area with small interfering RNA against galectin-3 facilitated fear memory and increased phosphorylated focal adhesion kinase (FAK levels, effects that were blocked by co-transfection of the FAK phosphorylation-defective mutant Flag-FAKY397F. Notably, levels of serine-phosphorylated galectin-3 were decreased by fear conditioning training. In addition, blockade of galectin-3 phosphorylation at Ser-6 facilitated fear memory, whereas constitutive activation of galectin-3 at Ser-6 impaired fear memory. Interestingly galectin-1 plays a role in fear-memory formation similar to that of galectin-3. Collectively, our data provide the first demonstration that galectin-3 is a novel negative regulator of memory formation that exerts its effects through both extracellular and intracellular mechanisms.

  2. With no lysine L-WNK1 isoforms are negative regulators of the K+-Cl- cotransporters.

    Science.gov (United States)

    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.

  3. The Hematopoietic Transcription Factor AML1 (RUNX1) Is Negatively Regulated by the Cell Cycle Protein Cyclin D3

    Science.gov (United States)

    Peterson, Luke F.; Boyapati, Anita; Ranganathan, Velvizhi; Iwama, Atsushi; Tenen, Daniel G.; Tsai, Schickwann; Zhang, Dong-Er

    2005-01-01

    The family of cyclin D proteins plays a crucial role in the early events of the mammalian cell cycle. Recent studies have revealed the involvement of AML1 transactivation activity in promoting cell cycle progression through the induction of cyclin D proteins. This information in combination with our previous observation that a region in AML1 between amino acids 213 and 289 is important for its function led us to investigate prospective proteins associating with this region. We identified cyclin D3 by a yeast two-hybrid screen and detected AML1 interaction with the cyclin D family by both in vitro pull-down and in vivo coimmunoprecipitation assays. Furthermore, we demonstrate that cyclin D3 negatively regulates the transactivation activity of AML1 in a dose-dependent manner by competing with CBFβ for AML1 association, leading to a decreased binding affinity of AML1 for its target DNA sequence. AML1 and its fusion protein AML1-ETO have been shown to shorten and prolong the mammalian cell cycle, respectively. In addition, AML1 promotes myeloid cell differentiation. Thus, our observations suggest that the direct association of cyclin D3 with AML1 functions as a putative feedback mechanism to regulate cell cycle progression and differentiation. PMID:16287839

  4. Negative regulation of miRNA-9 on oligodendrocyte lineage gene 1 during hypoxic-ischemic brain damage

    Institute of Scientific and Technical Information of China (English)

    Lijun Yang; Hong Cui; Ting Cao

    2014-01-01

    Oligodendrocyte lineage gene 1 plays a key role in hypoxic-ischemic brain damage and myelin repair. miRNA-9 is involved in the occurrence of many related neurological disorders. Bioin-formatics analysis demonstrated that miRNA-9 complementarily, but incompletely, bound oligodendrocyte lineage gene 1, but whether miRNA-9 regulates oligodendrocyte lineage gene 1 remains poorly understood. Whole brain slices of 3-day-old Sprague-Dawley rats were cultured and divided into four groups:control group;oxygen-glucose deprivation group (treatment with 8% O2+ 92%N2 and sugar-free medium for 60 minutes);transfection control group (after oxygen and glucose deprivation for 60 minutes, transfected with control plasmid) and miRNA-9 transfection group (after oxygen and glucose deprivation for 60 minutes, transfected with miRNA-9 plasmid). From the third day of transfection, and with increasing culture days, oligodendrocyte lineage gene 1 expression increased in each group, peaked at 14 days, and then decreased at 21 days. Real-time quantitative PCR results, however, demonstrated that oligoden-drocyte lineage gene 1 expression was lower in the miRNA-9 transfection group than that in the transfection control group at 1, 3, 7, 14, 21 and 28 days after transfection. Results suggested that miRNA-9 possibly negatively regulated oligodendrocyte lineage gene 1 in brain tissues during hypoxic-ischemic brain damage.

  5. Altered disease development in the eui mutants and Eui overexpressors indicates that gibberellins negatively regulate rice basal disease resistance.

    Science.gov (United States)

    Yang, Dong-Lei; Li, Qun; Deng, Yi-Wen; Lou, Yong-Gen; Wang, Mu-Yang; Zhou, Guo-Xing; Zhang, Ying-Ying; He, Zu-Hua

    2008-05-01

    Gibberellins (GAs) form a group of important plant tetracyclic diterpenoid hormones that are involved in many aspects of plant growth and development. Emerging evidence implicates that GAs also play roles in stress responses. However, the role of GAs in biotic stress is largely unknown. Here, we report that knockout or overexpression of the Elongated uppermost internode (Eui) gene encoding a GA deactivating enzyme compromises or increases, respectively, disease resistance to bacterial blight (Xanthomonas oryzae pv. oyrzae) and rice blast (Magnaporthe oryzae). Exogenous application of GA(3) and the inhibitor of GA synthesis (uniconazol) could increase disease susceptibility and resistance, respectively, to bacterial blight. Similarly, uniconazol restored disease resistance of the eui mutant and GA(3) decreased disease resistance of the Eui overexpressors to bacterial blight. Therefore, the change of resistance attributes to GA levels. In consistency with this, the GA metabolism genes OsGA20ox2 and OsGA2ox1 were down-regulated during pathogen challenge. We also found that PR1a induction was enhanced but the SA level was decreased in the Eui overexpressor, while the JA level was reduced in the eui mutant. Together, our current study indicates that GAs play a negative role in rice basal disease resistance, with EUI as a positive modulator through regulating the level of bioactive GAs.

  6. Identification of a novel human MD-2 splice variant that negatively regulates Lipopolysaccharide-induced TLR4 signaling.

    Science.gov (United States)

    Gray, Pearl; Michelsen, Kathrin S; Sirois, Cherilyn M; Lowe, Emily; Shimada, Kenichi; Crother, Timothy R; Chen, Shuang; Brikos, Constantinos; Bulut, Yonca; Latz, Eicke; Underhill, David; Arditi, Moshe

    2010-06-01

    Myeloid differentiation factor 2 (MD-2) is a secreted gp that assembles with TLR4 to form a functional signaling receptor for bacterial LPS. In this study, we have identified a novel alternatively spliced isoform of human MD-2, termed MD-2 short (MD-2s), which lacks the region encoded by exon 2 of the MD-2 gene. Similar to MD-2, MD-2s is glycosylated and secreted. MD-2s also interacted with LPS and TLR4, but failed to mediate LPS-induced NF-kappaB activation and IL-8 production. We show that MD-2s is upregulated upon IFN-gamma, IL-6, and TLR4 stimulation and negatively regulates LPS-mediated TLR4 signaling. Furthermore, MD-2s competitively inhibited binding of MD-2 to TLR4. Our study pinpoints a mechanism that may be used to regulate TLR4 activation at the onset of signaling and identifies MD-2s as a potential therapeutic candidate to treat human diseases characterized by an overly exuberant or chronic immune response to LPS.

  7. Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.

    Science.gov (United States)

    Rothschild, Daniel E; Zhang, Yao; Diao, Na; Lee, Christina K; Chen, Keqiang; Caswell, Clayton C; Slade, Daniel J; Helm, Richard F; LeRoith, Tanya; Li, Liwu; Allen, Irving C

    2017-02-01

    Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m(-/-) mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m(-/-) animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m(-/-) mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m(-/-) mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.

  8. Siglec-F-dependent negative regulation of allergen-induced eosinophilia depends critically on the experimental model.

    Science.gov (United States)

    McMillan, Sarah J; Richards, Hannah E; Crocker, Paul R

    2014-07-01

    Siglec-8 and siglec-F are paralogous membrane proteins expressed on human and murine eosinophils respectively. They bind similar sialylated and sulphated glycans and mediate eosinophil apoptosis when cross-linked with antibodies or glycan ligands. In models of allergic eosinophilic airway inflammation, siglec-F was shown previously to be important for negatively regulating eosinophilia. It was proposed that this was due to siglec-F-dependent apoptosis, triggered via engagement with ligands that are upregulated on bronchial epithelium. Our aim was to further investigate the functions of siglec-F by comparing two commonly used models of ovalbumin-induced airway inflammation that differ in the dose and route of administration of ovalbumin. In confirmation of published results, siglec-F-deficient mice had enhanced lung tissue eosinophilia in response to intranasal ovalbumin delivered every other day. However, following aerosolised ovalbumin delivered daily, there was no influence of siglec-F deficiency on lung eosinophilia. Expression of siglec-F ligands in lung tissues was similar in both models of allergen induced inflammation. These data demonstrate that siglec-F-dependent regulation of eosinophilia is subtle and depends critically on the model used. The findings also indicate that mechanisms other than ligand-induced apoptosis may be important in siglec-F-dependent suppression of eosinophilia. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  9. A20 negatively regulates T cell receptor signaling to NF-kappaB by cleaving Malt1 ubiquitin chains.

    Science.gov (United States)

    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.

  10. SAZ, a new SUPERMAN-like protein, negatively regulates a subset of ABA-responsive genes in Arabidopsis.

    Science.gov (United States)

    Jiang, Chang-Jie; Aono, Mitsuko; Tamaoki, Masanori; Maeda, Satoru; Sugano, Shoji; Mori, Masaki; Takatsuji, Hiroshi

    2008-02-01

    Arabidopsis SUPERMAN (SUP) and members of its family are plant-unique C(2)H(2)-type zinc finger genes that have been implicated in plant growth and development. In this paper, we report that a new SUP-family gene, designated as S A- and A BA-downregulated z inc finger gene (SAZ), is involved in the negative regulation of ABA-mediated signaling. SAZ-GUS fusion proteins were predominantly localized in the nuclei when they were transiently expressed in onion epidermal cells. SAZ transcripts were expressed in the leaves and pistils of very young flower buds. In young seedlings, SAZ expression was downregulated in response to environmental stresses such as drought, salt, ozone and ultraviolet-B irradiation. This downregulation was also observed in response to the phytohormones salicylic acid (SA) and abscisic acid (ABA). SA-responsive downregulation of SAZ was not observed in the npr1-1 mutant, indicating that this regulation is NPR1 dependent. RNAi-mediated knockdown of SAZ (SAZ-kd) resulted in elevated expression of the drought- and ABA-responsive genes rd29B and rab18 under unstressed conditions, and it enhanced the response of these genes to drought and ABA treatment. The expression of several other drought- and/or ABA-responsive genes was not affected by SAZ-kd. Based on these results, we propose that SAZ plays a role in repressing a subset of the ABA-mediated stress-responsive genes in unstressed conditions.

  11. HDAC1 negatively regulates Bdnf and Pvalb required for parvalbumin interneuron maturation in an experience-dependent manner.

    Science.gov (United States)

    Koh, Dawn X P; Sng, Judy C G

    2016-11-01

    During early postnatal development, neuronal circuits are sculpted by sensory experience provided by the external environment. This experience-dependent regulation of circuitry development consolidates the balance of excitatory-inhibitory (E/I) neurons in the brain. The cortical barrel-column that innervates a single principal whisker is used to provide a clear reference frame for studying the consolidation of E/I circuitry. Sensory deprivation of S1 at birth disrupts the consolidation of excitatory-inhibitory balance by decreasing inhibitory transmission of parvalbumin interneurons. The molecular mechanisms underlying this decrease in inhibition are not completely understood. Our findings show that epigenetic mechanisms, in particular histone deacetylation by histone deacetylases, negatively regulate the expression of brain-derived neurotrophic factor (Bdnf) and parvalbumin (Pvalb) genes during development, which are required for the maturation of parvalbumin interneurons. After whisker deprivation, increased histone deacetylase 1 expression and activity led to increased histone deacetylase 1 binding and decreased histone acetylation at Bdnf promoters I-IV and Pvalb promoter, resulting in the repression of Bdnf and Pvalb gene transcription. The decrease in Bdnf expression further affected parvalbumin interneuron maturation at layer II/III in S1, demonstrated by decreased parvalbumin expression, a marker for parvalbumin interneuron maturation. Knockdown of HDAC1 recovered Bdnf and Pvalb gene transcription and also prevented the decrease of inhibitory synapses accompanying whisker deprivation.

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

    Science.gov (United States)

    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.

  13. Altered Disease Development in the eui Mutants and Eui Overexpressors Indicates that Gibberellins Negatively Regulate Rice Basal Disease Resistance

    Institute of Scientific and Technical Information of China (English)

    Dong-Lei Yang; Qun Li; Yi-Wen Deng; Yong-Gen Lou; Mu-Yang Wang; Guo-Xing Zhou; Ying-Ying Zhang; Zu-Hua He

    2008-01-01

    Gibberellins (GAs) form a group of important plant tetracyclic diterpenoid hormones that are involved in many aspects of plant growth and development. Emerging evidence implicates that GAs also play roles in stress responses. However, the role of GAs in biotic stress is largely unknown. Here, we report that knockout or overexpression of the Elongated uppermost internode (Eui) gene encoding a GA deactivating enzyme compromises or increases, respectively, disease resistance to bacterial blight (Xanthomonas oryzae pv. oyrzae) and rice blast (Magnaporthe oryzae). Exogenous application of GA and the inhibitor of GA synthesis (uniconazol) could increase disease susceptibility and resistance, respectively, to bacterial blight. Similarly, uniconazol restored disease resistance of the eui mutant and GA3 decreased disease resistance of the Eui overexpressors to bacterial blight. Therefore, the change of resistance attributes to GA levels. In consistency with this, the GA metabolism genes OsGA2Oox2 and OsGA2oxl were down-regulated during pathogen challenge. We also found that PR1a induction was enhanced but the SA level was decreased in the Eui overexpressor, while the JA level was reduced in the eui mutant. Together, our current study indicates that GAs play a negative role in rice basal disease resistance, with EUI as a positive modulator through regulating the level of bioactive GAs.

  14. Continuous time Bayesian networks identify Prdm1 as a negative regulator of TH17 cell differentiation in humans.

    Science.gov (United States)

    Acerbi, Enzo; Viganò, Elena; Poidinger, Michael; Mortellaro, Alessandra; Zelante, Teresa; Stella, Fabio

    2016-03-15

    T helper 17 (TH17) cells represent a pivotal adaptive cell subset involved in multiple immune disorders in mammalian species. Deciphering the molecular interactions regulating TH17 cell differentiation is particularly critical for novel drug target discovery designed to control maladaptive inflammatory conditions. Using continuous time Bayesian networks over a time-course gene expression dataset, we inferred the global regulatory network controlling TH17 differentiation. From the network, we identified the Prdm1 gene encoding the B lymphocyte-induced maturation protein 1 as a crucial negative regulator of human TH17 cell differentiation. The results have been validated by perturbing Prdm1 expression on freshly isolated CD4(+) naïve T cells: reduction of Prdm1 expression leads to augmentation of IL-17 release. These data unravel a possible novel target to control TH17 polarization in inflammatory disorders. Furthermore, this study represents the first in vitro validation of continuous time Bayesian networks as gene network reconstruction method and as hypothesis generation tool for wet-lab biological experiments.

  15. Wen-Dan Decoction Improves Negative Emotions in Sleep-Deprived Rats by Regulating Orexin-A and Leptin Expression

    Directory of Open Access Journals (Sweden)

    Fengzhi Wu

    2014-01-01

    Full Text Available Wen-Dan Decoction (WDD, a formula of traditional Chinese medicine, has been clinically used for treating insomnia for approximately 800 years. However, the therapeutic mechanisms of WDD remain unclear. Orexin-A plays a key role in the sleep-wake cycle, while leptin function is opposite to orexin-A. Thus, orexin-A and leptin may be important factors in sleep disorders. In this study, 48 rats were divided into control, model, WDD-treated, and diazepam-treated groups. The model of insomnia was produced by sleep deprivation (SD for 14 days. The expressions of orexin-A, leptin, and their receptors in blood serum, prefrontal cortex, and hypothalamus were detected by enzyme-linked immunosorbent assay, immunohistochemistry, and real time PCR. Open field tests showed that SD increased both crossing movement (Cm and rearing-movement (Rm times. Orexin-A and leptin levels in blood serum increased after SD but decreased in brain compared to the control group. mRNA expressions of orexin receptor 1 and leptin receptor after SD were decreased in the prefrontal cortex but were increased in hypothalamus. WDD treatment normalized the behavior and upregulated orexin-A, leptin, orexin receptor 1 and leptin receptor in brain. The findings suggest that WDD treatment may regulate SD-induced negative emotions by regulating orexin-A and leptin expression.

  16. Tomato NAC transcription factor SlSRN1 positively regulates defense response against biotic stress but negatively regulates abiotic stress response.

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    Bo Liu

    Full Text Available Biotic and abiotic stresses are major unfavorable factors that affect crop productivity worldwide. NAC proteins comprise a large family of transcription factors that play important roles in plant growth and development as well as in responses to biotic and abiotic stresses. In a virus-induced gene silencing-based screening to identify genes that are involved in defense response against Botrytis cinerea, we identified a tomato NAC gene SlSRN1 (Solanum lycopersicum Stress-related NAC1. SlSRN1 is a plasma membrane-localized protein with transactivation activity in yeast. Expression of SlSRN1 was significantly induced by infection with B. cinerea or Pseudomonas syringae pv. tomato (Pst DC3000, leading to 6-8 folds higher than that in the mock-inoculated plants. Expression of SlSRN1 was also induced by salicylic acid, jasmonic acid and 1-amino cyclopropane-1-carboxylic acid and by drought stress. Silencing of SlSRN1 resulted in increased severity of diseases caused by B. cinerea and Pst DC3000. However, silencing of SlSRN1 resulted in increased tolerance against oxidative and drought stresses. Furthermore, silencing of SlSRN1 accelerated accumulation of reactive oxygen species but attenuated expression of defense genes after infection by B. cinerea. Our results demonstrate that SlSRN1 is a positive regulator of defense response against B. cinerea and Pst DC3000 but is a negative regulator for oxidative and drought stress response in tomato.

  17. Histone demethylase retinoblastoma binding protein 2 is overexpressed in hepatocellular carcinoma and negatively regulated by hsa-miR-212.

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    Xiuming Liang

    Full Text Available BACKGROUND: The H3K4 demethylase retinoblastoma binding protein 2 (RBP2 is involved in the pathogenesis of gastric cancer, but its role and regulation in hepatocellular carcinoma (HCC is unknown. We determined the function of RBP2 and its regulation in HCC in vitro and in human tissues. METHODS: We analyzed gene expression in 20 specimens each of human HCC and normal liver tissue by quantitative real-time PCR and immunohistochemistry. Proliferation was analyzed by foci formation and senescence by β-galactosidase staining. Promoter activity was detected by luciferase reporter assay. RESULTS: The expression of RBP2 was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, Homo sapiens miR-212 (hsa-miR-212, showed an opposite pattern. SiRNA knockdown of RBP2 significantly upregulated cyclin-dependent kinase inhibitors (CDKIs, with suppression of HCC cell proliferation and induction of senescence. Overexpression of hsa-miR-212 suppressed RBP2 expression, with inhibited cell proliferation and induced cellular senescence, which coincided with upregulated CDKIs; with low hsa-miR-212 expression, CDKIs were downregulated in HCC tissue. Inhibition of hsa-miR-212 expression upregulated RBP2 expression. Luciferase reporter assay detected the direct binding of hsa-miR-212 to the RBP2 3' UTR. CONCLUSIONS: RBP2 is overexpressed in HCC and negatively regulated by hsa-miR-212. The hsa-miR-212-RBP2-CDKI pathway may be important in the pathogenesis of HCC.

  18. The Hrs/Stam complex acts as a positive and negative regulator of RTK signaling during Drosophila development.

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    Hélène Chanut-Delalande

    Full Text Available BACKGROUND: Endocytosis is a key regulatory step of diverse signalling pathways, including receptor tyrosine kinase (RTK signalling. Hrs and Stam constitute the ESCRT-0 complex that controls the initial selection of ubiquitinated proteins, which will subsequently be degraded in lysosomes. It has been well established ex vivo and during Drosophila embryogenesis that Hrs promotes EGFR down regulation. We have recently isolated the first mutations of stam in flies and shown that Stam is required for air sac morphogenesis, a larval respiratory structure whose formation critically depends on finely tuned levels of FGFR activity. This suggest that Stam, putatively within the ESCRT-0 complex, modulates FGF signalling, a possibility that has not been examined in Drosophila yet. PRINCIPAL FINDINGS: Here, we assessed the role of the Hrs/Stam complex in the regulation of signalling activity during Drosophila development. We show that stam and hrs are required for efficient FGFR signalling in the tracheal system, both during cell migration in the air sac primordium and during the formation of fine cytoplasmic extensions in terminal cells. We find that stam and hrs mutant cells display altered FGFR/Btl localisation, likely contributing to impaired signalling levels. Electron microscopy analyses indicate that endosome maturation is impaired at distinct steps by hrs and stam mutations. These somewhat unexpected results prompted us to further explore the function of stam and hrs in EGFR signalling. We show that while stam and hrs together downregulate EGFR signalling in the embryo, they are required for full activation of EGFR signalling during wing development. CONCLUSIONS/SIGNIFICANCE: Our study shows that the ESCRT-0 complex differentially regulates RTK signalling, either positively or negatively depending on tissues and developmental stages, further highlighting the importance of endocytosis in modulating signalling pathways during development.

  19. HPK1 associates with SKAP-HOM to negatively regulate Rap1-mediated B-lymphocyte adhesion.

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    Sebastian Königsberger

    Full Text Available BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1 is a Ste20-related serine/threonine kinase activated by a range of environmental stimuli including genotoxic stress, growth factors, inflammatory cytokines and antigen receptor triggering. Being inducibly recruited to membrane-proximal signalling scaffolds to regulate NFAT, AP-1 and NFkappaB-mediated gene transcription in T-cells, the function of HPK1 in B-cells to date remains rather ill-defined. METHODOLOGY/PRINCIPAL FINDINGS: By using two loss of function models, we show that HPK1 displays a novel function in regulating B-cell integrin activity. Wehi 231 lymphoma cells lacking HPK1 after shRNA mediated knockdown exhibit increased basic activation levels of Ras-related protein 1 (Rap1, accompanied by a severe lymphocyte function-associated antigen-1 (LFA-1 dependent homotypic aggregation and increased adhesion to intercellular adhesion molecule 1 (ICAM-1. The observed phenotype of enhanced integrin activity is caused downstream of Src, by a signalling module independent of PI3K and PLC, involving HPK1, SKAP55 homologue (SKAP-HOM and Rap1-GTP-interacting adaptor molecule (RIAM. This alters actin dynamics and renders focal adhesion kinase (FAK constitutively phosphorylated. Bone marrow and splenic B-cell development of HPK1(-/- mice are largely unaffected, except age-related tendencies for increased splenic cellularity and BCR downregulation. In addition, naïve splenic knockout B-cells appear hyperresponsive to a range of stimuli applied ex vivo as recently demonstrated by others for T-cells. CONCLUSIONS/SIGNIFICANCE: We therefore conclude that HPK1 exhibits a dual function in B-cells by negatively regulating integrin activity and controlling cellular activation, which makes it an interesting candidate to study in pathological settings like autoimmunity and cancer.

  20. HNF-4alpha Negatively Regulates Hepcidin Expression Through BMPR1A in HepG2 Cells.

    Science.gov (United States)

    Shi, Wencai; Wang, Heyang; Zheng, Xuan; Jiang, Xin; Xu, Zheng; Shen, Hui; Li, Min

    2016-09-23

    Hepcidin synthesis is reported to be inadequate according to the body iron store in patients with non-alcoholic fatty liver disease (NAFLD) undergoing hepatic iron overload (HIO). However, the underlying mechanisms remain unclear. We hypothesize that hepatocyte nuclear factor-4α (HNF-4α) may negatively regulate hepcidin expression and contribute to hepcidin deficiency in NAFLD patients. The effect of HNF-4α on hepcidin expression was observed by transfecting specific HNF-4α small interfering RNA (siRNA) or plasmids into HepG2 cells. Both direct and indirect mechanisms involved in the regulation of HNF-4α on hepcidin were detected by real-time PCR, Western blotting, chromatin immunoprecipitation (chIP), and reporter genes. It was found that HNF-4α suppressed hepcidin messenger RNA (mRNA) and protein expressions in HepG2 cells, and this suppressive effect was independent of the potential HNF-4α response elements. Phosphorylation of SMAD1 but not STAT3 was inactivated by HNF-4α, and the SMAD4 response element was found essential to HNF-4α-induced hepcidin reduction. Neither inhibitory SMADs, SMAD6, and SMAD7 nor BMPR ligands, BMP2, BMP4, BMP6, and BMP7 were regulated by HNF-4α in HepG2 cells. BMPR1A, but not BMPR1B, BMPR2, ActR2A, ActR2B, or HJV, was decreased by HNF-4α, and HNF4α-knockdown-induced stimulation of hepcidin could be entirely blocked when BMPR1A was interfered with at the same time. In conclusion, the present study suggests that HNF-4α has a suppressive effect on hepcidin expression by inactivating the BMP pathway, specifically via BMPR1A, in HepG2 cells.

  1. Wnt4 activates the canonical β-catenin pathway and regulates negatively myostatin: functional implication in myogenesis.

    Science.gov (United States)

    Bernardi, Henri; Gay, Stephanie; Fedon, Yann; Vernus, Barbara; Bonnieu, Anne; Bacou, Francis

    2011-05-01

    Expression of Wnt proteins is known to be important for developmental processes such as embryonic pattern formation and determination of cell fate. Previous studies have shown that Wn4 was involved in the myogenic fate of somites, in the myogenic proliferation, and differentiation of skeletal muscle. However, the function of this factor in adult muscle homeostasis remains not well understood. Here, we focus on the roles of Wnt4 during C2C12 myoblasts and satellite cells differentiation. We analyzed its myogenic activity, its mechanism of action, and its interaction with the anti-myogenic factor myostatin during differentiation. Established expression profiles indicate clearly that both types of cells express a few Wnts, and among these, only Wnt4 was not or barely detected during proliferation and was strongly induced during differentiation. As attested by myogenic factors expression pattern analysis and fusion index determination, overexpression of Wnt4 protein caused a strong increase in satellite cells and C2C12 myoblast differentiation leading to hypertrophic myotubes. By contrast, exposure of satellite and C2C12 cells to small interfering RNA against Wnt4 strongly diminished this process, confirming the myogenic activity of Wnt4. Moreover, we reported that Wnt4, which is usually described as a noncanonical Wnt, activates the canonical β-catenin pathway during myogenic differentiation in both cell types and that this factor regulates negatively the expression of myostatin and the regulating pathways associated with myostatin. Interestingly, we found that recombinant myostatin was sufficient to antagonize the differentiation-promoting activities of Wnt4. Reciprocally, we also found that the genetic deletion of myostatin renders the satellite cells refractory to the hypertrophic effect of Wnt4. These results suggest that the Wnt4-induced decrease of myostatin plays a functional role during hypertrophy. We propose that Wnt4 protein may be a key factor that

  2. REVEILLE8 and PSEUDO-REPONSE REGULATOR5 Form a Negative Feedback Loop within the Arabidopsis Circadian Clock

    Science.gov (United States)

    Rawat, Reetika; Jones, Matthew A.; Schwartz, Jacob; Salemi, Michelle R.; Phinney, Brett S.; Harmer, Stacey L.

    2011-01-01

    Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE) in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE–binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms) is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE–containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms. PMID:21483796

  3. Mouse Sphingosine Kinase 1a Is Negatively Regulated through Conventional PKC-Dependent Phosphorylation at S373 Residue.

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    Yong-Seok Oh

    Full Text Available Sphingosine kinase is a lipid kinase that converts sphingosine into sphingosine-1-phosphate, an important signaling molecule with intracellular and extracellular functions. Although diverse extracellular stimuli influence cellular sphingosine kinase activity, the molecular mechanisms underlying its regulation remain to be clarified. In this study, we investigated the phosphorylation-dependent regulation of mouse sphingosine kinase (mSK isoforms 1 and 2. mSK1a was robustly phosphorylated in response to extracellular stimuli such as phorbol ester, whereas mSK2 exhibited a high basal level of phosphorylation in quiescent cells regardless of agonist stimulation. Interestingly, phorbol ester-induced phosphorylation of mSK1a correlated with suppression of its activity. Chemical inhibition of conventional PKCs (cPKCs abolished mSK1a phosphorylation, while overexpression of PKCα, a cPKC isoform, potentiated the phosphorylation, in response to phorbol ester. Furthermore, an in vitro kinase assay showed that PKCα directly phosphorylated mSK1a. In addition, phosphopeptide mapping analysis determined that the S373 residue of mSK1a was the only site phosphorylated by cPKC. Interestingly, alanine substitution of S373 made mSK1a refractory to the inhibitory effect of phorbol esters, whereas glutamate substitution of the same residue resulted in a significant reduction in mSK1a activity, suggesting the significant role of this phosphorylation event. Taken together, we propose that mSK1a is negatively regulated through cPKC-dependent phosphorylation at S373 residue.

  4. REVEILLE8 and PSEUDO-REPONSE REGULATOR5 form a negative feedback loop within the Arabidopsis circadian clock.

    Directory of Open Access Journals (Sweden)

    Reetika Rawat

    2011-03-01

    Full Text Available Circadian rhythms provide organisms with an adaptive advantage, allowing them to regulate physiological and developmental events so that they occur at the most appropriate time of day. In plants, as in other eukaryotes, multiple transcriptional feedback loops are central to clock function. In one such feedback loop, the Myb-like transcription factors CCA1 and LHY directly repress expression of the pseudoresponse regulator TOC1 by binding to an evening element (EE in the TOC1 promoter. Another key regulatory circuit involves CCA1 and LHY and the TOC1 homologs PRR5, PRR7, and PRR9. Purification of EE-binding proteins from plant extracts followed by mass spectrometry led to the identification of RVE8, a homolog of CCA1 and LHY. Similar to these well-known clock genes, expression of RVE8 is circadian-regulated with a dawn phase of expression, and RVE8 binds specifically to the EE. However, whereas cca1 and lhy mutants have short period phenotypes and overexpression of either gene causes arrhythmia, rve8 mutants have long-period and RVE8-OX plants have short-period phenotypes. Light input to the clock is normal in rve8, but temperature compensation (a hallmark of circadian rhythms is perturbed. RVE8 binds to the promoters of both TOC1 and PRR5 in the subjective afternoon, but surprisingly only PRR5 expression is perturbed by overexpression of RVE8. Together, our data indicate that RVE8 promotes expression of a subset of EE-containing clock genes towards the end of the subjective day and forms a negative feedback loop with PRR5. Thus RVE8 and its homologs CCA1 and LHY function close to the circadian oscillator but act via distinct molecular mechanisms.

  5. Regulation of triple-negative breast cancer cell metastasis by the tumor-suppressor liver kinase B1.

    Science.gov (United States)

    Rhodes, L V; Tate, C R; Hoang, V T; Burks, H E; Gilliam, D; Martin, E C; Elliott, S; Miller, D B; Buechlein, A; Rusch, D; Tang, H; Nephew, K P; Burow, M E; Collins-Burow, B M

    2015-10-05

    Liver kinase B1 (LKB1), also known as serine/threonine kinase 11 (STK11), has been identified as a tumor suppressor in many cancers including breast. Low LKB1 expression has been associated with poor prognosis of breast cancer patients, and we report here a significant association between loss of LKB1 expression and reduced patient survival specifically in the basal subtype of breast cancer. Owing to the aggressive nature of the basal subtype as evidenced by high incidences of metastasis, the purpose of this study was to determine if LKB1 expression could regulate the invasive and metastatic properties of this specific breast cancer subtype. Induction of LKB1 expression in basal-like breast cancer (BLBC)/triple-negative breast cancer cell lines, MDA-MB-231 and BT-549, inhibited invasiveness in vitro and lung metastatic burden in an orthotopic xenograft model. Further analysis of BLBC cells overexpressing LKB1 by unbiased whole transcriptomics (RNA-sequencing) revealed striking regulation of metastasis-associated pathways, including cell adhesion, extracellular matrix remodeling, and epithelial-to-mesenchymal transition (EMT). In addition, LKB1 overexpression inhibited EMT-associated genes (CDH2, Vimentin, Twist) and induced the epithelial cell marker CDH1, indicating reversal of the EMT phenotype in the MDA-MB-231 cells. We further demonstrated marked inhibition of matrix metalloproteinase 1 expression and activity via regulation of c-Jun through inhibition of p38 signaling in LKB1-expressing cells. Taken together, these data support future development of LKB1 inducing therapeutics for the suppression of invasion and metastasis of BLBC.

  6. TEAD1/4 exerts oncogenic role and is negatively regulated by miR-4269 in gastric tumorigenesis.

    Science.gov (United States)

    Zhou, Y; Huang, T; Zhang, J; Wong, C C; Zhang, B; Dong, Y; Wu, F; Tong, J H M; Wu, W K K; Cheng, A S L; Yu, J; Kang, W; To, K F

    2017-07-31

    TEA domain (TEAD) transcription factors are key components of the Hippo-YAP1 signaling pathway, but their functional role and regulatory mechanisms remain unclear. This study aims to comprehensively explore the expression pattern and functional role of TEAD family in gastric carcinogenesis and investigate its regulation by microRNAs (miRNAs). The mRNA and protein expression of TEAD family were examined by quantitative reverse transcription-PCR (qRT-PCR) and western blot. Their functional roles were determined by in vitro and in vivo studies. The clinicopathological association of TEAD4 in gastric cancer (GC) was studied using immunohistochemistry on tissue microarray. The prediction of miRNAs, which potentially target TEAD1/4, was performed by TargetScan and miRDB. The regulation of TEAD1/4 by miRNAs was confirmed by qRT-PCR, western blot and luciferase assays. TEAD1/4 were overexpressed in GC cell lines and primary GC tissues. Knockdown of TEAD1/4 induced a significant anticancer effect in vitro and in vivo. TEAD1 was confirmed to be a direct target of miR-377-3p and miR-4269, while TEAD4 was negatively regulated by miR-1343-3p and miR-4269. Among them, miR-4269 was the most effective inhibitor of TEAD1/4. Ectopic expression of these miRNAs substantiated their tumor-suppressive effects. In primary GC tumors, downregulation of miR-4269 was associated with poor disease-specific survival and showed a negative correlation with TEAD4. TEAD1 and TEAD4 are oncogenic factors, whose aberrant activation are, in part, mediated by the silence of miR-377-3p, miR-1343-3p and miR-4269. For the first time, the nuclear accumulated TEAD4 and downregulated miR-4269 are proposed to serve as novel prognostic biomarkers in GC.Oncogene advance online publication, 31 July 2017; doi:10.1038/onc.2017.257.

  7. Interferon-inducible IFI16, a negative regulator of cell growth, down-regulates expression of human telomerase reverse transcriptase (hTERT gene.

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    Lynda Li Song

    Full Text Available BACKGROUND: Increased levels of interferon (IFN-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22 in human normal prostate epithelial cells and diploid fibroblasts (HDFs are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT gene. METHODOLOGY/PRINCIPAL FINDINGS: We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT. CONCLUSIONS/SIGNIFICANCE: These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases.

  8. Interferon-Inducible IFI16, a Negative Regulator of Cell Growth, Down-Regulates Expression of Human Telomerase Reverse Transcriptase (hTERT) Gene

    Science.gov (United States)

    Shen, Hui; Duan, Xin; Alimirah, Fatouma; Choubey, Divaker

    2010-01-01

    Background Increased levels of interferon (IFN)-inducible IFI16 protein (encoded by the IFI16 gene located at 1q22) in human normal prostate epithelial cells and diploid fibroblasts (HDFs) are associated with the onset of cellular senescence. However, the molecular mechanisms by which the IFI16 protein contributes to cellular senescence-associated cell growth arrest remain to be elucidated. Here, we report that increased levels of IFI16 protein in normal HDFs and in HeLa cells negatively regulate the expression of human telomerase reverse transcriptase (hTERT) gene. Methodology/Principal Findings We optimized conditions for real-time PCR, immunoblotting, and telomere repeat amplification protocol (TRAP) assays to detect relatively low levels of hTERT mRNA, protein, and telomerase activity that are found in HDFs. Using the optimized conditions, we report that treatment of HDFs with inhibitors of cell cycle progression, such as aphidicolin or CGK1026, which resulted in reduced steady-state levels of IFI16 mRNA and protein, was associated with increases in hTERT mRNA and protein levels and telomerase activity. In contrast, knockdown of IFI16 expression in cells increased the expression of c-Myc, a positive regulator of hTERT expression. Additionally, over-expression of IFI16 protein in cells inhibited the c-Myc-mediated stimulation of the activity of hTERT-luc-reporter and reduced the steady-state levels of c-Myc and hTERT. Conclusions/Significance These data demonstrated that increased levels of IFI16 protein in HDFs down-regulate the expression of hTERT gene. Our observations will serve basis to understand how increased cellular levels of the IFI16 protein may contribute to certain aging-dependent diseases. PMID:20052289

  9. Flightless-I (FliI) is a potential negative regulator of the Toll pathway in Litopenaeus vannamei.

    Science.gov (United States)

    Zhang, Shuang; Qiu, Wei; Chen, Yong-gui; Yuan, Feng-Hua; Li, Chao-Zheng; Yan, Hui; Weng, Shao-Ping; He, Jian-Guo

    2015-02-01

    Flightless-I (FliI) is a protein negatively modulates the Toll-like receptor (TLR) pathway through interacting with Myeloid differentiation factor 88 (MyD88). To investigate the function of FliI in innate immune responses in invertebrates, Litopenaeus vannamei FliI (LvFliI) was identified and characterized. The full-length cDNA of LvFliI is 4, 304 bp long, with an open reading frame (ORF) encoding a putative protein of 1292 amino acids, including 12 leucine-rich repeat (LRR) domains at the N-terminus and 6 gelsolin homology (GEL) domains at the C-terminus. The LvFliI protein was located in the cytoplasm and LvFliI mRNA was constitutively expressed in healthy L. vannamei, with the highest expression level in the muscle. LvFliI could be up-regulated in hemocytes after lipopolysaccharide (LPS), poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV) challenges, suggesting a stimulation response of LvFliI to bacterial and immune stimulant challenges. Upon LPS stimulation, overexpression of LvFliI in Drosophila Schneider 2 cells led to downregulation of Drosophila and shrimp antimicrobial peptide (AMP) genes. Knockdown of LvFliI by RNA interference (RNAi) resulted in an increase of the expression of three shrimp AMP genes (PEN2, crustin, and Lyz1). However, the mortality rates of LvFliI-knockdown shrimp in response to V. parahaemolyticus, S. aureus or WSSV infections were not significantly different from those of the control group. Taken together, all the results suggested that LvFliI may play a negative role in TLR signaling response in L. vannamei.

  10. Keratin-6 driven ODC expression to hair follicle keratinocytes enhances stemness and tumorigenesis by negatively regulating Notch

    Energy Technology Data Exchange (ETDEWEB)

    Arumugam, Aadithya; Weng, Zhiping; Chaudhary, Sandeep C.; Afaq, Farrukh [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Elmets, Craig A. [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology, University of Alabama at Birmingham, Birmingham, AL 35294-0019 (United States); Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL 35294 (United States)

    2014-08-29

    Highlights: • Targeting ODC to hair follicle augments skin carcinogenesis and invasive SCCs. • Hair follicle ODC expands stem cell compartment carrying CD34{sup +}/K15{sup +}/p63{sup +} keratinocytes. • Negatively regulated Notch1 is associated with expansion of stem cell compartment. - Abstract: Over-expression of ornithine decarboxylase (ODC) is known to be involved in the epidermal carcinogenesis. However, the mechanism by which it enhances skin carcinogenesis remains undefined. Recently, role of stem cells localized in various epidermal compartments has been shown in the pathogenesis of skin cancer. To direct ODC expression in distinct epidermal compartments, we have developed keratin 6 (K6)-ODC/SKH-1 and keratin 14 (K14)-ODC/SKH-1 mice and employed them to investigate the role of ODC directed to these epidermal compartments on UVB-induced carcinogenesis. K6-driven ODC over-expression directed to outer root sheath (ORS) of hair follicle was more effective in augmenting tumorigenesis as compared to mice where K14-driven ODC expression was directed to inter-follicular epidermal keratinocytes. Chronically UVB-irradiated K6-ODC/SKH-1 developed 15 ± 2.5 tumors/mouse whereas K14-ODC/SKH-1 developed only 6.8 ± 1.5 tumors/mouse. K6-ODC/SKH-1 showed augmented UVB-induced proliferation and much higher pro-inflammatory responses than K14-ODC/SKH-1 mice. Tumors induced in K6-ODC/SKH-1 were rapidly growing, invasive and ulcerative squamous cell carcinoma (SCC) showing decreased expression of epidermal polarity marker E-cadherin and enhanced mesenchymal marker, fibronectin. Interestingly, the number of CD34/CK15/p63 positive stem-like cells was significantly higher in chronically UVB-irradiated K6-ODC/SKH-1 as compared to K14-ODC/SKH-1 mice. Reduced Notch1 expression was correlated with the expansion of stem cell compartment in these animals. However, other signaling pathways such as DNA damage response or mTOR signaling pathways were not significantly different in

  11. Ephrin-A2 and-A3 are negative regulators of the regenerative potential of Müller cells

    Institute of Scientific and Technical Information of China (English)

    Zhu Ruilin; Cho Kin-Sang; Chen Dong Feng; Yang Liu

    2014-01-01

    Background In a previous study,we demonstrated that ephrin-A2 and-A3 negatively regulate the growth of neural progenitor cells in the central nervous system.Adult mice deficient in ephrin-A2 and-A3 (A2-/-A3-/-) displayed active ongoing neurogenesis throughout the brain,and mice deficient in ephrin-A3 alone showed increased proliferation of ciliary epithelium derived retinal stem cells.This study aimed to detect that the increase in proliferation and neurogenic potential of Müller cells is influenced by the absence of ephrin-A2 and-A3.Methods We assessed the retinal and Müller cell expression of ephrin-As and their receptor and neural progenitor cell markers by immunostaining and real-time PCR.We cultured purified primary Müller cells derived from wild-type and A2-/-A3-/-mice in a defined culture medium that enables trans-differentiation of Müller cells into retinal neurons.To evaluate proliferating Müller cells in vivo,we injected 5'-ethylnyl-2'-deoxiuridine (EdU) intraperitoneally to adult mice.Results Expression of ephrin-A2/A3 and their receptor EphA4 were detected in the retinas of adult mice,with EphA4 expression particularly enriched in Müller cells.Müller cells of A2-/-A3-/-mice exhibited significantly elevated expression of retinal progenitor cell markers,Pax6 and Chx10,when compared with those from wild-type mice.Moreover,a higher percentage of Müller cells of A2-/-A3-/-mice trans-differentiated and became recoverin+ and β-Ⅲ-tublin+ in the culture than those from wild type mice.Strikingly,an increased number of EdU+ retinal cells was detected in the retinas of adult A2-/-A3-/-mice as compared with wild-type mice.Conclusions Ephrin-A2 and-A3 are negative regulators of the proliferative and neurogenic potentials of Müller cells.Manipulating ephrin-A signaling may thus represent a novel strategy for stimulating neuroregeneration from endogenous progenitors to participate in retinal repair in case of disease or damage.

  12. mTORC1 Is a Local, Postsynaptic Voltage Sensor Regulated by Positive and Negative Feedback Pathways

    Directory of Open Access Journals (Sweden)

    Farr Niere

    2017-05-01

    Full Text Available The mammalian/mechanistic target of rapamycin complex 1 (mTORC1 serves as a regulator of mRNA translation. Recent studies suggest that mTORC1 may also serve as a local, voltage sensor in the postsynaptic region of neurons. Considering biochemical, bioinformatics and imaging data, we hypothesize that the activity state of mTORC1 dynamically regulates local membrane potential by promoting and repressing protein synthesis of select mRNAs. Our hypothesis suggests that mTORC1 uses positive and negative feedback pathways, in a branch-specific manner, to maintain neuronal excitability within an optimal range. In some dendritic branches, mTORC1 activity oscillates between the “On” and “Off” states. We define this as negative feedback. In contrast, positive feedback is defined as the pathway that leads to a prolonged depolarized or hyperpolarized resting membrane potential, whereby mTORC1 activity is constitutively on or off, respectively. We propose that inactivation of mTORC1 increases the expression of voltage-gated potassium alpha (Kv1.1 and 1.2 and beta (Kvβ2 subunits, ensuring that the membrane resets to its resting membrane potential after experiencing increased synaptic activity. In turn, reduced mTORC1 activity increases the protein expression of syntaxin-1A and promotes the surface expression of the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA-type subunit 1 (GluN1 that facilitates increased calcium entry to turn mTORC1 back on. Under conditions such as learning and memory, mTORC1 activity is required to be high for longer periods of time. Thus, the arm of the pathway that promotes syntaxin-1A and Kv1 protein synthesis will be repressed. Moreover, dendritic branches that have low mTORC1 activity with increased Kv expression would balance dendrites with constitutively high mTORC1 activity, allowing for the neuron to maintain its overall activity level within an ideal operating range. Finally, such a model suggests that

  13. Negative and Positive mRNA Splicing Elements Act Competitively To Regulate Human Immunodeficiency Virus Type 1 Vif Gene Expression▿

    Science.gov (United States)

    Exline, C. M.; Feng, Z.; Stoltzfus, C. M.

    2008-01-01

    Over 40 different human immunodeficiency virus type 1 (HIV-1) mRNAs are produced by alternative splicing of the primary HIV-1 RNA transcripts. In addition, approximately half of the viral RNA remains unspliced and is used as genomic RNA and as mRNA for the Gag and Pol gene products. Regulation of splicing at the HIV-1 3′ splice sites (3′ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation occurs through the binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3′ss A1, which produces single-spliced vif mRNA and promotes the inclusion of HIV exon 2 into both completely and incompletely spliced viral mRNAs, is increased by optimizing the 5′ splice site (5′ss) downstream of exon 2 (5′ss D2). Here we show that the mutations within 5′ss D2 that are predicted to lower or increase the affinity of the 5′ss for U1 snRNP result in reduced or increased Vif expression, respectively. Splicing at 5′ss D2 was not necessary for the effect of 5′ss D2 on Vif expression. In addition, we have found that mutations of the GGGG motif proximal to the 5′ss D2 increase exon 2 inclusion and Vif expression. Finally, we report the presence of a novel exonic splicing enhancer (ESE) element within the 5′-proximal region of exon 2 that facilitates both exon inclusion and Vif expression. This ESE binds specifically to the cellular SR protein SRp75. Our results suggest that the 5′ss D2, the proximal GGGG silencer, and the ESE act competitively to determine the level of vif mRNA splicing and Vif expression. We propose that these positive and negative splicing elements act together to allow the accumulation of vif mRNA and unspliced HIV-1 mRNA, compatible with optimal virus replication. PMID:18272582

  14. Nontypeable Haemophilus influenzae-Induced MyD88 Short Expression Is Regulated by Positive IKKβ and CREB Pathways and Negative ERK1/2 Pathway

    Science.gov (United States)

    Andrews, Carla S.; Miyata, Masanori; Susuki-Miyata, Seiko; Lee, Byung-Cheol; Komatsu, Kensei; Li, Jian-Dong

    2015-01-01

    Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) are characterized by excessive inflammation and are exacerbated by nontypeable Haemophilus influenzae (NTHi). Airway epithelial cells mount the initial innate immune responses to invading pathogens and thus modulate inflammation. While inflammation is necessary to eliminate a pathogen, excessive inflammation can cause damage to the host tissue. Therefore, the inflammatory response must be tightly regulated and deciphering the signaling pathways involved in this response will enhance our understanding of the regulation of the host inflammatory response. NTHi binds to TLR2 and signal propagation requires the adaptor molecule myeloid differentiation factor 88 (MyD88). An alternative spliced form of MyD88 is called MyD88 short (MyD88s) and has been identified in macrophages and embryonic cell lines as a negative regulator of inflammation. However, the role of MyD88s in NTHi-induced inflammation in airway epithelial cells remains unknown. Here we show that NTHi induces MyD88s expression and MyD88s is a negative regulator of inflammation in airway epithelial cells. We further demonstrate that MyD88s is positively regulated by IKKβ and CREB and negatively regulated by ERK1/2 signaling pathways. Taken together these data indicate that airway inflammation is controlled in a negative feedback manner involving MyD88s and suggest that airway epithelial cells are essential to maintain immune homeostasis. PMID:26669856

  15. Nontypeable Haemophilus influenzae-Induced MyD88 Short Expression Is Regulated by Positive IKKβ and CREB Pathways and Negative ERK1/2 Pathway.

    Directory of Open Access Journals (Sweden)

    Carla S Andrews

    Full Text Available Airway diseases such as asthma and chronic obstructive pulmonary disease (COPD are characterized by excessive inflammation and are exacerbated by nontypeable Haemophilus influenzae (NTHi. Airway epithelial cells mount the initial innate immune responses to invading pathogens and thus modulate inflammation. While inflammation is necessary to eliminate a pathogen, excessive inflammation can cause damage to the host tissue. Therefore, the inflammatory response must be tightly regulated and deciphering the signaling pathways involved in this response will enhance our understanding of the regulation of the host inflammatory response. NTHi binds to TLR2 and signal propagation requires the adaptor molecule myeloid differentiation factor 88 (MyD88. An alternative spliced form of MyD88 is called MyD88 short (MyD88s and has been identified in macrophages and embryonic cell lines as a negative regulator of inflammation. However, the role of MyD88s in NTHi-induced inflammation in airway epithelial cells remains unknown. Here we show that NTHi induces MyD88s expression and MyD88s is a negative regulator of inflammation in airway epithelial cells. We further demonstrate that MyD88s is positively regulated by IKKβ and CREB and negatively regulated by ERK1/2 signaling pathways. Taken together these data indicate that airway inflammation is controlled in a negative feedback manner involving MyD88s and suggest that airway epithelial cells are essential to maintain immune homeostasis.

  16. Fentanyl inhibits the invasion and migration of colorectal cancer cells via inhibiting the negative regulation of Ets-1 on BANCR.

    Science.gov (United States)

    Li, Ai-xiang; Xin, Wen-qi; Ma, Chuan-gen

    2015-09-25

    Recent studies have shown the potential anti-tumor effect of fentanyl on colorectal cancer (CRC). However, its underling mechanism is still unclear. Since studies indicates the abnormal expression of transcription factor Ets-1 and BRAF-activated lncRNA (BANCR) in CRC progress, the relationship between Ets-1 and BANCR was investigated here to illustrate the fentanyl-induced mechanism on CRC in vitro. The expression levels of Ets-1 and BANCR were first detected in fentanyl-treated CRC cells. The interaction between Ets-1 and BANCR promoter was verified with chromatin immunoprecipitation assays, as well as corresponding acetylation of histones. The regulation of Ets-1 on BANCR expression was confirmed through luciferase assays and RT-PCR analysis. And, cell clone formation, cell migration and invasion were observed to evaluate the anti-tumor effects of fentanyl. Ets-1 overexpression or co-overexpression with BANCR was further performed by plasmids transfection to show the regulatory role of Ets-1 in fentanyl-induced mechanism. Fentanyl induced BANCR upregulation and Ets-1 downregulation in CRC cells. Further studies showed that Ets-1 negatively regulated BANCR expression via the deacetylation of histones H3 within BANCR promoter. Moreover, fentanyl induced less cell clone formation, as well as inhibited cell migration and invasion in vitro, while Ets-1 overexpression inhibited fentanyl-induced effects that could be reversed by BANCR co-overexpression. Fentanyl showed anti-tumor like effects on CRC cells, including less cell clone formation and inhibited cell migration and invasion. Furthermore, the regulatory role of Ets-1 on BANCR influenced fentanyl-induced mechanism, indicating their potential application in the therapeutic treatment of CRC. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Developmental signaling pathways regulating mammary stem cells and contributing to the etiology of triple-negative breast cancer.

    Science.gov (United States)

    Rangel, Maria Cristina; Bertolette, Daniel; Castro, Nadia P; Klauzinska, Malgorzata; Cuttitta, Frank; Salomon, David S

    2016-04-01

    Cancer has been considered as temporal and spatial aberrations of normal development in tissues. Similarities between mammary embryonic development and cell transformation suggest that the underlying processes required for mammary gland development are also those perturbed during various stages of mammary tumorigenesis and breast cancer (BC) development. The master regulators of embryonic development Cripto-1, Notch/CSL, and Wnt/β-catenin play key roles in modulating mammary gland morphogenesis and cell fate specification in the embryo through fetal mammary stem cells (fMaSC) and in the adult organism particularly within the adult mammary stem cells (aMaSC), which determine mammary progenitor cell lineages that generate the basal/myoepithelial and luminal compartments of the adult mammary gland. Together with recognized transcription factors and embryonic stem cell markers, these embryonic regulatory molecules can be inappropriately augmented during tumorigenesis to support the tumor-initiating cell (TIC)/cancer stem cell (CSC) compartment, and the effects of their deregulation may contribute for the etiology of BC, in particular the most aggressive subtype of BC, triple-negative breast cancer (TNBC). This in depth review will present evidence of the involvement of Cripto-1, Notch/CSL, and Wnt/β-catenin in the normal mammary gland morphogenesis and tumorigenesis, from fMaSC/aMaSC regulation to TIC generation and maintenance in TNBC. Specific therapies for treating TNBC by targeting these embryonic pathways in TICs will be further discussed, providing new opportunities to destroy not only the bulk tumor, but also TICs that initiate and promote the metastatic spread and recurrence of this aggressive subtype of BC.

  18. GRK2 negatively regulates IGF-1R signaling pathway and cyclins' expression in HepG2 cells.

    Science.gov (United States)

    Wei, Zhengyu; Hurtt, Reginald; Gu, Tina; Bodzin, Adam S; Koch, Walter J; Doria, Cataldo

    2013-09-01

    G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin-like growth factor 1 receptor (IGF-1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF-1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF-1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF-1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF-1R signaling pathway and cyclins' expression.

  19. Negative regulation of natural killer cell in tumor tissue and peripheral blood of oral squamous cell carcinoma.

    Science.gov (United States)

    Dutta, Anupam; Banerjee, Arunabha; Saikia, Nabajyoti; Phookan, Jyotirmoy; Baruah, Munindra Narayan; Baruah, Shashi

    2015-12-01

    Natural killer (NK) cells are the key lymphocytes in solid tumors. Its activity is regulated by both germline encoded receptors and cytokine microenvironment. We conducted a case-control study to investigate the activation status of NK cell in oral squamous cell carcinoma (OSCC). NK cell activation was assessed in context of NK cell cytotoxicity and transcript expression of NK cell receptors (NKp46 and KIRs) and NK cell associated cytokines (IL-1β, IL-2, IL-10, IL-12β, IL-15, IL-18, IL-21, IFN-γ, TNF-α and TGF-β). The results revealed possible mechanisms involved in reduced NK cell activation in peripheral circulation: quantitative deficiency of NK cell number and lowered cytotoxicity together with qualitative NK impairments caused by--(1) decreased expression of NK activating receptor NKp46, (2) increased expression of NK suppressive cytokines--IL-10 and TGF-β and (3) induction of FOXP3(+)CTLA4(+) suppressor cells. On the other hand, in the tumor tissue, escape of NK immune surveillance appeared to be modulated by upregulation of TGF-β and IL-10 together with downregulation of NK cell activating cytokines (IL-2, IL-12β, IL-15, IL-18, IL-21 and IFN-γ) and NK receptors (NKp46 and KIRs). In addition, our study supported the earlier contention that TNF-α and IL-1β expression levels may be used as markers of malignant transformation in oral leukoplakia. In conclusion, the study provided an insight into the negative regulation of NK cell in tumor tissue and peripheral blood of OSCC patients, which can be exploited to boost the current NK cell and cytokine based immunotherapy for the treatment of oral cancer.

  20. MicroRNA (miR396) negatively regulates expression of ceramidase-like genes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Dongmei Liu; Diqiu Yu

    2009-01-01

    MicroRNAs (miRNAs) are 21-23 nucleotide (nt), endogenous RNAs that regulate gene expression by targeting mRNAs for direct cleavage or translational repression in plants. In Arabidopsis, miR396 is encoded by two different loci (MIR396a and M1R396b) and sequence analysis suggests it may target three ceramidase-like genes (Atceramidase-like 1, Atceramidase-like 2 and Atceramidase-like 3). To demonstrate the biological function of miR396, we inserted the synthetic precursors, MIR396a or MIR396b, under the control of the enhanced cauliflower mosaic virus (CaMV) 35S promoter, into a plant transformation vector (pOCA30) and transformed the con-structs into Arabidopsis. The promoter increased miR396 levels by more than 2-fold, indicating appropriate maturation of the synthetic precursor MIR396a or MIR396b transcript in transgenic plants. Microarray analysis showed that the transcript levels of two ceramidase-like genes (Atceramidase-like 1, Atceramidase-like 2) were decreased by more than 2-fold and lactosylceramide 4-α-galactosyltransferase increased by more than 2-fold in transgenic plants compared with the empty vector-transformed plants. Northern blot analysis showed that the mRNA levels of the two ceramidase-like genes were significantly reduced in transgenic plants. These results indicated that miR396 probably plays a crucial role in the ceramide metabolism pathway by negatively regulating the expression of ceramidase-like genes in Arabidopsis.

  1. HRS1 acts as a negative regulator of abscisic acid signaling to promote timely germination of Arabidopsis seeds.

    Science.gov (United States)

    Wu, Chongming; Feng, Juanjuan; Wang, Ran; Liu, Hong; Yang, Huixia; Rodriguez, Pedro L; Qin, Huanju; Liu, Xin; Wang, Daowen

    2012-01-01

    In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H(+)-ATPase activity, than that of WT control. The plasmalemma H(+)-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H(+)-ATPase and the efficient elongation of LH and TZ cells.

  2. EDR2 negatively regulates salicylic acid-based defenses and cell death during powdery mildew infections of Arabidopsis thaliana

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    Nishimura Marc

    2007-07-01

    Full Text Available Abstract Background The hypersensitive necrosis response (HR of resistant plants to avirulent pathogens is a form of programmed cell death in which the plant sacrifices a few cells under attack, restricting pathogen growth into adjacent healthy tissues. In spite of the importance of this defense response, relatively little is known about the plant components that execute the cell death program or about its regulation in response to pathogen attack. Results We isolated the edr2-6 mutant, an allele of the previously described edr2 mutants. We found that edr2-6 exhibited an exaggerated chlorosis and necrosis response to attack by three pathogens, two powdery mildew and one downy mildew species, but not in response to abiotic stresses or attack by the bacterial leaf speck pathogen. The chlorosis and necrosis did not spread beyond inoculated sites suggesting that EDR2 limits the initiation of cell death rather than its spread. The pathogen-induced chlorosis and necrosis of edr2-6 was correlated with a stimulation of the salicylic acid defense pathway and was suppressed in mutants deficient in salicylic acid signaling. EDR2 encodes a novel protein with a pleckstrin homology and a StAR transfer (START domain as well as a plant-specific domain of unknown function, DUF1336. The pleckstrin homology domain binds to phosphatidylinositol-4-phosphate in vitro and an EDR2:HA:GFP protein localizes to endoplasmic reticulum, plasma membrane and endosomes. Conclusion EDR2 acts as a negative regulator of cell death, specifically the cell death elicited by pathogen attack and mediated by the salicylic acid defense pathway. Phosphatidylinositol-4-phosphate may have a role in limiting cell death via its effect on EDR2. This role in cell death may be indirect, by helping to target EDR2 to the appropriate membrane, or it may play a more direct role.

  3. MicroRNA-181b negatively regulates the proliferation of human epidermal keratinocytes in psoriasis through targeting TLR4.

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    Feng, Cheng; Bai, Ming; Yu, Nan-Ze; Wang, Xiao-Jun; Liu, Zeng

    2017-02-01

    Our study aims to explore the role of microRNA-181b (miR-181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty-eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non-lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real-time quantitative fluorescence polymerase chain reaction (RT-qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR-181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non-lesional skin respectively. The 3' untranslated region (3'UTR) of TLR4 combined with miR-181b was verified by a dual-luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR-181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non-lesional skin. In psoriatic lesional and non-lesional skin, TLR4-positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual-luciferase reporter assay verified that miR-181b targets TLR4. HEKs transfected with miR-181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR-181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR-181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR-181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4.

  4. Regulation of Toll-like receptors-mediated inflammation by immunobiotics in bovine intestinal epitheliocytes: role of signalling pathways and negative regulators

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    Julio eVillena

    2014-09-01

    Full Text Available Intestinal epithelial cells (IECs detect bacterial and viral associated-molecular-patterns (MAMPs via germline-encoded pattern-recognition receptors (PRRs and are responsible for maintaining immunetolerance to the communities of resident commensal bacteria while being also capable to mount immune responses against pathogens. Toll-like receptors (TLRs are a major class of PRRs expressed on IECs and immune cells, which are involved in the induction of both tolerance and inflammation. In the last decade, experimental and clinical evidence was generated to support the application of probiotics with immunoregulatory capacities (immunobiotics for the prevention and treatment of several gastrointestinal inflammatory disorders in which TLRs exert a significant role. The majority of these studies were performed in mouse and human cell lines and, despite the growing interest in the bovine immune system due to the economic importance of cattle as livestock, only few studies have been conducted on cattle. In this regard, our group have established a bovine intestinal epithelial (BIE cell line originally derived from fetal bovine intestinal epitheliocytes and used this cell line to evaluate the impact of immunobiotics in TLR-mediated inflammation. This review aims to summarize the current knowledge of the beneficial effects of immunobiotics in the regulation of intestinal inflammation/infection in cattle. Especially we discuss the role of TLRs and their negative regulators in both the inflammatory response nd the beneficial effects of immunobiotics in bovine IECs. This review article emphasizes the cellular and molecular interactions of immunobiotics with BIE cells through TLRs and gives the scientific basis for the development of immunomodulatory feed for bovine healthy development.

  5. OsFTIP1-Mediated Regulation of Florigen Transport in Rice Is Negatively Regulated by a Ubiquitin-like Domain Kinase OsUbDKγ4.

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    Song, Shiyong; Cheng, Ying; Liu, Lu; Wang, Yanwen; Bao, Shengjie; Zhou, Xuan; Teo, Zhi Wei Norman; Mao, Chuanzao; Gan, Yinbo; Yu, Hao

    2017-03-02

    Flowering time is a critical agronomic trait that determines successful seed production and adaptation of crop plants. Photoperiodic control of this process in flowering plants is mediated by the long-distance mobile signal called florigen partly encoded by FLOWERING LOCUS T (FT) in Arabidopsis and its orthologs in other plant species. Despite the progress in understanding FT transport in the dicot model Arabidopsis, the mechanisms of florigen transport in monocots, which provide most of the biomass in agriculture, are unknown. Here we show that rice FT-INTERACTING PROTEIN 1 (OsFTIP1), a member of the family of multiple C2 domain and transmembrane region proteins (MCTPs) and the closest ortholog of Arabidopsis FTIP1, is required for export of RICE FLOWERING LOCUS T 1 (RFT1) from companion cells to sieve elements. This affects RFT1 movement to the shoot apical meristem and its regulation of rice flowering time under long days. We further reveal that a ubiquitin-like domain kinase γ4, OsUbDKγ4, interacts with OsFTIP1, and modulates its degradation in leaves through the 26S proteasome, which in turn affects RFT1 transport to the SAM. Thus, dynamic modulation of OsFTIP1 abundance in leaves by a negative regulator OsUbDKγ4 is integral to the role of OsFTIP1 in mediating RFT1 transport in rice, and provide key evidence for a conserved role of FTIP1-like MCTPs in mediating florigen transport in flowering plants.

  6. A novel branched chain amino acids responsive transcriptional regulator, BCARR, negatively acts on the proteolytic system in Lactobacillus helveticus.

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    Taketo Wakai

    Full Text Available Transcriptional negative regulation of the proteolytic system of Lactobacillus helveticus CM4 in response to amino acids seems to be very important for the control of antihypertensive peptide production; however, it remains poorly understood. A 26-kDa protein with N-terminal cystathionine β-synthase domains (CBS domain protein, which seems to be involved in the regulatory system, was purified by using a DNA-sepharose bound 300-bp DNA fragment corresponding to the upstream regions of the six proteolytic genes that are down-regulated by amino acids. The CBS domain protein bound to a DNA fragment corresponding to the region upstream of the pepV gene in response to branched chain amino acids (BCAAs. The expression of the pepV gene in Escherichia coli grown in BCAA-enriched medium was repressed when the CBS domain protein was co-expressed. These results reveal that the CBS domain protein acts as a novel type of BCAA-responsive transcriptional regulator (BCARR in L. helveticus. From comparative analysis of the promoter regions of the six proteolysis genes, a palindromic AT-rich motif, 5'-AAAAANNCTWTTATT-3', was predicted as the consensus DNA motif for the BCARR protein binding. Footprint analysis using the pepV promotor region and gel shift analyses with the corresponding short DNA fragments strongly suggested that the BCARR protein binds adjacent to the pepV promoter region and affects the transcription level of the pepV gene in the presence of BCAAs. Homology search analysis of the C-terminal region of the BCARR protein suggested the existence of a unique βαββαβ fold structure that has been reported in a variety of ACT (aspartate kinase-chorismate mutase-tyrA domain proteins for sensing amino acids. These results also suggest that the sensing of BCAAs by the ACT domain might promote the binding of the BCARR to DNA sequences upstream of proteolysis genes, which affects the gene expression of the proteolytic system in L. helveticus.

  7. GlgS, described previously as a glycogen synthesis control protein, negatively regulates motility and biofilm formation in Escherichia coli.

    Science.gov (United States)

    Rahimpour, Mehdi; Montero, Manuel; Almagro, Goizeder; Viale, Alejandro M; Sevilla, Ángel; Cánovas, Manuel; Muñoz, Francisco J; Baroja-Fernández, Edurne; Bahaji, Abdellatif; Eydallin, Gustavo; Dose, Hitomi; Takeuchi, Rikiya; Mori, Hirotada; Pozueta-Romero, Javier

    2013-06-15

    Escherichia coli glycogen metabolism involves the regulation of glgBXCAP operon expression and allosteric control of the GlgC [ADPG (ADP-glucose) pyrophosphorylase]-mediated catalysis of ATP and G1P (glucose-1-phosphate) to ADPG linked to glycogen biosynthesis. E. coli glycogen metabolism is also affected by glgS. Though the precise function of the protein it encodes is unknown, its deficiency causes both reduced glycogen content and enhanced levels of the GlgC-negative allosteric regulator AMP. The transcriptomic analyses carried out in the present study revealed that, compared with their isogenic BW25113 wild-type strain, glgS-null (ΔglgS) mutants have increased expression of the operons involved in the synthesis of type 1 fimbriae adhesins, flagella and nucleotides. In agreement, ΔglgS cells were hyperflagellated and hyperfimbriated, and displayed elevated swarming motility; these phenotypes all reverted to the wild-type by ectopic glgS expression. Also, ΔglgS cells accumulated high colanic acid content and displayed increased ability to form biofilms on polystyrene surfaces. F-driven conjugation based on large-scale interaction studies of glgS with all the non-essential genes of E. coli showed that deletion of purine biosynthesis genes complement the glycogen-deficient, high motility and high biofilm content phenotypes of ΔglgS cells. Overall the results of the present study indicate that glycogen deficiency in ΔglgS cells can be ascribed to high flagellar propulsion and high exopolysaccharide and purine nucleotides biosynthetic activities competing with GlgC for the same ATP and G1P pools. Supporting this proposal, glycogen-less ΔglgC cells displayed an elevated swarming motility, and accumulated high levels of colanic acid and biofilm. Furthermore, glgC overexpression reverted the glycogen-deficient, high swarming motility, high colanic acid and high biofilm content phenotypes of ΔglgS cells to the wild-type. As on the basis of the present study Glg

  8. Inhibitory Effects of Robo2 on Nephrin: A Crosstalk between Positive and Negative Signals Regulating Podocyte Structure

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    Xueping Fan

    2012-07-01

    Full Text Available Robo2 is the cell surface receptor for the repulsive guidance cue Slit and is involved in axon guidance and neuronal migration. Nephrin is a podocyte slit-diaphragm protein that functions in the kidney glomerular filtration barrier. Here, we report that Robo2 is expressed at the basal surface of mouse podocytes and colocalizes with nephrin. Biochemical studies indicate that Robo2 forms a complex with nephrin in the kidney through adaptor protein Nck. In contrast to the role of nephrin that promotes actin polymerization, Slit2-Robo2 signaling inhibits nephrin-induced actin polymerization. In addition, the amount of F-actin associated with nephrin is increased in Robo2 knockout mice that develop an altered podocyte foot process structure. Genetic interaction study further reveals that loss of Robo2 alleviates the abnormal podocyte structural phenotype in nephrin null mice. These results suggest that Robo2 signaling acts as a negative regulator on nephrin to influence podocyte foot process architecture.

  9. Lactate dehydrogenase A negatively regulated by miRNAs promotes aerobic glycolysis and is increased in colorectal cancer.

    Science.gov (United States)

    Wang, Jian; Wang, Hui; Liu, Aifen; Fang, Changge; Hao, Jianguo; Wang, Zhenghui

    2015-08-14

    Reprogramming metabolism of tumor cells is a hallmark of cancer. Lactate dehydrogenase A (LDHA) is frequently overexpressed in tumor cells. Previous studies has shown higher levels of LDHA is related with colorectal cancer (CRC), but its role in tumor maintenance and underlying molecular mechanisms has not been established. Here, we investigated miRNAs-induced changes in LDHA expression. We reported that colorectal cancer express higher levels of LDHA compared with adjacent normal tissue. Knockdown of LDHA resulted in decreased lactate and ATP production, and glucose uptake. Colorectal cancer cells with knockdown of LDHA had much slower growth rate than control cells. Furthermore, we found that miR-34a, miR-34c, miR-369-3p, miR-374a, and miR-4524a/b target LDHA and regulate glycolysis in cancer cells. There is a negative correlation between these miRNAs and LDHA expression in colorectal cancer tissues. More importantly, we identified a genetic loci newly associated with increased colorectal cancer progression, rs18407893 at 11p15.4 (in 3'-UTR of LDHA), which maps to the seed sequence recognized by miR-374a. Cancer cells overexpressed miR-374a has decreased levels of LDHA compared with miR-374a-MUT (rs18407893 at 11p15.4). Taken together, these novel findings provide more therapeutic approaches to the Warburg effect and therapeutic targets of cancer energy metabolism.

  10. The Drosophila Prosecretory Transcription Factor dimmed Is Dynamically Regulated in Adult Enteroendocrine Cells and Protects Against Gram-Negative Infection.

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    Beebe, Katherine; Park, Dongkook; Taghert, Paul H; Micchelli, Craig A

    2015-05-20

    The endocrine system employs peptide hormone signals to translate environmental changes into physiological responses. The diffuse endocrine system embedded in the gastrointestinal barrier epithelium is one of the largest and most diverse endocrine tissues. Furthermore, it is the only endocrine tissue in direct physical contact with the microbial environment of the gut lumen. However, it remains unclear how this sensory epithelium responds to specific pathogenic challenges in a dynamic and regulated manner. We demonstrate that the enteroendocrine cells of the adult Drosophila melanogaster midgut display a transient, sensitive, and systemic induction of the prosecretory factor dimmed (dimm) in response to the Gram-negative pathogen Pseudomonas entomophila (Pe). In enteroendocrine cells, dimm controls the levels of the targets Phm, dcat-4, and the peptide hormone, Allatostatin A. Finally, we identify dimm as a host factor that protects against Pe infection and controls the expression of antimicrobial peptides. We propose that dimm provides "gain" in enteroendocrine output during the adaptive response to episodic pathogen exposure. Copyright © 2015 Beebe et al.

  11. TINA interacts with the NIMA kinase in Aspergillus nidulans and negatively regulates astral microtubules during metaphase arrest.

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    Osmani, Aysha H; Davies, Jonathan; Oakley, C Elizabeth; Oakley, Berl R; Osmani, Stephen A

    2003-08-01

    The tinA gene of Aspergillus nidulans encodes a protein that interacts with the NIMA mitotic protein kinase in a cell cycle-specific manner. Highly similar proteins are encoded in Neurospora crassa and Aspergillus fumigatus. TINA and NIMA preferentially interact in interphase and larger forms of TINA are generated during mitosis. Localization studies indicate that TINA is specifically localized to the spindle pole bodies only during mitosis in a microtubule-dependent manner. Deletion of tinA alone is not lethal but displays synthetic lethality in combination with the anaphase-promoting complex/cyclosome mutation bimE7. At the bimE7 metaphase arrest point, lack of TINA enhanced the nucleation of bundles of cytoplasmic microtubules from the spindle pole bodies. These microtubules interacted to form spindles joined in series via astral microtubules as revealed by live cell imaging. Because TINA is modified and localizes to the spindle pole bodies at mitosis, and lack of TINA causes enhanced production of cytoplasmic microtubules at metaphase arrest, we suggest TINA is involved in negative regulation of the astral microtubule organizing capacity of the spindle pole bodies during metaphase.

  12. RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer.

    Science.gov (United States)

    Pascual-Vargas, Patricia; Cooper, Samuel; Sero, Julia; Bousgouni, Vicky; Arias-Garcia, Mar; Bakal, Chris

    2017-03-01

    In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population.

  13. The role of ELK3 to regulate peritumoral lymphangiogenesis and VEGF-C production in triple negative breast cancer cells.

    Science.gov (United States)

    Oh, Nuri; Park, Ji-In; Park, Ji-Hoon; Kim, Kwang-Soo; Lee, Dong Ryul; Park, Kyung-Soon

    2017-03-18

    Tumor-induced lymphangiogenesis, a major conduit for cancer cell dissemination from the primary tumor site to lymph nodes and beyond, eventually leads to metastasis in cancer patients. Given the recent evidence revealing that the suppression of ELK3 inhibits the metastasis of triple-negative breast cancer cells, we aimed to study the underlying mechanism of impaired metastasis in ELK3-suppressed MDA-MB-231 cells (ELK3 KD) with regard to lymphangiogenesis. We found that the secretome of ELK3 KD cells inhibited tube formation, whereas it promoted the migration and invasion of lymphatic endothelial cells (LECs) in vitro. In vivo analysis revealed that peritumoral lymphatic vessels were not developed around the xenografted tumors of ELK3 KD. We further revealed that the suppression of NF-κB signaling in ELK3 KD was the primary cause of the reduced VEGFC expression. Taken together, we suggest that ELK3 is an upstream regulator of the NF-κB signaling pathway, the inhibition of which leads to the suppression of peritumoral lymphatic vessel development, possibly due to a low VEGFC expression.

  14. Phosphorylation of Nlp by Plk1 negatively regulates its dynein-dynactin-dependent targeting to the centrosome.

    Science.gov (United States)

    Casenghi, Martina; Barr, Francis A; Nigg, Erich A

    2005-11-01

    When cells enter mitosis the microtubule (MT) network undergoes a profound rearrangement, in part due to alterations in the MT nucleating and anchoring properties of the centrosome. Ninein and the ninein-like protein (Nlp) are centrosomal proteins involved in MT organisation in interphase cells. We show that the overexpression of these two proteins induces the fragmentation of the Golgi, and causes lysosomes to disperse toward the cell periphery. The ability of Nlp and ninein to perturb the cytoplasmic distribution of these organelles depends on their ability to interact with the dynein-dynactin motor complex. Our data also indicate that dynactin is required for the targeting of Nlp and ninein to the centrosome. Furthermore, phosphorylation of Nlp by the polo-like kinase 1 (Plk1) negatively regulates its association with dynactin. These findings uncover a mechanism through which Plk1 helps to coordinate changes in MT organisation with cell cycle progression, by controlling the dynein-dynactin-dependent transport of centrosomal proteins.

  15. Phosphodiesterase 2 negatively regulates adenosine-induced transcription of the tyrosine hydroxylase gene in PC12 rat pheochromocytoma cells.

    Science.gov (United States)

    Makuch, Edyta; Kuropatwa, Marianna; Kurowska, Ewa; Ciekot, Jaroslaw; Klopotowska, Dagmara; Matuszyk, Janusz

    2014-07-05

    Adenosine induces expression of the tyrosine hydroxylase (TH) gene in PC12 cells. However, it is suggested that atrial natriuretic peptide (ANP) inhibits expression of this gene. Using real-time PCR and luciferase reporter assays we found that ANP significantly decreases the adenosine-induced transcription of the TH gene. Results of measurements of cyclic nucleotide concentrations indicated that ANP-induced accumulation of cGMP inhibits the adenosine-induced increase in cAMP level. Using selective phosphodiesterase 2 (PDE2) inhibitors and a synthetic cGMP analog activating PDE2, we found that PDE2 is involved in coupling the ANP-triggered signal to the cAMP metabolism. We have established that ANP-induced elevated levels of cGMP as well as cGMP analog stimulate hydrolytic activity of PDE2, leading to inhibition of adenosine-induced transcription of the TH gene. We conclude that ANP mediates negative regulation of TH gene expression via stimulation of PDE2-dependent cAMP breakdown in PC12 cells.

  16. Genome-wide profiling of AP-1-regulated transcription provides insights into the invasiveness of triple-negative breast cancer.

    Science.gov (United States)

    Zhao, Chunyan; Qiao, Yichun; Jonsson, Philip; Wang, Jian; Xu, Li; Rouhi, Pegah; Sinha, Indranil; Cao, Yihai; Williams, Cecilia; Dahlman-Wright, Karin

    2014-07-15

    Triple-negative breast cancer (TNBC) is an aggressive clinical subtype accounting for up to 20% of all breast cancers, but its malignant determinants remain largely undefined. Here, we show that in TNBC the overexpression of Fra-1, a component of the transcription factor AP-1, offers prognostic potential. Fra-1 depletion or its heterodimeric partner c-Jun inhibits the proliferative and invasive phenotypes of TNBC cells in vitro. Similarly, RNAi-mediated attenuation of Fra-1 or c-Jun reduced cellular invasion in vivo in a zebrafish tumor xenograft model. Exploring the AP-1 cistrome and the AP-1-regulated transcriptome, we obtained insights into the transcriptional regulatory networks of AP-1 in TNBC cells. Among the direct targets identified for Fra-1/c-Jun involved in proliferation, adhesion, and cell-cell contact, we found that AP-1 repressed the expression of E-cadherin by transcriptional upregulation of ZEB2 to stimulate cell invasion. Overall, this work illuminates the pathways through which TNBC cells acquire invasive and proliferative properties.

  17. Negative regulation of opioid receptor-G protein-Ca2+ channel pathway by the nootropic nefiracetam.

    Science.gov (United States)

    Yoshii, Mitsunobu; Furukawa, Taiji; Ogihara, Yoshiyasu; Watabe, Shigeo; Shiotani, Tadashi; Ishikawa, Yasuro; Nishimura, Masao; Nukada, Toshihide

    2004-10-01

    It has recently been reported that nefiracetam, a nootropic agent, is capable of attenuating the development of morphine dependence and tolerance in mice. The mechanism of this antimorphine action is not clear. The present study was designed to address this issue using Xenopus oocytes expressing delta-opioid receptors, G proteins (G(i3alpha) or G(o1alpha)), and N-type (alpha1B) Ca2+ channels. Membrane currents through Ca2+ channels were recorded from the oocytes under voltage-clamp conditions. The Ca2+ channel currents were reduced reversibly by 40-60% in the presence of 1 microM leucine-enkephalin (Leu-Enk). The Leu-Enk-induced current inhibition was recovered promptly by nefiracetam (1 microM), while control currents in the absence of Leu-Enk were not influenced by nefiracetam. A binding assay revealed that 3H-nefiracetam preferentially bound to the membrane fraction of oocytes expressing G(i3alpha). When delta-opioid receptors were coexpressed, the binding was significantly increased. However, an additional expression of alpha1B Ca2+ channels decreased the binding. The results suggest that nefiracetam preferentially binds to G(i3alpha) associated with delta-opioid receptors, thereby inhibiting the association of G proteins with Ca2+ channels. In conclusion, nefiracetam negatively regulates the inhibitory pathway of opioid receptor-G protein-Ca2+ channel.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Jessica Diring

    Full Text Available 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.

  20. The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

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    Min Wang

    Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

  1. RNAi screens for Rho GTPase regulators of cell shape and YAP/TAZ localisation in triple negative breast cancer

    Science.gov (United States)

    Pascual-Vargas, Patricia; Cooper, Samuel; Sero, Julia; Bousgouni, Vicky; Arias-Garcia, Mar; Bakal, Chris

    2017-01-01

    In order to metastasise, triple negative breast cancer (TNBC) must make dynamic changes in cell shape. The shape of all eukaryotic cells is regulated by Rho Guanine Nucleotide Exchange Factors (RhoGEFs), which activate Rho-family GTPases in response to mechanical and informational cues. In contrast, Rho GTPase-activating proteins (RhoGAPs) inhibit Rho GTPases. However, which RhoGEFs and RhoGAPS couple TNBC cell shape to changes in their environment is very poorly understood. Moreover, whether the activity of particular RhoGEFs and RhoGAPs become dysregulated as cells evolve the ability to metastasise is not clear. Towards the ultimate goal of identifying RhoGEFs and RhoGAPs that are essential for TNBC metastasis, we performed an RNAi screen to isolate RhoGEFs and RhoGAPs that contribute to the morphogenesis of the highly metastatic TNBC cell line LM2, and its less-metastatic parental cell line MDA-MB-231. For ~6 million cells from each cell line, we measured 127 different features following the depletion of 142 genes. Using a linear classifier scheme we also describe the morphological heterogeneity of each gene-depleted population. PMID:28248929

  2. Positive and negative regulation of FcepsilonRI-mediated signaling by the adaptor protein LAB/NTAL.

    Science.gov (United States)

    Zhu, Minghua; Liu, Yan; Koonpaew, Surapong; Granillo, Olivia; Zhang, Weiguo

    2004-10-18

    Linker for activation of B cells (LAB, also called NTAL; a product of wbscr5 gene) is a newly identified transmembrane adaptor protein that is expressed in B cells, NK cells, and mast cells. Upon BCR activation, LAB is phosphorylated and interacts with Grb2. LAB is capable of rescuing thymocyte development in LAT-deficient mice. To study the in vivo function of LAB, LAB-deficient mice were generated. Although disruption of the Lab gene did not affect lymphocyte development, it caused mast cells to be hyperresponsive to stimulation via the FcepsilonRI, evidenced by enhanced Erk activation, calcium mobilization, degranulation, and cytokine production. These data suggested that LAB negatively regulates mast cell function. However, mast cells that lacked both linker for activation of T cells (LAT) and LAB proteins had a more severe block in FcepsilonRI-mediated signaling than LAT(-/-) mast cells, demonstrating that LAB also shares a redundant function with LAT to play a positive role in FcepsilonRI-mediated signaling.

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

    Science.gov (United States)

    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.

  4. Salinomycin activates AMP-activated protein kinase-dependent autophagy in cultured osteoblastoma cells: a negative regulator against cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Lun-qing Zhu

    Full Text Available BACKGROUND: The malignant osteoblastoma has poor prognosis, thus the search for novel and more efficient chemo-agents against this disease is urgent. Salinomycin induces broad anti-cancer effects both in vivo and in vitro, however, its role in osteoblastoma is still not clear. KEY FINDINGS: Salinomycin induced both apoptosis and autophagy in cultured U2OS and MG-63 osteoblastoma cells. Inhibition of autophagy by 3-methyladenine (3-MA, or by RNA interference (RNAi of light chain 3B (LC3B, enhanced salinomycin-induced cytotoxicity and apoptosis. Salinomycin induced a profound AMP-activated protein kinase (AMPK activation, which was required for autophagy induction. AMPK inhibition by compound C, or by AMPKα RNAi prevented salinomycin-induced autophagy activation, while facilitating cancer cell death and apoptosis. On the other hand, the AMPK agonist AICAR promoted autophagy activation in U2OS cells. Salinomycin-induced AMPK activation was dependent on reactive oxygen species (ROS production in osteoblastoma cells. Antioxidant n-acetyl cysteine (NAC significantly inhibited salinomycin-induced AMPK activation and autophagy induction. CONCLUSIONS: Salinomycin activates AMPK-dependent autophagy in osteoblastoma cells, which serves as a negative regulator against cell apoptosis. AMPK-autophagy inhibition might be a novel strategy to sensitize salinomycin's effect in cancer cells.

  5. Canonical Transient Receptor Potential (TRPC) 1 Acts as a Negative Regulator for Vanilloid TRPV6-mediated Ca2+ Influx*

    Science.gov (United States)

    Schindl, Rainer; Fritsch, Reinhard; Jardin, Isaac; Frischauf, Irene; Kahr, Heike; Muik, Martin; Riedl, Maria Christine; Groschner, Klaus; Romanin, Christoph

    2012-01-01

    TRP proteins mostly assemble to homomeric channels but can also heteromerize, preferentially within their subfamilies. The TRPC1 protein is the most versatile member and forms various TRPC channel combinations but also unique channels with the distantly related TRPP2 and TRPV4. We show here a novel cross-family interaction between TRPC1 and TRPV6, a Ca2+ selective member of the vanilloid TRP subfamily. TRPV6 exhibited substantial co-localization and in vivo interaction with TRPC1 in HEK293 cells, however, no interaction was observed with TRPC3, TRPC4, or TRPC5. Ca2+ and Na+ currents of TRPV6-overexpressing HEK293 cells are significantly reduced by co-expression of TRPC1, correlating with a dramatically suppressed plasma membrane targeting of TRPV6. In line with their intracellular retention, remaining currents of TRPC1 and TRPV6 co-expression resemble in current-voltage relationship that of TRPV6. Studying the N-terminal ankyrin like repeat domain, structurally similar in the two proteins, we have found that these cytosolic segments were sufficient to mediate a direct heteromeric interaction. Moreover, the inhibitory role of TRPC1 on TRPV6 influx was also maintained by expression of only its N-terminal ankyrin-like repeat domain. Our experiments provide evidence for a functional interaction of TRPC1 with TRPV6 that negatively regulates Ca2+ influx in HEK293 cells. PMID:22932896

  6. The LIN-15A and LIN-56 transcriptional regulators interact to negatively regulate EGF/Ras signaling in Caenorhabditis elegans vulval cell-fate determination.

    Science.gov (United States)

    Davison, Ewa M; Saffer, Adam M; Huang, Linda S; DeModena, John; Sternberg, Paul W; Horvitz, H Robert

    2011-03-01

    The restricted expression of epidermal growth factor (EGF) family ligands is important for proper development and for preventing cancerous growth in mammals. In Caenorhabditis elegans, the class A and B synthetic multivulva (synMuv) genes redundantly repress expression of lin-3 EGF to negatively regulate Ras-mediated vulval development. The class B synMuv genes encode proteins homologous to components of the NuRD and Myb-MuvB/dREAM transcriptional repressor complexes, indicating that they likely silence lin-3 EGF through chromatin remodeling. The two class A synMuv genes cloned thus far, lin-8 and lin-15A, both encode novel proteins. The LIN-8 protein is nuclear. We have characterized the class A synMuv gene lin-56 and found it to encode a novel protein that shares a THAP-like C(2)CH motif with LIN-15A. Both the LIN-56 and LIN-15A proteins localize to nuclei. Wild-type levels of LIN-56 require LIN-15A, and wild-type levels and/or localization of LIN-15A requires LIN-56. Furthermore, LIN-56 and LIN-15A interact in the yeast two-hybrid system. We propose that LIN-56 and LIN-15A associate in a nuclear complex that inhibits vulval specification by repressing lin-3 EGF expression.

  7. A tomato (Solanum lycopersicum) APETALA2/ERF gene, SlAP2a, is a negative regulator of fruit ripening

    National Research Council Canada - National Science Library

    Chung, Mi‐Young; Vrebalov, Julia; Alba, Rob; Lee, JeMin; McQuinn, Ryan; Chung, Jae‐Dong; Klein, Patricia; Giovannoni, James

    2010-01-01

    .... Tomato is a model for biology and genetics regulating specific ripening pathways including ethylene, carotenoids and cell wall metabolism in addition to upstream signaling and transcriptional regulators. Ripening...

  8. Effect of Negative Pressure on Proliferation, Virulence Factor Secretion, Biofilm Formation, and Virulence-Regulated Gene Expression of Pseudomonas aeruginosa In Vitro

    Directory of Open Access Journals (Sweden)

    Guo-Qi Wang

    2016-01-01

    Full Text Available Objective. To investigate the effect of negative pressure conditions induced by NPWT on P. aeruginosa. Methods. P. aeruginosa was cultured in a Luria–Bertani medium at negative pressure of −125 mmHg for 24 h in the experimental group and at atmospheric pressure in the control group. The diameters of the colonies of P. aeruginosa were measured after 24 h. ELISA kit, orcinol method, and elastin-Congo red assay were used to quantify the virulence factors. Biofilm formation was observed by staining with Alexa Fluor® 647 conjugate of concanavalin A (Con A. Virulence-regulated genes were determined by quantitative RT-PCR. Results. As compared with the control group, growth of P. aeruginosa was inhibited by negative pressure. The colony size under negative pressure was significantly smaller in the experimental group than that in the controls (p<0.01. Besides, reductions in the total amount of virulence factors were observed in the negative pressure group, including exotoxin A, rhamnolipid, and elastase. RT-PCR results revealed a significant inhibition in the expression level of virulence-regulated genes. Conclusion. Negative pressure could significantly inhibit the growth of P. aeruginosa. It led to a decrease in the virulence factor secretion, biofilm formation, and a reduction in the expression level of virulence-regulated genes.

  9. Mothers' depressive symptoms predict both increased and reduced negative reactivity: aversion sensitivity and the regulation of emotion.

    Science.gov (United States)

    Dix, Theodore; Moed, Anat; Anderson, Edward R

    2014-07-01

    This study examined whether, as mothers' depressive symptoms increase, their expressions of negative emotion to children increasingly reflect aversion sensitivity and motivation to minimize ongoing stress or discomfort. In multiple interactions over 2 years, negative affect expressed by 319 mothers and their children was observed across variations in mothers' depressive symptoms, the aversiveness of children's immediate behavior, and observed differences in children's general negative reactivity. As expected, depressive symptoms predicted reduced maternal negative reactivity when child behavior was low in aversiveness, particularly with children who were high in negative reactivity. Depressive symptoms predicted high negative reactivity and steep increases in negative reactivity as the aversiveness of child behavior increased, particularly when high and continued aversiveness from the child was expected (i.e., children were high in negative reactivity). The findings are consistent with the proposal that deficits in parenting competence as depressive symptoms increase reflect aversion sensitivity and motivation to avoid conflict and suppress children's aversive behavior.

  10. The impact of emotion regulation and illness-focused coping strategies on the relation of illness-related negative emotions to subjective health.

    Science.gov (United States)

    Karademas, Evangelos C; Tsalikou, Calliope; Tallarou, Maria-Christina

    2011-04-01

    In this study we examined whether emotion regulation and illness-focused coping strategies mediate and/ or moderate the relation of illness-related negative emotions to patients' subjective health. One hundred and thirty-five cardiac patients participated in the study. Illness-focused coping strategies were found to mediate the relation of emotions to physical functioning, whereas emotion regulation strategies mediated the relation to psychological well-being. Moreover, an emotion regulation strategy (i.e. emotion suppression) and two illness-focused coping strategies (instrumental coping and adherence) moderated the two relationships. These findings suggest that both emotion regulation and illness-focused coping strategies are integral parts of the illness-related negative emotions-health relationship.

  11. Thyroid hormone negatively regulates CDX2 and SOAT2 mRNA expression via induction of miRNA-181d in hepatic cells

    Energy Technology Data Exchange (ETDEWEB)

    Yap, Chui Sun; Sinha, Rohit Anthony [Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857 (Singapore); Ota, Sho; Katsuki, Masahito [Department of Molecular Endocrinology, Tohoku University Graduate School of Medicine, Seiryo-machi, Aoba-ku, Sendai 980-8575 (Japan); Yen, Paul Michael, E-mail: paul.yen@duke-nus.edu.sg [Cardiovascular and Metabolic Disorders, Duke-NUS Graduate Medical School, 8, College Road, Singapore 169857 (Singapore)

    2013-11-01

    Highlights: •Thyroid hormone induces miR-181d expression in human hepatic cells and mouse livers. •Thyroid hormone downregulates CDX2 and SOAT2 (or ACAT2) via miR-181d. •miR-181d reduces cholesterol output from human hepatic cells. -- Abstract: Thyroid hormones (THs) regulate transcription of many metabolic genes in the liver through its nuclear receptors (TRs). Although the molecular mechanisms for positive regulation of hepatic genes by TH are well understood, much less is known about TH-mediated negative regulation. Recently, several nuclear hormone receptors were shown to downregulate gene expression via miRNAs. To further examine the potential role of miRNAs in TH-mediated negative regulation, we used a miRNA microarray to identify miRNAs that were directly regulated by TH in a human hepatic cell line. In our screen, we discovered that miRNA-181d is a novel hepatic miRNA that was regulated by TH in hepatic cell culture and in vivo. Furthermore, we identified and characterized two novel TH-regulated target genes that were downstream of miR-181d signaling: caudal type homeobox 2 (CDX2) and sterol O-acyltransferase 2 (SOAT2 or ACAT2). CDX2, a known positive regulator of hepatocyte differentiation, was regulated by miR-181d and directly activated SOAT2 gene expression. Since SOAT2 is an enzyme that generates cholesteryl esters that are packaged into lipoproteins, our results suggest miR-181d plays a significant role in the negative regulation of key metabolic genes by TH in the liver.

  12. Estrogen receptor beta participate in the regulation of metabolizm of extracellular matrix in estrogen alpha negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Mariusz Kuźmicki

    2010-01-01

    Full Text Available The biology of breast cancer is closely releted to sex steroid hormones. Estrogen receptor beta is overexpressed in around 70% breast cancer cases, referrd to as "ER positive". Estrogens bind to estrogen receptor and stimulate the transcription of genes involved in control of cell proliferation. Moreover, estrogens may induce growth factors and components of extracellular matrix and interact with them in a complex manner. Extracellular matrix and integrins play an important role in cell functions and their aberrant expressions are implicated in breast cancer development, invasion and metastasis. ER beta is certainly associated with more differentiated tumors, while evidence of role of ER beta is controversial. The highly invasive breast cancer ER beta negative cell line MDA-MB 231 can be the model of exam the role of ER beta in breast cancer. The aim of this study was to examine the role of activation of ER beta on the metabolism of the extracellular matrix and the expression of beta-1 integrin in the breast cancer cell line MDA-MB 231. The cells were exposed on the estradiol, tamoxifen, raloxifen and genisteina in dose dependent concentrations. To determine the relative rate of collagen syntesis we measured the time-dependent reduction of collagen-bound radioactivity after pulse-chase labeling with [3 H] prolina by Peterkofsky methods. The expression of beta-1 integrin was determine by Western blot analysis. The activity of MMP2 and 9 were measured using gelatin zymography with an image analysis system. Our data suggest on the role of estrogen receptor beta on the metabolism of extracellular matrix in the breast cancer line MDA - MB 231. Estradiol and SERMs regulate the expression of ECM proteins: collagen, integrins and enhance activity of metaloproteinases 2 and 9.

  13. Estrogen receptor beta participate in the regulation of metabolizm of extracellular matrix in estrogen alpha negative breast cancer.

    Science.gov (United States)

    Leśniewska, Monika; Miltyk, Wojciech; Swiatecka, Jolanta; Tomaszewska, Małgorzata; Kuźmicki, Mariusz; Pałka, Jerzy; Wołczyński, Sławomir

    2009-01-01

    The biology of breast cancer is closely releted to sex steroid hormones. Estrogen receptor beta is overexpressed in around 70% breast cancer cases, referrd to as "ER positive". Estrogens bind to estrogen receptor and stimulate the transcription of genes involved in control of cell proliferation. Moreover, estrogens may induce growth factors and components of extracellular matrix and interact with them in a complex manner. Extracellular matrix and integrins play an important role in cell functions and their aberrant expressions are implicated in breast cancer development, invasion and metastasis. ER beta is certainly associated with more differentiated tumors, while evidence of role of ER beta is controversial. The highly invasive breast cancer ER beta negative cell line MDA-MB 231 can be the model of exam the role of ER beta in breast cancer. The aim of this study was to examine the role of activation of ER beta on the metabolism of the extracellular matrix and the expression of beta-1 integrin in the breast cancer cell line MDA-MB 231. The cells were exposed on the estradiol, tamoxifen, raloxifen and genisteina in dose dependent concentrations. To determine the relative rate of collagen syntesis we measured the time-dependent reduction of collagen-bound radioactivity after pulse-chase labeling with [3 H] prolina by Peterkofsky methods. The expression of beta-1 integrin was determine by Western blot analysis. The activity of MMP2 and 9 were measured using gelatin zymography with an image analysis system. Our data suggest on the role of estrogen receptor beta on the metabolism of extracellular matrix in the breast cancer line MDA - MB 231. Estradiol and SERMs regulate the expression of ECM proteins: collagen, integrins and enhance activity of metaloproteinases 2 and 9.

  14. ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.

  15. Negative Fgf8-Bmp2 feed-back is regulated by miR-130 during early cardiac specification.

    Science.gov (United States)

    Lopez-Sanchez, Carmen; Franco, Diego; Bonet, Fernando; Garcia-Lopez, Virginio; Aranega, Amelia; Garcia-Martinez, Virginio

    2015-10-01

    It is known that secreted proteins from the anterior lateral endoderm, FGF8 and BMP2, are involved in mesodermal cardiac differentiation, which determines the first cardiac field, defined by the expression of the earliest specific cardiac markers Nkx-2.5 and Gata4. However, the molecular mechanisms responsible for early cardiac development still remain unclear. At present, microRNAs represent a novel layer of complexity in the regulatory networks controlling gene expression during cardiovascular development. This paper aims to study the role of miR130 during early cardiac specification. Our model is focused on developing chick at gastrula stages. In order to identify those regulatory factors which are involved in cardiac specification, we conducted gain- and loss-of-function experiments in precardiac cells by administration of Fgf8, Bmp2 and miR130, through in vitro electroporation technique and soaked beads application. Embryos were subjected to in situ hybridization, immunohistochemistry and qPCR procedures. Our results reveal that Fgf8 suppresses, while Bmp2 induces, the expression of Nkx-2.5 and Gata4. They also show that Fgf8 suppresses Bmp2, and vice versa. Additionally, we observed that Bmp2 regulates miR-130 -a putative microRNA that targets Erk1/2 (Mapk1) 3'UTR, recognizing its expression in precardiac cells which overlap with Erk1/2 pattern. Finally, we evidence that miR-130 is capable to inhibit Erk1/2 and Fgf8, resulting in an increase of Bmp2, Nkx-2.5 and Gata4. Our data present miR-130 as a necessary linkage in the control of Fgf8 signaling, mediated by Bmp2, establishing a negative feed-back loop responsible to achieve early cardiac specification.

  16. Connective tissue growth factor reacts as an IL-6/STAT3-regulated hepatic negative acute phase protein

    Institute of Scientific and Technical Information of China (English)

    Olav A Gressner; Ieva Peredniene; Axel M Gressner

    2011-01-01

    AIM: To investigate the mechanisms involved in a possible modulator role of interleukin (IL)-6 signalling on CYR61-CTGF-NOV (CCN) 2/connective tissue growth factor (CTGF) expression in hepatocytes (PC) and to look for a relation between serum concentrations of these two parameters in patients with acute inflammation.METHODS: Expression of CCN2/CTGF, p-STAT3, p-Smad 3/1 and p-Smad2 was examined in primary freshly isolated rat or cryo-preserved human PC exposed to various stimuli by Western blotting, electrophoretic mobility shift assay (EMSA), reporter-gene-assays and reversetranscriptase polymerase chain reaction.RESULTS: IL-6 strongly down-regulated CCN2/CTGF protein and mRNA expression in PC, enhanceable by extracellular presence of the soluble IL-6 receptor gp80,and supported by an inverse relation between IL-6 and CCN2/CTGF concentrations in patients' sera. The inhibition of TGFβ1 driven CCN2/CTGF expression by IL-6 did not involve a modulation of Smad2 (and Smad1/3)signalling. However, the STAT3 SH2 domain binding peptide, a selective inhibitor of STAT3 DNA binding activity, counteracted the inhibitory effect of IL-6 on CCN2/CTGF expression much more pronounced than pyrrolidine-dithiocarbamate, an inhibitor primarily of STAT3 phosphorylation. An EMSA confirmed STAT3 binding to the proposed proximal STAT binding site in the CCN2 /CTGF promoter.CONCLUSION: CCN2/CTGF is identified as a hepatocellular negative acute phase protein which is downregulated by IL-6 via the STAT3 pathway through interaction on the DNA binding level.

  17. Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

    Directory of Open Access Journals (Sweden)

    Jin-Gyoung Jung

    2014-10-01

    Full Text Available The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2