Science.gov

Sample records for ap-1 transcriptional activity

  1. The AP-1 transcription factor homolog Pf-AP-1 activates transcription of multiple biomineral proteins and potentially participates in Pinctada fucata biomineralization

    PubMed Central

    Zheng, Xiangnan; Cheng, Minzhang; Xiang, Liang; Liang, Jian; Xie, Liping; Zhang, Rongqing

    2015-01-01

    Activator protein-1 (AP-1) is an important bZIP transcription factor that regulates a series of physiological processes by specifically activating transcription of several genes, and one of its well-chartered functions in mammals is participating in bone mineralization. We isolated and cloned the complete cDNA of a Jun/AP-1 homolog from Pinctada fucata and called it Pf-AP-1. Pf-AP-1 had a highly conserved bZIP region and phosphorylation sites compared with those from mammals. A tissue distribution analysis showed that Pf-AP-1 was ubiquitously expressed in P. fucata and the mRNA level of Pf-AP-1 is extremely high in mantle. Pf-AP-1 expression was positively associated with multiple biomineral proteins in the mantle. The luciferase reporter assay in a mammalian cell line showed that Pf-AP-1 significantly up-regulates the transcriptional activity of the promoters of KRMP, Pearlin, and Prisilkin39. Inhibiting the activity of Pf-AP-1 depressed the expression of multiple matrix proteins. Pf-AP-1 showed a unique expression pattern during shell regeneration and pearl sac development, which was similar to the pattern observed for biomineral proteins. These results suggest that the Pf-AP-1 AP-1 homolog is an important transcription factor that regulates transcription of several biomineral proteins simultaneously and plays a role in P. fucata biomineralization, particularly during pearl and shell formation. PMID:26404494

  2. YY1 represses human papillomavirus type 16 transcription by quenching AP-1 activity.

    PubMed Central

    O'Connor, M J; Tan, S H; Tan, C H; Bernard, H U

    1996-01-01

    YY1 is a multifunctional transcription factor that has been shown to regulate the expression of a number of cellular and viral genes, including the human papillomavirus (HPV) oncogenes E6 and E7. In this study, we have analyzed the YY1-mediated repression of the HPV type 16 (HPV-16) E6-E7 promoter. A systematic analysis to identify YY1 sites present in the HPV-16 long control region showed that of 30 potential YY1 binding motifs, 24 bound purified recombinant YY1 protein, but only 10 of these were able to bind YY1 when nuclear extracts of HeLa cells were used. Of these, only a cluster of five sites, located in the vicinity of an AP-1 motif, were found to be responsible for repressing the HPV-16 P97 promoter. All five sites were required for repression, the mutation of any one site giving rise to a four- to sixfold increase in transcriptional activity. The target for YY1-mediated repression was identified as being a highly conserved AP-1 site, and we propose that AP-1 may represent a common target for YY1 repression. We also provide data demonstrating that YY1 can bind the transcriptional coactivator CREB-binding protein and propose a potentially novel mechanism by which YY1 represses AP-1 activity as a result of this YY1-CREB-binding protein interaction. PMID:8794287

  3. Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development

    PubMed Central

    Vonhoff, Fernando; Kuehn, Claudia; Blumenstock, Sonja; Sanyal, Subhabrata; Duch, Carsten

    2013-01-01

    Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. We combine genetic manipulation, imaging and quantitative dendritic architecture analysis in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dα7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter we demonstrate that AP-1 transcriptional activity is downstream of Dα7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. Our results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies. PMID:23293292

  4. Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

    PubMed Central

    Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

    2015-01-01

    The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

  5. Activation of transcription factor AP-1 and mitogen-activated protein kinases in aniline-induced splenic toxicity

    SciTech Connect

    Khan, M. Firoze . E-mail: mfkhan@utmb.edu; Kannan, Subburaj; Wang Jianling

    2006-01-15

    Signaling mechanisms in aniline-induced fibrogenic and/or tumorigenic response in the spleen are not known. Previous studies have shown that aniline exposure leads to iron accumulation and oxidative stress in the spleen, which may cause activation of redox-sensitive transcription factors and regulate the transcription of genes involved in fibrosis and/or tumorigenesis. To test this, male SD rats were treated with 0.5 mmol/kg/day aniline via drinking water for 30 days, and activation of transcription factor AP-1 was determined in the splenocyte nuclear extracts (NEs). AP-1 DNA-binding activity in the NEs of freshly isolated splenocytes from aniline-treated rats increased in comparison to the controls, as determined by electrophoretic mobility shift assay (EMSA). AP-1 binding was also determined in the NEs of cultured splenocytes (2 h and 24 h), which showed even a greater increase in binding activity at 2 h. The specificity of AP-1 binding for relevant DNA motifs was confirmed by competition EMSA and by supershift EMSA using antibodies specific to c-Jun and c-Fos. To further explore the signaling mechanisms in the AP-1 activation, phosphorylation patterns of mitogen-activated protein kinases (MAPKs) were pursued. Aniline exposure induced increases in the phosphorylation of the three classes of MAPKs: extracellular-signal-regulated kinase (ERK 1/2), c-Jun N-terminal kinase (JNK 1/2), and p38 MAPKs. Furthermore, TGF-{beta}1 mRNA expression showed a 3-fold increase in the spleens of aniline-treated rats. These observations suggest a strong association among MAPK phosphorylation, AP-1 activation, and enhanced TGF-{beta}1 gene expression. The observed sequence of events subsequent to aniline exposure could regulate genes that lead to fibrogenic and/or tumorigenic response in the spleen.

  6. Retinoic acid-induced AP-1 transcriptional activity regulates B16 mouse melanoma growth inhibition and differentiation.

    PubMed

    Huang, Ying; Boskovic, Goran; Niles, Richard M

    2003-02-01

    Retinoic acid (RA) inhibits growth and induces differentiation of B16 mouse melanoma cells. These effects are accompanied by a large increase in PKCalpha mRNA and protein levels and surprisingly an increase in activating protein-1 (AP-1) transcriptional activity. To further investigate the RA-induced AP-1 activity we established clones of B16 cells stably expressing an AP-1-luciferase reporter gene. Treatment of these clones with phorbol dibutyrate increased AP-1 activity which peaked at 2-4 h and returned to baseline level by 24 h. In contrast, RA treatment resulted in a slow increase in AP-1 activity that reached a maximum level at 48 h and was maintained for the duration of the treatment. We tested the importance of the RA-induced AP-1 activity by establishing clones which stably express a dominant negative fos gene (A-fos) and have greatly diminished AP-1 activity. Growth rates of untreated A-fos expressing cells were similar to wt B16 and clones not expressing A-fos. However, clones expressing the dominant-negative fos had a markedly decreased sensitivity to RA-induced inhibition of anchorage-dependent and -independent growth. Treatment of wt B16 cells for 48 h with RA increased melanin production by two to fourfold, but this effect was completely lost in the A-fos clones. The ability of RA to induce RARbeta and PKCalpha expression was retained in A-fos clones, suggesting that A-fos was not interfering with RAR transcription activation functions. We tested whether the RA-induced AP-1 activity might be mediated by the ERK1/2 MAPK pathway. Inhibition of ERK1/2 phosphorylation stimulated AP-1 activity, which was not additive to that induced by RA. This finding raises the possibility that this MAPK pathway may be a target of retinoid action. Our observations suggest that AP-1 transcriptional activity induced by RA likely plays an important role in the biological changes mediated by this retinoid in B16 melanoma cells. PMID:12494454

  7. Transcription factor AP-1 in esophageal squamous cell carcinoma: Alterations in activity and expression during Human Papillomavirus infection

    PubMed Central

    2009-01-01

    Background Esophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths in Jammu and Kashmir (J&K) region of India. A substantial proportion of esophageal carcinoma is associated with infection of high-risk HPV type 16 and HPV18, the oncogenic expression of which is controlled by host cell transcription factor Activator Protein-1 (AP-1). We, therefore, have investigated the role of DNA binding and expression pattern of AP-1 in esophageal cancer with or without HPV infection. Methods Seventy five histopathologically-confirmed esophageal cancer and an equal number of corresponding adjacent normal tissue biopsies from Kashmir were analyzed for HPV infection, DNA binding activity and expression of AP-1 family of proteins by PCR, gel shift assay and immunoblotting respectively. Results A high DNA binding activity and elevated expression of AP-1 proteins were observed in esophageal cancer, which differed between HPV positive (19%) and HPV negative (81%) carcinomas. While JunB, c-Fos and Fra-1 were the major contributors to AP-1 binding activity in HPV negative cases, Fra-1 was completely absent in HPV16 positive cancers. Comparison of AP-1 family proteins demonstrated high expression of JunD and c-Fos in HPV positive tumors, but interestingly, Fra-1 expression was extremely low or nil in these tumor tissues. Conclusion Differential AP-1 binding activity and expression of its specific proteins between HPV - positive and HPV - negative cases indicate that AP-1 may play an important role during HPV-induced esophageal carcinogenesis. PMID:19758438

  8. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites.

    PubMed

    Fernández-Calero, Tamara; Flouriot, Gilles; Marín, Mónica

    2016-01-01

    Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1.S everal ERα synonymous polymorphisms have been identified and efforts to understand their implications have been made. Nevertheless effects of synonymous polymorphisms are still neglected. This chapter focuses on the experimental procedure employed in order to characterize the transcriptional activity of a synonymous polymorphism of the ERα (rs746432) called Alanine 87 (Ala87). Activity of both WT and Ala87 ERα isoforms on transcriptional pathways can be analyzed in transiently transfected cells using different reporter constructs. ERα efficiency on the classical genomic pathway can be analyzed by determining its transactivation activity on an ERE-driven thymidine kinase (TK) promoter controlling the expression of the luciferase reporter gene. Transcriptional activity through the indirect genomic pathway can be analyzed by employing an AP-1 DNA response element-driven promoter also controlling the expression of luciferase reporter gene. PMID:26585143

  9. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription.

    PubMed

    Busch, S J; Martin, G A; Barnhart, R L; Mano, M; Cardin, A D; Jackson, R L

    1992-01-01

    Heparin blocks the phorbol ester-induced progression of nontransformed cells through the G0/G1 phase (Wright, T.C., L.A. Pukac, J.J. Castellot, M.J. Karnovsky, R.A. Levine, H.-Y. Kim-Park, and J. Campisi. 1989. Proc. Natl. Acad. Sci. USA. 86: 3199-3203) or G1 to S phase (Reilly, C. F., M. S. Kindy, K. E. Brown, R. D. Rosenberg, and G. E Sonenshein. 1989. J. Biol. Chem. 264:6990-6995) of the cell cycle. Cell cycle arrest was associated with decreased levels of stage-specific mRNAs suggesting transcriptional regulation of cell growth. In the present report, we show that heparin selectively repressed TPA-inducible AP-1-mediated gene expression. Heparin-induced trans-repression was observed in primary vascular smooth muscle cells, as well as in the transformed HeLa cell line and in nondifferentiated F9 teratocarcinoma cells. Inhibition of AP-1-mediated trans-activation occurred with heparin and pentosan polysulfate but not with chondroitin sulfate A or C. Heparin-binding peptides or heparitinase I addition to nuclear lysates of heparin-treated cells allowed enhanced recovery of endogenous AP-1-specific DNA binding activity. We propose a model in which nuclear glycosaminoglycans play a trans-regulatory role in altering the patterns of inducible gene expression. PMID:1730747

  10. Transcriptional suppression of IL-27 production by Mycobacterium tuberculosis-activated p38 MAPK via inhibition of AP-1 binding.

    PubMed

    Zhang, Jidong; Qian, Xuesong; Ning, Huan; Eickhoff, Christopher S; Hoft, Daniel F; Liu, Jianguo

    2011-05-15

    Mycobacterium tuberculosis remains a major global challenge to human health care, and the mechanisms of how M. tuberculosis evades host immune surveillance to favor its survival are still largely unknown. In this study, we found that bacillus Calmette-Guérin (BCG) and viable M. tuberculosis as well as M. tuberculosis lysates could activate IL-27 expression in human and mouse macrophages by induction of p28 subunit transcription. However, in parallel with these effects, BCG and M. tuberculosis lysate stimulation of macrophages induced activation of p38 MAPK signaling molecules MLK3/MKK3/MK2 to prevent maximal IL-27 production. M. tuberculosis lysate-induced p28 transcription was dependent on MyD88 signaling pathway. AP-1/c-Fos was shown to bind directly to the p28 promoter and induce p28 expression after M. tuberculosis lysate stimulation. Overexpression of p38α inhibited the binding of c-Fos to the p28 promoter but had no effect on c-Fos protein expression or phosphorylation in response to M. tuberculosis lysate stimulation. Furthermore, blockade of p38 by SB203580 enhanced M. tuberculosis-induced AP-1 binding to the p28 promoter. Importantly, we show that adding exogenous IL-27 to increase the levels produced by PBMCs stimulated with live mycobacteria enhanced the ability of BCG-expanded T cells to inhibit intracellular mycobacterial growth in human macrophages. Taken together, our data demonstrate that mycobacterial stimulation induces both IL-27 production and p38 MAPK activation. Strategies designed to tip the balance toward positive regulation of p28 induction by mycobacteria could lead to enhanced protective tuberculosis immunity. PMID:21482740

  11. The Forkhead Transcription Factor FOXK2 Promotes AP-1-Mediated Transcriptional Regulation

    PubMed Central

    Ji, Zongling; Donaldson, Ian J.; Liu, Jingru; Hayes, Andrew; Zeef, Leo A. H.

    2012-01-01

    The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression. PMID:22083952

  12. A cluster region of AP-1 responsive elements is required for transcriptional activity of mouse ODC gene by hepatocyte growth factor.

    PubMed

    Bianchi, Laura; Tacchini, Lorenza; Matteucci, Emanuela; Desiderio, Maria Alfonsina

    2002-05-01

    Ornithine decarboxylase (ODC) activity is regulated by a variety of mechanisms including transcription, translation, and RNA and protein half-life. Since in mouse B16-F1 melanoma cells an early and remarkable (about 6-fold) increase in steady state mRNA levels was observed after hepatocyte growth factor (HGF) treatment, we investigated the transcriptional regulation of mouse ODC promoter. Transient transfection of various ODC-luciferase promoter constructs into the B16-Fl cells in combination with electrophoretic mobility shift assays identified the HGF-responsive element as a cluster of three AP-1 binding sites (-1660 to -1572). Even if each site differs from the canonical TPA responsive element for one nucleotide, only the first two AP-1 consensus sequences seemed to be functional since allowed DNA-binding activity of nuclear proteins after HGF treatment. Comparison of the results of transfection assays with the pOD2.5-luc (2.5 kb gene fragment) and with the construct deprived of the AP-1 cluster pOD-B-luc showed that this 50 bp region was required for ODC transactivating activity in response to HGF. Since in B16-F1 cells HGF increased AP-1 activity and the mRNA expression of various AP-1 subunits, we may conclude that HGF-induced transcription of mouse ODC was largely due to triggering of AP-1 pathway. PMID:12054494

  13. Small Molecule Inhibitors Targeting Activator Protein 1 (AP-1)

    PubMed Central

    2015-01-01

    Activator protein 1 (AP-1) is a pivotal transcription factor that regulates a wide range of cellular processes including proliferation, apoptosis, differentiation, survival, cell migration, and transformation. Accumulating evidence supports that AP-1 plays an important role in several severe disorders including cancer, fibrosis, and organ injury, as well as inflammatory disorders such as asthma, psoriasis, and rheumatoid arthritis. AP-1 has emerged as an actively pursued drug discovery target over the past decade. Excitingly, a selective AP-1 inhibitor T-5224 (51) has been investigated in phase II human clinical trials. Nevertheless, no effective AP-1 inhibitors have yet been approved for clinical use. Despite significant advances achieved in understanding AP-1 biology and function, as well as the identification of small molecules modulating AP-1 associated signaling pathways, medicinal chemistry efforts remain an urgent need to yield selective and efficacious AP-1 inhibitors as a viable therapeutic strategy for human diseases. PMID:24831826

  14. Transcriptional activation of the fra-1 gene by AP-1 is mediated by regulatory sequences in the first intron.

    PubMed Central

    Bergers, G; Graninger, P; Braselmann, S; Wrighton, C; Busslinger, M

    1995-01-01

    Constitutive expression of c-Fos, FosB, Fra-1, or c-Jun in rat fibroblasts leads to up-regulation of the immediate-early gene fra-1. Using the posttranslational FosER induction system, we demonstrate that this AP-1-dependent stimulation of fra-1 expression is rapid, depends on a functional DNA-binding domain of FosER, and is a general phenomenon observed in different cell types. In vitro mutagenesis and functional analysis of the rat fra-1 gene in stably transfected Rat-1A-FosER fibroblasts indicated that basal and AP-1-regulated expression of the fra-1 gene depends on regulatory sequences in the first intron which comprise a consensus AP-1 site and two AP-1-like elements. We have also investigated the transactivating and transforming properties of the Fra-1 protein to address the significance of fra-1 up-regulation. The entire Fra-1 protein fused to the DNA-binding domain of Ga14 is shown to lack any transactivation function, and yet it possesses oncogenic potential, as overexpression of Fra-1 in established rat fibroblasts results in anchorage-independent growth in vitro and tumor development in athymic mice, fra-1 is therefore not only induced by members of the Fos family, but its gene product may also contribute to cellular transformation by these proteins. Together, these data identify fra-1 as a unique member of the fos gene family which is under positive control by AP-1 activity. PMID:7791782

  15. Insulin-stimulated expression of c-fos, fra1 and c-jun accompanies the activation of the activator protein-1 (AP-1) transcriptional complex.

    PubMed Central

    Griffiths, M R; Black, E J; Culbert, A A; Dickens, M; Shaw, P E; Gillespie, D A; Tavaré, J M

    1998-01-01

    The activator protein-1 (AP-1) transcriptional complex is made up of members of the Fos (c-Fos, FosB, Fra1, Fra2) and Jun (c-Jun, JunB, JunD) families and is stimulated by insulin in several cell types. The mechanism by which insulin activates this complex is not well understood but it is dependent on the activation of the Erk1 and Erk2 isoforms of mitogen-activated protein kinases. In the current study we show that the AP-1 complex isolated from insulin-stimulated cells contained c-Fos, Fra1, c-Jun and JunB. The activation of the AP-1 complex by insulin was accompanied by (i) a transient increase in c-fos expression, and the transactivation of the ternary complex factors Elk1 and Sap1a, in an Erk1/Erk2-dependent fashion; (ii) a substantial increase in the expression of Fra1 protein and mRNA, which was preceded by a transient decrease in its electrophoretic mobility upon SDS/PAGE, indicative of phosphorylation; and (iii) a sustained increase in c-jun expression without increasing c-Jun phosphorylation on serines 63 and 73 or activation of the stress-activated kinase JNK/SAPK. In conclusion, insulin appears to stimulate the activity of the AP-1 complex primarily through a change in the abundance of the components of this complex, although there may be an additional role for Fra1 phosphorylation. PMID:9742208

  16. Activin A enhances MMP-7 activity via the transcription factor AP-1 in an esophageal squamous cell carcinoma cell line.

    PubMed

    Yoshinaga, Keiji; Mimori, Koshi; Inoue, Hiroshi; Kamohara, Yukio; Yamashita, Keishi; Tanaka, Fumiaki; Mori, Masaki

    2008-09-01

    Activin A, a member of the transforming growth factor beta (TGF-beta) superfamily, is often overexpressed in solid carcinomas. We have previously reported that the expression of activin A is associated with lymph node metastasis in esophageal cancer. In the current study, our goal was to clarify the molecular mechanisms underlying the aggressive behavior of tumors expressing high levels of activin A. Using cDNA microarrays, the gene expression profile of a human esophageal carcinoma cell line (KYSE170) stably transfected with activin betaA (Act-betaA, a subunit of activin A) was compared with those of control human esophageal carcinoma cell lines. We found that expression of MMP-7 was higher in the Act-betaA transfectants than in the control cells. To reveal the mechanism of expression of MMP-7 mediated by activin A, we evaluated mRNA expression of MMP-7 and Act-betaA with or without activin A neutralizing antibody, using real-time PCR and Northern blot analysis. We also performed promoter analysis of the MMP-7 promoter and assessed c-Jun and Smad2/3 expression. MMP-7 expression in the transfectants was correlated with the level of Act-betaA expression and was reduced by activin A neutralizing antibody. The Act-betaA transfectants had higher MMP-7 promoter activity than control cells. MMP-7 promoter activity was not affected by mutation in the Smad binding site, while mutation of the AP-1 binding site did reduce activity. Moreover, the expression of c-Jun was increased in Act-betaA transfectants. These results indicate that activin A may modulate the expression of MMP-7 via AP-1 and not through Smad2/3. PMID:18695873

  17. Activation of AP-1 and nuclear factor-kappaB transcription factors is involved in hydrogen peroxide-induced apoptotic cell death of oligodendrocytes.

    PubMed

    Vollgraf, U; Wegner, M; Richter-Landsberg, C

    1999-12-01

    H2O2-induced onset and execution of programmed cell death in mature rat brain oligodendrocytes in culture is accompanied by the induction and nuclear translocation of the transcription factors AP-1 and nuclear factor-kappaB (NF-kappaB), both of which have been discussed as regulators of cell death and survival. Supershift analysis of nuclear extracts indicated that the AP-1 complex consists of c-Jun, c-Fos, JunD, and possibly JunB proteins, and that the NF-kappaB complex contains p50, p65, and c-Rel proteins. The first signs of DNA fragmentation were seen already during the first hour after the treatment. DNA fragmentation could be prevented by the antioxidants pyrrolidine dithiocarbamate and vitamin E, by the nuclease inhibitor aurintricarboxylic acid, and by preincubation with the iron chelator deferoxamine (DFO). Additionally, DFO protected oligodendrocytes from H2O2-induced cytotoxic effects as assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and suppressed the formation of free radicals. DFO alone led to a slight increase and in combination with H2O2 synergistically induced DNA-binding activities of AP-1 and NF-kappaB in oligodendrocytes. Our data suggest that although low levels of H2O2 directly activate AP-1 and NF-kappaB and might contribute to signal transduction pathways promoting cell survival, the formation and action of hydroxyl radicals promote cell death mechanisms that can be attenuated by the iron chelator DFO. PMID:10582611

  18. Terminalia catappa Exerts Antimetastatic Effects on Hepatocellular Carcinoma through Transcriptional Inhibition of Matrix Metalloproteinase-9 by Modulating NF-κB and AP-1 Activity.

    PubMed

    Yeh, Chao-Bin; Hsieh, Ming-Ju; Hsieh, Yih-Shou; Chien, Ming-Hsien; Lin, Pen-Yuan; Chiou, Hui-Ling; Yang, Shun-Fa

    2012-01-01

    High mortality and morbidity rates for hepatocellular carcinoma (HCC) in Taiwan primarily result from uncontrolled tumor metastasis. Previous studies have identified that Terminalia catappa leaf extracts (TCE) exert hepatoprotective, antioxidative, antiinflammatory, anticancer, and antimetastatic activities. However, the effects of TCE on HCC and the underlying molecular mechanisms of its activities have yet to be fully elucidated. The present study's findings demonstrate that TCE concentration dependently inhibits human HCC migration/invasion. Zymographic and western blot analyses revealed that TCE inhibited the activities and expression of matrix metalloproteinase-9 (MMP-9). Assessment of mRNA levels, using reverse transcriptase polymerase chain reaction (PCR) and real-time PCR, and promoter assays confirmed the inhibitory effects of TCE on MMP-9 expression in HCC cells. The inhibitory effects of TCE on MMP-9 proceeded by upregulating tissue inhibitor of metalloproteinase-1 (TIMP-1), as well as suppressing nuclear translocation and DNA binding activity of nuclear factor-kappa B (NF-κB) and activating protein-1 (AP-1) on the MMP-9 promoter in Huh7 cells. In conclusion, TCE inhibits MMP-9 expression and HCC cell metastasis and, thus, has potential use as a chemopreventive agent. Its inhibitory effects are associated with downregulation of the binding activities of the transcription factors NF-κB and AP-1. PMID:23258989

  19. Sp1 binds two sites in the CD11c promoter in vivo specifically in myeloid cells and cooperates with AP1 to activate transcription.

    PubMed Central

    Noti, J D; Reinemann, B C; Petrus, M N

    1996-01-01

    The leukocyte integrin gene, CD11c, is transcriptionally regulated and is expressed predominantly on differentiated cells of the myelomonocytic lineage. In this study we have demonstrated that the regions -72 to -63 and -132 to -104 of the CD11c promoter contain elements responsible for phorbol ester-induced differentiation of the myeloid cell line HL60. DNase I footprinting analysis revealed that these regions can bind purified Sp1, and supershift analysis with Sp1 antibody confirmed that Sp1 in HL60 nuclear extracts could bind these regions. Transfection analysis of CD11c promoter-chloramphenicol acetyltransferase constructs containing deletions of these Sp1-binding sites revealed that these sites are essential for expression of the CD11c gene in HL60 cells but not in the T-cell line Molt4 or the cervical carcinoma cell line HeLa. Moreover, cotransfection of pPacSp1 along with these CD11c promoter-chloramphenicol acetyltransferase constructs into Sp1-deficient Drosophila Schneider 2 cells verified that these sites are essential for Sp1-dependent expression of the CD11c promoter. In vivo genomic footprinting revealed that Sp1 contacts the CD11c promoter within the regions -69 to -63 and -116 to -105 in phorbol 12-myristate 13-acetate-differentiated HL60 cells but not in undifferentiated HL60 cells or in Molt4 or HeLa cells. Cotransfection assays in HL60 cells revealed that Sp1 acts synergistically with Ap1 to activate CD11c. Further, both Sp1 sites are capable of cooperating with AP1. In vitro DNase I footprinting analysis with purified Sp1 and c-jun proteins showed that Sp1 binding could facilitate binding of c-jun. We propose that myeloid-specific expression of the CD11c promoter and is facilitated by cooperative interaction between the Sp1- and Ap1-binding sites. PMID:8649405

  20. An Estrogen Receptor-α/p300 Complex Activates the BRCA-1 Promoter at an AP-1 Site That Binds Jun/Fos Transcription Factors: Repressive Effects of p53 on BRCA-1 Transcription1

    PubMed Central

    Jeffy, Brandon D; Hockings, Jennifer K; Kemp, Michael Q; Morgan, Sherif S; Hager, Jill A; Beliakoff, Jason; Whitesell, Luke J; Bowden, G. Timothy; Romagnolo, Donato F

    2005-01-01

    Abstract One of the puzzles in cancer predisposition is that women carrying BRCA-1 mutations preferentially develop tumors in epithelial tissues of the breast and ovary. Moreover, sporadic breast tumors contain lower levels of BRCA-1 in the absence of mutations in the BRCA-1 gene. The problem of tissue specificity requires analysis of factors that are unique to tissues of the breast. For example, the expression of estrogen receptor-α (ERα) is inversely correlated with breast cancer risk, and 90% of BRCA-1 tumors are negative for ERα. Here, we show that estrogen stimulates BRCA-1 promoter activity in transfected cells and the recruitment of ERα and its cofactor p300 to an AP-1 site that binds Jun/Fos transcription factors. The recruitment of ERα/p300 coincides with accumulation in the S-phase of the cell cycle and is antagonized by the antiestrogen tamoxifen. Conversely, we document that overexpression of wild-type p53 prevents the recruitment of ERα to the AP-1 site and represses BRCA-1 promoter activity. Taken together, our findings support a model in which an ERα/AP-1 complex modulates BRCA-1 transcription under conditions of estrogen stimulation. Conversely, the formation of this transcription complex is abrogated in cells overexpressing p53. PMID:16229810

  1. The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

    PubMed Central

    Antinore, M J; Birrer, M J; Patel, D; Nader, L; McCance, D J

    1996-01-01

    The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway. Images PMID:8617242

  2. Tiron Inhibits UVB-Induced AP-1 Binding Sites Transcriptional Activation on MMP-1 and MMP-3 Promoters by MAPK Signaling Pathway in Human Dermal Fibroblasts

    PubMed Central

    Zhang, Chao; Zhao, Mei; Zhang, Quan-Wu; Gao, Feng-Hou

    2016-01-01

    Recent research found that Tiron was an effective antioxidant that could act as the intracellular reactive oxygen species (ROS) scavenger or alleviate the acute toxic metal overload in vivo. In this study, we investigated the inhibitory effect of Tiron on matrix metalloproteinase (MMP)-1 and MMP-3 expression in human dermal fibroblast cells. Western blot and ELISA analysis revealed that Tiron inhibited ultraviolet B (UVB)-induced protein expression of MMP-1 and MMP-3. Real-time quantitative PCR confirmed that Tiron could inhibit UVB-induced mRNA expression of MMP-1 and MMP-3. Furthermore, Tiron significantly blocked UVB-induced activation of the MAPK signaling pathway and activator protein (AP)-1 in the downstream of this transduction pathway in fibroblasts. Through the AP-1 binding site mutation, it was found that Tiron could inhibit AP-1-induced upregulation of MMP-1 and MMP-3 expression through blocking AP-1 binding to the AP-1 binding sites in the MMP-1 and MMP-3 promoter region. In conclusion, Tiron may be a novel antioxidant for preventing and treating skin photoaging UV-induced. PMID:27486852

  3. Activation of mitogen-activated protein kinases and AP-1 transcription factor in ovotoxicity induced by 4-vinylcyclohexene diepoxide in rats.

    PubMed

    Hu, Xiaoming; Flaws, Jodi A; Sipes, I Glenn; Hoyer, Patricia B

    2002-09-01

    Previous studies have demonstrated that ovotoxicity induced in small preantral (primordial and primary) ovarian follicles by 4-vinylcyclohexene diepoxide (VCD) in rats is likely via acceleration of the normal process of atresia (apoptosis). This acceleration is associated with increased activities of caspase cascades, changes in subcellular distribution of Bcl-2 family members, and alteration of estrogen receptor-mediated signaling pathways. The present study was designed to investigate possible effects of VCD dosing on the mitogen-activated protein kinases (MAPK)/AP-1 signaling pathways in rat ovarian small follicles. Female F344 rats were given a single dose of VCD (80 mg/kg i.p., 1 day--a time when ovotoxicity has not been initiated) or dosed daily for 10 or 15 days (80 mg/kg i.p.; 10 days--a time when the earliest signs of impending follicular destruction is seen, 15 days--a time when significant ovotoxicity is underway). Four hours following the final dose, ovaries and livers were collected. Ovarian small (25-100 microm) and large (100-250 microm) preantral follicles were isolated, and cytosolic or nuclear extracts were prepared from follicles and livers for analyses. Activities of MAPKs, including extracellular signal-regulated kinase, c-Jun N-terminal protein kinase (JNK), and p38 kinase, were determined in follicular and liver cytosolic extracts, and AP-1 DNA binding activity was determined in follicular and liver nuclear extracts. Compared with control, a single dose of VCD caused a decrease in JNK activity and an increase of AP-1 binding activity in isolated small ovarian follicles. After repeated daily dosing with VCD for 10 or 15 days, JNK and p38 kinase activities in small ovarian follicles were increased (p38 kinase: 1.64 +/- 0.14 for 10 days, 1.48 +/- 0.11 for 15 days, VCD/control, P < 0.01; JNK: 1.44 +/- 0.11 for 10 days, 1.37 +/- 0.06 for 15 days, VCD/control, P < 0.01) and AP-1 binding activity in small ovarian follicles was decreased (10 days, 0

  4. Thrombin induces endothelial arginase through AP-1 activation.

    PubMed

    Zhu, Weifei; Chandrasekharan, Unni M; Bandyopadhyay, Smarajit; Morris, Sidney M; DiCorleto, Paul E; Kashyap, Vikram S

    2010-04-01

    Arterial thrombosis is a common disease leading to severe ischemia beyond the obstructing thrombus. Additionally, endothelial dysfunction at the site of thrombosis can be rescued by l-arginine supplementation or arginase blockade in several animal models. Exposure of rat aortic endothelial cells (RAECs) to thrombin upregulates arginase I mRNA and protein levels. In this study, we further investigated the molecular mechanism of thrombin-induced arginase changes in endothelial cells. Thrombin strikingly increased arginase I promoter and enzyme activity in primary cultured RAECs. Using different deletion and point mutations of the promoter, we demonstrated that the activating protein-1 (AP-1) consensus site located at -3,157 bp in the arginase I promoter was a thrombin-responsive element. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that upon thrombin stimulation, c-Jun and activating transcription factor-2 (ATF-2) bound to the AP-1 site, which initiated the transactivation. Moreover, loss-of-function studies using small interfering RNA confirmed that recruitment of these two transcription factors to the AP-1 site was required for thrombin-induced arginase upregulation. In the course of defining the signaling pathway leading to the activation of AP-1 by thrombin, we found thrombin-induced phosphorylation of stress-activated protein kinase/c-Jun-NH(2)-terminal kinase (SAPK/JNK or JNK1/2/3) and p38 mitogen-activated protein kinase, which were followed by the phosphorylation of both c-Jun and ATF-2. These findings reveal the basis for thrombin induction of endothelial arginase I and indicate that arginase inhibition may be an attractive therapeutic alternative in the setting of arterial thrombosis and its associated endothelial dysfunction. PMID:20032511

  5. NF-κB and AP-1 Connection: Mechanism of NF-κB-Dependent Regulation of AP-1 Activity

    PubMed Central

    Fujioka, Shuichi; Niu, Jiangong; Schmidt, Christian; Sclabas, Guido M.; Peng, Bailu; Uwagawa, Tadashi; Li, Zhongkui; Evans, Douglas B.; Abbruzzese, James L.; Chiao, Paul J.

    2004-01-01

    Nuclear factor κB (NF-κB) and activator protein 1 (AP-1) transcription factors regulate many important biological and pathological processes. Activation of NF-κB is regulated by the inducible phosphorylation of NF-κB inhibitor IκB by IκB kinase. In contrast, Fos, a key component of AP-1, is primarily transcriptionally regulated by serum responsive factors (SRFs) and ternary complex factors (TCFs). Despite these different regulatory mechanisms, there is an intriguing possibility that NF-κB and AP-1 may modulate each other, thus expanding the scope of these two rapidly inducible transcription factors. To determine whether NF-κB activity is involved in the regulation of fos expression in response to various stimuli, we analyzed activity of AP-1 and expression of fos, fosB, fra-1, fra-2, jun, junB, and junD, as well as AP-1 downstream target gene VEGF, using MDAPanc-28 and MDAPanc-28/IκBαM pancreatic tumor cells and wild-type, IKK1−/−, and IKK2−/− murine embryonic fibroblast cells. Our results show that elk-1, a member of TCFs, is one of the NF-κB downstream target genes. Inhibition of NF-κB activity greatly decreased expression of elk-1. Consequently, the reduced level of activated Elk-1 protein by extracellular signal-regulated kinase impeded constitutive, serum-, and superoxide-inducible c-fos expression. Thus, our study revealed a distinct and essential role of NF-κB in participating in the regulation of elk-1, c-fos, and VEGF expression. PMID:15314185

  6. Arsenite suppression of involucrin transcription through AP1 promoter sites in cultured human keratinocytes

    SciTech Connect

    Sinitsyna, Nadezda N.; Reznikova, Tatiana V.; Qin Qin; Song, Hyukhwan; Phillips, Marjorie A.; Rice, Robert H.

    2010-03-15

    While preserving keratinocyte proliferative ability, arsenite suppresses cellular differentiation markers by preventing utilization of AP1 transcriptional response elements. In present experiments, arsenite had a dramatic effect in electrophoretic mobility supershift analysis of proteins binding to an involucrin promoter AP1 response element. Without arsenite treatment, binding of JunB and Fra1 was readily detected in nuclear extracts from preconfluent cultures and was not detected a week after confluence, while c-Fos was detected only after confluence. By contrast, band shift of nuclear extracts from arsenite treated cultures showed only JunB and Fra1 binding in postconfluent as well as preconfluent cultures. Immunoblotting of cell extracts showed that arsenite treatment prevented the loss of Fra1 and the increase in c-Fos proteins that occurred after confluence in untreated cultures. Chromatin immunoprecipitation assays demonstrated substantial reduction of c-Fos and acetylated histone H3 at the proximal and distal AP1 response elements in the involucrin promoter and of coactivator p300 at the proximal element. Alteration of AP1 transcription factors was also examined in response to treatment with four metal containing compounds (chromate, vanadate, hemin, divalent cadmium) that also suppress involucrin transcription. These agents all influenced transcription at AP1 elements in a transcriptional reporter assay, but exhibited less effect than arsenite on binding activity assessed by mobility shift and chromatin immunoprecipitation and displayed variable effects on AP1 protein levels. These findings help trace a mechanism by which transcriptional effects of arsenite become manifest and help rationalize the unique action of arsenite, compared to the other agents, to preserve proliferative ability.

  7. Antioxidant-induced changes of the AP-1 transcription complex are paralleled by a selective suppression of human papillomavirus transcription.

    PubMed Central

    Rösl, F; Das, B C; Lengert, M; Geletneky, K; zur Hausen, H

    1997-01-01

    Considering the involvement of a redox-regulatory pathway in the expression of human papillomaviruses (HPVs), HPV type 16 (HPV-16)-immortalized human keratinocytes were treated with the antioxidant pyrrolidine-dithiocarbamate (PDTC). PDTC induces elevated binding of the transcription factor AP-1 to its cognate recognition site within the viral regulatory region. Despite of increased AP-1 binding, normally indispensable for efficient HPV-16 transcription, viral gene expression was selectively suppressed at the level of initiation of transcription. Electrophoretic mobility supershift assays showed that the composition of the AP-1 complex, predominantly consisting of Jun homodimers in untreated cells, was altered. Irrespective of enhanced c-fos expression, c-jun was phosphorylated and became primarily heterodimerized with fra-1, which was also induced after PDTC incubation. Additionally, there was also an increased complex formation between c-jun and junB. Because both fra-1 and junB overexpression negatively interferes with c-jun/c-fos trans-activation of AP-1-responsive genes, our results suggest that the observed block in viral transcription is mainly the consequence of an antioxidant-induced reconstitution of the AP-1 transcription complex. Since expression of the c-jun/c-fos gene family is tightly regulated during cellular differentiation, defined reorganization of a central viral transcription factor may represent a novel mechanism controlling the transcription of pathogenic HPVs during keratinocyte differentiation and in the progression to cervical cancer. PMID:8985358

  8. Transcription Factor AP1 Potentiates Chromatin Accessibility and Glucocorticoid Receptor Binding

    PubMed Central

    Biddie, Simon C.; John, Sam; Sabo, Pete J.; Thurman, Robert E.; Johnson, Thomas A.; Schiltz, R. Louis; Miranda, Tina B.; Sung, Myong-Hee; Trump, Saskia; Lightman, Stafford L.; Vinson, Charles; Stamatoyannopoulos, John A.; Hager, Gordon L.

    2011-01-01

    Summary Ligand-dependent transcription by the nuclear receptor glucocorticoid receptor (GR) is mediated by interactions with co-regulators. The role of these interactions in determining selective binding of GR to regulatory elements remains unclear. Recent findings indicate a large fraction of genomic GR binding coincides with chromatin that is accessible prior to hormone treatment, suggesting that receptor binding is dictated by proteins that maintain chromatin in an open state. Combining DNaseI accessibility and chromatin immunoprecipitation with high-throughput sequencing, we identify the activator protein 1 (AP1) as a major partner for productive GR-chromatin interactions. AP1 is critical for GR-regulated transcription and recruitment to co-occupied regulatory elements, illustrating an extensive AP1-GR interaction network. Importantly, the maintenance of baseline chromatin accessibility facilitates GR recruitment and is dependent on AP1 binding. We propose a model where the basal occupancy of transcription factors act to prime chromatin and direct inducible transcription factors to select regions in the genome. PMID:21726817

  9. Bcl-3 Expression Promotes Cell Survival following Interleukin-4 Deprivation and Is Controlled by AP1 and AP1-Like Transcription Factors

    PubMed Central

    Rebollo, Angelita; Dumoutier, Laure; Renauld, Jean-Christophe; Zaballos, Angel; Ayllón, Verónica; Martínez-A., Carlos

    2000-01-01

    We have analyzed the interleukin-4 (IL-4)-triggered mechanisms implicated in cell survival and show here that IL-4 deprivation induces apoptotic cell death but does not modulate Bcl-2 or Bcl-x expression. Since Bcl-x expression is insufficient to ensure cell survival in the absence of IL-4, we speculate that additional molecules replace the antiapoptotic role of Bcl-2 and Bcl-x in an alternative IL-4-triggered pathway. Cell death is associated with Bcl-3 downregulation and Bcl-3 expression blocks IL-4 deprivation-induced apoptosis, suggesting that Bcl-3 acts as a survival factor in the absence of growth factor. To characterize the IL-4-induced regulation of murine Bcl-3 expression, we cloned the promoter of this gene. Sequencing of the promoter showed no TATA box element but did reveal binding sites for AP1, AP1-like, and SP1 transcription factors. Retardation gels showed that IL-4 specifically induces AP1 and AP1-like binding activity and that mutation of these binding sites abolishes the IL-4-induced Bcl-3 promoter activity, suggesting that these transcription factors are important in Bcl-3 promoter transactivation. IL-4 deprivation induces downregulation of Jun expression and upregulation of Fos expression, both of which are proteins involved in the formation of AP1 and AP1-like transcription factors. Overexpression of Jun family proteins transactivates the promoter and restores Bcl-3 expression in the absence of IL-4 stimulation. Taken together, these data describe a new biological role for Bcl-3 and define the regulatory pathway implicated in Bcl-3 expression. PMID:10779330

  10. GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation.

    PubMed Central

    Wu, A L; Moye-Rowley, W S

    1994-01-01

    Changes in gene dosage of the YAP1 gene, encoding the yAP-1 transcriptional regulatory protein, cause profound alterations in cellular drug and metal resistance. Previous studies on yAP-1 action in yeast cells have used the AP-1 response element (ARE) from simian virus 40 as an artificial site for yAP-1-mediated transcriptional activation. No authentic yeast target sites for control of gene expression by yAP-1 are known. Here we show that the GSH1 gene, encoding gamma-glutamylcysteine synthetase, is transcriptionally responsive to the yAP-1 protein. GSH1 encodes the rate-limiting step in yeast glutathione biosynthesis and contains within its promoter region a DNA element that matches the ARE in 11 of 12 positions. The GSH1 yAP-1 response element (YRE) was recognized by yAP-1 protein in vitro. Northern (RNA) blot analysis showed that GSH1 mRNA levels were responsive to YAP1 gene dosage. A site-directed mutation in the YRE that blocked yAP-1 binding in vitro prevented the mutant GSH1 promoter from responding to elevation in YAP1 gene dosage. A delta gsh1 mutant strain was constructed and unable to grow in the absence of exogenous glutathione. A mutant GSH1 gene lacking the YRE was unable to confer normal cadmium tolerance, although other yAP-1-mediated phenotypes remained normal. Thus, GSH1 is one of several genes that are transcriptionally controlled by yAP-1 and influence drug resistance. Images PMID:7915005

  11. ANKRD1 acts as a transcriptional repressor of MMP13 via the AP-1 site.

    PubMed

    Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E; Davidson, Jeffrey M

    2014-04-01

    The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1(-/-) (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1(fl/fl) (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

  12. ANKRD1 Acts as a Transcriptional Repressor of MMP13 via the AP-1 Site

    PubMed Central

    Almodóvar-García, Karinna; Kwon, Minjae; Samaras, Susan E.

    2014-01-01

    The transcriptional cofactor ANKRD1 is sharply induced during wound repair, and its overexpression enhances healing. We recently found that global deletion of murine Ankrd1 impairs wound contraction and enhances necrosis of ischemic wounds. A quantitative PCR array of Ankrd1−/− (KO) fibroblasts indicated that ANKRD1 regulates MMP genes. Yeast two-hybrid and coimmunoprecipitation analyses associated ANKRD1 with nucleolin, which represses AP-1 activation of MMP13. Ankrd1 deletion enhanced both basal and phorbol 12-myristate 13-acetate (PMA)-induced MMP13 promoter activity; conversely, Ankrd1 overexpression in control cells decreased PMA-induced MMP13 promoter activity. Ankrd1 reconstitution in KO fibroblasts decreased MMP13 mRNA, while Ankrd1 knockdown increased these levels. MMP13 mRNA and protein were elevated in intact skin and wounds of KO versus Ankrd1fl/fl (FLOX) mice. Electrophoretic mobility shift assay gel shift patterns suggested that additional transcription factors bind to the MMP13 AP-1 site in the absence of Ankrd1, and this concept was reinforced by chromatin immunoprecipitation analysis as greater binding of c-Jun to the AP-1 site in extracts from FLOX versus KO fibroblasts. We propose that ANKRD1, in association with factors such as nucleolin, represses MMP13 transcription. Ankrd1 deletion additionally relieved MMP10 transcriptional repression. Nuclear ANKRD1 appears to modulate extracellular matrix remodeling by MMPs. PMID:24515436

  13. MEKK1 regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability.

    PubMed

    Cuevas, Bruce D; Uhlik, Mark T; Garrington, Timothy P; Johnson, Gary L

    2005-01-27

    Activator protein 1 (AP-1) transcription factor dimers are composed of Jun, Fos, and ATF member proteins, but the mechanisms that determine AP-1 composition are not clearly defined and the function of specific dimers is not well understood. MEKK1 is a mitogen-activated protein kinase (MAPK) kinase kinase and an ubiquitin ligase that regulates both the extracellular signal-regulated kinase 1/2 and the c-Jun amino-terminal kinase. Herein, we demonstrate that MEKK1 regulates the AP-1 protein repertoire. Both FGF-2 and phorbol ester-inducible urokinase-type plasminogen activator (uPA) expression requires AP-1 binding to an enhancer element in the uPA promoter, and we have previously shown that FGF-2 or PMA induction of uPA expression is strongly dependent on MEKK1. JunB mRNA is significantly increased in MEKK1-/- cells, demonstrating that MEKK1 suppresses JunB mRNA expression. Upregulation of JunB expression in MEKK1-/- cells forms an inhibitory AP-1 complex that binds to the uPA promoter and inhibits uPA transcription. MEKK1 also regulates Fra-2 protein stability by inducing Fra-2 ubiquitination and degradation. MEKK1 regulates AP-1-dependent gene expression by regulating the expression, activity and degradation of component members of the AP-1 complex. Controlling the repertoire of a transcription factor complex is a newly defined function for an MAPK kinase kinase. PMID:15558021

  14. A functional activating protein 1 (AP-1) site regulates matrix metalloproteinase 2 (MMP-2) transcription by cardiac cells through interactions with JunB-Fra1 and JunB-FosB heterodimers.

    PubMed Central

    Bergman, Marina R; Cheng, Sunfa; Honbo, Norman; Piacentini, Lucia; Karliner, Joel S; Lovett, David H

    2003-01-01

    Enhanced synthesis of a specific matrix metalloproteinase, MMP-2, has been demonstrated in experimental models of ventricular failure and in cardiac extracts from patients with ischaemic cardiomyopathy. Cultured neonatal rat cardiac fibroblasts and myocytes were used to analyse the determinants of MMP-2 synthesis, including the effects of hypoxia. Culture of rat cardiac fibroblasts for 24 h in 1% oxygen enhanced MMP-2 synthesis by more than 5-fold and augmented the MMP-2 synthetic responses of these cells to endothelin-1, angiotensin II and interleukin 1beta. A series of MMP-2 promoter-luciferase constructs were used to map the specific enhancer element(s) that drive MMP-2 transcription in cardiac cells. Deletion studies mapped a region of potent transactivating function within the 91 bp region from -1433 to -1342 bp, the activity of which was increased by hypoxia. Oligonucleotides from this region were cloned in front of a heterologous simian-virus-40 (SV40) promoter and mapped the enhancer activity to a region between -1410 and -1362 bp that included a potential activating protein 1 (AP-1)-binding sequence, C(-1394)CTGACCTCC. Site-specific mutagenesis of the core TGAC sequence (indicated in bold) eliminated the transactivating activity within the -1410 to -1362 bp sequence. Electrophoretic mobility shift assays (EMSAs) using the -1410 to -1362 bp oligonucleotide and rat cardiac fibroblast nuclear extracts demonstrated specific nuclear-protein binding that was eliminated by cold competitor oligonucleotide, but not by the AP-1-mutated oligonucleotide. Antibody-supershift EMSAs of nuclear extracts from normoxic rat cardiac fibroblasts demonstrated Fra1 and JunB binding to the -1410 to -1362 bp oligonucleotide. Nuclear extracts isolated from hypoxic rat cardiac fibroblasts contained Fra1, JunB and also included FosB. Co-transfection of cardiac fibroblasts with Fra1-JunB and FosB-JunB expression plasmids led to significant increases in transcriptional activity. These

  15. Procyanidin B2 3,3″-di-O-gallate, a biologically active constituent of grape seed extract, induces apoptosis in human prostate cancer cells via targeting NF-κB, Stat3 and AP1 transcription factors

    PubMed Central

    Tyagi, Alpna; Raina, Komal; Shrestha, Suraj Prakash; Miller, Bettina; Thompson, John A.; Wempe, Michael F.; Agarwal, Rajesh; Agarwal, Chapla

    2014-01-01

    Recently, we identified procyanidin B2 3,3″-di-O-gallate (B2G2) as most active constituent of grape seed extract (GSE) for efficacy against prostate cancer (PCa). Isolating large quantities of B2G2 from total GSE is labor intensive and expensive, thereby limiting both efficacy and mechanistic studies with this novel anti-cancer agent. Accordingly, here we synthesized gram-scale quantities of B2G2, compared it with B2G2 isolated from GSE for possible equivalent biological activity, and conducted mechanistic studies. Both B2G2 preparations inhibited cell growth, decreased clonogenicity, and induced cell cycle arrest and apoptotic death, comparable to each other, in various human PCa cell lines. Mechanistic studies focusing on transcription factors involved in apoptotic and survival pathways revealed that B2G2 significantly inhibits NF-κB and AP1 transcriptional activity and nuclear translocation of Stat3 in PCa cell lines, irrespective of their functional androgen receptor status. B2G2 also decreased survivin expression which is regulated by NF-κB, AP1 and Stat3, and increased cleaved PARP level. In summary, we report B2G2 chemical synthesis at gram-quantity with equivalent biological efficacy against human PCa cell lines and same molecular targeting profiles at key transcription factors level. The synthetic B2G2 will stimulate more research on prostate and possibly other malignancies in preclinical models and clinical translation. PMID:24191894

  16. Activation of an AP1-Like Transcription Factor of the Maize Pathogen Cochliobolus heterostrophus in Response to Oxidative Stress and Plant Signals

    SciTech Connect

    Lev, Sophie; hadar, Ruthi; Amedeo, Paolo; Baker, Scott E.; Yoder, Olen; Horwitz, Benjamin A.

    2005-02-01

    Redox sensing is a ubiquitous mechanism regulating cellular activity. Fungal pathogens face reactive oxygen species produced by the host plant's oxidative burst in addition to endogenous reactive oxygen species produced during aerobic metabolism. An array of preformed and induced detoxifying enzymes, including superoxide dismutase, catalases, and peroxidases, could allow fungi to infect plants despite the oxidative burst. We isolated a gene (CHAP1) encoding a redox-regulated transcription factor in Cochliobolus heterostrophus, a fungal pathogen of maize. CHAP1 is a bZIP protein that possesses two cysteine-rich domains structurally and functionally related to Saccharomyces cerevisiae YAP1. Deletion of CHAP1 in C. heterostrophus resulted in decreased resistance to oxidative stress caused by hydrogen peroxide and menadione, but the virulence of chap1 mutants was unaffected. Upon activation by oxidizing agents or plant signals, a green fluorescent protein (GFP)-CHAP1 fusion protein became localized in the nucleus. Expression of genes encoding antioxidant proteins was induced in the wild type but not in chap1 mutants. Activation of CHAP1 occurred from the earliest stage of plant infection, in conidial germ tubes on the leaf surface, and persisted during infection. Late in the course of infection, after extensive necrotic lesions were formed, GFP-CHAP1 redistributed to the cytosol in hyphae growing on the leaf surface. Localization of CHAP1 to the nucleus may, through changes in the redox state of the cell, provide a mechanism linking extracellular cues to transcriptional regulation during the plant-pathogen interaction.

  17. ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED ARSENICALS

    EPA Science Inventory

    ACTIVATION OF AP-1 IN UROTSA CELLS BY METHYLATED TRIVALENT ARSENICALS. Z Drobna1, I Jaspers2, D J Thomas3 and M Styblo1. 1Department of Pediatrics; 2Center for Environmental Medicine and Lung Biology, University of North Carolina at Chapel Hill, NC, USA; 3US EPA, RTP, NC, USA.

  18. Glabridin inhibits migration and invasion by transcriptional inhibition of matrix metalloproteinase 9 through modulation of NF-κB and AP-1 activity in human liver cancer cells

    PubMed Central

    Hsieh, Ming-Ju; Lin, Chiao-Wen; Yang, Shun-Fa; Chen, Mu-Kuan; Chiou, Hui-Ling

    2014-01-01

    BACKGROUND AND PURPOSE High mortality and morbidity rates for hepatocellular carcinoma in Taiwan primarily result from uncontrolled tumour metastasis. Glabridin, a prenylated isoflavonoid of licorice (Glycyrrhiza glabra) roots, is associated with a wide range of biological properties, such as regulation of energy metabolism, oestrogenic, neuroprotective, anti-osteoporotic and skin whitening. However, the effect of glabridin on the metastasis of tumour cells has not been clarified. EXPERIMENTAL APPROACH A wound healing model and Boyden chamber assays in vitro were used to determine the effects of glabridin on the migration and invasion of human hepatocellular carcinoma (HHC) cells. Western blot analysis, gelatin zymography, real-time PCR and promoter assays were used to evaluate the inhibitory effects of glabridin on matrix metalloproteinase 9 (MMP9) expression in these cells. KEY RESULTS Glabridin significantly inhibited migration/invasion capacities of HCC cells, Huh7 and Sk-Hep-1, cell lines that have low cytotoxicity in vitro, even at high concentrations. Western blot analysis and gelatin zymography showed that glabridin inhibited the expression, activities and protein levels of MMP9 and the phosphorylation of ERK1/2 and JNK1/2. These inhibitory effects were associated with an up-regulation of tissue inhibitor of metalloproteinase-1 and a down-regulation of the transcription factors NF-κB and activator protein 1 signalling pathways. Finally, the administration of glabridin effectively suppressed the tumour formation in the hepatoma xenograft model in vivo. CONCLUSION AND IMPLICATIONS Glabridin inhibited the invasion of human HCC cells and may have potential as a chemopreventive agent against liver cancer metastasis. PMID:24641665

  19. Transcriptional regulation of endothelial nitric oxide synthase expression in uterine artery endothelial cells by c-Jun/AP-1

    PubMed Central

    Qian, Xiao-Xian; Mata-Greenwood, Eugenia; Liao, Wu Xiang; Zhang, Honghai; Zheng, Jing; Chen, Dong-bao

    2007-01-01

    Despite extensive studies have shown that increased endothelial nitric oxide synthase (NOS3) expression in the uterine artery endothelial cells (UAEC) plays a key role in uterine vasodilatation, the molecular mechanism controlling NOS3 expression in UAEC is unknown. According to the sheep NOS3 promoter sequence isolated in our laboratory, we hypothesize that the activator protein-1 (AP-1) site in the proximal sheep NOS3 promoter (TGAGTCA, -682 to -676) is important for NOS3 expression. We developed a c-Jun adenoviral expression system to overexpress c-Jun protein into UAEC to investigate the effects of c-Jun/AP-1 on NOS3 expression. Basal levels of c-Jun protein and mRNA were detected in UAEC. C-Jun protein was overexpressed in a concentration and time-dependent fashion in UAEC infected with sense c-Jun (S-c-Jun), but not sham and antisense c-Jun (A-c-Jun) adenoviruses. Infection with S-c-Jun adenovirus (25 MOI, multiplicity of infection) resulted in efficient c-Jun protein overexpression in UAEC up to 3 days. In S-c-Jun, but not sham and A-c-Jun adenovirus infected UAEC, NOS3 mRNA and protein levels were increased (P<0.05) compared to noninfected controls. Increased NOS3 expression was associated with increased total NOS activity. Transient transfections showed that c-Jun overexpression augmented the transactivation of the sheep NOS3 promoter-driven luciferase/reporter constructs with the AP-1 site but not of deletion constructs without the AP-1 site. When the AP-1 site was mutated, c-Jun failed to trans-activate the sheep NOS3 promoter. AP-1 DNA binding activity also increased in c-Jun overexpressed UAEC. Lastly, the pharmacological AP-1 activator phorbol myristate acetate increased AP-1 binding, trans-activated the wild-type but not the AP-1 mutant NOS3 promoter and dose-dependently stimulated UAEC NOS3 and c-Jun protein expression. Hence, our data show that c-Jun/AP-1 regulates NOS3 transcription involving the proximal AP-1 site in the 5′-regulatory region of

  20. Overexpression of members of the AP-1 transcriptional factor family from an early stage of renal carcinogenesis and inhibition of cell growth by AP-1 gene antisense oligonucleotides in the Tsc2 gene mutant (Eker) rat model.

    PubMed

    Urakami, S; Tsuchiya, H; Orimoto, K; Kobayashi, T; Igawa, M; Hino, O

    1997-12-01

    We previously isolated subtracted cDNA clones for genes having increased expression in Tsc2 gene mutant (Eker) rat renal carcinomas (RCs). Among them, fra-1 encoding a transcriptional factor activator protein 1 (AP-1) was identified. We have therefore investigated whether other members of the AP-1 transcription factor family might also be involved in renal carcinogenesis in the Eker rat model. In the present study, overexpression of fra-1, fra-2, c-jun, junB, and junD mRNAs was demonstrated in RCs by Northern blot analysis. Interestingly, AP-1 proteins were highly expressed even in the earliest preneoplastic lesions (e.g., phenotypically altered tubules) as suggested by immunohistochemistry. Moreover, 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-binding activity of AP-1 proteins was observed in RC cell extracts by electrophoretic mobility shift assay. As a next step, we transfected antisense oligonucleotides targeting AP-1 genes into RC cells and demonstrated that their growth was strongly inhibited. Thus, the data suggest that overexpression of AP-1 genes might play a crucial role in renal carcinogenesis in the Eker rat model. PMID:9405228

  1. Staphylococcus aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB transcription factors in bovine mammary gland fibroblasts.

    PubMed

    Wu, Jianmei; Ding, Yulin; Bi, Yannan; Wang, Yi; Zhi, Yu; Wang, Jinling; Wang, Fenglong

    2016-06-01

    Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland. To better understand the importance of bovine mammary fibroblasts (BMFBs) and the roles of the TLR-NF-κB and TLR-AP-1 signaling pathways in the regulation of S. aureus-associated mastitis and mammary fibosis, BMFBs cultured in vitro were stimulated with different concentrations of heat-inactivated S. aureus to analyze the gene and protein expression of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), transforming growth factor beta 1 (TGF-β1), basic fibroblast growth factor (bFGF) as well as the protein expression of nuclear factor-kappa B (NF-κB) and activation protein-1 (AP-1) by means of quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1 and bFGF expression. The results indicated that, in addition to increasing mRNA and protein expression of TLR2 and TLR4, S. aureus could also upregulate TGF-β1 and bFGF mRNA expression and secretion through the activation of NF-κB and AP-1. The increase in TGF-β1 and bFGF expression was shown to be inhibited by AP-1- and NF-κB-specific inhibitors. Taken together, S. aureus induces TGF-β1 and bFGF expression through the activation of AP-1 and NF-κB in BMFBs. This information offers new potential targets for the treatment of bovine mammary fibrosis. PMID:26948281

  2. Luteolin, a flavonoid, inhibits AP-1 activation by basophils

    SciTech Connect

    Hirano, Toru; Higa, Shinji; Arimitsu, Junsuke; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Matsuda, Hisashi; Yoshikawa, Masayuki; Maezaki, Naoyoshi; Tanaka, Tetsuaki; Kawase, Ichiro; Tanaka, Toshio . E-mail: ttanak@imed3.med.osaka-u.ac.jp

    2006-02-03

    Flavonoids including luteolin, apigenin, and fisetin are inhibitors of IL-4 synthesis and CD40 ligand expression by basophils. This study was done to search for compounds with greater inhibitory activity of IL-4 expression and to clarify the molecular mechanisms through which flavonoids inhibit their expression. Of the 37 flavonoids and related compounds examined, ayanin, luteolin, and apigenin were the strongest inhibitors of IL-4 production by purified basophils in response to anti-IgE antibody plus IL-3. Luteolin did not suppress Syk or Lyn phosphorylation in basophils, nor did suppress p54/46 SAPK/JNK, p38 MAPK, and p44/42 MAPK activation by a basophilic cell line, KU812 cells, stimulated with A23187 and PMA. However, luteolin did inhibit phosphorylation of c-Jun and DNA binding activity of AP-1 in nuclear lysates from stimulated KU812 cells. These results provide a fundamental structure of flavonoids for IL-4 inhibition and demonstrate a novel action of flavonoids that suppresses the activation of AP-1.

  3. Modulation of AP-1 activity by the human progesterone receptor in endometrial adenocarcinoma cells.

    PubMed Central

    Bamberger, A M; Bamberger, C M; Gellersen, B; Schulte, H M

    1996-01-01

    The composite transcription factor activating protein 1 (AP-1) integrates various mitogenic signals in a large number of cell types, and is therefore a major regulator of cell proliferation. In the normal human endometrium, proliferation and differentiation alternate in a cyclic fashion, with progesterone being largely implicated in the latter process. However, the effects of progesterone and the progesterone receptor (hPR) on AP-1 activity in the human endometrium are not known. To address this issue, HEC-1-B endometrial adenocarcinoma cells, which are devoid of hPR, were transfected with luciferase reporter constructs driven by two different AP-1-dependent promoters. Unexpectedly, cotransfection of hPR caused a marked induction of luciferase activity in the absence of ligand on both promoters. The magnitude of this induction was similar to that observed in response to the phorbol ester TPA. Addition of ligand reversed the stimulating effect of the unliganded hPR on AM activity in these cells. These effects were specific for hPR, and were not observed with either human estrogen receptor or human glucocorticoid receptor. Furthermore, they strictly depended on the presence of AP-1-responsive sequences within target promoters. Finally, the described effects of hPR on AP-1 activity were shown to be cell-type specific, because they could not be demonstrated in SKUT-1-B, JEG-3, and COS-7 cells. To our knowledge this is the first report of an unliganded steroid receptor stimulating AP-1 activity. This effect and its reversal in the presence of ligand suggest a novel mechanism, through which hPR can act as a key regulator of both proliferation and differentiation in the human endometrium. PMID:8650238

  4. Multiple Protein Kinases via Activation of Transcription Factors NF-κB, AP-1 and C/EBP-δ Regulate the IL-6/IL-8 Production by HIV-1 Vpr in Astrocytes

    PubMed Central

    Gangwani, Mohitkumar R.; Kumar, Anil

    2015-01-01

    Neurocognitive impairments affect a substantial population of HIV-1 infected individuals despite the success of anti-retroviral therapy in controlling viral replication. Astrocytes are emerging as a crucial cell type that might be playing a very important role in the persistence of neuroinflammation seen in patients suffering from HIV-1 associated neurocognitive disorders. HIV-1 viral proteins including Vpr exert neurotoxicity through direct and indirect mechanisms. Induction of IL-8 in microglial cells has been shown as one of the indirect mechanism through which Vpr reduces neuronal survival. We show that HIV-1 Vpr induces IL-6 and IL-8 in astrocytes in a time-dependent manner. Additional experiments utilizing chemical inhibitors and siRNA revealed that HIV-1 Vpr activates transcription factors NF-κB, AP-1 and C/EBP-δ via upstream protein kinases PI3K/Akt, p38-MAPK and Jnk-MAPK leading to the induction of IL-6 and IL-8 in astrocytes. We demonstrate that one of the mechanism for neuroinflammation seen in HIV-1 infected individuals involves induction of IL-6 and IL-8 by Vpr in astrocytes. Understanding the molecular pathways involved in the HIV-1 neuroinflammation would be helpful in the design of adjunct therapy to ameliorate some of the symptoms associated with HIV-1 neuropathogenesis. PMID:26270987

  5. AP-1 activity during normal human keratinocyte differentiation: evidence for a cytosolic modulator of AP-1/DNA binding.

    PubMed

    Briata, P; D'Anna, F; Franzi, A T; Gherzi, R

    1993-01-01

    Increased levels of c-fos and c-jun expression have been observed in differentiating epithelial cells. However, no data are available on activator protein 1 (AP-1) activity during keratinocyte differentiation. In this work we investigated c-fos and c-jun gene expression and AP-1-(12-O-tetradecanoylphorbol-13-acetate)-responsive enhancer element (TRE) binding activity during keratinocyte differentiation utilizing both authentic and in culture-reconstituted human epidermis. We demonstrate that: (i) in reconstituted epidermis, non-differentiated and differentiated keratinocytes express equivalent levels of c-Jun, while in reconstituted epidermis permanently grafted onto athymic mice, as well as in authentic epidermis, c-Jun is predominantly expressed in the granular layer of the tissue. Equivalent levels of c-fos expression have been found in all the layers of both reconstituted and authentic epidermis. (ii) Nuclear extracts from cultures enriched in differentiated keratinocytes display an 80-90% reduction of AP-1 activity when compared to extracts from cultures enriched in nondifferentiated cells. (iii) Cytosolic extracts obtained from cultures enriched in differentiated cells reduce, in a concentration-dependent manner, the AP-1 activity present in nuclear extracts of both mammalian and Drosophila cells. (iv) The specific TRE binding activity of a recombinant c-Jun protein is significantly reduced by cytosolic extracts of differentiated keratinocytes, while the specific DNA binding of the purified recombinant human homeoprotein HOX4B is not. (v) The dephosphorylation, by alkaline phosphatase, of cytosolic extracts increases the inhibitory activity already present or makes evident a latent activity. PMID:8416791

  6. Apocynin increases glutathione synthesis and activates AP-1 in alveolar epithelial cells.

    PubMed

    Lapperre, T S; Jimenez, L A; Antonicelli, F; Drost, E M; Hiemstra, P S; Stolk, J; MacNee, W; Rahman, I

    1999-01-25

    Apocynin (4-hydroxy-3-methoxy-acetophenone) is a potent intracellular inhibitor of superoxide anion production in neutrophils. In this study, we studied the effect of apocynin on the regulation of the antioxidant glutathione (GSH) and activation of the transcription factor AP-I in human alveolar epithelial cells (A549). Apocynin enhanced intracellular GSH by increasing gamma-glutamylcysteine synthetase activity in A549 cells. Apocynin also increased the expression of gamma-GCS heavy subunit mRNA. This was associated with increased AP-1 DNA binding as measured by the electrophoretic mobility shift assay. These data indicate that apocynin displays antioxidant properties, in part, by increasing glutathione synthesis through activation of AP-1. PMID:9989612

  7. TAK1 regulates NF-{Kappa}B and AP-1 activation in airway epithelial cells following RSV infection

    SciTech Connect

    Dey, Nilay; Liu Tianshuang; Garofalo, Roberto P.; Casola, Antonella

    2011-09-30

    Respiratory syncytial virus (RSV) is the most common cause of epidemic respiratory diseases in infants and young children. RSV infection of airway epithelial cells induces the expression of immune/inflammatory genes through the activation of a subset of transcription factors, including Nuclear Factor-{kappa}B (NF-{kappa}B) and AP-1. In this study, we have investigated the signaling pathway leading to activation of these two transcription factors in response to RSV infection. Our results show that IKK{beta} plays a key role in viral-induced NF-{kappa}B activation, while JNK regulates AP-1-dependent gene transcription, as demonstrated by using kinase inactive proteins and chemical inhibitors of the two kinases. Inhibition of TAK1 activation, by overexpression of kinase inactive TAK1 or using cells lacking TAK1 expression, significantly reduced RSV-induced NF-{kappa}B and AP-1 nuclear translocation and DNA-binding activity, as well as NF-{kappa}B-dependent gene expression, identifying TAK1 as an important upstream signaling molecule regulating RSV-induced NF-{kappa}B and AP-1 activation. - Highlights: > IKK{beta} is a major kinase involved in RSV-induced NF-{kappa}B activation. > JNK regulates AP-1-dependent gene transcription in RSV infection. > TAK1 is a critical upstream signaling molecule for both pathways in infected cells.

  8. Structural and biochemical changes underlying a keratoderma-like phenotype in mice lacking suprabasal AP1 transcription factor function

    PubMed Central

    Rorke, E A; Adhikary, G; Young, C A; Rice, R H; Elias, P M; Crumrine, D; Meyer, J; Blumenberg, M; Eckert, R L

    2015-01-01

    Epidermal keratinocyte differentiation on the body surface is a carefully choreographed process that leads to assembly of a barrier that is essential for life. Perturbation of keratinocyte differentiation leads to disease. Activator protein 1 (AP1) transcription factors are key controllers of this process. We have shown that inhibiting AP1 transcription factor activity in the suprabasal murine epidermis, by expression of dominant-negative c-jun (TAM67), produces a phenotype type that resembles human keratoderma. However, little is understood regarding the structural and molecular changes that drive this phenotype. In the present study we show that TAM67-positive epidermis displays altered cornified envelope, filaggrin-type keratohyalin granule, keratin filament, desmosome formation and lamellar body secretion leading to reduced barrier integrity. To understand the molecular changes underlying this process, we performed proteomic and RNA array analysis. Proteomic study of the corneocyte cross-linked proteome reveals a reduction in incorporation of cutaneous keratins, filaggrin, filaggrin2, late cornified envelope precursor proteins, hair keratins and hair keratin-associated proteins. This is coupled with increased incorporation of desmosome linker, small proline-rich, S100, transglutaminase and inflammation-associated proteins. Incorporation of most cutaneous keratins (Krt1, Krt5 and Krt10) is reduced, but incorporation of hyperproliferation-associated epidermal keratins (Krt6a, Krt6b and Krt16) is increased. RNA array analysis reveals reduced expression of mRNA encoding differentiation-associated cutaneous keratins, hair keratins and associated proteins, late cornified envelope precursors and filaggrin-related proteins; and increased expression of mRNA encoding small proline-rich proteins, protease inhibitors (serpins), S100 proteins, defensins and hyperproliferation-associated keratins. These findings suggest that AP1 factor inactivation in the suprabasal

  9. Structural and biochemical changes underlying a keratoderma-like phenotype in mice lacking suprabasal AP1 transcription factor function.

    PubMed

    Rorke, E A; Adhikary, G; Young, C A; Rice, R H; Elias, P M; Crumrine, D; Meyer, J; Blumenberg, M; Eckert, R L

    2015-01-01

    Epidermal keratinocyte differentiation on the body surface is a carefully choreographed process that leads to assembly of a barrier that is essential for life. Perturbation of keratinocyte differentiation leads to disease. Activator protein 1 (AP1) transcription factors are key controllers of this process. We have shown that inhibiting AP1 transcription factor activity in the suprabasal murine epidermis, by expression of dominant-negative c-jun (TAM67), produces a phenotype type that resembles human keratoderma. However, little is understood regarding the structural and molecular changes that drive this phenotype. In the present study we show that TAM67-positive epidermis displays altered cornified envelope, filaggrin-type keratohyalin granule, keratin filament, desmosome formation and lamellar body secretion leading to reduced barrier integrity. To understand the molecular changes underlying this process, we performed proteomic and RNA array analysis. Proteomic study of the corneocyte cross-linked proteome reveals a reduction in incorporation of cutaneous keratins, filaggrin, filaggrin2, late cornified envelope precursor proteins, hair keratins and hair keratin-associated proteins. This is coupled with increased incorporation of desmosome linker, small proline-rich, S100, transglutaminase and inflammation-associated proteins. Incorporation of most cutaneous keratins (Krt1, Krt5 and Krt10) is reduced, but incorporation of hyperproliferation-associated epidermal keratins (Krt6a, Krt6b and Krt16) is increased. RNA array analysis reveals reduced expression of mRNA encoding differentiation-associated cutaneous keratins, hair keratins and associated proteins, late cornified envelope precursors and filaggrin-related proteins; and increased expression of mRNA encoding small proline-rich proteins, protease inhibitors (serpins), S100 proteins, defensins and hyperproliferation-associated keratins. These findings suggest that AP1 factor inactivation in the suprabasal

  10. Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation

    SciTech Connect

    Wang, Pingzhang; Sun, Bo; Hao, Dongxia; Zhang, Xiujun; Shi, Taiping; Ma, Dalong

    2010-04-16

    Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174{Delta}TM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

  11. TNF¿ and GM-CSF-induced activation of the CAEV promoter is independent of AP-1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Caprine arthritis encephalitis virus transcription is under the control of the viral promoter within the long terminal repeat. Previous studies with the closely related maedi visna lentivirus have indicated that viral transcription is dependent upon the AP-1 transcription factor. Other studies hav...

  12. Functional erythroid promoters created by interaction of the transcription factor GATA-1 with CACCC and AP-1/NFE-2 elements.

    PubMed Central

    Walters, M; Martin, D I

    1992-01-01

    We have investigated interactions between the erythroid transcription factor GATA-1 and factors binding two cis-acting elements commonly linked to GATA sites in erythroid control elements. GATA-1 is present at all stages of erythroid differentiation, is necessary for erythropoiesis, and binds sites in all erythroid control elements. However, minimal promoters containing GATA-1 sites are inactive when tested in erythroid cells. Based on this observation, two erythroid cis elements, here termed CACCC and AP-1/NFE-2, were linked to GATA sites in minimal promoters. None of the elements linked only to a TATA box created an active promoter, but GATA sites linked to either CACCC or AP-1/NFE-2 elements formed strong erythroid promoters. A mutation of T to C at position -175 in the gamma-globin promoter GATA site, associated with hereditary persistence of fetal hemoglobin (HPFH), increased expression of these promoters in both fetal and adult cells. A construct bearing the beta-globin CACCC element was more active in adult and less active in fetal erythroid cells, when compared with the gamma-globin CACCC element. These studies suggest that erythroid control elements are formed by the interactions of at least three transcription factors, none of which functions alone. Images PMID:1438231

  13. Heparin (GAG-hed) inhibits LCR activity of Human Papillomavirus type 18 by decreasing AP1 binding

    PubMed Central

    Villanueva, Rita; Morales-Peza, Néstor; Castelán-Sánchez, Irma; García-Villa, Enrique; Tapia, Rocio; Cid-Arregui, Ángel; García-Carrancá, Alejandro; López-Bayghen, Esther; Gariglio, Patricio

    2006-01-01

    Background High risk HPVs are causative agents of anogenital cancers. Viral E6 and E7 genes are continuously expressed and are largely responsible for the oncogenic activity of these viruses. Transcription of the E6 and E7 genes is controlled by the viral Long Control Region (LCR), plus several cellular transcription factors including AP1 and the viral protein E2. Within the LCR, the binding and activity of the transcription factor AP1 represents a key regulatory event in maintaining E6/E7 gene expression and uncontrolled cell proliferation. Glycosaminoglycans (GAGs), such as heparin, can inhibit tumour growth; they have also shown antiviral effects and inhibition of AP1 transcriptional activity. The purpose of this study was to test the heparinoid GAG-hed, as a possible antiviral and antitumoral agent in an HPV18 positive HeLa cell line. Methods Using in vivo and in vitro approaches we tested GAG-hed effects on HeLa tumour cell growth, cell proliferation and on the expression of HPV18 E6/E7 oncogenes. GAG-hed effects on AP1 binding to HPV18-LCR-DNA were tested by EMSA. Results We were able to record the antitumoral effect of GAG-hed in vivo by using as a model tumours induced by injection of HeLa cells into athymic female mice. The antiviral effect of GAG-hed resulted in the inhibition of LCR activity and, consequently, the inhibition of E6 and E7 transcription. A specific diminishing of cell proliferation rates was observed in HeLa but not in HPV-free colorectal adenocarcinoma cells. Treated HeLa cells did not undergo apoptosis but the percentage of cells in G2/M phase of the cell cycle was increased. We also detected that GAG-hed prevents the binding of the transcription factor AP1 to the LCR. Conclusion Direct interaction of GAG-hed with the components of the AP1 complex and subsequent interference with its ability to correctly bind specific sites within the viral LCR may contribute to the inhibition of E6/E7 transcription and cell proliferation. Our data

  14. The E3 ubiquitin ligase Trim7 mediates c-Jun/AP-1 activation by Ras signalling

    PubMed Central

    Chakraborty, Atanu; Diefenbacher, Markus E.; Mylona, Anastasia; Kassel, Olivier; Behrens, Axel

    2015-01-01

    The c-Jun/AP-1 transcription factor controls key cellular behaviours, including proliferation and apoptosis, in response to JNK and Ras/MAPK signalling. While the JNK pathway has been well characterised, the mechanism of activation by Ras was elusive. Here we identify the uncharacterised ubiquitin ligase Trim7 as a critical component of AP-1 activation via Ras. We found that MSK1 directly phosphorylates Trim7 in response to direct activation by the Ras–Raf–MEK–ERK pathway, and this modification stimulates Trim7 E3 ubiquitin ligase activity. Trim7 mediates Lys63-linked ubiquitination of the AP-1 coactivator RACO-1, leading to RACO-1 protein stabilisation. Consequently, Trim7 depletion reduces RACO-1 levels and AP-1-dependent gene expression. Moreover, transgenic overexpression of Trim7 increases lung tumour burden in a Ras-driven cancer model, and knockdown of Trim7 in established xenografts reduces tumour growth. Thus, phosphorylation-ubiquitination crosstalk between MSK1, Trim7 and RACO-1 completes the long sought-after mechanism linking growth factor signalling and AP-1 activation. PMID:25851810

  15. Dioscorea nipponica Makino inhibits migration and invasion of human oral cancer HSC-3 cells by transcriptional inhibition of matrix metalloproteinase-2 through modulation of CREB and AP-1 activity.

    PubMed

    Chien, Ming-Hsien; Ying, Tsung-Ho; Hsieh, Yih-Shou; Chang, Yu-Chao; Yeh, Chia-Ming; Ko, Jiunn-Liang; Lee, Wen-Sen; Chang, Jer-Hua; Yang, Shun-Fa

    2012-03-01

    Oral cancer mortality has increased during the last decade due to the difficulties in treating related metastasis. Dioscorea nipponica Makino, a popular folk medicine, exerts anti-obesity and anti-inflammation properties. However, the effect of this folk medicine on metastasis of oral cancer has yet to be fully elucidated. The present study demonstrates that D. nipponica extracts (DNE), at a range of concentrations (0-50 μg/mL), concentration-dependently inhibited migration/invasion capacities of human oral cancer cells, HSC-3, without cytotoxic effects. The anti-migration effect of DNE was also observed in two other OSCC cell lines, Ca9-22 and Cal-27. Zymography, real time PCR, and Western blotting analyses revealed that DNE inhibited matrix metalloproteinase-2 (MMP-2) enzyme activity, and RNA and protein expression. The inhibitory effects of DNE on MMP-2 proceeded by up-regulating tissue inhibitor of metalloproteinase-2 (TIMP-2), as well as suppressing nuclear translocation and DNA binding activity of cAMP response element-binding (CREB) and activating protein-1 (AP-1) on the MMP-2 promoter in HSC-3 cells. In conclusion, DNE inhibited the invasion of oral cancer cells and may have potential use as a chemopreventive agent against oral cancer metastasis. PMID:22210353

  16. Synthetic glucocorticoids that dissociate transactivation and AP-1 transrepression exhibit antiinflammatory activity in vivo.

    PubMed

    Vayssière, B M; Dupont, S; Choquart, A; Petit, F; Garcia, T; Marchandeau, C; Gronemeyer, H; Resche-Rigon, M

    1997-08-01

    Some of the most potent antiinflammatory and immunosuppressive agents are synthetic glucocorticoids. However, major side effects severely limit their therapeutic use. The development of improved glucocorticoid-based drugs will require the separation of beneficial from deleterious effects. One possibility toward this goal is to try to dissociate two main activities of glucocorticoids, i.e. transactivation and transrepression. Screening of a library of compounds using transactivation and AP-1 transrepression models in transiently transfected cells identified dissociated glucocorticoids, which exert strong AP-1 inhibition but little or no transactivation. Importantly, despite high ligand binding affinity, the prototypic dissociated compound, RU24858, acted as a weak agonist and did not efficiently antagonize dexamethasone-induced transcription in transfected cells. Similar results were obtained in hepatic HTC cells for the transactivation of the endogenous tyrosine amino transferase gene (TAT), which encodes one of the enzymes involved in the glucocorticoid-dependent stimulation of neoglucogenesis. To investigate whether dissociated glucocorticoids retained the antiinflammatory and immunosuppressive potential of classic glucocorticoids, several in vitro and in vivo models were used. Indeed, secretion of the proinflammatory lymphokine interleukin-1beta was severely inhibited by dissociated glucocorticoids in human monocytic THP 1 cells. Moreover, in two in vivo models, these compounds exerted an antiinflammatory and immunosuppressive activity as potent as that of the classic glucocorticoid prednisolone. These results may lead to an improvement of antiinflammatory and immunosuppressive therapies and provide a novel concept for drug discovery. PMID:9259316

  17. The p38 SAPK pathway regulates the expression of the MMP-9 collagenase via AP-1-dependent promoter activation.

    PubMed

    Simon, C; Simon, M; Vucelic, G; Hicks, M J; Plinkert, P K; Koitschev, A; Zenner, H P

    2001-12-10

    The invasive phenotype of cancers critically depends on the expression of proteases such as the M(R) 92,000 type IV collagenase (MMP-9). Several growth factors and oncogenes were found to increase promoter activity and as a consequence protease expression. This frequently requires the activation of the transcription factor AP-1 by signal transduction cascades such as the ERK and JNK pathways. We have previously demonstrated that the tumor promoter TPA can induce MMP-9 expression via a third signaling cascade, the p38 pathway. Considering that TPA is a potent activator of AP-1, we hypothesized that this transcription factor might also be required for p38 pathway-dependent MMP-9 regulation. While dominant negative p38 and MKK-6 mutants reduced MMP-9 promoter activity in CAT assays, a construct encoding an activating mutation in the MKK-6 protein potently stimulated it. This was mediated via 144 bp of the 5'flanking region of the wild-type promoter, which contains an AP-1 site at -79. Both point mutations in this motif and the expression of a c-jun protein lacking its transactivation domain and therefore acting as a dominant negative AP-1 mutant abrogated MKK-6-dependent promoter stimulation. Finally SB 203580, a specific p38 pathway inhibitor, reduced MMP-9 expression/secretion and in vitro invasion of cancer cells. Thus, our results provide evidence that also the third SAPK/MAPK signaling cascade, the p38 signal transduction pathway, stimulates MMP-9 expression in an AP-1-dependent fashion. PMID:11716547

  18. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity.

    PubMed Central

    Xanthoudakis, S; Curran, T

    1992-01-01

    Fos and Jun form a heterodimeric complex that regulates gene transcription by binding to the activator protein-1 (AP-1) DNA sequence motif. Previously, we demonstrated that the DNA-binding activity of Fos and Jun is regulated in vitro by a novel redox (reduction-oxidation) mechanism. Reduction of a conserved cysteine (cys) residue in the DNA-binding domains of Fos and Jun by chemical reducing agents or by a nuclear redox factor stimulates DNA-binding activity. Here, we describe purification and characterization of a 37 kDa protein (Ref-1) corresponding to the redox factor. Although Ref-1 does not bind to the AP-1 site in association with Fos and Jun, it partially copurifies with a subset of AP-1 proteins. Purified Ref-1 protein stimulates AP-1 DNA-binding activity through the conserved Cys residues in Fos and Jun, but it does not alter the DNA-binding specificity of Fos and Jun. Ref-1 may represent a novel redox component of the signal transduction processes that regulate eukaryotic gene expression. Images PMID:1537340

  19. Omega 3 but not omega 6 fatty acids inhibit AP-1 activity and cell transformation in JB6 cells

    PubMed Central

    Liu, Guangming; Bibus, Douglas M.; Bode, Ann M.; Ma, Wei-Ya; Holman, Ralph T.; Dong, Zigang

    2001-01-01

    Epidemiological and animal-based investigations have indicated that the development of skin cancer is in part associated with poor dietary practices. Lipid content and subsequently the derived fatty acid composition of the diet are believed to play a major role in the development of tumorigenesis. Omega 3 (ω3) fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can effectively reduce the risk of skin cancer whereas omega 6 (ω6) fatty acids such as arachidonic acid (AA) reportedly promote risk. To investigate the effects of fatty acids on tumorigenesis, we performed experiments to examine the effects of the ω3 fatty acids EPA and DHA and of the ω6 fatty acid AA on phorbol 12-tetradecanoate 13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced transcription activator protein 1 (AP-1) transactivation and on the subsequent cellular transformation in a mouse epidermal JB6 cell model. DHA treatment resulted in marked inhibition of TPA- and EGF-induced cell transformation by inhibiting AP-1 transactivation. EPA treatment also inhibited TPA-induced AP-1 transactivation and cell transformation but had no effect on EGF-induced transformation. AA treatment had no effect on either TPA- or EGF-induced AP-1 transactivation or transformation, but did abrogate the inhibitory effects of DHA on TPA- or EGF-induced AP-1 transactivation and cell transformation in a dose-dependent manner. The results of this study demonstrate that the inhibitory effects of ω3 fatty acids on tumorigenesis are more significant for DHA than for EPA and are related to an inhibition of AP-1. Similarly, because AA abrogates the beneficial effects of DHA, the dietary ratio of ω6 to ω3 fatty acids may be a significant factor in mediating tumor development. PMID:11416221

  20. AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

    PubMed

    Qiao, Yichun; Shiue, Chiou-Nan; Zhu, Jian; Zhuang, Ting; Jonsson, Philip; Wright, Anthony P H; Zhao, Chunyan; Dahlman-Wright, Karin

    2015-04-10

    The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression. PMID:25762639

  1. Collagen XVI Induces Expression of MMP9 via Modulation of AP-1 Transcription Factors and Facilitates Invasion of Oral Squamous Cell Carcinoma

    PubMed Central

    Bedal, Konstanze B.; Grässel, Susanne; Oefner, Peter J.; Reinders, Joerg; Reichert, Torsten E.; Bauer, Richard

    2014-01-01

    Collagen XVI belongs to the family of fibril-associated collagens with interrupted triple helices (FACIT). It is overexpressed during the progression of oral squamous cell carcinoma (OSCC). The present data show a strong collagen XVI-dependent induction of MMP9 and an increase in OSCC cell invasion. We found activated integrin-linked kinase (ILK) in a complex with kindlin-1 and activation of protein kinase B (PKB/Akt) to be responsible for MMP9 induction. Inhibition of the formation of focal adhesions reduced MMP9 expression. Moreover, collagen XVI overexpressing OSCC cell clones (COLXVI cell clones) transfected with vectors containing different MMP9 promoter fragments adjacent to a luciferase reporter revealed an increase in luciferase signal dependent on AP-1 binding sites. Deletion of the AP-1 binding site 98 bp upstream of the reported transcription start site and inhibition of AP-1 with Tanshinone IIA resulted in decreased MMP9 expression. The AP-1 subunit JunB showed differential expression between COLXVI cell clones and mock control cells. Additionally, mass spectrometric analysis of immunoprecipitates revealed that c-Fos interacted strongly with dyskerin in COLXVI cell clones compared to mock controls. PMID:24466237

  2. Suppression of albumin enhancer activity by H-ras and AP-1 in hepatocyte cell lines.

    PubMed Central

    Hu, J; Isom, H C

    1994-01-01

    We demonstrated, using a transient transfection assay, that the albumin enhancer increased the expression of the albumin promoter in a highly differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ras oncogene. A transient cotransfection assay showed that T24ras and normal c-Ha-ras were each able to inhibit the activity of the albumin enhancer in an immortal hepatocyte cell line. DNase I footprinting and gel mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transformed cells. ras also did not diminish the expression of HNF1 alpha, C/EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identified within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Subsequent functional assays showed that overexpression of c-jun and c-fos inhibited the activity of the albumin enhancer. Site-directed mutagenesis of the AP-1 binding sites in the albumin enhancer partially abrogated the suppressing effect of ras and c-jun/c-fos on the enhancer. These functional studies therefore supported the results of the structural studies with AP-1. We conclude that the activity of the albumin enhancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP-1. Images PMID:8114691

  3. Redox Regulation of an AP-1-Like Transcription Factor, YapA, in the Fungal Symbiont Epichloë festucae

    PubMed Central

    Cartwright, Gemma M.

    2013-01-01

    One of the central regulators of oxidative stress in Saccharomyces cerevisiae is Yap1, a bZIP transcription factor of the AP-1 family. In unstressed cells, Yap1 is reduced and cytoplasmic, but in response to oxidative stress, it becomes oxidized and accumulates in the nucleus. To date, there have been no reports on the role of AP-1-like transcription factors in symbiotic fungi. An ortholog of Yap1, named YapA, was identified in the genome of the grass symbiont Epichloë festucae and shown to complement an S. cerevisiae Δyap1 mutant. Hyphae of the E. festucae ΔyapA strain were sensitive to menadione and diamide but resistant to H2O2, KO2, and tert-butyl hydroperoxide (t-BOOH). In contrast, conidia of the ΔyapA strain were very sensitive to H2O2 and failed to germinate. Using a PcatA-eGFP degron-tagged reporter, YapA was shown to be required for expression of a spore-specific catalase gene, catA. Although YapA-EGFP localized to the nucleus in response to host reactive oxygen species during seedling infection, there was no difference in whole-plant and cellular phenotypes of plants infected with the ΔyapA strain compared to the wild-type strain. Homologs of the S. cerevisiae and Schizosaccharomyces pombe redox-sensing proteins (Gpx3 and Tpx1, respectively) did not act as redox sensors for YapA in E. festucae. In response to oxidative stress, YapA-EGFP localized to the nuclei of E. festucae ΔgpxC, ΔtpxA, and ΔgpxC ΔtpxA cells to the same degree as that in wild-type cells. These results show that E. festucae has a robust system for countering oxidative stress in culture and in planta but that Gpx3- or Tpx1-like thiol peroxidases are dispensable for activation of YapA. PMID:23893078

  4. Activation of NF-κB and AP-1 Mediates Hyperproliferation by Inducing β-Catenin and c-Myc in Helicobacter pylori-Infected Gastric Epithelial Cells

    PubMed Central

    Byun, Eunyoung; Park, Bohye; Lim, Joo Weon

    2016-01-01

    Purpose In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. Materials and Methods Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. Results H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. Conclusion H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells. PMID:26996564

  5. AP-1/IRF-3 Targeted Anti-Inflammatory Activity of Andrographolide Isolated from Andrographis paniculata.

    PubMed

    Shen, Ting; Yang, Woo Seok; Yi, Young-Su; Sung, Gi-Ho; Rhee, Man Hee; Poo, Haryoung; Kim, Mi-Yeon; Kim, Kyung-Woon; Kim, Jong Heon; Cho, Jae Youl

    2013-01-01

    Andrographolide (AG) is an abundant component of plants of the genus Andrographis and has a number of beneficial properties including neuroprotective, anticancer, anti-inflammatory, and antidiabetic effects. Despite numerous pharmacological studies, the precise mechanism of AG is still ambiguous. Thus, in the present study, we investigated the molecular mechanisms of AG and its target proteins as they pertain to anti-inflammatory responses. AG suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2), as well as the mRNA abundance of inducible NO synthase (iNOS), tumor necrosis factor-alpha (TNF- α ), cyclooxygenase (COX)-2, and interferon-beta (IFN- β ) in a dose-dependent manner in both lipopolysaccharide- (LPS-) activated RAW264.7 cells and peritoneal macrophages. AG also substantially ameliorated the symptoms of LPS-induced hepatitis and EtOH/HCl-induced gastritis in mice. Based on the results of luciferase reporter gene assays, kinase assays, and measurement of nuclear levels of transcription factors, the anti-inflammatory effects of AG were found to be clearly mediated by inhibition of both (1) extracellular signal-regulated kinase (ERK)/activator protein (AP)-1 and (2) I κ B kinase ε (IKK ε )/interferon regulatory factor (IRF)-3 pathways. In conclusion, we detected a novel molecular signaling pathway by which AG can suppress inflammatory responses. Thus, AG is a promising anti-inflammatory drug with two pharmacological targets. PMID:23840248

  6. DIFFERENTIAL ACTIVATION OF AP-1 IN HUMAN BLADDER EPITHELIAL CELLS BY INORGANIC AND METHYLATED ARSENICALS

    EPA Science Inventory

    Differential Activation of AP-1 in Human Bladder Epithelial Cells by Inorganic and Methylated Arsenicals

    Zuzana Drobna, Ilona Jaspers, David J. Thomas, and Miroslav Styblo

    ABSTRACT

    Epidemiological studies have linked chronic ingestion of drinking water contai...

  7. AP-1 Transcription Factors c-FOS and c-JUN Mediate GnRH-Induced Cadherin-11 Expression and Trophoblast Cell Invasion.

    PubMed

    Peng, Bo; Zhu, Hua; Ma, Liyang; Wang, Yan-Ling; Klausen, Christian; Leung, Peter C K

    2015-06-01

    GnRH is expressed in first-trimester human placenta and increases cell invasion in extravillous cytotrophoblasts (EVTs). Invasive phenotypes have been reported to be regulated by transcription factor activator protein 1 (AP-1) and mesenchymal cadherin-11. The aim of our study was to investigate the roles of AP-1 components (c-FOS/c-JUN) and cadherin-11 in GnRH-induced cell invasion in human EVT cells. Phosphorylated c-FOS and phosphorylated c-JUN were detected in the cell column regions of human first-trimester placental villi by immunohistochemistry. GnRH treatment increased c-FOS, c-JUN, and cadherin-11 mRNA and protein levels in immortalized EVT (HTR-8/SVneo) cells. Moreover, GnRH treatment induced c-FOS and c-JUN protein phosphorylation and nuclear accumulation. Pretreatment with antide, a GnRH antagonist, attenuated GnRH-induced cadherin-11 expression. Importantly, basal and GnRH-induced cadherin-11 expression and cell invasion were reduced by small interfering RNA-mediated knockdown of c-FOS, c-JUN, and cadherin-11 in HTR-8/SVneo cells. Our results suggest that GnRH induces the expression and phosphorylation of the AP-1 transcription factors c-FOS and c-JUN in trophoblast cells, which contributes to GnRH-induced elevation of cadherin-11 expression and cell invasion. PMID:25794160

  8. bZIP transcription factor CgAP1 is essential for oxidative stress tolerance and full virulence of the poplar anthracnose fungus Colletotrichum gloeosporioides.

    PubMed

    Sun, Yingjiao; Wang, Yonglin; Tian, Chengming

    2016-10-01

    Yeast AP1 transcription factor is a regulator of oxidative stress response. Here, we report the identification and characterization of CgAP1, an ortholog of YAP1 in poplar anthracnose fungus Colletotrichum gloeosporioides. The expression of CgAP1 was highly induced by reactive oxygen species. CgAP1 deletion mutants displayed enhanced sensitivity to oxidative stress compared with the wild-type strain, and their poplar leaf virulence was obviously reduced. However, the mutants exhibited no obvious defects in aerial hyphal growth, conidia production, and appressoria formation. CgAP1::eGFP fusion protein localized to the nucleus after TBH (tert-Butyl hydroperoxide) treatment, suggesting that CgAP1 functions as a redox sensor in C. gloeosporioides. In addition, CgAP1 prevented the accumulation of ROS during early stages of biotrophic growth. CgAP1 also acted as a positive regulator of several ROS-related genes (i.e., Glr1, Hyr1, and Cyt1) involved in the antioxidative response. These results highlight the key regulatory role of CgAP1 transcription factor in oxidative stress response and provide insights into the function of ROS detoxification in virulence of C. gloeosporioides. PMID:27544415

  9. The AP-1 Transcription Factor c-Jun Prevents Stress-Imposed Maladaptive Remodeling of the Heart

    PubMed Central

    Windak, Renata; Müller, Julius; Felley, Allison; Akhmedov, Alexander; Wagner, Erwin F.; Pedrazzini, Thierry; Sumara, Grzegorz; Ricci, Romeo

    2013-01-01

    Systemic hypertension increases cardiac workload and subsequently induces signaling networks in heart that underlie myocyte growth (hypertrophic response) through expansion of sarcomeres with the aim to increase contractility. However, conditions of increased workload can induce both adaptive and maladaptive growth of heart muscle. Previous studies implicate two members of the AP-1 transcription factor family, junD and fra-1, in regulation of heart growth during hypertrophic response. In this study, we investigate the function of the AP-1 transcription factors, c-jun and c-fos, in heart growth. Using pressure overload-induced cardiac hypertrophy in mice and targeted deletion of Jun or Fos in cardiomyocytes, we show that c-jun is required for adaptive cardiac hypertrophy, while c-fos is dispensable in this context. c-jun promotes expression of sarcomere proteins and suppresses expression of extracellular matrix proteins. Capacity of cardiac muscle to contract depends on organization of principal thick and thin filaments, myosin and actin, within the sarcomere. In line with decreased expression of sarcomere-associated proteins, Jun-deficient cardiomyocytes present disarrangement of filaments in sarcomeres and actin cytoskeleton disorganization. Moreover, Jun-deficient hearts subjected to pressure overload display pronounced fibrosis and increased myocyte apoptosis finally resulting in dilated cardiomyopathy. In conclusion, c-jun but not c-fos is required to induce a transcriptional program aimed at adapting heart growth upon increased workload. PMID:24039904

  10. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation.

    PubMed

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical "molecular switch" to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  11. Photodynamic activation as a molecular switch to promote osteoblast cell differentiation via AP-1 activation

    PubMed Central

    Kushibiki, Toshihiro; Tu, Yupeng; Abu-Yousif, Adnan O.; Hasan, Tayyaba

    2015-01-01

    In photodynamic therapy (PDT), cells are impregnated with a photosensitizing agent that is activated by light irradiation, thereby photochemically generating reactive oxygen species (ROS). The amounts of ROS produced depends on the PDT dose and the nature of the photosensitizer. Although high levels of ROS are cytotoxic, at physiological levels they play a key role as second messengers in cellular signaling pathways, pluripotency, and differentiation of stem cells. To investigate further the use of photochemically triggered manipulation of such pathways, we exposed mouse osteoblast precursor cells and rat primary mesenchymal stromal cells to low-dose PDT. Our results demonstrate that low-dose PDT can promote osteoblast differentiation via the activation of activator protein-1 (AP-1). Although PDT has been used primarily as an anti-cancer therapy, the use of light as a photochemical “molecular switch” to promote differentiation should expand the utility of this method in basic research and clinical applications. PMID:26279470

  12. AP-1-Targeting Anti-Inflammatory Activity of the Methanolic Extract of Persicaria chinensis

    PubMed Central

    Son, Young-Jin; Baek, Kwang-Soo; Yang, Woo Seok; Park, Jae Gwang; Kim, Han Gyung; Chung, Woo-Jae; Yoon, Keejung; Lee, Sang Yeol; Kim, Jong-Hoon

    2015-01-01

    In traditional Chinese medicine, Persicaria chinensis L. has been prescribed to cure numerous inflammatory disorders. We previously analyzed the bioactivity of the methanol extract of this plant (Pc-ME) against LPS-induced NO and PGE2 in RAW264.7 macrophages and found that it prevented HCl/EtOH-induced gastric ulcers in mice. The purpose of the current study was to explore the molecular mechanism by which Pc-ME inhibits activator protein- (AP-) 1 activation pathway and mediates its hepatoprotective activity. To investigate the putative therapeutic properties of Pc-ME against AP-1-mediated inflammation and hepatotoxicity, lipopolysaccharide- (LPS-) stimulated RAW264.7 and U937 cells, a monocyte-like human cell line, and an LPS/D-galactosamine- (D-GalN-) induced acute hepatitis mouse model were employed. The expression of LPS-induced proinflammatory cytokines including interleukin- (IL-) 1β, IL-6, and tumor necrosis factor-α (TNF-α) was significantly diminished by Pc-ME. Moreover, Pc-ME reduced AP-1 activation and mitogen-activated protein kinase (MAPK) phosphorylation in both LPS-stimulated RAW264.7 cells and differentiated U937 cells. Additionally, we highlighted the hepatoprotective and curative effects of Pc-ME pretreated orally in a mouse model of LPS/D-GalN-intoxicated acute liver injury by demonstrating the significant reduction in elevated serum AST and ALT levels and histological damage. Therefore, these results strongly suggest that Pc-ME could function as an antihepatitis remedy suppressing MAPK/AP-1-mediated inflammatory events. PMID:25878717

  13. Expression profiling of genes regulated by Fra-1/AP-1 transcription factor during bleomycin-induced pulmonary fibrosis

    PubMed Central

    2013-01-01

    Background The Fra-1/AP-1 transcription factor regulates the expression of genes controlling various processes including migration, invasion, and survival as well as extracellular remodeling. We recently demonstrated that loss of Fra-1 leads to exacerbated bleomycin-induced pulmonary fibrosis, accompanied by enhanced expression of various inflammatory and fibrotic genes. To better understand the molecular mechanisms by which Fra-1 confers protection during bleomycin-induced lung injury, genome-wide mRNA expression profiling was performed. Results We found that Fra-1 regulates gene expression programs that include: 1) several cytokines and chemokines involved in inflammation, 2) several genes involved in the extracellular remodeling and cell adhesion, and 3) several genes involved in programmed cell death. Conclusion Loss of Fra-1 leads to the enhanced expression of genes regulating inflammation and immune responses and decreased the expression of genes involved in apoptosis, suggesting that this transcription factor distinctly modulates early pro-fibrotic cellular responses. PMID:23758685

  14. CARMA1- and MyD88-dependent activation of Jun/ATF-type AP-1 complexes is a hallmark of ABC diffuse large B-cell lymphomas

    PubMed Central

    Juilland, Mélanie; Gonzalez, Montserrat; Erdmann, Tabea; Banz, Yara; Jevnikar, Zala; Hailfinger, Stephan; Tzankov, Alexandar; Grau, Michael; Lenz, Georg; Novak, Urban

    2016-01-01

    A hallmark of the diffuse large B-cell lymphoma (DLBCL) of the activated B-cell (ABC) type, a molecular subtype characterized by adverse outcome, is constitutive activation of the transcription factor nuclear factor–κB (NF-κB), which controls expression of genes promoting cellular survival and proliferation. Much less, however, is known about the role of the transcription factor activator protein-1 (AP-1) in ABC DLBCL. Here, we show that AP-1, like NF-κB, was controlled by constitutive activation of the B-cell receptor signaling component caspase recruitment domain-containing membrane-associated guanylate kinase 1 (CARMA1) and/or the Toll-like receptor signaling component myeloid differentiation primary response gene 88 (MyD88) in ABC DLBCL cell lines. In contrast to germinal center (GC) B-cell (GCB) DLBCL, ABC DLBCL cell lines expressed high levels of the AP-1 family members c-Jun, JunB, and JunD, which formed heterodimeric complexes with the AP-1 family members activating transcription factor (ATF) 2, ATF3, and ATF7. Inhibition of these complexes by a dominant-negative approach led to impaired growth of a majority of ABC DLBCL cell lines. Individual silencing of c-Jun, ATF2, or ATF3 decreased cellular survival and revealed c-Jun/ATF2-dependent control of ATF3 expression. As a consequence, ATF3 expression was much higher in ABC vs GCB DLBCL cell lines. Samples derived from DLBCL patients showed a clear trend toward high and nuclear ATF3 expression in nodal DLBCL of the non-GC or ABC subtype. These findings identify the activation of AP-1 complexes of the Jun/ATF-type as an important element controlling the growth of ABC DLBCL. PMID:26747248

  15. Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity.

    PubMed

    Wang, Yan; Lu, Chengrong; Wei, Handong; Wang, Na; Chen, Xiaoxiao; Zhang, Lingqiang; Zhai, Yun; Zhu, Yunping; Lu, Yinglin; He, Fuchu

    2004-08-13

    Hepatopoietin (HPO)/augmenter of liver regeneration (ALR) is a specific hepatotrophic growth factor, which plays a key role in liver regeneration. Our previous study indicated that HPO executes its function by an inter-reactive network of the autocrine, paracrine and endocrine pathways. Recently, we have demonstrated that intracellular HPO interacts with Jun activation domain-binding protein 1 (JAB1) and leads to potentiation of activating protein-1 (AP-1) activity in a MAPK independent fashion. JAB1 is the fifth subunit of the COP9 signalosome (CSN), which is first identified as a suppressor of plant morphogenesis. A protein complex kinase activity associated with the CSN has been reported but not identified yet. In this report, we investigated further the association of HPO with the whole CSN. HPO exists in a complex with the eight-component CSN, both when purified from glycerol gradient centrifugation and when reciprocal immunoprecipitated from the lysates of transfected COS-7 cells. Intracellular HPO colocalizes with endogenous CSN in nucleus of hepatic cells. In addition, intracellular function of HPO that increases the phosphorylation of c-Jun leading to potentiate the AP-1 activity is inhibited by curcumin, a potent inhibitor of CSN-associated kinase. Taken together, these results elucidate a novel relationship of intracellular growth factor, HPO with large protein complex, CSN, which suggests a possible linkage between CSN and liver regeneration. PMID:15304329

  16. CREB, ATF, and AP-1 transcription factors regulate IFN-gamma secretion by human T cells in response to mycobacterial antigen.

    PubMed

    Samten, Buka; Townsend, James C; Weis, Steven E; Bhoumik, Anindita; Klucar, Peter; Shams, Homayoun; Barnes, Peter F

    2008-08-01

    IFN-gamma production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-gamma, -73 to -48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-gamma in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-gamma upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-gamma proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-gamma and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-gamma proximal promoter and that they up-regulate IFN-gamma transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation. PMID:18641343

  17. Interleukin 1β and tumor necrosis factor α promote hFOB1.19 cell viability via activating AP1

    PubMed Central

    Ying, Hongliang; Li, Qiang; Zhao, Changfu

    2016-01-01

    Bone trauma healing is a complex physiological process, which may involve the function of various inflammatory cytokines. Our study aimed to explore the roles of inflammatory cytokines in bone trauma healing and reveal the potential mechanism. Concentrations of interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α) in peripheral blood serum of bone trauma patients after surgery were determined by ELISA. The human osteoblast hFOB1.19 cell line was cultured to determine the effect of these cytokines in cell viability using MTT assay. In addition, luciferase reporter assay was performed to investigate the activator protein 1 (AP1) transcriptional activity, and small interfering RNA was transfected to inhibit FOS, a component of AP1 molecule. IL-6, IL-1β and TNF-α exhibited higher level in patients with more severe bone traumas after surgery. IL-1β and TNF-α, but not IL-6, induced a significant increase of hFOB1.19 viability after three days of treatment (P < 0.05). IL-1β and TNF-α could activate AP1 transcriptional activity in hFOB1.19 cells (P < 0.001), but the activation was inhibited when cells were pretreated with inhibitor of JNKs, SP600125 (P < 0.001). Besides, the effect of IL-1β and TNF-α on promoting viability was significantly inhibited after knockdown of FOS. These findings indicated that IL-1β and TNF-α played an important role in promoting osteoblast viability via the activation of AP1 transcriptional activity, which was likely to involve the JNK/MAPK signaling pathway. Modulating inflammatory cytokines is a potential strategy for improving the outcome of bone trauma healing. PMID:27347349

  18. The AP-1 transcription factor component Fosl2 potentiates the rate of myocardial differentiation from the zebrafish second heart field.

    PubMed

    Jahangiri, Leila; Sharpe, Michka; Novikov, Natasha; González-Rosa, Juan Manuel; Borikova, Asya; Nevis, Kathleen; Paffett-Lugassy, Noelle; Zhao, Long; Adams, Meghan; Guner-Ataman, Burcu; Burns, Caroline E; Burns, C Geoffrey

    2016-01-01

    The vertebrate heart forms through successive phases of cardiomyocyte differentiation. Initially, cardiomyocytes derived from first heart field (FHF) progenitors assemble the linear heart tube. Thereafter, second heart field (SHF) progenitors differentiate into cardiomyocytes that are accreted to the poles of the heart tube over a well-defined developmental window. Although heart tube elongation deficiencies lead to life-threatening congenital heart defects, the variables controlling the initiation, rate and duration of myocardial accretion remain obscure. Here, we demonstrate that the AP-1 transcription factor, Fos-like antigen 2 (Fosl2), potentiates the rate of myocardial accretion from the zebrafish SHF. fosl2 mutants initiate accretion appropriately, but cardiomyocyte production is sluggish, resulting in a ventricular deficit coupled with an accumulation of SHF progenitors. Surprisingly, mutant embryos eventually correct the myocardial deficit by extending the accretion window. Overexpression of Fosl2 also compromises production of SHF-derived ventricular cardiomyocytes, a phenotype that is consistent with precocious depletion of the progenitor pool. Our data implicate Fosl2 in promoting the progenitor to cardiomyocyte transition and uncover the existence of regulatory mechanisms to ensure appropriate SHF-mediated cardiomyocyte contribution irrespective of embryonic stage. PMID:26732840

  19. Human lung and bladder carcinoma tumors as compared to their adjacent normal tissue have elevated AP-1 activity associated with the retinoblastoma gene promoter.

    PubMed

    Linardopoulos, S; Papadakis, E; Delakas, D; Theodosiou, V; Cranidis, A; Spandidos, D A

    1993-01-01

    Examination of the nucleotide sequence of the retinoblastoma (Rb) promoter revealed the presence of a DNA region highly homologous to the recognition site for the cellular transcription factor AP-1. A pair of complementary oligonucleotides containing the AP-1 site was synthesized and used in gel retardation assays to determine the role of the AP-1 protein in the regulation of the Rb gene expression. Using nuclear extracts from Hela cells as well as from lung and bladder tumors, we found specific binding of the AP-1 protein to this oligonucleotide. This binding is elevated in Hela cells, in 10/13 lung and 3/8 bladder tumors as compared to adjacent normal tissue. These results suggest that AP-1 could be implicated in Rb gene transcriptional regulation through its interaction with the AP-1 binding site of the Rb gene promoter. PMID:8476221

  20. Alpha-melanocyte-stimulating hormone modulates activation of NF-kappa B and AP-1 and secretion of interleukin-8 in human dermal fibroblasts.

    PubMed

    Böhm, M; Schulte, U; Kalden, H; Luger, T A

    1999-10-20

    Alpha-melanocyte-stimulating hormone (alpha-MSH) has evolved as a mediator of diverse biological activities in an ever-growing number of non-melanocytic cell types. One mechanism by which alpha-MSH exerts its effects is modulation of AP-1 and NF-kappa B. These two transcription factors also play an important role in fibroblasts, in extracellular matrix composition, and in cytokine expression. By use of electric mobility shift assays, we demonstrate that alpha-MSH (10(-6) to 10(-14) M) activates AP-1 in human dermal fibroblasts, whereas coincubation with interleukin-1 beta (IL-1 beta) results in suppression of its activation. alpha-MSH also induces activation of NF-kappa B but does not modulate DNA binding on costimulation with IL-1 beta. Since AP-1 and NF-kappa B are key elements in controlling interleukin-8 (IL-8) transcription, human fibroblasts were treated with alpha-MSH and IL-1 beta for 24 hours, and cytokine levels in the supernatants were measured by ELISA. alpha-MSH alone had little effect, whereas coincubation with IL-1 beta led to marked downregulation of IL-8 secretion (at most 288 +/- 152 ng/mL) when compared to treatment with IL-1 beta alone (919 +/- 157 ng/mL). Our results indicate that alpha-MSH exerts modulatory effects on the activation of NF-kappa B and AP-1, and that it can regulate chemokine secretion in human dermal fibroblasts. These effects of alpha-MSH may have important regulatory functions in extracellular matrix composition, wound healing, or angiogenesis. PMID:10816661

  1. Silibinin suppresses PMA-induced MMP-9 expression by blocking the AP-1 activation via MAPK signaling pathways in MCF-7 human breast carcinoma cells.

    PubMed

    Lee, Syng-Ook; Jeong, Yun-Jeong; Im, Hyo Gwon; Kim, Cheorl-Ho; Chang, Young-Chae; Lee, In-Seon

    2007-03-01

    Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. In this study, we examined the inhibitory effect of silibinin, a flavonoid antioxidant from milk thistle (Silybum marianum L.) on PMA-induced MMP-9 expression in MCF-7 human breast carcinoma cells. Silibinin significantly and selectively suppressed PMA-induced MMP-9 expression in MCF-7. Silibinin has been found to inhibit PMA-induced MMP-9 gene transcriptional activity by blocking the activation of AP-1 via MAPK signaling pathways. Moreover, the Matrigel invasion assay showed that silibinin reduces PMA-induced invasion of MCF-7 cells. These results suggest that silibinin represents a potential anti-metastatic agent suppressing PMA-induced cancer cell invasion through the specific inhibition of AP-1-dependent MMP-9 gene expression. PMID:17214970

  2. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation

    SciTech Connect

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-08

    Highlights: • SHQA increases PPARα/γ transactivation and inhibits MMP-2/-9 expression. • SHQA inhibits TNFα-induced AP-1 and MAPK signaling. • SHQA inhibits TNFα-induced p65 translocation and IκBα phosphorylation. • SHQA inhibits TNFα-induced AP-1 and NF-κB signaling via PPARα. - Abstract: Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ’s role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα.

  3. The nuclear factor YY1 suppresses the human gamma interferon promoter through two mechanisms: inhibition of AP1 binding and activation of a silencer element.

    PubMed Central

    Ye, J; Cippitelli, M; Dorman, L; Ortaldo, J R; Young, H A

    1996-01-01

    Our group has previously reported that the nuclear factor Yin-Yang 1 (YY1), a ubiquitous DNA-binding protein, is able to interact with a silencer element (BE) in the gamma interferon (IFN-gamma) promoter region. In this study, we demonstrated that YY1 can directly inhibit the activity of the IFN-gamma promoter by interacting with multiple sites in the promoter. In cotransfection assays, a YY1 expression vector significantly inhibited IFN-gamma promoter activity. Mutation of the YY1 binding site in the native IFN-gamma promoter was associated with an increase in the IFN-gamma promoter activity. Analysis of the DNA sequences of the IFN-gamma promoter revealed a second functional YY1 binding site (BED) that overlaps with an AP1 binding site. In this element, AP1 enhancer activity was suppressed by YY1. Since the nuclear level of YY1 does not change upon cell activation, our data support a model that the nuclear factor YY1 acts to suppress basal IFN-gamma transcription by interacting with the promoter at multiple DNA binding sites. This repression can occur through two mechanisms: (i) cooperation with an as-yet-unidentified AP2-like repressor protein and (ii) competition for DNA binding with the transactivating factor AP1. PMID:8756632

  4. A new APE1/Ref-1-dependent pathway leading to reduction of NF-kappaB and AP-1, and activation of their DNA-binding activity.

    PubMed

    Ando, Kozue; Hirao, Satoshi; Kabe, Yasuaki; Ogura, Yuji; Sato, Iwao; Yamaguchi, Yuki; Wada, Tadashi; Handa, Hiroshi

    2008-08-01

    APE1/Ref-1 is thought to be a multifunctional protein involved in reduction-oxidation (redox) regulation and base excision DNA repair, and is required for early embryonic development in mice. APE1/Ref-1 has redox activity and AP endonuclease activity, and is able to enhance DNA-binding activity of several transcription factors, including NF-kappaB, AP-1 and p53, through reduction of their critical cysteine residues. However, it remains elusive exactly how APE1/Ref-1 carries out its essential functions in vivo. Here, we show that APE1/Ref-1 not only reduces target transcription factors directly but also facilitates their reduction by other reducing molecules such as glutathione or thioredoxin. The new activity of APE1/Ref-1, termed redox chaperone activity, is exerted at concentration significantly lower than that required for its redox activity and is neither dependent on its redox activity nor on its AP endonuclease activity. We also show evidence that redox chaperone activity of APE1/Ref-1 is critical to NF-kappaB-mediated gene expression in human cells and is mediated through its physical association with target transcription factors. Thus, APE1/Ref-1 may play multiple roles in an antioxidative stress response pathway through its different biochemical activities. These findings also provide new insight into the mechanism of intracellular redox regulation. PMID:18586825

  5. Delta FosB and AP-1-mediated transcription modulate cannabinoid CB1 receptor signaling and desensitization in striatal and limbic brain regions

    PubMed Central

    Lazenka, Matthew F.; David, Bethany G.; Lichtman, Aron H.; Nestler, Eric J.; Selley, Dana E.; Sim-Selley, Laura J.

    2014-01-01

    Repeated Δ9-tetrahydrocannabinol (THC) administration produces cannabinoid type 1 receptor (CB1R) desensitization and downregulation, as well as tolerance to its in vivo pharmacological effects. However, the magnitude of CB1R desensitization varies by brain region, with CB1Rs in the striatum and its output nuclei undergoing less desensitization than other regions. A growing body of data indicates that regional differences in CB1R desensitization are produced, in part, by THC-mediated induction of the stable transcription factor, ΔFosB, and subsequent regulation of CB1Rs. The purpose of the present study was to determine whether THC-mediated induction of ΔFosB in the striatum inhibits CB1R desensitization in the striatum and output nuclei. This hypothesis was tested using bitransgenic mice with inducible expression of ΔFosB or ΔcJun, a dominant negative inhibitor of AP-1-mediated transcription, in specific forebrain regions. Mice were treated repeatedly with escalating doses of THC or vehicle for 6.5 days, and CB1R-mediated G-protein activation was assessed using CP55,940-stimulated [35S]GTPγS autoradiography. Overexpression of ΔFosB in striatal dopamine type 1 receptor-containing (D1R) medium spiny neurons (MSNs) attenuated CB1R desensitization in the substantia nigra, ventral tegmental area (VTA) and amygdala. Expression of ΔcJun in striatal D1R- and dopamine type 2 receptor (D2R)-containing MSNs enhanced CB1R desensitization in the caudate-putamen and attenuated desensitization in the hippocampus and VTA. THC-mediated in vivo pharmacological effects were then assessed in ΔcJun-expressing mice. Tolerance to THC-mediated hypomotility was enhanced in ΔcJun-expressing mice. These data reveal that ΔFosB and possibly other AP-1 binding proteins regulate CB1R signaling and adaptation in the striatum and limbic system. PMID:25093286

  6. Sargahydroquinoic acid inhibits TNFα-induced AP-1 and NF-κB signaling in HaCaT cells through PPARα activation.

    PubMed

    Jeon, Youngsic; Jung, Yujung; Kim, Min Cheol; Kwon, Hak Cheol; Kang, Ki Sung; Kim, Yong Kee; Kim, Su-Nam

    2014-08-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors and expressed in various cell types in the skin, including keratinocytes, fibroblasts and infiltrating immune cells. Thus, their ligands are targets for the treatment of various skin disorders, such as photo-aging and chronological aging of skin. Intensive studies have revealed that PPARα/γ functions in photo-aging and age-related inflammation by regulating matrix metalloproteinases (MMPs) via activator protein-1 (AP-1) and nuclear factor kappa B (NF-κB). However, the detailed mechanism of PPARα/γ's role in skin aging has not yet been elucidated. In this study, we confirmed that sargahydroquinoic acid (SHQA) as a PPARα/γ ligand significantly decreased Tumor Necrosis Factor-alpha (TNFα)-induced MMP-2/-9 expression by downregulating TNFα-induced transcription factors, subsequently reducing IκBα degradation and blocking NF-κB p65 nuclear translocation in HaCaT human epidermal keratinocyte cells. Treatment of cells with SHQA and GW6471 (PPARα antagonist) not bisphenol A diglycidyl ether (PPARγ antagonists), reversed the effect on TNFα-induced inflammatory signaling pathway activation. Taken together, our data suggest that SHQA inhibit TNFα-induced MMP-2/-9 expression and age-related inflammation by suppressing AP-1 and NF-κB pathway via PPARα. PMID:25019995

  7. S100P/RAGE signaling regulates microRNA-155 expression via AP-1 activation in colon cancer

    PubMed Central

    Onyeagucha, Benjamin Chidi; Mercado-Pimentel, Melania E.; Hutchison, Jennifer; Flemington, Erik K.; Nelson, Mark A.

    2013-01-01

    Accumulating evidence indicates that elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these observations, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Ectopic S100P up-regulated miR-155 levels in human colon cancer cells. Conversely, knockdown of S100P resulted in a decrease in miR-155 levels. Exogenous S100P induced miR-155 expression, but blockage of the RAGE with anti-RAGE antibody suppressed the induction of miR-155 by exogenous S100P. Attenuation of AP-1 activation through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppressed miR-155 induction by exogenous S100P. Also, S100P treatment stimulated the enrichment of c-Fos, an AP-1 family member, at the miR-155 host gene promoter site. Finally, a functional study demonstrated that miR-155 knockdown decreases colon cancer cell growth, motility, and invasion. Altogether, these data demonstrate that the expression of miR-155 is regulated by S100P and is dependent on RAGE activation and stimulation of AP-1. PMID:23693020

  8. Immunohistochemical analysis of the expression of cellular transcription NFκB (p65), AP-1 (c-Fos and c-Jun), and JAK/STAT in leprosy.

    PubMed

    Silva, Luciana Mota; Hirai, Kelly Emi; de Sousa, Jorge Rodrigues; de Souza, Juarez; Fuzii, Hellen Thais; Dias, Leonidas Braga; Carneiro, Francisca Regina Oliveira; de Souza Aarão, Tinara Leila; Quaresma, Juarez Antonio Simões

    2015-05-01

    Leprosy is a disease whose clinical spectrum depends on the cytokine patterns produced during the early stages of the immune response. The main objective of this study was to describe the activation pattern of cellular transcription factors and to correlate these factors with the clinical forms of leprosy. Skin samples were obtained from 16 patients with the tuberculoid (TT) form and 14 with the lepromatous (LL) form. The histologic sections were immunostained with anti-c-Fos and anti-c-Jun monoclonal antibodies for investigation of AP-1, anti-NFκB p65 for the study of NFκB, and anti-JAK2, STAT1, STAT3, and STAT4 for investigation of the JAK/STAT pathway. Cells expressing STAT1 were more frequent in the TT form than in LL lesions (P = .0096), in agreement with the protective immunity provided by IFN-γ. STAT4 was also more highly expressed in the TT form than in the LL form (P = .0098). This transcription factor is essential for the development of a Th1 response because it is associated with interleukin-12. NFκB (p65) and STAT4 expression in the TT form showed a strong and significant correlation (r = 0.7556 and P = .0007). A moderate and significant correlation was observed between JAK2 and STAT4 in the TT form (r = 0.6637 and P = .0051), with these factors responding to interleukin-12 in Th1 profiles. The results suggest that STAT1, JAK2, and NFκB, together with STAT4, contribute to the development of cell-mediated immunity, which is able to contain the proliferation of Mycobacterium leprae. PMID:25771902

  9. Surgical Intervention for Symptomatic Benign Prostatic Hyperplasia is Correlated With Expression of the AP-1 Transcription Factor Network

    PubMed Central

    Lin-Tsai, Opal; Clark, Peter E.; Miller, Nicole L.; Fowke, Jay H.; Hameed, Omar; Hayward, Simon W.; Strand, Douglas W.

    2014-01-01

    BACKGROUND Approximately one-third of patients fail medical treatment for benign prostatic hyperplasia and associated lower urinary tract symptoms (BPH/LUTS) requiring surgical intervention. Our purpose was to establish a molecular characterization for patients undergoing surgical intervention for LUTS to address therapeutic deficiencies. METHODS Clinical, molecular, and histopathological profiles were analyzed in 26 patients undergoing surgery for severe LUTS. Incidental transitional zone nodules were isolated from 37 patients with mild symptoms undergoing radical prostatectomy. Clinical parameters including age, prostate volume, medication, prostate specific antigen, symptom score, body mass index, and incidence of diabetes were collected. Multivariate logistic regression analysis with adjustments for potential confounding variables was used to examine associations between patient clinical characteristics and molecular targets identified through molecular profiling. RESULTS Compared to incidental BPH, progressive symptomatic BPH was associated with increased expression of the activating protein-1 transcription factor/chemokine network. As expected, inverse correlations were drawn between androgen receptor levels and age, as well as between 5α-reductase inhibitor (5ARI) treatment and tissue prostate specific antigen levels; however, a novel association was also drawn between 5ARI treatment and increased c-FOS expression. CONCLUSIONS This study provides molecular evidence that a network of pro-inflammatory activating protein-1 transcription factors and associated chemokines are highly enriched in symptomatic prostate disease, a profile that molecularly categorizes with many other chronic autoimmune diseases. Because 5ARI treatment was associated with increased c-FOS expression, future studies should explore whether increased activating protein-1 proteins are causal factors in the development of symptomatic prostate disease, inflammation or resistance to

  10. Cdk3-promoted epithelial-mesenchymal transition through activating AP-1 is involved in colorectal cancer metastasis

    PubMed Central

    Tang, Na; Li, Yuejin; Peng, Zhengke; Lu, Chengrong; Dong, Zigang; Tang, Faqing

    2016-01-01

    Cyclin dependent kinase-3 (Cdk3) is a positive regulator of the G1 mammalian cell cycle phase. Cdk3 is involved in cancer progression, but very little is known about its mechanism in cancer development and progression. Herein, we found that Cdk3 increased colorectal cancer metastasis through promoting epithelial-mesenchymal transition (EMT) shift. Cdk3 was found to highly express in metastatic cancer and induce cell motility and invasion. Cdk3 was shown to phosphorylate c-Jun at Ser 63 and Ser 73 in vitro and ex vivo. Cdk3-phosphorylated c-Jun at Ser 63 and Ser 73 resulted in an increased AP-1 activity. Ectopic expression of Cdk3 promoted colorectal cancer from epithelial to mesenchymal transition conjugating AP-1 activation, while AP-1 inhibition dramatically decreased Cdk3-increased EMT shift. These results showed that the Cdk3/c-Jun signaling axis mediating epithelial-mesenchymal transition plays an important role in colorectal cancer metastasis. PMID:26755651

  11. Radical Scavenging Activity-Based and AP-1-Targeted Anti-Inflammatory Effects of Lutein in Macrophage-Like and Skin Keratinocytic Cells

    PubMed Central

    Oh, Jueun; Kim, Ji Hye; Park, Jae Gwang; Yi, Young-Su; Park, Kye Won; Rho, Ho Sik; Lee, Min-Seuk; Yoo, Jae Won; Kang, Seung-Hyun; Hong, Yong Deog; Shin, Song Seok; Cho, Jae Youl

    2013-01-01

    Lutein is a naturally occurring carotenoid with antioxidative, antitumorigenic, antiangiogenic, photoprotective, hepatoprotective, and neuroprotective properties. Although the anti-inflammatory effects of lutein have previously been described, the mechanism of its anti-inflammatory action has not been fully elucidated. Therefore, in the present study, we aimed to investigate the regulatory activity of lutein in the inflammatory responses of skin-derived keratinocytes or macrophages and to elucidate the mechanism of its inhibitory action. Lutein significantly reduced several skin inflammatory responses, including increased expression of interleukin-(IL-) 6 from LPS-treated macrophages, upregulation of cyclooxygenase-(COX-) 2 from interferon-γ/tumor necrosis-factor-(TNF-) α-treated HaCaT cells, and the enhancement of matrix-metallopeptidase-(MMP-) 9 level in UV-irradiated keratinocytes. By evaluating the intracellular signaling pathway and the nuclear transcription factor levels, we determined that lutein inhibited the activation of redox-sensitive AP-1 pathway by suppressing the activation of p38 and c-Jun-N-terminal kinase (JNK). Evaluation of the radical and ROS scavenging activities further revealed that lutein was able to act as a strong anti-oxidant. Taken together, our findings strongly suggest that lutein-mediated AP-1 suppression and anti-inflammatory activity are the result of its strong antioxidative and p38/JNK inhibitory activities. These findings can be applied for the preparation of anti-inflammatory and cosmetic remedies for inflammatory diseases of the skin. PMID:23533312

  12. Overexpression of cyclin D1-CDK4 in silica-induced transformed cells is due to activation of ERKs, JNKs/AP-1 pathway.

    PubMed

    Shen, Fuhai; Fan, Xueyun; Liu, Bingci; Jia, Xiaowei; Du, Hongju; You, Baorong; Ye, Meng; Huang, Chuanshu; Shi, Xianglin

    2006-01-25

    Silica has been known to be a factor inducing acute injury and chronic pulmonary fibrosis. Silica has also been listed as a human carcinogen in 1996 by International Agency for Research on Cancer (IARC). However, the molecular mechanisms involved its pathologic effects are not well understood. In these studies, we found that exposure of human embryonic lung fibroblasts (HELF) to crystalline silica could cause increases in activation of extracellular signal-regulated kinases (ERKs), p38K, and c-Jun NH2-terminal amino kinases (JNKs), and HELF transformation. Interestingly, silica-induced transformation of HELF (S-HELF) led to increases in activated levels of ERKs and p46 of JNKs, and decrease in p38K activation, and no effect on activation of p54 of JNKs, as compared with those in parental HELF. Further studies showed that there are differential effects of ERKs, JNKs and p38K, as well as their downstream transcription factor AP-1, in regulation of expression of cyclin D1 and CDK4 and cell cycle alternations induced by silica. Cyclin D1 and CDK4 were increased in S-HELF as compared with those in HELF. Inhibition of ERKs activation by AG126, JNK by SP600125, and AP-1 by curcumin could reduced the induction of cyclin D1 and CDK4. There is no significant difference for cell cycle distribution between groups. These results demonstrate that ERKs and JNKs, but not p38K is responsible for induction of cyclin D1 and CDK4 in S-HELF, suggesting that overexpression of cyclin D1 and CDK4 caused by silica is mediated by ERK, JNK/AP-1signaling pathway. PMID:16125882

  13. Benzyl alcohol derivatives from the mushroom Hericium erinaceum attenuate LPS-stimulated inflammatory response through the regulation of NF-κB and AP-1 activity.

    PubMed

    Noh, Hyung Jun; Yoon, Ju Young; Kim, Geum Sook; Lee, Seung Eun; Lee, Dae Young; Choi, Je Hun; Kim, Seung Yu; Kang, Ki Sung; Cho, Jae Youl; Kim, Ki Hyun

    2014-10-01

    On the search for anti-inflammatory compounds from natural Korean medicinal sources, a bioassay-guided fractionation and chemical investigation of the MeOH extract from the fruiting bodies of Hericium erinaceum resulted in the isolation and identification of five benzyl alcohol derivatives (1-5). In this study, their anti-inflammatory effects on lipopolysaccharide (LPS)-induced production of pro-inflammatory mediators were examined using RAW 264.7 macrophage cells. The structures of isolates were identified by comparing their spectroscopic data with previously reported values. The analysis of their inhibitory activities on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW 264.7 macrophage cells showed that erinacerin B (2) and hericenone E (4) decreased the levels of NO and PGE2 production in a concentration-dependent manner. Next, this study was performed to examine their mechanism of action on the regulation of NO and PGE2 production. Compounds 2 and 4 were found to block the LPS-induced phosphorylation of two major inflammatory transcription factors, NF-κB (p65/p50) and AP-1 (c-Jun and c-Fos). Taken together, these results suggest that down-regulation of LPS-induced NO and PGE2 production by compounds 2 and 4 is mediated through the modulation of NF-κB and AP-1 activation in macrophage cells. These results impact the development of potential health products for preventing and treating inflammatory diseases. PMID:25090632

  14. Nucleolin binds specifically to an AP-1 DNA sequence and represses AP1-dependent transactivation of the matrix metalloproteinase-13 gene.

    PubMed

    Samuel, Shaija; Twizere, Jean-Claude; Beifuss, Katherine K; Bernstein, Lori R

    2008-01-01

    Transcriptional regulation via activator protein-1 (AP-1) protein binding to AP-1 binding sites within gene promoter regions of AP-1 target genes plays a key role in controlling cellular invasion, proliferation, and oncogenesis, and is important to pathogenesis of arthritis and cardiovascular disease. To identify new proteins that interact with the AP-1 DNA binding site, we performed the DNA affinity chromatography-based Nucleotide Affinity Preincubation Specificity TEst of Recognition (NAPSTER) assay, and discovered a 97 kDa protein that binds in vitro to a minimal AP-1 DNA sequence element. Mass spectrometric fragmentation sequencing determined that p97 is nucleolin. Immunoblotting of DNA affinity-purified material with anti-nucleolin antibodies confirmed this identification. Nucleolin also binds the AP-1 site in gel shift assays. Nucleolin interacts in NAPSTER with the AP-1 site within the promoter sequence of the metalloproteinase-13 gene (MMP-13), and binds in vivo in chromatin immunoprecipitation assays in the vicinity of the AP-1 site in the MMP-13 promoter. Overexpression of nucleolin in human HeLa cervical carcinoma cells significantly represses AP-1 dependent gene transactivation of a minimal AP-1 reporter construct and of an MMP-13 promoter reporter sequence. This is the first report of nucleolin binding and transregulation at the AP-1 site. PMID:17626252

  15. Reciprocal effects of varicella-zoster virus (VZV) and AP1: activation of jun, fos and ATF-2 after VZV infection and their importance for the regulation of viral genes.

    PubMed

    Rahaus, Markus; Wolff, Manfred H

    2003-03-01

    Varicella-zoster virus, an alpha-herpesvirus that is pathogenic for man, encodes its own transcription activators, but ubiquitous cellular transcription factors are of great importance, too. Performing quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) we found an increase of transcription of AP1 components jun, fos and of ATF-2 at different times post infection (p.i.). Jun and ATF-2 proteins were detected in infected cells. To study general effects of AP1 in the regulation of VZV encoded genes, oligonucleotide transfections were performed to knockout jun and ATF-2 transcription followed by infection with cell free VZV. RT-PCR analyses of ORFs 4, 9, 21, 29 and 68 belonging to all three kinetic classes of genes and containing different combinations of AP1/TRE and ATF/CREB sites in their respective promoters were carried out. In all cases a reduction of viral transcription was found. Virions produced after this procedure were still infectious, but the amount of newly synthesized virions was clearly reduced. PMID:12606072

  16. Identification of a functional transcriptional factor AP-1 site in the sheep interferon tau gene that mediates a response to PMA in JEG3 cells.

    PubMed Central

    Yamaguchi, H; Ikeda, Y; Moreno, J I; Katsumura, M; Miyazawa, T; Takahashi, E; Imakawa, K; Sakai, S; Christenson, R K

    1999-01-01

    To examine regulatory mechanisms of sheep interferon tau (oIFNtau) gene expression, potential enhancer/silencer elements of the oIFNtau gene were examined using a transient transfection system with oIFNtau gene-chloramphenicol acetyltransferase (oIFNtau-CAT) reporter constructs in human choriocarcinoma cells, JEG3. Experiments with 5'-deletion constructs revealed that the upstream regions from bases -654 to -607 and from bases -606 to -555 were essential for oIFNtau gene expression. In a heterologous transcriptional system in which the upstream regions of oIFNtau were inserted in front of simian virus 40 (SV40) promoter, the regions between bases -654 and -555 were determined as being the enhancer region required for oIFNtau-SV40-CAT transactivation. A subsequent study with the oIFNtau-CAT constructs lacking the upstream region between bases -542 and -124 revealed that, in addition to the further upstream region between bases -1000 and -654, the sequences from bases -543 to -452 seemed to act as silencer regions. The oIFNtau-CAT constructs with site-specific mutagenesis revealed that multiple enhancer elements existed between bases -654 and -555 of the oIFNtau gene. On the basis of nucleotide sequence analysis, there are numerous sites between bases -654 and -555 to which potential transcriptional factors, AP-1, GATA and GATA-related proteins, could bind. Furthermore, gel mobility-shift assays revealed that AP-1 or other nuclear factors could bind to these elements. In co-transfection studies, the expression of c-Jun plus c-Fos enhanced the transactivation of oIFNtau-CAT but the expression of GATA-1, GATA-2 or GATA-3 did not. Taken together, these results suggest that the upstream region between bases -654 and -555 could be considered as the enhancer region for oIFNtau gene transactivation. PMID:10359663

  17. Characterization of the human granulocyte-macrophage colony-stimulating factor gene promoter: an AP1 complex and an Sp1-related complex transactivate the promoter activity that is suppressed by a YY1 complex.

    PubMed Central

    Ye, J; Zhang, X; Dong, Z

    1996-01-01

    It is well documented that a repeated CATT element in the human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter is required for promoter activity. However, the transcription factors that are able to transactivate this enhancer element remain unidentified. Recently, we have found that nuclear factor YY1 can interact with the enhancer element. Here, we report that in addition to YY1, two other nuclear factors have been identified in the DNA-protein complexes formed by the CATT oligonucleotide and the Jurkat T-cell nuclear protein. One of these factors is AP1, and the other one is an Sp1-related protein. Results from transient transfection of Jurkat T cells have revealed that formation of both AP1 and the Sp1-related complex is required for the full enhancer activity of the CATT element. This result is supported by cotransfection of a c-jun expression vector and mutational analysis of the AP1 site or the Sp1-related protein binding site. In contrast, formation of the YY1 complex suppresses enhancer activity, since deletion of the YY1 complex induces an augmentation of the enhancer activity and overexpression of YY1 results in an attenuation of the enhancer activity. Results from the mechanism study have revealed that YY1 is able to inhibit transactivation mediated by either AP1 or the Sp1-related protein, and YY1 suppressive activity is DNA binding dependent. Taken together, these data support the ideas that AP1 and the Sp1-related nuclear protein are required for transactivation of the human GM-CSF gene promoter and that YY1 can suppress transactivation of the promoter even under inducible conditions. PMID:8524292

  18. The Role of JNK and p38 MAPK Activities in UVA-Induced Signaling Pathways Leading to AP-1 Activation and c-Fos Expression1

    PubMed Central

    Silvers, Amy L; Bachelor, Michael A; Bowden, G Timothy

    2003-01-01

    Abstract To further delineate ultraviolet A (UVA) signaling pathways in the human keratinocyte cell line HaCaT, we examined the potential role of mitogen-activated protein kinases (MAPKs) in UVA-induced activator protein-1 (AP-1) transactivation and c-Fos expression. UVA-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK) proteins was detected immediately after irradiation and disappeared after approximately 2 hours. Conversely, phosphorylation of extracellular signal-regulated kinase was significantly inhibited for up to 1 hour post-UVA irradiation. To examine the role of p38 and JNK MAPKs in UVA-induced AP-1 and c-fos transactivations, the selective pharmacologic MAPK inhibitors, SB202190 (p38 inhibitor) and SP600125 (JNK inhibitor), were used to independently treat stably transfected HaCaT cells in luciferase reporter assays. Both SB202190 and SP600125 dose-dependently inhibited UVA-induced AP-1 and c-fos transactivations. SB202190 (0.25–0.5 µM) and SP600125 (62–125 nM) treatments also primarily inhibited UVA-induced c-Fos expression. These results demonstrated that activation of both JNK and p38 play critical role in UVA-mediated AP-1 transactivation and c-Fos expression in these human keratinocyte cells. Targeted inhibition of these MAPKs with their selective pharmacologic inhibitors may be effective chemopreventive strategies for UVA-induced nonmelanoma skin cancer. PMID:14511403

  19. 12-O-Tetradecanoylphorbol-13-Acetate Induces Up-Regulated Transcription of Variant 1 but Not Variant 2 of VIL2 in Esophageal Squamous Cell Carcinoma Cells via ERK1/2/AP-1/Sp1 Signaling

    PubMed Central

    Zhang, Xiao-Dan; Xie, Jian-Jun; Liao, Lian-Di; Long, Lin; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

    2015-01-01

    The membrane-cytoskeleton link organizer ezrin may be the most “dramatic” tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the VIL2 gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human VIL2 in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, VIL2 V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of VIL2 V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of VIL2 V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of VIL2 V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce VIL2 V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the VIL2 V1 promoter, and that VIL2 is an important TPA-induced effector. PMID:25915860

  20. AP-1 family members act with Sox9 to promote chondrocyte hypertrophy.

    PubMed

    He, Xinjun; Ohba, Shinsuke; Hojo, Hironori; McMahon, Andrew P

    2016-08-15

    An analysis of Sox9 binding profiles in developing chondrocytes identified marked enrichment of an AP-1-like motif. Here, we have explored the functional interplay between Sox9 and AP-1 in mammalian chondrocyte development. Among AP-1 family members, Jun and Fosl2 were highly expressed within prehypertrophic and early hypertrophic chondrocytes. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) showed a striking overlap in Jun- and Sox9-bound regions throughout the chondrocyte genome, reflecting direct binding of each factor to the same enhancers and a potential for protein-protein interactions within AP-1- and Sox9-containing complexes. In vitro reporter analysis indicated that direct co-binding of Sox9 and AP-1 at target motifs promoted gene activity. By contrast, where only one factor can engage its DNA target, the presence of the other factor suppresses target activation consistent with protein-protein interactions attenuating transcription. Analysis of prehypertrophic chondrocyte removal of Sox9 confirmed the requirement of Sox9 for hypertrophic chondrocyte development, and in vitro and ex vivo analyses showed that AP-1 promotes chondrocyte hypertrophy. Sox9 and Jun co-bound and co-activated a Col10a1 enhancer in Sox9 and AP-1 motif-dependent manners consistent with their combined action promoting hypertrophic gene expression. Together, the data support a model in which AP-1 family members contribute to Sox9 action in the transition of chondrocytes to the hypertrophic program. PMID:27471255

  1. Two peptides, TsAP-1 and TsAP-2, from the venom of the Brazilian yellow scorpion, Tityus serrulatus: evaluation of their antimicrobial and anticancer activities.

    PubMed

    Guo, Xiaoxiao; Ma, Chengbang; Du, Qiang; Wei, Ran; Wang, Lei; Zhou, Mei; Chen, Tianbao; Shaw, Chris

    2013-09-01

    Here we report two novel 17-mer amidated linear peptides (TsAP-1 and TsAP-2) whose structures were deduced from cDNAs cloned from a venom-derived cDNA library of the Brazilian yellow scorpion, Tityus serrulatus. Both mature peptides were structurally-characterised following their location in chromatographic fractions of venom and synthetic replicates of each were subjected to a range of biological assays. The peptides were each active against model test micro-organisms but with different potencies. TsAP-1 was of low potency against all three test organisms (MICs 120-160 μM), whereas TsAP-2 was of high potency against the Gram-positive bacterium, Staphylococcus aureus (MIC 5 μM) and the yeast, Candida albicans (10 μM). Haemolytic activity of TsAP-1 was low (4% at 160 μM) and in contrast, that of TsAP-2 was considerably higher (18% at 20 μM). Substitution of four neutral amino acid residues with Lys residues in each peptide had dramatic effects on their antimicrobial potencies and haemolytic activities, particularly those of TsAP-1. The MICs of the enhanced cationic analogue (TsAP-S1) were 2.5 μM for S. aureus/C. albicans and 5 μM for E. coli but with an associated large increase in haemolytic activity (30% at 5 μM). The same Lys residue substitutions in TsAP-2 produced a dramatic effect on its MIC for E. coli lowering this from >320 μM to 5 μM. TsAP-1 was ineffective against three of the five human cancer cell lines tested while TsAP-2 inhibited the growth of all five. Lys residue substitution of both peptides enhanced their potency against all five cell lines with TsAp-S2 being the most potent with IC50 values ranging between 0.83 and 2.0 μM. TsAP-1 and TsAP-2 are novel scorpion venom peptides with broad spectrum antimicrobial and anticancer cell activities the potencies of which can be significantly enhanced by increasing their cationicity. PMID:23770440

  2. A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling.

    PubMed

    Yang, Fan; Nam, Sangkil; Zhao, Robin; Tian, Yan; Liu, Lucy; Horne, David A; Jove, Richard

    2013-11-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma. PMID:24025361

  3. A novel synthetic derivative of the natural product berbamine inhibits cell viability and induces apoptosis of human osteosarcoma cells, associated with activation of JNK/AP-1 signaling

    PubMed Central

    Yang, Fan; Nam, Sangkil; Zhao, Robin; Tian, Yan; Liu, Lucy; Horne, David A; Jove, Richard

    2013-01-01

    Osteosarcoma is the most common primary bone tumor in children and adolescents. There is a critical need to find more potent drugs for patients with metastatic or recurrent disease. Berbamine (BBM) is a natural compound derived from the Berberis amurensis plants. BBM and its derivatives have been shown to have antitumor effects in several cancers. Here, we report that a novel synthetic berbamine derivative, BBMD3, inhibits cell viability and induces apoptosis of G292, KHOS, and MG-63 human osteosarcoma cells. Induction of apoptosis in these tumor cells depends on activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP). Since pan-caspase inhibitor (Z-VAD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) could block the cleavage of PARP, the apoptosis induced by BBMD3 is through intrinsic signaling pathway. BBMD3 increased phosphorylation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), resulting in increase of phosphorylated c-Jun and total c-Fos, the major components of transcriptional factor AP-1. JNK inhibitor could partially suppress antitumor effect of BBMD3 on osteosarcoma cells. BBMD3 increased the production of reactive oxygen species (ROS) and ROS scavenger, N-acetylcysteine (NAC), could block the phosphorylation of JNK and c-Jun induced by BBMD3. BBMD3 increased the expression of the pro-apototic gene Bad, associated with apoptosis induction. Finally, BBMD3 also decreased the expression of cyclin D1 and D2, the positive cell cycle regulators, which is correlated with growth inhibition in osteosarcoma cells. Collectively, these findings indicate that BBMD3 is a potentially promising drug for the treatment of human osteosarcoma. PMID:24025361

  4. Advanced glycation end products upregulate lysyl oxidase and endothelin-1 in human aortic endothelial cells via parallel activation of ERK1/2-NF-κB and JNK-AP-1 signaling pathways.

    PubMed

    Adamopoulos, Christos; Piperi, Christina; Gargalionis, Antonios N; Dalagiorgou, Georgia; Spilioti, Eliana; Korkolopoulou, Penelope; Diamanti-Kandarakis, Evanthia; Papavassiliou, Athanasios G

    2016-04-01

    Endothelial dysfunction involves deregulation of the key extracellular matrix (ECM) enzyme lysyl oxidase (LOX) and the vasoconstrictor protein, endothelin-1 (ET-1), whose gene expression can be modulated by the transcriptional activators nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1). Advanced glycation end products (AGEs) present an aggravating factor of endothelial dysfunction which upon engagement to their receptor RAGE induce upregulation of mitogen-activated protein kinases (MAPKs), leading to NF-κB and AP-1 potentiation. We hypothesized that AGEs could induce NF-κΒ- and AP-1-dependent regulation of LOX and ET-1 expression via the AGE/RAGE/MAPK signaling axis. Western blot, real-time qRT-PCR, FACS analysis and electrophoretic mobility-shift assays were employed in human aortic endothelial cells (HAECs) following treatment with AGE-bovine serum albumin (AGE-BSA) to investigate the signaling pathway towards this hypothesis. Furthermore, immunohistochemical analysis of AGEs, RAGE, LOX and ET-1 expression was conducted in aortic endothelium of a rat experimental model exposed to high- or low-AGE content diet. HAECs exposed to AGE-BSA for various time points exhibited upregulation of LOX and ET-1 mRNA levels in a dose- and time-dependent manner. Exposure of HAECs to AGE-BSA also showed specific elevation of phospho(p)-ERK1/2 and p-JNK levels in a dose- and time-dependent fashion. AGE administration significantly increased NF-κΒ- and AP-1-binding activity to both LOX and ET-1 cognate promoter regions. Moreover, LOX and ET-1 overexpression in rat aortic endothelium upon high-AGE content diet confirmed the functional interrelation of these molecules. Our findings demonstrate that AGEs trigger NF-κΒ- and AP-1-mediated upregulation of LOX and ET-1 via the AGE/RAGE/MAPK signaling cascade in human endothelial cells, thus contributing to distorted endothelial homeostasis by impairing endothelial barrier function, altering ECM biomechanical properties

  5. Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK1 cells.

    PubMed Central

    Lee, J S; von der Ahe, D; Kiefer, B; Nagamine, Y

    1993-01-01

    Urokinase-type plasminogen activator (uPA) is an extracellular protease and expressed in various cells that exhibit dynamic changes in cell morphology, suggesting a link between cytoskeletal reorganization (CSR) and uPA expression. CSR can be induced by pharmacological agents, such as by colchicine for microtubule cytoskeleton and by cytochalasin for microfilament cytoskeleton. Using these agents, we previously showed that CSR induced the uPA gene in LLC-PK1 cells independently of the protein kinase C and cAMP-dependent protein kinase. Here we show that the induction of the uPA gene by CSR is mediated by the activation of c-Jun which interacts with an AP-1-like site located 2 kb upstream of the uPA gene. 12-O-tetradecanoylphorbol 13-acetate (TPA) induces the uPA gene through the same elements, but additionally utilizes an adjacent PEA3 element and induces c-fos. Furthermore, CSR induces a greater accumulation and a more pronounced phosphorylation of c-Jun than TPA induction. AP-1 is a positive regulator of growth and oncogenesis, and CSR is an integral part of these processes. Our results provide a view how CSR and AP-1 could be coupled in these processes. We also show that TPA and CSR act synergistically, suggesting a model where an initial activation signal could be amplified by CSR. Images PMID:8346015

  6. A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1–dependent Golgi–plasma membrane trafficking

    PubMed Central

    Parmar, Hirendrasinh B.; Duncan, Roy

    2016-01-01

    The reovirus fusion–associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell–cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN–plasma membrane transport. PMID:26941330

  7. A novel tribasic Golgi export signal directs cargo protein interaction with activated Rab11 and AP-1-dependent Golgi-plasma membrane trafficking.

    PubMed

    Parmar, Hirendrasinh B; Duncan, Roy

    2016-04-15

    The reovirus fusion-associated small transmembrane (FAST) proteins comprise a unique family of viral membrane fusion proteins dedicated to inducing cell-cell fusion. We recently reported that a polybasic motif (PBM) in the cytosolic tail of reptilian reovirus p14 FAST protein functions as a novel tribasic Golgi export signal. Using coimmunoprecipitation and fluorescence resonance energy transfer (FRET) assays, we now show the PBM directs interaction of p14 with GTP-Rab11. Overexpression of dominant-negative Rab11 and RNA interference knockdown of endogenous Rab11 inhibited p14 plasma membrane trafficking and resulted in p14 accumulation in the Golgi complex. This is the first example of Golgi export to the plasma membrane that is dependent on the interaction of membrane protein cargo with activated Rab11. RNA interference and immunofluorescence microscopy further revealed that p14 Golgi export is dependent on AP-1 (but not AP-3 or AP-4) and that Rab11 and AP-1 both colocalize with p14 at the TGN. Together these results imply the PBM mediates interactions of p14 with activated Rab11 at the TGN, resulting in p14 sorting into AP1-coated vesicles for anterograde TGN-plasma membrane transport. PMID:26941330

  8. Interleukin-18 induces EMMPRIN expression in primary cardiomyocytes via JNK/Sp1 signaling and MMP-9 in part via EMMPRIN and through AP-1 and NF-κB activation

    PubMed Central

    Reddy, Venkatapuram Seenu; Prabhu, Sumanth D.; Mummidi, Srinivas; Valente, Anthony J.; Venkatesan, Balachandar; Shanmugam, Prakashsrinivasan; Delafontaine, Patrice

    2010-01-01

    IL-18 and the extracellular matrix metalloproteinase (MMP) inducer (EMMPRIN) stimulate the expression of proinflammatory cytokines and MMPs and are elevated in myocardial hypertrophy, remodeling, and failure. Here, we report several novel findings in primary cardiomyocytes treated with IL-18. First, IL-18 activated multiple transcription factors, including NF-κB (p50 and p65), activator protein (AP)-1 (cFos, cJun, and JunD), GATA, CCAAT/enhancer-binding protein, myocyte-specific enhancer-binding factor, interferon regulatory factor-1, p53, and specific protein (Sp)-1. Second, IL-18 induced EMMPRIN expression via myeloid differentiation primary response gene 88/IL-1 receptor-associated kinase/TNF receptor-associated factor-6/JNK-dependent Sp1 activation. Third, IL-18 induced a number of MMP genes, particularly MMP-9, at a rapid rate as well as tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-3 at a slower rate. Finally, the IL-18 induction of MMP-9 was mediated in part via EMMPRIN and through JNK- and ERK-dependent AP-1 activation and p38 MAPK-dependent NF-κB activation. These results suggest that the elevated expression of IL-18 during myocardial injury and inflammation may favor EMMPRIN and MMP induction and extracellular matrix degradation. Therefore, targeting IL-18 or its signaling pathways may be of potential therapeutic benefit in adverse remodeling. PMID:20693392

  9. Activation of TGF-β1 promoter by hepatitis C virus-induced AP-1 and Sp1: role of TGF-β1 in hepatic stellate cell activation and invasion.

    PubMed

    Presser, Lance D; McRae, Steven; Waris, Gulam

    2013-01-01

    Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-β1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-β1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-κB and STAT-3 are involved in TGF-β1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-β1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-β1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-β1 is critical for the induction of alpha smooth muscle actin (α-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-β1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-β1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-β1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection. PMID:23437118

  10. S100B/RAGE-dependent activation of microglia via NF-kappaB and AP-1 Co-regulation of COX-2 expression by S100B, IL-1beta and TNF-alpha.

    PubMed

    Bianchi, Roberta; Giambanco, Ileana; Donato, Rosario

    2010-04-01

    Extracellular S100B is known to affect astrocytic, neuronal and microglial activities, with different effects depending on its concentration. Whereas at relatively low concentrations S100B exerts trophic effects on neurons and astrocytes, at relatively high concentrations the protein causes neuronal apoptosis and activates astrocytes and microglia, thus potentially representing an endogenous factor implicated in neuroinflammation. We have reported that RAGE ligation by S100B in BV-2 microglia results in the upregulation of expression of the pro-inflammatory cyclo-oxygenase 2 (COX-2) via parallel Ras-Cdc42-Rac1-dependent activation of c-Jun NH(2) terminal protein kinase (JNK) and Ras-Rac1-dependent stimulation of NF-kappaB transcriptional activity. We show here that: (1) S100B also stimulates AP-1 transcriptional activity in microglia via RAGE-dependent activation of JNK; (2) S100B upregulates IL-1beta and TNF-alpha expression in microglia via RAGE engagement; and (3) S100B/RAGE-induced upregulation of COX-2, IL-1beta and TNF-alpha expression requires the concurrent activation of NF-kappaB and AP-1. We also show that S100B synergizes with IL-1beta and TNF-alpha to upregulate on COX-2 expression in microglia. Given the crucial roles of COX-2, IL-1beta and TNF-alpha in the inflammatory response, we propose that, by engaging RAGE, S100B might play an important role in microglia activation in the course of brain damage. PMID:18599158

  11. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells

    PubMed Central

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-01-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3′-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA–protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1–TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  12. Terminal deoxynucleotidyl transferase is down-regulated by AP-1-like regulatory elements in human lymphoid cells.

    PubMed

    Peralta-Zaragoza, Oscar; Recillas-Targa, Félix; Madrid-Marina, Vicente

    2004-02-01

    Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that catalyses the incorporation of deoxyribonucleotides into the 3'-hydroxyl end of DNA templates and is thought to increase junctional diversity of antigen receptor genes. TdT is expressed only on immature lymphocytes and acute lymphoblastic leukaemia cells and its transcriptional expression is tightly regulated. We had previously found that protein kinase C (PKC) activation down-regulates TdT expression. PKC-activation induces the synthesis of the Fos and Jun proteins, known as the major components of activation protein 1 (AP-1) transcriptional factor implicated in transcriptional control. Here we report the identification of several DNA-protein interactions within the TdT promoter region in non-TdT expressing human cells. Sequence analysis revealed the presence of a putative AP-1-like DNA-binding site, suggesting that AP-1 may play a relevant role in TdT transcriptional regulation. Using a different source of nuclear extracts and the AP-1-TdT motif as a probe we identified several DNA-protein retarded complexes in electrophoretic mobility shift assays. Super-band shifting analysis using an antibody against c-Jun protein confirmed that the main interaction is produced by a nuclear factor that belongs to the AP-1 family transcription factors. Our findings suggest that the TdT gene expression is down-regulated, at least in part, through AP-1-like transcription factors. PMID:15027905

  13. Two tobacco AP1-like gene promoters with highly specific, tightly regulated and uniquely expressed activity during floral transition, initiation and development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biotech engineering of agronomic traits requires an array of highly specific and tightly regulated promoters in flower or other tissues. In this study, we isolated and characterized two tobacco AP1-like promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using GUS reporter and tissue-speci...

  14. Characterization of quinolone antibacterial-induced convulsions and increases in nuclear AP-1 DNA- and CRE-binding activities in mouse brain.

    PubMed

    Ito, Y; Ishige, K; Aizawa, M; Fukuda, H

    1999-05-01

    The quinolone antibacterials enoxacin and norfloxacin (2.5 mg/kg, i.v.) provoked clonic convulsions in mice treated concomitantly with biphenylacetic acid (BPAA, 100 mg/kg, i.p.), a major metabolite of the nonsteroidal anti-inflammatory drug fenbufen. Gel-shift assays showed that enoxacin-induced convulsions resulted in increases in nuclear activator protein 1 (AP-1) DNA- and cyclic AMP responsive element (CRE)-binding activities in the cerebral cortex and hippocampus, but not in other regions, such as the cerebellum and thalamus. In contrast, ofloxacin and levofloxacin, at the same doses, in the presence of BPAA did not evoke convulsions or increase these DNA-binding activities. Administration of these quinolones and BPAA alone elicited neither convulsions nor increases in these DNA-binding activities. These results suggest that the increased nuclear AP-1 DNA- and CRE-binding activities in the cerebral cortex and hippocampus induced by quinolones with BPAA correlated with seizure activities and that these brain regions play pivotal roles in quinolone-induced convulsions. PMID:10340309

  15. Laa1p, a Conserved AP-1 Accessory Protein Important for AP-1 Localization in Yeast

    PubMed Central

    Fernández, G. Esteban

    2006-01-01

    AP-1 and Gga adaptors participate in clathrin-mediated protein transport between the trans-Golgi network and endosomes. Both adaptors contain homologous domains that act to recruit accessory proteins involved in clathrin-coated vesicle formation, but the spectrum of known adaptor-binding partners is limited. This study describes an evolutionarily conserved protein of Saccharomyces cerevisiae, Laa1p (Yjl207cp), that interacts and functions specifically with AP-1. Deletion of LAA1, when combined with a conditional mutation in clathrin heavy chain or deletion of GGA genes, accentuated growth defects and increased disruption of clathrin-dependent α-factor maturation and transport of carboxypeptidase Y to the vacuole. In contrast, such genetic interactions were not observed between deletions of LAA1 and AP-1 subunit genes. Laa1p preferentially interacted with AP-1 compared with Gga proteins by glutathione S-transferase-fusion affinity binding and coimmunoprecipitations. Localization of AP-1 and Laa1p, but not Gga proteins, was highly sensitive to brefeldin A, an inhibitor of ADP-ribosylation factor (Arf) activation. Importantly, deletion of LAA1 caused mislocalization of AP-1, especially in cells at high density (postdiauxic shift), but it did not affect Gga protein distribution. Our results identify Laa1p as a new determinant of AP-1 localization, suggesting a model in which Laa1p and Arf cooperate to direct stable association of AP-1 with appropriate intracellular membranes. PMID:16687571

  16. Transcriptional activators in yeast

    PubMed Central

    2006-01-01

    Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested ∼6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix–loop–helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2). PMID:16464826

  17. Low concentrations of copper in drinking water increase AP-1 binding in the brain.

    PubMed

    Lung, Shyang; Li, Huihui; Bondy, Stephen C; Campbell, Arezoo

    2015-12-01

    Copper (Cu) in trace amounts is essential for biological organisms. However, dysregulation of the redox-active metal has been implicated in different neurological disorders such as Wilson's, Menkes', Alzheimer's, and Parkinson's diseases. Since many households use Cu tubing in the plumbing system, and corrosion causes the metal to leach into the drinking water, there may be adverse effects on the central nervous system connected with low-level chronic exposure. The present study demonstrates that treatment with a biologically relevant concentration of Cu for 3 months significantly increases activation of the redox-modulated transcription factor AP-1 in mouse brains. This was independent of an upstream kinase indicated in AP-1 activation. Another redox-active transcription factor, NF-κB, was not significantly modified by the Cu exposure. These results indicate that the effect of Cu on AP-1 is unique and may involve direct modulation of DNA binding. PMID:23719850

  18. TGF-β2 induces Grb2 to recruit PI3-K to TGF-RII that activates JNK/AP-1-signaling and augments invasiveness of Theileria-transformed macrophages

    PubMed Central

    Haidar, Malak; Whitworth, Jessie; Noé, Gaelle; Liu, Wang Qing; Vidal, Michel; Langsley, Gordon

    2015-01-01

    Theileria-infected macrophages display many features of cancer cells such as heightened invasive capacity; however, the tumor-like phenotype is reversible by killing the parasite. Moreover, virulent macrophages can be attenuated by multiple in vitro passages and so provide a powerful model to elucidate mechanisms related to transformed macrophage virulence. Here, we demonstrate that in two independent Theileria-transformed macrophage cell lines Grb2 expression is down-regulated concomitant with loss of tumor virulence. Using peptidimer-c to ablate SH2 and SH3 interactions of Grb2 we identify TGF-receptor II and the p85 subunit of PI3-K, as Grb2 partners in virulent macrophages. Ablation of Grb2 interactions reduces PI3-K recruitment to TGF-RII and decreases PIP3 production, and dampens JNK phosphorylation and AP-1-driven transcriptional activity down to levels characteristic of attenuated macrophages. Loss of TGF-R>PI3-K>JNK>AP-1 signaling negatively impacts on virulence traits such as reduced JAM-L/ITG4A and Fos-B/MMP9 expression that contribute to virulent macrophage adhesion and invasiveness. PMID:26511382

  19. Inhibition of transcriptional activity of c-JUN by SIRT1

    SciTech Connect

    Gao Zhanguo; Ye Jianping

    2008-11-28

    c-JUN is a major component of heterodimer transcription factor AP-1 (Activator Protein-1) that activates gene transcription in cell proliferation, inflammation and stress responses. SIRT1 (Sirtuin 1) is a histone deacetylase that controls gene transcription through modification of chromatin structure. However, it is not clear if SIRT1 regulates c-JUN activity in the control of gene transcription. Here, we show that SIRT1 associated with c-JUN in co-immunoprecipitation of whole cell lysate, and inhibited the transcriptional activity of c-JUN in the mammalian two hybridization system. SIRT1 was found in the AP-1 response element in the matrix metalloproteinase-9 (MMP9) promoter DNA leading to inhibition of histone 3 acetylation as shown in a ChIP assay. The SIRT1 signal was reduced by the AP-1 activator PMA, and induced by the SIRT1 activator Resveratrol in the promoter DNA. SIRT1-mediaetd inhibition of AP-1 was demonstrated in the MMP9 gene expression at the gene promoter, mRNA and protein levels. In mouse embryonic fibroblast (MEF) with SIRT1 deficiency (SIRT1{sup -/-}), mRNA and protein of MMP9 were increased in the basal condition, and the inhibitory activity of Resveratrol was significantly attenuated. Glucose-induced MMP9 expression was also inhibited by SIRT1 in response to Resveratrol. These data consistently suggest that SIRT1 directly inhibits the transcriptional activity of AP-1 by targeting c-JUN.

  20. Exogenous avian leukosis virus-induced activation of the ERK/AP1 pathway is required for virus replication and correlates with virus-induced tumorigenesis

    PubMed Central

    Dai, Manman; Feng, Min; Ye, Yu; Wu, Xiaochan; Liu, Di; Liao, Ming; Cao, Weisheng

    2016-01-01

    A proteomics approach was used to reveal the up-regulated proteins involved in the targeted mitogen-activated protein kinase (MAPK) signal transduction pathway in DF-1 cells after ALV subgroup J (ALV-J) infection. Next, we found that ALV-J CHN06 strain infection of DF-1 cells correlated with extracellular signal-regulated kinase 2 (ERK2) activation, which was mainly induced within 15 min, a very early stage of infection, and at a late infection stage, from 108 h to 132 h post-infection. Infection with other ALV subgroup (A/B) strains also triggered ERK/MAPK activation. Moreover, when activating ERK2, ALV subgroups A, B and J simultaneously induced the phosphorylation of c-Jun, an AP1 family member and p38 activation but had no obvious effect on JNK activation at either 15 min or 120 h. Interestingly, only PD98059 inhibited the ALV-induced c-Jun phosphorylation while SP600125 or SB203580 had no influence on c-Jun activation. Furthermore, the viral gp85 and gag proteins were found to contribute to ERK2/AP1 activation. Additionally, the specific ERK inhibitor, PD980509, significantly suppressed ALV replication, as evidenced by extremely low levels of ALV promoter activity and ALV-J protein expression. In vivo analysis of ERK2 activation in tumor cells derived from ALV-J-infected chicken demonstrated a strong correlation between ERK/MAPK activation and virus-associated tumorigenesis. PMID:26754177

  1. Effect Of Simulated Microgravity On Activated T Cell Gene Transcription

    NASA Technical Reports Server (NTRS)

    Morrow, Maureen A.

    2003-01-01

    Studies of T lymphocytes under the shear stress environment of clinorotation have demonstrated an inhibition of activation in response to TCR mediated signaling. These results mimic those observed during space flight. This work investigates the molecular signaling events of T lymphocyte activation with clinorotation. Purified human T lymphocytes and the T cell clone Jurkat exhibit an uncoupling of signaling as mediated through the TCR. Activation of the transcription factor AP-1 is inhibited while activation of NFAT occurs. NFAT dephosphorylation and activation is dependent on sustained Ca(++) influx. Alternatively, AP-1, which consists of two transcription factors, jun and fos, is activated by PKC and Ras mediated pathways. TCR signaling is known to be dependent on cytoskeletal rearrangements, in particular, raft aggregation is critical. Raft aggregation, as mediated through GM, crosslinking, overcomes the inhibition of T lymphocyte activation with clinorotation, indicating that the block is occurring upstream of raft aggregation. Clinorotation is shown to have an effect similar to a weak TCR signal.

  2. Dimerumic Acid Inhibits SW620 Cell Invasion by Attenuating H2O2-Mediated MMP-7 Expression via JNK/C-Jun and ERK/C-Fos Activation in an AP-1-Dependent Manner

    PubMed Central

    Ho, Bing-Ying; Wu, Yao-Ming; Chang, King-Jen; Pan, Tzu-Ming

    2011-01-01

    Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) in the tumor microenvironment play important roles in tumor invasion and metastasis. Recently, ROS have been reported to cause a significant increase in the production and expression of matrix metalloproteinase (MMP)-7, which is closely correlated with metastatic colorectal cancer. The present study was undertaken to evaluate the scavenging activity of dimerumic acid (DMA) for H2O2 isolated from Monascus-fermented rice to investigate the inhibitory effects of DMA on the invasive potential of SW620 human colon cancer cells, and to explore the mechanisms underlying both these phenomena. Our results showed that increased MMP-7 expression due to H2O2 exposure was mediated by activation of mitogen-activated protein kinases (MAPKs) such as Jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK), and p38 kinase. DMA pretreatment suppressed activation of H2O2-mediated MAPK pathways and cell invasion. Moreover, H2O2-triggered MMP-7 production was demonstrated via JNK/c-Jun and ERK/c-Fos activation in an activating protein 1 (AP-1)-dependent manner. Taken together, these results suggest that DMA suppresses H2O2-induced cell invasion by inhibiting AP-1-mediated MMP-7 gene transcription via the JNK/c-Jun and ERK/c-Fos signaling pathways in SW620 human colon cancer cells. Our data suggest that DMA may be useful in minimizing the development of colorectal metastasis. In the future, DMA supplementation may be a beneficial antioxidant to enhance surgical outcomes. PMID:21814482

  3. HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

    NASA Astrophysics Data System (ADS)

    Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

    2014-03-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.

  4. HIV-1 Nef Induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

    PubMed Central

    Liu, Xun; Shah, Ankit; Gangwani, Mohitkumar R.; Silverstein, Peter S.; Fu, Mingui; Kumar, Anil

    2014-01-01

    The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes. PMID:24658403

  5. Coenzyme Q0 regulates NFκB/AP-1 activation and enhances Nrf2 stabilization in attenuation of LPS-induced inflammation and redox imbalance: Evidence from in vitro and in vivo studies.

    PubMed

    Yang, Hsin-Ling; Lin, Ming-Wei; Korivi, Mallikarjuna; Wu, Jia-Jiuan; Liao, Chun-Huei; Chang, Chia-Ting; Liao, Jiunn-Wang; Hseu, You-Cheng

    2016-02-01

    Coenzyme Q (CoQ) analogs with variable number of isoprenoid units have been demonstrated as anti-inflammatory and antioxidant/pro-oxidant molecules. In this study we used CoQ0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone, zero isoprenoid side-chains), a novel quinone derivative, and investigated its molecular actions against LPS-induced inflammation and redox imbalance in murine RAW264.7 macrophages and mice. In LPS-stimulated macrophages, non-cytotoxic concentrations of CoQ0 (2.5-10 μM) inhibited iNOS/COX-2 protein expressions with subsequent reductions of NO, PGE2, TNF-α and IL-1β secretions. This inhibition was reasoned by suppression of NFκB (p65) activation, and inhibition of AP-1 (c-Jun., c-Fos, ATF2) translocation. Our findings indicated that IKKα-mediated I-κB degradation and MAPK-signaling are involved in regulation of NFκB/AP-1 activation. Furthermore, CoQ0 triggered HO-1 and NQO-1 genes through increased Nrf2 nuclear translocation and Nrf2/ARE-signaling. This phenomenon was confirmed by diminished CoQ0 protective effects in Nrf2 knockdown cells, where LPS-induced NO, PGE2, TNF-α and IL-1β productions remained high. Molecular evidence revealed that CoQ0 enhanced Nrf2 steady-state level at both transcriptional and translational levels. CoQ0-induced Nrf2 activation appears to be regulated by ROS-JNK-signaling cascades, as evidenced by suppressed Nrf2 activation upon treatment with pharmacological inhibitors of ROS (N-acetylcysteine) and JNK (SP600125). Besides, oral administration of CoQ0 (5 mg/kg) suppressed LPS-induced (1 mg/kg) induction of iNOS/COX-2 and TNF-α/IL-1β through tight regulation of NFκB/Nrf2 signaling in mice liver and spleen. Our findings conclude that pharmacological actions of CoQ0 are mediated via inhibition of NFκB/AP-1 activation and induction of Nrf2/ARE-signaling. Owing to its potent anti-inflammatory and antioxidant properties, CoQ0 could be a promising candidate to treat inflammatory disorders. PMID:26548719

  6. Transcriptomic analysis by RNA-seq reveals AP-1 pathway as key regulator that green tea may rely on to inhibit lung tumorigenesis.

    PubMed

    Pan, Jing; Zhang, Qi; Xiong, Donghai; Vedell, Peter; Yan, Ying; Jiang, Hui; Cui, Peng; Ding, Feng; Tichelaar, Jay W; Wang, Yian; Lubet, Ronald A; You, Ming

    2014-01-01

    Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects. PMID:24343902

  7. Identification and characterization a novel transcription factor activator protein-1 in the sea cucumber Apostichopus japonicus.

    PubMed

    Yang, Limeng; Li, Chenghua; Chang, Yaqing; Gao, Yinxue; Wang, Yi; Wei, Jing; Song, Jian; Sun, Ping

    2015-08-01

    The transcription factor activator protein-1 (AP-1) is an important gene expression regulator with typical Jun and region-leucine zipper (bZIP) domains and can respond to a plethora of physiological and pathological stimulus. In this study, we identified a novel AP-1 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjAP-1). The full-length of AjAP-1 was of 2944 bp including a 5' untranslated region (UTR) of 201 bp, a 3' UTR of 1753 bp and a putative open reading frame of 990 bp encoding a polypeptide of 329 amino acid residues. Two representative domains of Jun and bZIP as well as two nuclear localization signals (NLSs) were also detected in deduced amino acid of AjAP-1. Spatial distribution expression indicated that AjAP-1 was ubiquitously expressed in all examined tissues with predominant expression in the body wall, moderate in the tube feet, respiratory tree and colemocytes and slightly weak in the intestine and longitudinal muscle. Time-course expression analysis in intestine and coelomocytes revealed that AjAP-1 both reached its peak expression at 4 h after Vibrio splendidus challenge with a 2.6 and 8.2-fold increase compared to their control groups, respectively. Taken together, all these results suggested that AjAP-1 was a novel immune factor and might be involved in the processes of anti-bacteria response in sea cucumber. PMID:26093208

  8. Bamboo extract reduces interleukin 6 (IL-6) overproduction under lipotoxic conditions through inhibiting the activation of NF-κB and AP-1 pathways.

    PubMed

    Higa, Jason K; Panee, Jun

    2011-07-01

    Interleukin 6 (IL-6) is an inflammatory cytokine overexpressed in obese individuals that contributes to the development of diseases such as insulin resistance, type 2 diabetes, and cardiovascular disease. This study investigated the inhibitory effect of an extract from the bamboo Phyllostachys edulis (BEX) on lipotoxicity-induced over-production of IL-6 in metabolic cell lines. Palmitic acid (PA, 0.4mM) was used to induce lipotoxicity in murine C2C12, 3T3-L1, and Hepa6 cells. Both intra- and extra-cellular protein concentrations of IL-6 were measured in the three cell lines after PA treatment with or without the presence of BEX using cytometric bead assays. IL-6 mRNA levels were quantified using real-time PCR, and nuclear concentrations of c-fos, p50 and p65 proteins were measured using DNA-binding ELISA in 3T3-L1 cells. Lipotoxicity increased IL-6 protein concentration in both cytosol and media collected from myoblast and myotube C2C12, as well as preadipose and adipose 3T3-L1, and the presence of BEX (0.5%, v/v) effectively inhibited this overproduction. IL-6 protein expression in hepatic Hepa6 cells was less affected by lipotoxicity. BEX significantly ameliorated PA-induced upregulation of IL-6 mRNA, which correlated with a reduction in nuclear translocation of p50, p65, and c-fos proteins with the presence of BEX, indicating inhibition of NF-κB and AP-1 activation. In summary, BEX inhibits lipotoxicity-induced IL-6 overproduction in muscle and adipose cell lines through the NF-κB and AP-1 pathways, implicating a potential application of this natural product as a cost-effective anti-inflammation nutraceutical. PMID:21474329

  9. Citrus bergamia Juice Extract Attenuates β-Amyloid-Induced Pro-Inflammatory Activation of THP-1 Cells Through MAPK and AP-1 Pathways

    PubMed Central

    Currò, Monica; Risitano, Roberto; Ferlazzo, Nadia; Cirmi, Santa; Gangemi, Chiara; Caccamo, Daniela; Ientile, Riccardo; Navarra, Michele

    2016-01-01

    Flavonoids have been shown to be effective in protecting against age-related cognitive and motor decline in both in vitro and in vivo models. Recently, a flavonoid-rich extract of Citrus bergamia juice (BJe) has been shown to display anti-oxidant and anti-inflammatory properties against LPS-induced activation of human THP-1 monocytes. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer’s disease. To this aim we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against amyloid-beta1–42 (Aβ1−42) -mediated inflammation. Exposure of THP-1 cells to Aβ1−42 significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aβ1−42 raises AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. Our data indicate that BJe may effectively counteract the pro-inflammatory activation of monocytes/microglial cells exposed to amyloid fibrils, suggesting a promising role as a natural drug against neuroinflammatory processes. PMID:26853104

  10. Citrus bergamia Juice Extract Attenuates β-Amyloid-Induced Pro-Inflammatory Activation of THP-1 Cells Through MAPK and AP-1 Pathways.

    PubMed

    Currò, Monica; Risitano, Roberto; Ferlazzo, Nadia; Cirmi, Santa; Gangemi, Chiara; Caccamo, Daniela; Ientile, Riccardo; Navarra, Michele

    2016-01-01

    Flavonoids have been shown to be effective in protecting against age-related cognitive and motor decline in both in vitro and in vivo models. Recently, a flavonoid-rich extract of Citrus bergamia juice (BJe) has been shown to display anti-oxidant and anti-inflammatory properties against LPS-induced activation of human THP-1 monocytes. In the light of these observations, we wondered whether BJe may be beneficial against neuroinflammatory processes, such as those observed in Alzheimer's disease. To this aim we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against amyloid-beta1-42 (Aβ1-42) -mediated inflammation. Exposure of THP-1 cells to Aβ1-42 significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aβ1-42 raises AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. Our data indicate that BJe may effectively counteract the pro-inflammatory activation of monocytes/microglial cells exposed to amyloid fibrils, suggesting a promising role as a natural drug against neuroinflammatory processes. PMID:26853104

  11. Opposing Effects of Zac1 and Curcumin on AP-1-Regulated Expressions of S100A7

    PubMed Central

    Chu, Yu-Wen; Liu, Shu-Ting; Cheng, Hsiao-Chun; Huang, Shih-Ming; Chang, Yung-Lung; Chiang, Chien-Ping; Liu, Ying-Chun; Wang, Wei-Ming

    2015-01-01

    ZAC, an encoding gene mapped at chromosome 6q24-q25 within PSORS1, was previously found over-expressed in the lower compartment of the hyperplastic epidermis in psoriatic lesions. Cytokines produced in the inflammatory dermatoses may drive AP-1 transcription factor to induce responsive gene expressions. We demonstrated that mZac1 can enhance AP-1-responsive S100A7 expression of which the encoding gene was located in PSORS4 with HaCaT keratinocytes. However, the mZac1-enhanced AP-1 transcriptional activity was suppressed by curcumin, indicating the anti-inflammatory property of this botanical agent and is exhibited by blocking the AP-1-mediated cross-talk between PSORS1 and PSORS4. Two putative AP-1-binding sites were found and demonstrated to be functionally important in the regulation of S100A7 promoter activity. Moreover, we found curcumin reduced the DNA-binding activity of AP-1 to the recognition element located in the S100A7 promoter. The S100A7 expression was found to be upregulated in the lesioned epidermis of atopic dermatitis and psoriasis, which is where this keratinocyte-derived chemoattractant engaged in the pro-inflammatory feedback loop. Understanding the regulatory mechanism of S100A7 expression will be helpful to develop therapeutic strategies for chronic inflammatory dermatoses via blocking the reciprocal stimuli between the inflammatory cells and keratinocytes. PMID:26633653

  12. CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway.

    PubMed

    Huang, Juin-Hua; Lin, Ching-Yu; Wu, Sheng-Yang; Chen, Wen-Yu; Chu, Ching-Liang; Brown, Gordon D; Chuu, Chih-Pin; Wu-Hsieh, Betty A

    2015-07-01

    Collaboration between heterogeneous pattern recognition receptors (PRRs) leading to synergistic coordination of immune response is important for the host to fight against invading pathogens. Although complement receptor 3 (CR3) and Dectin-1 are major PRRs to detect fungi, crosstalk between these two receptors in antifungal immunity is largely undefined. Here we took advantage of Histoplasma capsulatum which is known to interact with both CR3 and Dectin-1 and specific particulate ligands to study the collaboration of CR3 and Dectin-1 in macrophage cytokine response. By employing Micro-Western Array (MWA), genetic approach, and pharmacological inhibitors, we demonstrated that CR3 and Dectin-1 act collaboratively to trigger macrophage TNF and IL-6 response through signaling integration at Syk kinase, allowing subsequent enhanced activation of Syk-JNK-AP-1 pathway. Upon engagement, CR3 and Dectin-1 colocalize and form clusters on lipid raft microdomains which serve as a platform facilitating their cooperation in signaling activation and cytokine production. Furthermore, in vivo studies showed that CR3 and Dectin-1 cooperatively participate in host defense against disseminated histoplasmosis and instruct adaptive immune response. Taken together, our findings define the mechanism of receptor crosstalk between CR3 and Dectin-1 and demonstrate the importance of their collaboration in host defense against fungal infection. PMID:26132276

  13. Baculovirus p35 gene is oppositely regulated by P53 and AP-1 like factors in Spodoptera frugiperda

    SciTech Connect

    Mohareer, Krishnaveni; Sahdev, Sudhir; Hasnain, Seyed E.

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer Baculovirus p35 is regulated by both viral and host factors. Black-Right-Pointing-Pointer Baculovirus p35 is negatively regulated by SfP53-like factor. Black-Right-Pointing-Pointer Baculovirus p35 is positively regulated by SfAP-1-like factor. -- Abstract: Baculovirus p35 belongs to the early class of genes of AcMNPV and requires viral factors like Immediate Early protein-1 for its transcription. To investigate the role of host factors in regulating p35 gene expression, the putative transcription factor binding sites were examined in silico and the role of these factors in influencing the transcription of p35 gene was assessed. We focused our studies on AP-1 and P53-like factors, which are activated under oxidative stress conditions. The AP-1 motif is located at -1401 while P53 motif is at -1912 relative to p35 translation start site. The predicted AP-1 and P53 elements formed specific complexes with Spodoptera frugiperda nuclear extracts. Both AP-1 and P53 motif binding proteins were down regulated as a function of AcMNPV infection in Spodoptera cells. To address the question whether during an oxidative outburst, the p35 transcription is enhanced; we investigated the role of these oxidative stress induced host transcription factors in influencing p35 gene transcription. Reporter assays revealed that AP-1 element enhances the transcription of p35 by a factor of two. Interestingly, P53 element appears to repress the transcription of p35 gene.

  14. In simple synthetic promoters YY1-induced DNA bending is important in transcription activation and repression.

    PubMed Central

    Kim, J; Shapiro, D J

    1996-01-01

    Depending on promoter context, YY1 can activate or repress transcription, or provide a site for transcription initiation. To investigate whether the ability of YY1 to induce DNA bending influenced its ability to activate and repress transcription, simple synthetic promoters were constructed in which the YY1 binding site was inserted between the TATA box and either the NF1 or AP1 recognition sequences. In transient transfections of COS cells, the NF1YY1TATA and NF1RYY1TATA promoters exhibited a dramatic 15-20-fold increase in correctly initiated transcription. These promoters exhibited even larger 60-80-fold increases in transcription in HeLa cells. Neither multiple copies of the YY1 binding site alone, nor placement of a YY1 site upstream of the NF1 site activated transcription. Deletion of 4 bp between the NF1 and YY1 sites, which changes the phase of the DNA bends, abolished the 16-fold activation of transcription by NF1YY1TATA. Insertion of the YY1 site between the AP1 site and the TATA box decreased transcription approximately 3-fold. Replacing the YY1 binding site with an intrinsic DNA bending sequence mimicked this transcription repression. Sequences of similar length which do not bend DNA fail to repress AP1-mediated transcription. Gel mobility shift assays were used to show that binding of YY1 to its recognition sequence did not repress binding of AP1 to its recognition sequences. Our data indicate that YY1-induced DNA bending may activate and repress transcription by changing the spatial relationships between transcription activators and components of the basal transcription apparatus. PMID:8932392

  15. Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling

    PubMed Central

    2011-01-01

    Background Mushrooms are well recognized for their culinary properties as well as for their potency to enhance immune response. In the present study, we evaluated anti-inflammatory properties of an edible oyster mushroom (Pleurotus ostreatus) in vitro and in vivo. Methods RAW264.7 murine macrophage cell line and murine splenocytes were incubated with the oyster mushroom concentrate (OMC, 0-100 μg/ml) in the absence or presence of lipopolysacharide (LPS) or concanavalin A (ConA), respectively. Cell proliferation was determined by MTT assay. Expression of cytokines and proteins was measured by ELISA assay and Western blot analysis, respectively. DNA-binding activity was assayed by the gel-shift analysis. Inflammation in mice was induced by intraperitoneal injection of LPS. Results OMC suppressed LPS-induced secretion of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6), and IL-12p40 from RAW264.7 macrophages. OMC inhibited LPS-induced production of prostaglandin E2 (PGE2) and nitric oxide (NO) through the down-regulation of expression of COX-2 and iNOS, respectively. OMC also inhibited LPS-dependent DNA-binding activity of AP-1 and NF-κB in RAW264.7 cells. Oral administration of OMC markedly suppressed secretion of TNF-α and IL-6 in mice challenged with LPS in vivo. Anti-inflammatory activity of OMC was confirmed by the inhibition of proliferation and secretion of interferon-γ (IFN-γ), IL-2, and IL-6 from concanavalin A (ConA)-stimulated mouse splenocytes. Conclusions Our study suggests that oyster mushroom possesses anti-inflammatory activities and could be considered a dietary agent against inflammation. The health benefits of the oyster mushroom warrant further clinical studies. PMID:21575254

  16. Identification of GATA2 and AP-1 activator elements within the enhancer VNTR occurring in intron 5 of the human SIRT3 gene

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human SIRT3 gene contains an intronic VNTR enhancer. A T > C transition occurring in the second repeat of each VNTR allele implies the presence/absence of a putative GATA binding motif. A partially overlapping AP-1 site, not affected by the transition, was also identified. Aims of the present study ...

  17. Anti-cancer effect of snake venom toxin through down regulation of AP-1 mediated PRDX6 expression

    PubMed Central

    Son, Dong Ju; Song, Ho Sub; Kim, Jung Hyun; Ko, Seong Cheol; Song, Min Jong; Lee, Won Hyoung; Yoon, Joo Hee; Ham, Young Wan; Han, Sang Bae; Hong, Jin Tae

    2015-01-01

    Snake venom toxin (SVT) from Vipera lebetina turanica contains a mixture of different enzymes and proteins. Peroxiredoxin 6 (PRDX6) is known to be a stimulator of lung cancer cell growth. PRDX6 is a member of peroxidases, and has calcium-independent phospholipase A2 (iPLA2) activities. PRDX6 has an AP-1 binding site in its promoter region of the gene. Since AP-1 is implicated in tumor growth and PRDX6 expression, in the present study, we investigated whether SVT inhibits PRDX6, thereby preventing human lung cancer cell growth (A549 and NCI-H460) through inactivation of AP-1. A docking model study and pull down assay showed that SVT completely fits on the basic leucine zipper (bZIP) region of c-Fos of AP-1. SVT (0–10 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decreased cIAP and Bcl2 expression via inactivation of AP-1. In an xenograft in vivo model, SVT (0.5 mg/kg and 1 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression, but increased expression of proapoptotic proteins. These data indicate that SVT inhibits tumor growth via inhibition of PRDX6 activity through interaction with its transcription factor AP-1. PMID:26061816

  18. Rb binds c-Jun and activates transcription.

    PubMed Central

    Nead, M A; Baglia, L A; Antinore, M J; Ludlow, J W; McCance, D J

    1998-01-01

    The retinoblastoma protein (Rb) acts as a critical cell-cycle regulator and loss of Rb function is associated with a variety of human cancer types. Here we report that Rb binds to members of the AP-1 family of transcription factors, including c-Jun, and stimulates c-Jun transcriptional activity from an AP-1 consensus sequence. The interaction involves the leucine zipper region of c-Jun and the B pocket of Rb as well as a C-terminal domain. We also present evidence that the complexes are found in terminally differentiating keratinocytes and cells entering the G1 phase of the cell cycle after release from serum starvation. The human papillomavirus type 16 E7 protein, which binds to both c-Jun and Rb, inhibits the ability of Rb to activate c-Jun. The results provide evidence of a role for Rb as a transcriptional activator in early G1 and as a potential modulator of c-Jun expression during keratinocyte differentiation. PMID:9545246

  19. Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation.

    PubMed

    Fan, Bin; Dun, Sai-Hong; Gu, Jian-Qiu; Guo, Yang; Ikuyama, Shoichiro

    2015-01-01

    Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia. PMID:26367267

  20. Pycnogenol Attenuates the Release of Proinflammatory Cytokines and Expression of Perilipin 2 in Lipopolysaccharide-Stimulated Microglia in Part via Inhibition of NF-κB and AP-1 Activation

    PubMed Central

    Fan, Bin; Dun, Sai-Hong; Gu, Jian-Qiu; Guo, Yang; Ikuyama, Shoichiro

    2015-01-01

    Over activation of microglia results in the production of proinflammatory agents that have been implicated in various brain diseases. Pycnogenol is a patented extract from French maritime pine bark (Pinus pinaster Aiton) with strong antioxidant and anti-inflammatory potency. The present study investigated whether pycnogenol may be associated with the production of proinflammatory mediators in lipopolysaccharide-stimulated BV2 (mouse-derived) microglia. It was found that pycnogenol treatment was dose-dependently associated with significantly less release of nitricoxide (NO), TNF-α, IL-6 and IL-1β, and lower levels of intercellular adhesion molecule1 (ICAM-1) and perilipin 2 (PLIN2). Furthermore, this effect was replicated in primary brain microglia. Levels of inducible NO synthase mRNA and protein were attenuated, whereas there was no change in the production of the anti-inflammatory cytokine IL-10. Further evidence indicated that pycnogenol treatment led to the suppression of NF-κB activation through inhibition of p65 translocation into the nucleus and inhibited DNA binding of AP-1, suggesting that these proinflammatory factors are associated with NF-κB and AP-1. We conclude that pycnogenol exerts anti-inflammatory effects through inhibition of the NF-κB and AP-1pathway, and may be useful as a therapeutic agent in the prevention of diseases caused by over activation of microglia. PMID:26367267

  1. Functional Characterization of OsMADS18, a Member of the AP1/SQUA Subfamily of MADS Box Genes1[w

    PubMed Central

    Fornara, Fabio; Pařenicová, Lucie; Falasca, Giuseppina; Pelucchi, Nilla; Masiero, Simona; Ciannamea, Stefano; Lopez-Dee, Zenaida; Altamura, Maria Maddalena; Colombo, Lucia; Kater, Martin M.

    2004-01-01

    MADS box transcription factors controlling flower development have been isolated and studied in a wide variety of organisms. These studies have shown that homologous MADS box genes from different species often have similar functions. OsMADS18 from rice (Oryza sativa) belongs to the phylogenetically defined AP1/SQUA group. The MADS box genes of this group have functions in plant development, like controlling the transition from vegetative to reproductive growth, determination of floral organ identity, and regulation of fruit maturation. In this paper we report the functional analysis of OsMADS18. This rice MADS box gene is widely expressed in rice with its transcripts accumulated to higher levels in meristems. Overexpression of OsMADS18 in rice induced early flowering, and detailed histological analysis revealed that the formation of axillary shoot meristems was accelerated. Silencing of OsMADS18 using an RNA interference approach did not result in any visible phenotypic alteration, indicating that OsMADS18 is probably redundant with other MADS box transcription factors. Surprisingly, overexpression of OsMADS18 in Arabidopsis caused a phenotype closely resembling the ap1 mutant. We show that the ap1 phenotype is not caused by down-regulation of AP1 expression. Yeast two-hybrid experiments showed that some of the natural partners of AP1 interact with OsMADS18, suggesting that the OsMADS18 overexpression phenotype in Arabidopsis is likely to be due to the subtraction of AP1 partners from active transcription complexes. Thus, when compared to AP1, OsMADS18 during evolution seems to have conserved the mechanistic properties of protein-protein interactions, although it cannot complement the AP1 function. PMID:15299121

  2. Down-Regulation of NF-κB Target Genes by the AP-1 and STAT Complex during the Innate Immune Response in Drosophila

    PubMed Central

    Kim, Lark Kyun; Choi, Un Yung; Cho, Hwan Sung; Lee, Jung Seon; Lee, Wook-bin; Kim, Jihyun; Jeong, Kyoungsuk; Shim, Jaewon; Kim-Ha, Jeongsil; Kim, Young-Joon

    2007-01-01

    The activation of several transcription factors is required for the elimination of infectious pathogens via the innate immune response. The transcription factors NF-κB, AP-1, and STAT play major roles in the synthesis of immune effector molecules during innate immune responses. However, the fact that these immune responses can have cytotoxic effects requires their tight regulation to achieve restricted and transient activation, and mis-regulation of the damping process has pathological consequences. Here we show that AP-1 and STAT are themselves the major inhibitors responsible for damping NF-κB–mediated transcriptional activation during the innate immune response in Drosophila. As the levels of dAP-1 and Stat92E increase due to continuous immune signaling, they play a repressive role by forming a repressosome complex with the Drosophila HMG protein, Dsp1. The dAP-1–, Stat92E-, and Dsp1-containing complexes replace Relish at the promoters of diverse immune effector genes by binding to evolutionarily conserved cis-elements, and they recruit histone deacetylase to inhibit transcription. Reduction by mutation of dAP-1, Stat92E, or Dsp1 results in hyperactivation of Relish target genes and reduces the viability of bacterially infected flies despite more efficient pathogen clearance. These defects are rescued by reducing the Relish copy number, thus confirming that mis-regulation of Relish, not inadequate activation of dAP-1, Stat92E, or Dsp1 target genes, is responsible for the reduced survival of the mutants. We conclude that an inhibitory effect of AP-1 and STAT on NF-κB is required for properly balanced immune responses and appears to be evolutionarily conserved. PMID:17803358

  3. Structural basis of transcription activation.

    PubMed

    Feng, Yu; Zhang, Yu; Ebright, Richard H

    2016-06-10

    Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme. PMID:27284196

  4. Regulation of steatohepatitis and PPARγ signaling by distinct AP-1 dimers

    PubMed Central

    Hasenfuss, Sebastian C.; Bakiri, Latifa; Thomsen, Martin K.; Williams, Evan G.; Auwerx, Johan; Wagner, Erwin F.

    2014-01-01

    Summary Non-alcoholic fatty liver disease (NAFLD) affects up to 30% of the adult population in Western societies, yet the underlying molecular pathways remain poorly understood. Here, we identify the dimeric Activator Protein 1 as a regulator of NAFLD. The Fos-related antigen 1 (Fra-1) and 2 (Fra-2) prevent dietary NAFLD by inhibiting pro-steatotic PPARγ signaling. Moreover, established NAFLD and the associated liver damage can be efficiently reversed by hepatocyte-specific Fra-1 expression. In contrast, c-Fos promotes PPARγ expression, while c-Jun exerts opposing, dimer-dependent functions. Interestingly, JunD was found to be essential for PPARγ signaling and NAFLD development. This unique antagonistic regulation of PPARγ by distinct AP-1 dimers occurs at the transcriptional level and establishes AP-1 as a link between obesity, hepatic lipid metabolism and NAFLD. PMID:24411941

  5. Regulation of steatohepatitis and PPARγ signaling by distinct AP-1 dimers.

    PubMed

    Hasenfuss, Sebastian C; Bakiri, Latifa; Thomsen, Martin K; Williams, Evan G; Auwerx, Johan; Wagner, Erwin F

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) affects up to 30% of the adult population in Western societies, yet the underlying molecular pathways remain poorly understood. Here, we identify the dimeric Activator Protein 1 as a regulator of NAFLD. Fos-related antigen 1 (Fra-1) and Fos-related antigen 2 (Fra-2) prevent dietary NAFLD by inhibiting prosteatotic PPARγ signaling. Moreover, established NAFLD and the associated liver damage can be efficiently reversed by hepatocyte-specific Fra-1 expression. In contrast, c-Fos promotes PPARγ expression, while c-Jun exerts opposing, dimer-dependent functions. Interestingly, JunD was found to be essential for PPARγ signaling and NAFLD development. This unique antagonistic regulation of PPARγ by distinct AP-1 dimers occurs at the transcriptional level and establishes AP-1 as a link between obesity, hepatic lipid metabolism, and NAFLD. PMID:24411941

  6. JUNB/AP-1 controls IFN-γ during inflammatory liver disease

    PubMed Central

    Thomsen, Martin K.; Bakiri, Latifa; Hasenfuss, Sebastian C.; Hamacher, Rainer; Martinez, Lola; Wagner, Erwin F.

    2013-01-01

    Understanding the molecular pathogenesis of inflammatory liver disease is essential to design efficient therapeutic approaches. In hepatocytes, the dimeric transcription factor c-JUN/AP-1 is a major mediator of cell survival during hepatitis, although functions for other JUN proteins in liver disease are less defined. Here, we found that JUNB was specifically expressed in human and murine immune cells during acute liver injury. We analyzed the molecular function of JUNB in experimental models of hepatitis, including administration of concanavalin A (ConA) or α-galactosyl-ceramide, which induce liver inflammation and injury. Mice specifically lacking JUNB in hepatocytes displayed a mild increase in ConA-induced liver damage. However, targeted deletion of Junb in immune cells and hepatocytes protected against hepatitis in experimental models that involved NK/NKT cells. The absence of JUNB in immune cells decreased IFN-γ expression and secretion from NK and NKT cells, leading to reduced STAT1 pathway activation. Systemic IFN-γ treatment or adenovirus-based IRF1 delivery to Junb-deficient mice restored hepatotoxicity, and we demonstrate that Ifng is a direct transcriptional target of JUNB. These findings demonstrate that JUNB/AP-1 promotes cell death during acute hepatitis by regulating IFN-γ production in NK and NKT cells and thus functionally antagonizes the hepatoprotective function of c-JUN/AP-1 in hepatocytes. PMID:24200694

  7. Fra-2/AP-1 controls bone formation by regulating osteoblast differentiation and collagen production.

    PubMed

    Bozec, Aline; Bakiri, Latifa; Jimenez, Maria; Schinke, Thorsten; Amling, Michael; Wagner, Erwin F

    2010-09-20

    The activator protein-1 (AP-1) transcription factor complex, in particular the Fos proteins, is an important regulator of bone homeostasis. Fra-2 (Fosl2), a Fos-related protein of the AP-1 family, is expressed in bone cells, and newborn mice lacking Fra-2 exhibit defects in chondrocytes and osteoclasts. Here we show that Fra-2-deficient osteoblasts display a differentiation defect both in vivo and in vitro. Moreover, Fra-2-overexpressing mice are osteosclerotic because of increased differentiation of osteoblasts, which appears to be cell autonomous. Importantly, the osteoblast-specific osteocalcin (Oc) gene and collagen1α2 (col1α2) are transcriptional targets of Fra-2 in both murine and human bone cells. In addition, Fra-2, Oc, and col1 are expressed in stromal cells of human chondroblastic and osteoblastic osteosarcomas (Os's) as well as during osteoblast differentiation of human Os cell lines. These findings reveal a novel function of Fra-2/AP-1 as a positive regulator of bone and matrix formation in mice and humans. PMID:20837772

  8. The JNK/AP-1 pathway upregulates expression of the recycling endosome rab11a gene in B cells transformed by Theileria.

    PubMed

    Lizundia, Regina; Chaussepied, Marie; Naissant, Bernina; Masse, Guillemette X; Quevillon, Emmanuel; Michel, Fréderique; Monier, Solange; Weitzman, Jonathan B; Langsley, Gordon

    2007-08-01

    Lymphocyte transformation induced by Theileria parasites involves constitutive activation of c-Jun N-terminal kinase (JNK) and the AP-1 transcription factor. We found that JNK/AP-1 activation is associated with elevated levels of Rab11 protein in Theileria-transformed B cells. We show that AP-1 regulates rab11a promoter activity in B cells and that the induction of c-Jun activity in mouse fibroblasts also leads to increased transcription of the endogenous rab11a gene, consistent with it being an AP-1 target. Pharmacological inhibition of the JNK pathway reduced Rab11 protein levels and endosome recycling of transferrin receptor (TfR) and siRNA knockdown of JNK1 and Rab11A levels also reduced TfR surface expression. We propose a model, where activation of the JNK/AP-1 pathway during cell transformation might assure that the regulation of recycling endosomes is co-ordinated with cell-cycle progression. This might be achieved via the simultaneous upregulation of the cell cycle machinery (e.g. cyclin D1) and the recycling endosome regulators (e.g. Rab11A). PMID:17388783

  9. Molecular Basis for Enhancement of the Meiotic DMCI Recombinase by RAD51AP1

    SciTech Connect

    Dray, Eloise; Dunlop, Myun Hwa; Kauppi, Liisa; San Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2010-11-05

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 Associated Protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional co-operation is dependent on complex formation between DMC1 and RAD51AP1, and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci co-localize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination.

  10. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells.

    PubMed

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT. PMID:26160345

  11. On involvement of transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells, activator protein-1 and signal transducer and activator of transcription-3 in photodynamic therapy-induced death of crayfish neurons and satellite glial cells

    NASA Astrophysics Data System (ADS)

    Berezhnaya, Elena; Neginskaya, Marya; Kovaleva, Vera; Sharifulina, Svetlana; Ischenko, Irina; Komandirov, Maxim; Rudkovskii, Mikhail; Uzdensky, Anatoly B.

    2015-07-01

    Photodynamic therapy (PDT) is currently used in the treatment of brain tumors. However, not only malignant cells but also neighboring normal neurons and glial cells are damaged during PDT. In order to study the potential role of transcription factors-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), activator protein (AP-1), and signal transducer and activator of transcription-3 (STAT-3)-in photodynamic injury of normal neurons and glia, we photosensitized the isolated crayfish mechanoreceptor consisting of a single sensory neuron enveloped by glial cells. Application of different inhibitors and activators showed that transcription factors NF-κB (inhibitors caffeic acid phenethyl ester and parthenolide, activator betulinic acid), AP-1 (inhibitor SR11302), and STAT-3 (inhibitors stattic and cucurbitacine) influenced PDT-induced death and survival of neurons and glial cells in different ways. These experiments indicated involvement of NF-κB in PDT-induced necrosis of neurons and apoptosis of glial cells. However, in glial cells, it played the antinecrotic role. AP-1 was not involved in PDT-induced necrosis of neurons and glia, but mediated glial apoptosis. STAT-3 was involved in PDT-induced apoptosis of glial cells and necrosis of neurons and glia. Therefore, signaling pathways that regulate cell death and survival in neurons and glial cells are different. Using various inhibitors or activators of transcription factors, one can differently influence the sensitivity and resistance of neurons and glial cells to PDT.

  12. Quercetin modulates NF-kappa B and AP-1/JNK pathways to induce cell death in human hepatoma cells.

    PubMed

    Granado-Serrano, Ana Belén; Martín, María Angeles; Bravo, Laura; Goya, Luis; Ramos, Sonia

    2010-01-01

    Quercetin, a dietary flavonoid, has been shown to possess anticarcinogenic properties, but the precise molecular mechanisms of action are not thoroughly elucidated. The aim of this study was to investigate the regulatory effect of quercetin (50 microM) on two main transcription factors (NF-kappa B and AP-1) related to survival/proliferation pathways in a human hepatoma cell line (HepG2) over time. Quercetin induced a significant time-dependent inactivation of the NF-kappa B pathway consistent with a downregulation of the NF-kappa B binding activity (from 15 min onward). These features were in concert with a time-dependent activation (starting at 15 min and maintained up to 18 h) of the AP-1/JNK pathway, which played an important role in the control of the cell death induced by the flavonoid and contributed to the regulation of survival/proliferation (AKT, ERK) and death (caspase-3, p38, unbalance of Bcl-2 proapoptotic and antiapoptotic proteins) signals. These data suggest that NF-kappa B and AP-1 play a main role in the tight regulation of survival/proliferation pathways exerted by quercetin and that the sustained JNK/AP-1 activation and inhibition of NF-kappa B provoked by the flavonoid induced HepG2 death. PMID:20358477

  13. Creating small transcription activating RNAs.

    PubMed

    Chappell, James; Takahashi, Melissa K; Lucks, Julius B

    2015-03-01

    We expanded the mechanistic capability of small RNAs by creating an entirely synthetic mode of regulation: small transcription activating RNAs (STARs). Using two strategies, we engineered synthetic STAR regulators to disrupt the formation of an intrinsic transcription terminator placed upstream of a gene in Escherichia coli. This resulted in a group of four highly orthogonal STARs that had up to 94-fold activation. By systematically modifying sequence features of this group, we derived design principles for STAR function, which we then used to forward engineer a STAR that targets a terminator found in the Escherichia coli genome. Finally, we showed that STARs could be combined in tandem to create previously unattainable RNA-only transcriptional logic gates. STARs provide a new mechanism of regulation that will expand our ability to use small RNAs to construct synthetic gene networks that precisely control gene expression. PMID:25643173

  14. Baicalein Attenuates Oxidative Stress-Induced Expression of Matrix Metalloproteinase-1 by Regulating the ERK/JNK/AP-1 Pathway in Human Keratinocytes

    PubMed Central

    Kim, Ki Cheon; Kang, Sam Sik; Lee, Jongsung; Park, Deokhoon; Hyun, Jin Won

    2012-01-01

    The matrix metalloproteinase (MMP) family is involved in the breakdown of the extracellular matrix during normal physiological processes such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes such as pathological aging, arthritis, and metastasis. Oxidative conditions generate reactive oxygen species (ROS) (e.g., hydrogen peroxide [H2O2]) in cells, which subsequently induce the synthesis of matrix metalloproteinase-1 (MMP-1). MMP-1, an interstitial collagenase, in turn stimulates an aging phenomenon. In this study, baicalein (5,6,7-trihydroxyflavone) was investigated for its in vitro activity against H2O2-induced damage using a human skin keratinocyte model. Baicalein pretreatment significantly inhibited H2O2-induced up-regulation of MMP-1 mRNA, MMP-1 protein expression and MMP-1 activity in cultured HaCaT keratinocytes. In addition, baicalein decreased the transcriptional activity of activator protein-1 (AP-1) and the expression of c-Fos and c-Jun, both components of the heterodimeric AP-1 transcription factor. Furthermore, baicalein reduced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK), which are upstream of the AP-1 transcription factor. The results of this study suggest that baicalein is involved in the inhibition of oxidative stress-induced expression of MMP-1 via inactivation of the ERK/JNK/AP-1 signaling pathway. PMID:24116275

  15. Negative regulation of the rat stromelysin gene promoter by retinoic acid is mediated by an AP1 binding site.

    PubMed Central

    Nicholson, R C; Mader, S; Nagpal, S; Leid, M; Rochette-Egly, C; Chambon, P

    1990-01-01

    Stromelysin is a member of the metalloproteinase family which plays an important role in extracellular matrix remodelling during many normal and disease processes. We show here that in polyomavirus-transformed rat embryo fibroblast cells (PyT21), the transcription from the stromelysin gene is repressed by the vitamin A derivative retinoic acid (RA). Furthermore, expression vectors encoding the human RA receptors hRAR-alpha, hRAR-beta and hRAR-gamma repress chloramphenicol acetyltransferase (CAT) expression from stromelysin promoter-CAT gene expression vectors in RA-treated PyT21 and human HeLa cells, as determined by transient transfection assays. Through mutation and deletion analysis, we show that the RA dependent repression is mediated by a 25 bp region from nucleotide positions -72 to -48 of the rat stromelysin 5'-flanking DNA sequence. Further mutation analysis of this region indicates that the DNA sequence required for RA dependent repression colocalizes with an AP1 binding site which is essential for promoter activity. We show also that RA represses the transcriptional activity of a reporter gene containing a TPA responding AP1 binding site driving the HSV tk promoter. Thus the RAR-RA complex appears to repress transcription of the stromelysin gene by blocking activation by positive regulatory factors. However, we found no evidence supporting the possibility that the RA dependent repression could be due to RAR binding to the AP1 binding site or to the AP1 components c-fos and c-jun. Images Fig. 1. Fig. 2. Fig. 4. Fig. 6. Fig. 7. Fig. 8. PMID:2176152

  16. Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer

    PubMed Central

    Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

    2015-01-01

    In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers. PMID:25932049

  17. Curcumin modulates cellular AP-1, NF-kB, and HPV16 E6 proteins in oral cancer.

    PubMed

    Mishra, Alok; Kumar, Rakesh; Tyagi, Abhishek; Kohaar, Indu; Hedau, Suresh; Bharti, Alok C; Sarker, Subhodeep; Dey, Dipankar; Saluja, Daman; Das, Bhudev

    2015-01-01

    In this study, we investigated the effects of the natural antioxidant curcumin on the HPV16-positive oral carcinoma cell line 93VU147T and demonstrated that curcumin is not only a potent inhibitor for the activity of host nuclear transcription factors AP-1 and NF-kB but it also selectively suppresses transcription of the HPV16/E6 oncogene during the carcinogenic process in oral cancer cells. This study suggests a therapeutic potential of curcumin for high-risk human papilloma virus (HPV)-infected oral cancers. PMID:25932049

  18. Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins.

    PubMed

    Luna, Milene S A; Hortencio, Thiago M A; Ferreira, Zulma S; Yamanouye, Norma

    2009-05-01

    The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NFkappaB and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NFkappaB and AP-1. Activation of NFkappaB and AP-1 depended on phospholipase C and protein kinase A. Activation of NFkappaB also depended on protein kinase C. Isoprenaline activated both NFkappaB and AP-1, and phenylephrine activated NFkappaB and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production. PMID:19411547

  19. Linking Smads and transcriptional activation.

    PubMed

    Inman, Gareth J

    2005-02-15

    TGF-beta1 (transforming growth factor-beta1) is the prototypical member of a large family of pleiotropic cytokines that regulate diverse biological processes during development and adult tissue homoeostasis. TGF-beta signals via membrane bound serine/threonine kinase receptors which transmit their signals via the intracellular signalling molecules Smad2, Smad3 and Smad4. These Smads contain conserved MH1 and MH2 domains separated by a flexible linker domain. Smad2 and Smad3 act as kinase substrates for the receptors, and, following phosphorylation, they form complexes with Smad4 and translocate to the nucleus. These Smad complexes regulate gene expression and ultimately determine the biological response to TGF-beta. In this issue of the Biochemical Journal, Wang et al. have shown that, like Smad4, the linker domain of Smad3 contains a Smad transcriptional activation domain. This is capable of recruiting the p300 transcriptional co-activator and is required for Smad3-dependent transcriptional activation. This study raises interesting questions about the nature and regulation of Smad-regulated gene activation and elevates the status of the linker domain to rival that of the much-lauded MH1 and MH2 domains. PMID:15702493

  20. AP-1-directed human T cell leukemia virus type 1 viral gene expression during monocytic differentiation.

    PubMed

    Grant, Christian; Jain, Pooja; Nonnemacher, Michael; Flaig, Katherine E; Irish, Bryan; Ahuja, Jaya; Alexaki, Aikaterini; Alefantis, Timothy; Wigdahl, Brian

    2006-09-01

    Human T cell leukemia virus type 1 (HTLV-1) has previously been shown to infect antigen-presenting cells and their precursors in vivo. However, the role these important cell populations play in the pathogenesis of HTLV-1-associated myelopathy/tropical spastic paraparesis or adult T cell leukemia remains unresolved. To better understand how HTLV-1 infection of these important cell populations may potentially impact disease progression, the regulation of HTLV-1 viral gene expression in established monocytic cell lines was examined. U-937 promonocytic cells transiently transfected with a HTLV-1 long-terminal repeat (LTR) luciferase construct were treated with phorbol 12-myristate 13-acetate (PMA) to induce cellular differentiation. PMA-induced cellular differentiation resulted in activation of basal and Tax-mediated transactivation of the HTLV-1 LTR. In addition, electrophoretic mobility shift analyses demonstrated that PMA-induced cellular differentiation induced DNA-binding activity of cellular transcription factors to Tax-responsive element 1 (TRE-1) repeat II. Supershift analyses revealed that factors belonging to the activator protein 1 (AP-1) family of basic region/leucine zipper proteins (Fra-1, Fra-2, JunB, and JunD) were induced to bind to TRE-1 repeat II during cellular differentiation. Inhibition of AP-1 DNA-binding activity by overexpression of a dominant-negative c-Fos mutant (A-Fos) in transient expression analyses resulted in severely decreased levels of HTLV-1 LTR activation in PMA-induced U-937 cells. These results have suggested that following infection of peripheral blood monocytes, HTLV-1 viral gene expression may become up-regulated by AP-1 during differentiation into macrophages or dendritic cells. PMID:16829632

  1. LDL immune complexes stimulate LDL receptor expression in U937 histiocytes via extracellular signal-regulated kinase and AP-1.

    PubMed

    Fu, Yuchang; Huang, Yan; Bandyopadhyay, Sumita; Virella, Gabriel; Lopes-Virella, Maria F

    2003-07-01

    We have previously shown that LDL-containing immune complexes (LDL-ICs) induce up-regulation of LDL receptor (LDLR) expression in human macrophages. The present study further investigated the molecular mechanisms leading to LDLR up-regulation by LDL-ICs as well as the signaling pathways involved. Results showed that treatment of U937 histiocytes with LDL-ICs did not increase the precursors and the cleaved forms of sterol-regulatory element binding proteins (SREBPs) 1a and 2, suggesting that SREBPs may not be involved in LDLR up-regulation by LDL-ICs. Promoter deletion and mutation studies showed that the AP-1 binding sites were essential for LDL-IC-stimulated LDLR expression. Electrophoretic mobility shift assays further demonstrated that LDL-ICs stimulated transcription factor AP-1 activity. Studies assessing the signaling pathways involved in LDLR up-regulation by LDL-ICs showed that the up-regulation of LDLR was extracellular signal-regulated kinase (ERK) dependent. In conclusion, the present study shows that LDL-ICs up-regulate LDLR expression via the ERK signaling pathway and the AP-1 motif-dependent transcriptional activation. PMID:12730303

  2. Inhibition of AP-1 by Sulforaphane Involves Interaction with Cysteine in the cFos DNA-Binding Domain; Implications for Chemoprevention of UVB-Induced Skin Cancer

    PubMed Central

    Dickinson, Sally E.; Melton, Tania F.; Olson, Erik R.; Zhang, Jian; Saboda, Kathylynn; Bowden, G. Timothy

    2009-01-01

    Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables which has been linked to decreased risk of certain cancers. Although the role of SFN in the induction of the transcription factor Nrf2 has been studied extensively, there is also evidence that inhibition of the transcription factor AP-1 may contribute to the chemopreventive properties of this compound. In this study, we show for the first time that SFN is effective at reducing the multiplicity and tumor burden of UVB-induced squamous cell carcinomas (SCCs) in a mouse model utilizing co-treatment with the compound and the carcinogen. We also show that SFN pretreatment is able to reduce the activity of AP-1 luciferase in the skin of transgenic mice after UVB. Chromatin immunoprecipitation analysis verified that a main constituent of the AP-1 dimer, cFos, is inhibited from binding to the AP-1 DNA binding site by SFN. EMSA analysis of nuclear proteins also show that SFN and diamide, both known to react with cysteine amino acids, are effective at inhibiting AP-1 from binding to its response element. Using truncated recombinant cFos and cJun we show that mutation of critical cysteines in the DNA binding domain of these proteins (Cys154 in cFos and Cys272 in cJun) results in loss of sensitivity to both SFN and diamide in EMSA analysis. Together, these data indicate that inhibition of AP-1 activity may be an important molecular mechanism in chemoprevention of SCC by SFN. PMID:19671797

  3. Signalling in inflammatory skin disease by AP-1 (Fos/Jun).

    PubMed

    Uluçkan, Özge; Guinea-Viniegra, Juan; Jimenez, Maria; Wagner, Erwin F

    2015-01-01

    Skin inflammation is a physiological reaction to tissue injury, pathogen invasion and irritants. During this process, innate and/or adaptive immune cells are activated and recruited to the site of inflammation to either promote or suppress inflammation. The sequential recruitment and activation of immune cells is modulated by a combination of cytokines and chemokines, which are regulated by transcription factors, such as AP-1 (Fos/Jun), NF-κB, NFATs, and STATs. Here we review the present evidence and the underlying mechanisms of how Jun/AP-1 proteins control skin inflammation. Genetically engineered mouse models (GEMMs) in which AP-1 proteins are deleted in the epidermis have revealed that these proteins control cytokine expression at multiple levels. Constitutive epidermal deletion of JunB in mice leads to a multi-organ disease characterised by increased levels of pro-inflammatory cytokines. These JunB-deficient mutant mice display several phenotypes from skin inflammation to a G-CSF-dependent myeloproliferative disease, as well as kidney atrophy and bone loss, reminiscent of psoriasis and systemic lupus erythematosus. Importantly, epidermal deletion of both JunB and c-Jun in an inducible manner in adult mice leads to a psoriasis-like disease, in which the epidermal proteome expression profile is comparable to the one from psoriasis patient samples. In this GEMM and in psoriasis patient-derived material, S100A8/A9-dependent C3/CFB complement activation, as well as a miR-21-dependent TIMP-3/TACE pathway leading to TNF-α shedding, plays causal roles in disease development. The newly identified therapeutic targets from GEMMs together with investigations in human patient samples open up new avenues for therapeutic interventions for psoriasis and related inflammatory skin diseases. PMID:26458100

  4. Scorpion Venom Heat-Resistant Peptide Attenuates Glial Fibrillary Acidic Protein Expression via c-Jun/AP-1.

    PubMed

    Cao, Zhen; Wu, Xue-Fei; Peng, Yan; Zhang, Rui; Li, Na; Yang, Jin-Yi; Zhang, Shu-Qin; Zhang, Wan-Qin; Zhao, Jie; Li, Shao

    2015-11-01

    Scorpion venom has been used in the Orient to treat central nervous system diseases for many years, and the protein/peptide toxins in Buthus martensii Karsch (BmK) venom are believed to be the effective components. Scorpion venom heat-resistant peptide (SVHRP) is an active component of the scorpion venom extracted from BmK. In a previous study, we found that SVHRP could inhibit the formation of a glial scar, which is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, in the epileptic hippocampus. However, the cellular and molecular mechanisms underlying this process remain to be clarified. The results of the present study indicate that endogenous GFAP expression in primary rat astrocytes was attenuated by SVHRP. We further demonstrate that the suppression of GFAP was primarily mediated by inhibiting both c-Jun expression and its binding with AP-1 DNA binding site and other factors at the GFAP promoter. These results support that SVHRP contributes to reducing GFAP at least in part by decreasing the activity of the transcription factor AP-1. In conclusion, the effects of SVHRP on astrocytes with respect to the c-Jun/AP-1 signaling pathway in vitro provide a practical basis for studying astrocyte activation and inhibition and a scientific basis for further studies of traditional medicine. PMID:26134308

  5. The regulation of hepcidin expression by serum treatment: requirements of the BMP response element and STAT- and AP-1-binding sites.

    PubMed

    Kanamori, Yohei; Murakami, Masaru; Matsui, Tohru; Funaba, Masayuki

    2014-11-10

    Expression of hepcidin, a central regulator of systemic iron metabolism, is transcriptionally regulated by the bone morphogenetic protein (BMP) pathway. However, the factors other than the BMP pathway also participate in the regulation of hepcidin expression. In the present study, we show that serum treatment increased hepcidin expression and transcription without inducing the phosphorylation of Smad1/5/8 in primary hepatocytes, HepG2 cells or Hepa1-6 cells. Co-treatment with LDN-193189, an inhibitor of the BMP type I receptor, abrogated this hepcidin induction. Reporter assays using mutated reporters revealed the involvement of the BMP response element-1 (BMP-RE1) and signal transducers and activator of transcription (STAT)- and activator protein (AP)-1-binding sites in serum-induced hepcidin transcription in HepG2 cells. Serum treatment induced the expression of the AP-1 components c-fos and junB in primary hepatocytes and HepG2 cells. Forced expression of c-fos or junB enhanced the response of hepcidin transcription to serum treatment. By contrast, the expression of dominant negative (dn)-c-fos and dn-junB decreased hepcidin transcription. The present study reveals that serum contains factors stimulating hepcidin transcription. Basal BMP activity is essential for the serum-induced hepcidin transcription, although serum treatment does not stimulate the BMP pathway. The induction of c-fos and junB by serum treatment stimulates hepcidin transcription, through possibly cooperation with BMP-mediated signaling. Considering that AP-1 is induced by various stimuli, the present results suggest that hepcidin expression is regulated by more diverse factors than had been previously considered. PMID:25151311

  6. Molecular basis for enhancement of the meiotic DMC1 recombinase by RAD51 associated protein 1 (RAD51AP1)

    PubMed Central

    Dray, Eloïse; Dunlop, Myun Hwa; Kauppi, Liisa; Filippo, Joseph San; Wiese, Claudia; Tsai, Miaw-Sheue; Begovic, Sead; Schild, David; Jasin, Maria; Keeney, Scott; Sung, Patrick

    2011-01-01

    Homologous recombination is needed for meiotic chromosome segregation, genome maintenance, and tumor suppression. RAD51AP1 (RAD51 associated protein 1) has been shown to interact with and enhance the recombinase activity of RAD51. Accordingly, genetic ablation of RAD51AP1 leads to enhanced sensitivity to and also chromosome aberrations upon DNA damage, demonstrating a role for RAD51AP1 in mitotic homologous recombination. Here we show physical association of RAD51AP1 with the meiosis-specific recombinase DMC1 and a stimulatory effect of RAD51AP1 on the DMC1-mediated D-loop reaction. Mechanistic studies have revealed that RAD51AP1 enhances the ability of the DMC1 presynaptic filament to capture the duplex-DNA partner and to assemble the synaptic complex, in which the recombining DNA strands are homologously aligned. We also provide evidence that functional cooperation is dependent on complex formation between DMC1 and RAD51AP1 and that distinct epitopes in RAD51AP1 mediate interactions with RAD51 and DMC1. Finally, we show that RAD51AP1 is expressed in mouse testes, and that RAD51AP1 foci colocalize with a subset of DMC1 foci in spermatocytes. These results suggest that RAD51AP1 also serves an important role in meiotic homologous recombination. PMID:21307306

  7. Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats.

    PubMed

    Yeung, B G; Payne, G S

    2001-08-01

    Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-binding sites were defined in the beta1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the gamma subunit. When introduced into chromosomal genes, point mutations in the beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The beta1 mutations or the gamma truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when beta1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and alpha-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo. PMID:11489214

  8. Growth inhibition of non-small cell lung cancer cells by AP-1 blockade using a cJun dominant-negative mutant.

    PubMed

    Shimizu, Y; Kinoshita, I; Kikuchi, J; Yamazaki, K; Nishimura, M; Birrer, M J; Dosaka-Akita, H

    2008-03-11

    cJun, a major constituent of AP-1 transcription factor transducing multiple mitogen growth signals, is frequently overexpressed in non-small cell lung cancers (NSCLCs). The purpose of this study is to determine the effects of AP-1 blockade on the growth of NSCLC cells using a cJun dominant-negative mutant, TAM67. Transiently transfected TAM67 inhibited AP-1 transcriptional activity in NSCLC cell lines, NCI-H1299 (H1299), A549 and NCI-H520 (H520). The colony-forming efficiency of H1299 and A549 was reduced by TAM67, while that of H520 was not. To elucidate the effects of TAM67 on the growth of H1299, we established H1299 clone cells that expressed TAM67 under the control of a doxycycline-inducible promoter. In the H1299 clone cells, the induced TAM67 inhibited anchorage-dependent growth by promoting G1 cell-cycle block, but not by apoptosis. The induced TAM67 decreased the expression of a cell-cycle regulatory protein, cyclin A. TAM67 also inhibited anchorage-independent growth of these cells. Furthermore, TAM67 reduced growth of established xenograft tumours from these cells in nude mice. These results suggest that AP-1 plays an essential role in the growth of at least some of NSCLC cells. PMID:18283312

  9. Loss of JUNB/AP-1 promotes invasive prostate cancer

    PubMed Central

    Thomsen, M K; Bakiri, L; Hasenfuss, S C; Wu, H; Morente, M; Wagner, E F

    2015-01-01

    Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16Ink4a and p21CIP1 in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression. PMID:25526087

  10. Loss of JUNB/AP-1 promotes invasive prostate cancer.

    PubMed

    Thomsen, M K; Bakiri, L; Hasenfuss, S C; Wu, H; Morente, M; Wagner, E F

    2015-04-01

    Prostate cancer is a frequent cause of male death in the Western world. Relatively few genetic alterations have been identified, likely owing to disease heterogeneity. Here, we show that the transcription factor JUNB/AP-1 limits prostate cancer progression. JUNB expression is increased in low-grade prostate cancer compared with normal human prostate, but downregulated in high-grade samples and further decreased in all metastatic samples. To model the hypothesis that this downregulation is functionally significant, we genetically inactivated Junb in the prostate epithelium of mice. When combined with Pten (phosphatase and tensin homologue) loss, double-mutant mice were prone to invasive cancer development. Importantly, invasive tumours also developed when Junb and Pten were inactivated in a small cell population of the adult anterior prostate by topical Cre recombinase delivery. The resulting tumours displayed strong histological similarity with human prostate cancer. Loss of JunB expression led to increased proliferation and decreased senescence, likely owing to decreased p16(Ink4a) and p21(CIP1) in epithelial cells. Furthermore, the tumour stroma was altered with increased osteopontin and S100 calcium-binding protein A8/9 expression, which correlated with poor prognoses in patients. These data demonstrate that JUNB/AP-1 cooperates with PTEN signalling as barriers to invasive prostate cancer, whose concomitant genetic or epigenetic suppression induce malignant progression. PMID:25526087

  11. AP-1/σ1A and AP-1/σ1B adaptor-proteins differentially regulate neuronal early endosome maturation via the Rab5/Vps34-pathway

    PubMed Central

    Candiello, Ermes; Kratzke, Manuel; Wenzel, Dirk; Cassel, Dan; Schu, Peter

    2016-01-01

    The σ1 subunit of the AP-1 clathrin-coated-vesicle adaptor-protein complex is expressed as three isoforms. Tissues express σ1A and one of the σ1B and σ1C isoforms. Brain is the tissue with the highest σ1A and σ1B expression. σ1B-deficiency leads to severe mental retardation, accumulation of early endosomes in synapses and fewer synaptic vesicles, whose recycling is slowed down. AP-1/σ1A and AP-1/σ1B regulate maturation of these early endosomes into multivesicular body late endosomes, thereby controlling synaptic vesicle protein transport into a degradative pathway. σ1A binds ArfGAP1, and with higher affinity brain-specific ArfGAP1, which bind Rabex-5. AP-1/σ1A-ArfGAP1-Rabex-5 complex formation leads to more endosomal Rabex-5 and enhanced, Rab5GTP-stimulated Vps34 PI3-kinase activity, which is essential for multivesicular body endosome formation. Formation of AP-1/σ1A-ArfGAP1-Rabex-5 complexes is prevented by σ1B binding of Rabex-5 and the amount of endosomal Rabex-5 is reduced. AP-1 complexes differentially regulate endosome maturation and coordinate protein recycling and degradation, revealing a novel molecular mechanism by which they regulate protein transport besides their established function in clathrin-coated-vesicle formation. PMID:27411398

  12. Involvement of Ras/Raf/AP-1 in BMP-4 signaling during Xenopus embryonic development.

    PubMed Central

    Xu, R H; Dong, Z; Maeno, M; Kim, J; Suzuki, A; Ueno, N; Sredni, D; Colburn, N H; Kung, H F

    1996-01-01

    Previously, we elucidated the role of bone morphogenetic protein 4 (BMP-4) in the dorsal-ventral patterning of the Xenopus embryo by using a dominant negative mutant of the BMP-4 receptor (DN-BR). The present paper describes the involvement of Ras, Raf, and activator protein 1 (AP-1) in BMP-4 signaling during Xenopus embryonic development. The AP-1 activity was determined by injecting an AP-1-dependent luciferase reporter gene into two-cell-stage Xenopus embryos and measuring the luciferase activity at various developmental stages. We found that injection of BMP-4 mRNA increased AP-1 activity, whereas injection of DN-BR mRNA inhibited AP-1 activity. Similar inhibitory effects were seen with injection of mRNAs encoding dominant negative mutants of c-Ha-Ras, c-Raf, or c-Jun. These results suggest that the endogenous AP-1 activity is regulated by BMP-4/Ras/Raf/Jun signals. We next investigated the effects of Ras/Raf/AP-1 signals on the biological functions of BMP-4. DN-BR-induced dorsalization of the embryo, revealed by the formation of a secondary body axis or dorsalization of the ventral mesoderm explant analyzed by histological and molecular criteria, was significantly reversed by coinjection of [Val12]Ha-Ras, c-Raf, or c-Jun mRNA. Furthermore, the BMP-4-stimulated erythroid differentiation in the ventral mesoderm was substantially inhibited by coinjection with the dominant negative c-Ha-Ras, c-Raf, or c-Jun mutant. Our results suggest the involvement of Ras/Raf/AP-1 in the BMP-4 signaling pathway. Images Fig. 2 Fig. 3 Fig. 4 PMID:8570644

  13. Concurrent BMP7 and FGF9 signalling governs AP-1 function to promote self-renewal of nephron progenitor cells

    PubMed Central

    Muthukrishnan, Sree Deepthi; Yang, Xuehui; Friesel, Robert; Oxburgh, Leif

    2015-01-01

    Self-renewal of nephron progenitor cells (NPCs) is governed by BMP, FGF and WNT signalling. Mechanisms underlying cross-talk between these pathways at the molecular level are largely unknown. Here we delineate the pathway through which the proliferative BMP7 signal is transduced in NPCs in the mouse. BMP7 activates the MAPKs TAK1 and JNK to phosphorylate the transcription factor JUN, which in turn governs transcription of AP-1-element containing G1-phase cell cycle regulators such as Myc and Ccnd1 to promote NPC proliferation. Conditional inactivation of Tak1 or Jun in cap mesenchyme causes identical phenotypes characterized by premature depletion of NPCs. While JUN is regulated by BMP7, we find that its partner FOS is regulated by FGF9. We demonstrate that BMP7 and FGF9 coordinately regulate AP-1 transcription to promote G1-S cell cycle progression and NPC proliferation. Our findings identify a molecular mechanism explaining the important cooperation between two major NPC self-renewal pathways. PMID:26634297

  14. Forcing FAK into Transcriptional Activity.

    PubMed

    Lietha, Daniel

    2016-08-01

    Focal adhesion kinase (FAK) has known signaling roles in cytoplasmic adhesion structures, but was recently shown to act as a transcriptional regulator in the nucleus. In this issue of Structure, Cardoso et al. (2016) report that mechanical forces translocate FAK to the nucleus of cardiomyocytes, and provide structural insights into how FAK interacts with the MEF2 transcription factor to control cardiac hypertrophy. PMID:27486913

  15. Transcriptional activation in yeast cells lacking transcription factor IIA.

    PubMed Central

    Chou, S; Chatterjee, S; Lee, M; Struhl, K

    1999-01-01

    The general transcription factor IIA (TFIIA) forms a complex with TFIID at the TATA promoter element, and it inhibits the function of several negative regulators of the TATA-binding protein (TBP) subunit of TFIID. Biochemical experiments suggest that TFIIA is important in the response to transcriptional activators because activation domains can interact with TFIIA, increase recruitment of TFIID and TFIIA to the promoter, and promote isomerization of the TFIID-TFIIA-TATA complex. Here, we describe a double-shut-off approach to deplete yeast cells of Toa1, the large subunit of TFIIA, to <1% of the wild-type level. Interestingly, such TFIIA-depleted cells are essentially unaffected for activation by heat shock factor, Ace1, and Gal4-VP16. However, depletion of TFIIA causes a general two- to threefold decrease of transcription from most yeast promoters and a specific cell-cycle arrest at the G2-M boundary. These results indicate that transcriptional activation in vivo can occur in the absence of TFIIA. PMID:10581267

  16. AP-1 Inhibition by SR 11302 Protects Human Hepatoma HepG2 Cells from Bile Acid-Induced Cytotoxicity by Restoring the NOS-3 Expression

    PubMed Central

    González-Rubio, Sandra; Linares, Clara I.; Aguilar-Melero, Patricia; Rodríguez-Perálvarez, Manuel; Montero-Álvarez, José L.

    2016-01-01

    The harmful effects of bile acid accumulation occurring during cholestatic liver diseases have been associated with oxidative stress increase and endothelial nitric oxide synthase (NOS-3) expression decrease in liver cells. We have previously reported that glycochenodeoxycholic acid (GCDCA) down-regulates gene expression by increasing SP1 binding to the NOS-3 promoter in an oxidative stress dependent manner. In the present study, we aimed to investigate the role of transcription factor (TF) AP-1 on the NOS-3 deregulation during GCDCA-induced cholestasis. The cytotoxic response to GCDCA was characterized by 1) the increased expression and activation of TFs cJun and c-Fos; 2) a higher binding capability of these at position -666 of the NOS-3 promoter; 3) a decrease of the transcriptional activity of the promoter and the expression and activity of NOS-3; and 4) the expression increase of cyclin D1. Specific inhibition of AP-1 by the retinoid SR 11302 counteracted the cytotoxic effects induced by GCDCA while promoting NOS-3 expression recovery and cyclin D1 reduction. NOS activity inhibition by L-NAME inhibited the protective effect of SR 11302. Inducible NOS isoform was no detected in this experimental model of cholestasis. Our data provide direct evidence for the involvement of AP-1 in the NOS-3 expression regulation during cholestasis and define a critical role for NOS-3 in regulating the expression of cyclin D1 during the cell damage induced by bile acids. AP-1 appears as a potential therapeutic target in cholestatic liver diseases given its role as a transcriptional repressor of NOS-3. PMID:27490694

  17. Role of hippocampal activity-induced transcription in memory consolidation.

    PubMed

    Eagle, Andrew L; Gajewski, Paula A; Robison, Alfred J

    2016-08-01

    Experience-dependent changes in the strength of connections between neurons in the hippocampus (HPC) are critical for normal learning and memory consolidation, and disruption of this process drives a variety of neurological and psychiatric diseases. Proper HPC function relies upon discrete changes in gene expression driven by transcription factors (TFs) induced by neuronal activity. Here, we describe the induction and function of many of the most well-studied HPC TFs, including cyclic-AMP response element binding protein, serum-response factor, AP-1, and others, and describe their role in the learning process. We also discuss the known target genes of many of these TFs and the purported mechanisms by which they regulate long-term changes in HPC synaptic strength. Moreover, we propose that future research in this field will depend upon unbiased identification of additional gene targets for these activity-dependent TFs and subsequent meta-analyses that identify common genes or pathways regulated by multiple TFs in the HPC during learning or disease. PMID:27180338

  18. n-Butyrate inhibits Jun NH(2)-terminal kinase activation and cytokine transcription in mast cells

    SciTech Connect

    Diakos, Christos; Prieschl, Eva E.; Saeemann, Marcus D.; Boehmig, Georg A.; Csonga, Robert; Sobanov, Yury; Baumruker, Thomas; Zlabinger, Gerhard J. . E-mail: gerhard.zlabinger@meduniwien.ac.at

    2006-10-20

    Mast cells are well known to contribute to type I allergic conditions but only recently have been brought in association with chronic relapsing/remitting autoimmune diseases such as celiac disease and ulcerative colitis. Since the bacterial metabolite n-butyrate is considered to counteract intestinal inflammation we investigated the effects of this short chain fatty acid on mast cell activation. Using RNAse protection assays and reporter gene technology we show that n-butyrate downregulates TNF-{alpha} transcription. This correlates with an impaired activation of the Jun NH(2)-terminal kinase (JNK) but not other MAP kinases such as ERK and p38 that are largely unaffected by n-butyrate. As a consequence, we observed a decreased nuclear activity of AP-1 and NF-AT transcription factors. These results indicate that n-butyrate inhibits critical inflammatory mediators in mast cells by relatively selectively targeting the JNK signalling.

  19. Quassinoid Inhibition of AP-1 Function Does Not Correlate with Cytotoxicity or Protein Synthesis Inhibition†

    PubMed Central

    Beutler, John A.; Kang, Moon-Il; Robert, Francis; Clement, Jason A.; Pelletier, Jerry; Colburn, Nancy H.; McKee, Tawnya C.; Goncharova, Ekaterina; McMahon, James B.; Henrich, Curtis J.

    2010-01-01

    Several quassinoids were identified in a high-throughput screening assay as inhibitors of the transcription factor AP-1. Further biological characterization revealed that while their effect was not specific to AP-1, protein synthesis inhibition and cell growth assays were inconsistent with a mechanism of simple protein synthesis inhibition. Numerous plant extracts from the plant family Simaroubaceae were also identified in the same screen; bioassay-guided fractionation of one extract (Ailanthus triphylla) yielded two known quassinoids, ailanthinone (3) and glaucarubinone (4), which were also identified in the pure compound screening procedure. PMID:19199792

  20. Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter.

    PubMed Central

    Oliviero, S; Struhl, K

    1991-01-01

    Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription. Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites. It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions. Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites. Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites. Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter. This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery). Images PMID:1898773

  1. A CRE/AP-1-Like Motif Is Essential for Induced Syncytin-2 Expression and Fusion in Human Trophoblast-Like Model

    PubMed Central

    Vargas, Amandine; Rassart, Éric; Barbeau, Benoit

    2015-01-01

    Syncytin-2 is encoded by the envelope gene of Endogenous Retrovirus-FRD (ERVFRD-1) and plays a critical role in fusion of placental trophoblasts leading to the formation of the multinucleated syncytiotrophoblast. Its expression is consequently regulated in a strict manner. In the present study, we have identified a forskolin-responsive region located between positions -300 to -150 in the Syncytin-2 promoter region. This 150 bp region in the context of a minimal promoter mediated an 80-fold induction of promoter activity following forskolin stimulation. EMSA analyses with competition experiments with nuclear extracts from forskolin-stimulated BeWo cells demonstrated that the -211 to -177 region specifically bound two forskolin-induced complexes, one of them containing a CRE/AP-1-like motif. Site-directed mutagenesis of the CRE/AP-1 binding site in the context of the Syncytin-2 promoter or a heterologous promoter showed that this motif was mostly essential for forskolin-induced promoter activity. Transfection experiments with dominant negative mutants and constitutively activated CREB expression vectors in addition to Chromatin Immunoprecipitation suggested that a CREB family member, CREB2 was binding and acting through the CRE/AP-1 motif. We further demonstrated the binding of JunD to this same motif. Similar to forskolin and soluble cAMP, CREB2 and JunD overexpression induced Syncytin-2 promoter activity in a CRE/AP-1-dependent manner and Syncytin-2 expression. In addition, BeWo cell fusion was induced by both CREB2 and JunD overexpression, while being repressed following silencing of either gene. These results thereby demonstrate that induced expression of Syncytin-2 is highly dependent on the interaction of bZIP-containing transcription factors to a CRE/AP-1 motif and that this element is important for the regulation of Syncytin-2 expression, which results in the formation of the peripheral syncytiotrophoblast layer. PMID:25781974

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

    PubMed

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

    2007-01-12

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

  3. Over-expression of the PaAP1 gene from sweet cherry (Prunus avium L.) causes early flowering in Arabidopsis thaliana.

    PubMed

    Wang, Jing; Zhang, Xiaoming; Yan, Guohua; Zhou, Yu; Zhang, Kaichun

    2013-02-15

    A homologue of SQUAMOSA/APETALA1, designated PaAP1, was isolated from Prunus avium by reverse transcription-PCR (RT-PCR). The full length of PaAP1 cDNA is 753 bp, and it codes for a polypeptide of 250 amino acid residues. Sequence comparison revealed that PaAP1 belongs to the MADS-box gene family. Phylogenetic analysis indicated that PaAP1 shared the highest identity with SQUA/AP1 homologues from Prunus serrulata. Real-time fluorescence quantitative PCR analysis showed that PaAP1 was expressed at high levels in petal, sepal, style, and flower buds, which was slightly different from the expression pattern of AP1 of Arabidopsis thaliana. To characterize the functions of PaAP1, we assessed Arabidopsis transformed with 35S::PaAP1. A total of 8 transgenic T(1) lines with an early flowering phenotype were obtained, and a 3:1 segregation ratio of flowering time was observed in the T(2) generation of 4 lines. This study provides the first functional analysis of an SQUA/AP1 homolog from P. avium and suggests that PaAP1 is potentially useful for shortening the juvenile period in sweet cherry. PMID:23206932

  4. TAF7 (TAFII55) plays a role in the transcription activation by c-Jun.

    PubMed

    Munz, Christine; Psichari, Eleni; Mandilis, Dimitris; Lavigne, Anne-Claire; Spiliotaki, Maria; Oehler, Thomas; Davidson, Irwin; Tora, Laszlo; Angel, Peter; Pintzas, Alexander

    2003-06-13

    c-Jun is a member of the AP-1 family of transcription factors regulating expression of specific target genes in a variety of cellular processes including proliferation, stress response, and tumorigenicity. In the present study we have analyzed the mechanism of c-Jun function as a transactivator with respect to members of the basal transcription machinery, TATA-binding protein-associated factors (TAFs). We show that one member of the family, human TAF7 (formerly TAFII55), physically interacts with c-Jun through two independent interaction domains, within the N- and C-terminal part of c-Jun. Interaction in vitro correlates with enhanced transactivation function of c-Jun in HEK293 and COS cells in the presence of increasing amounts of TAF7. TAF7 interacts preferentially with DNA-bound phosphorylated c-Jun, suggesting that TAF7 represents a novel c-Jun co-activator mediating activation of AP-1 target genes in response to extracellular signals. PMID:12676957

  5. AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

    PubMed Central

    Burgess, Jason; Jauregui, Miluska; Tan, Julie; Rollins, Janet; Lallet, Sylvie; Leventis, Peter A.; Boulianne, Gabrielle L.; Chang, Henry C.; Le Borgne, Roland; Krämer, Helmut; Brill, Julie A.

    2011-01-01

     Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing “glue granules” that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1– and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules. PMID:21490149

  6. An AP-1 binding site in the enhancer/core element of the HIV-1 promoter controls the ability of HIV-1 to establish latent infection.

    PubMed

    Duverger, Alexandra; Wolschendorf, Frank; Zhang, Mingce; Wagner, Fredric; Hatcher, Brandon; Jones, Jennifer; Cron, Randall Q; van der Sluis, Renee M; Jeeninga, Rienk E; Berkhout, Ben; Kutsch, Olaf

    2013-02-01

    Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon. PMID:23236059

  7. An AP-1 Binding Site in the Enhancer/Core Element of the HIV-1 Promoter Controls the Ability of HIV-1 To Establish Latent Infection

    PubMed Central

    Duverger, Alexandra; Wolschendorf, Frank; Zhang, Mingce; Wagner, Fredric; Hatcher, Brandon; Jones, Jennifer; Cron, Randall Q.; van der Sluis, Renee M.; Jeeninga, Rienk E.; Berkhout, Ben

    2013-01-01

    Following integration, HIV-1 in most cases produces active infection events; however, in some rare instances, latent infection events are established. The latter have major clinical implications, as latent infection allows the virus to persist despite antiretroviral therapy. Both the cellular factors and the viral elements that potentially determine whether HIV-1 establishes active or latent infection events remain largely elusive. We detail here the contribution of different long terminal repeat (LTR) sequences for the establishment of latent HIV-1 infection. Using a panel of full-length replication-competent virus constructs that reflect naturally occurring differences of HIV-1 subtype-specific LTRs and targeted LTR mutants, we found the primary ability of HIV-1 to establish latent infection in this system to be controlled by a four-nucleotide (nt) AP-1 element just upstream of the NF-κB element in the viral promoter. Deletion of this AP-1 site mostly deprived HIV-1 of the ability to establish latent HIV-1 infection. Extension of this site to a 7-nt AP-1 sequence massively promoted latency establishment, suggesting that this promoter region represents a latency establishment element (LEE). Given that these minimal changes in a transcription factor binding site affect latency establishment to such large extent, our data support the notion that HIV-1 latency is a transcription factor restriction phenomenon. PMID:23236059

  8. Cyclin D1 transcriptional activation in MCL.

    PubMed

    Beà, Sílvia

    2014-03-27

    In this issue of Blood, Allinne et al propose the nucleolin-dependent activation of the translocated CCND1 allele in mantle cell lymphoma (MCL) because of its relocalization to a transcriptionally favorable area in the perinucleolar region. PMID:24677400

  9. Activation of the Arabidopsis B class homeotic genes by APETALA1.

    PubMed

    Ng, M; Yanofsky, M F

    2001-04-01

    Proper development of petals and stamens in Arabidopsis flowers requires the activities of APETALA3 (AP3) and PISTILLATA (PI), whose transcripts can be detected in the petal and stamen primordia. Localized expression of AP3 and PI requires the activities of at least three genes: APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO). It has been proposed that UFO provides spatial cues and that LFY specifies competence for AP3 and PI expression in the developing flower. To understand the epistatic relationship among AP1, LFY, and UFO in regulating AP3 and PI expression, we generated two versions of AP1 that have strong transcriptional activation potential. Genetic and molecular analyses of transgenic plants expressing these activated AP1 proteins show that the endogenous AP1 protein acts largely as a transcriptional activator in vivo and that AP1 specifies petals by regulating the spatial domains of AP3 and PI expression through UFO. PMID:11283333

  10. Chromatin insulation by a transcriptional activator

    PubMed Central

    Sutter, Nathan B.; Scalzo, David; Fiering, Steven; Groudine, Mark; Martin, David I. K.

    2003-01-01

    In eukaryotic genomes, transcriptionally active regions are interspersed with silent chromatin that may repress genes in its vicinity. Chromatin insulators are elements that can shield a locus from repressive effects of flanking chromatin. Few such elements have been characterized in higher eukaryotes, but transcriptional activating elements are an invariant feature of active loci and have been shown to suppress transgene silencing. Hence, we have assessed the ability of a transcriptional activator to cause chromatin insulation, i.e., to relieve position effects at transgene integration sites in cultured cells. The transgene contained a series of binding sites for the metal-inducible transcriptional activator MTF, linked to a GFP reporter. Clones carrying single integrated transgenes were derived without selection for expression, and in most clones the transgene was silent. Induction of MTF resulted in transition of the transgene from the silent to the active state, prolongation of the active state, and a marked narrowing of the range of expression levels at different genomic sites. At one genomic site, prolonged induction of MTF resulted in suppression of transgene silencing that persisted after withdrawal of the induction stimulus. These results are consistent with MTF acting as a chromatin insulator and imply that transcriptional activating elements can insulate active loci against chromatin repression. PMID:12547916

  11. Rhizoma Coptidis Inhibits LPS-Induced MCP-1/CCL2 Production in Murine Macrophages via an AP-1 and NFκB-Dependent Pathway

    PubMed Central

    Remppis, Andrew; Bea, Florian; Greten, Henry Johannes; Buttler, Annette; Wang, Hongjie; Zhou, Qianxing; Preusch, Michael R.; Enk, Ronny; Ehehalt, Robert; Katus, Hugo; Blessing, Erwin

    2010-01-01

    Introduction. The Chinese extract Rhizoma coptidis is well known for its anti-inflammatory, antioxidative, antiviral, and antimicrobial activity. The exact mechanisms of action are not fully understood. Methods. We examined the effect of the extract and its main compound, berberine, on LPS-induced inflammatory activity in a murine macrophage cell line. RAW 264.7 cells were stimulated with LPS and incubated with either Rhizoma coptidis extract or berberine. Activation of AP-1 and NFκB was analyzed in nuclear extracts, secretion of MCP-1/CCL2 was measured in supernatants. Results. Incubation with Rhizoma coptidis and berberine strongly inhibited LPS-induced monocyte chemoattractant protein (MCP)-1 production in RAW cells. Activation of the transcription factors AP-1 and NFκB was inhibited by Rhizoma coptidis in a dose- and time-dependent fashion. Conclusions. Rhizoma coptidis extract inhibits LPS-induced MCP-1/CCL2 production in vitro via an AP-1 and NFκB-dependent pathway. Anti-inflammatory action of the extract is mediated mainly by its alkaloid compound berberine. PMID:20652055

  12. Elk1 and AP-1 sites in the TBP promoter mediate alcohol-induced deregulation of Pol III-dependent genes

    PubMed Central

    Zhong, Qian; Shi, Ganggang; Zhang, Yanmei; Levy, Daniel; Zhong, Shuping

    2013-01-01

    The major risk factors for hepatocellular carcinoma (HCC) are chronic liver diseases that include hepatitis B, hepatitis C, alcoholic liver disease and non-alcoholic steatohepatitis. However, the mechanisms of alcohol-associated HCC remain to be elucidated. The products of RNA Pol III (RNA polymerase III) dependent genes are elevated in both transformation cells and tumor cells. TBP (TATA-box binding protein) is a central transcription factor, which regulates Pol I, Pol II and Pol III gene activity. Our studies have demonstrated that alcohol increases TBP expression and Pol III gene transcription to promote liver tumor formation. We continue to investigate how ethanol mediates TBP expression. Here, we report that ethanol induces TBP promoter activity and the induction is ethanol dose dependent. Blocking the JNK1 pathway by a chemical inhibitor and siRNA reduce this ethanol-induced activity. Furthermore, mutating G>A at a −46bp Elk1 binding site of the TBP promoter or mutating AP-1 binding site at −37bp (A>G) and −38bp (C>T) reduces the TBP promoter activity. Mutation of both Elk1 and AP-1 binding sites dramatically represses this induction. Together, these studies demonstrate that, for the first time, alcohol increases Pol III gene transcription through a response element, which is composed of the overlapping the Elk1 and AP-1 binding sites of the TBP promoter. It suggests that these binding sites may play a critical role in alcohol-induced deregulation of Pol III genes in liver tumor development. PMID:23454483

  13. Calcium-dependent Nr4a1 expression in mouse Leydig cells requires distinct AP1/CRE and MEF2 elements.

    PubMed

    Abdou, Houssein S; Robert, Nicholas M; Tremblay, Jacques J

    2016-04-01

    The nuclear receptor NR4A1 is expressed in steroidogenic Leydig cells where it plays pivotal roles by regulating the expression of several genes involved in steroidogenesis and male sex differentiation including Star, HSD3B2, and Insl3 Activation of the cAMP and Ca(2+) signaling pathways in response to LH stimulation leads to a rapid and robust activation of Nr4a1 gene expression that requires the Ca(2+)/CAMKI pathway. However, the downstream transcription factor(s) have yet to be characterized. To identify potential Ca(2+)/CaM effectors responsible for hormone-induced Nr4a1 expression, MA-10 Leydig cells were treated with forskolin to increase endogenous cAMP levels, dantrolene to inhibit endoplasmic reticulum Ca(2+) release, and W7 to inhibit CaM activity. We identified Ca(2+)-responsive elements located in the discrete regions of the Nr4a1 promoter, which contain binding sites for several transcription factors such as AP1, CREB, and MEF2. We found that one of the three AP1/CRE sites located at -255 bp is the most responsive to the Ca(2+) signaling pathway as are the two MEF2 binding sites at -315 and -285 bp. Furthermore, we found that the hormone-induced recruitment of phospho-CREB and of the co-activator p300 to the Nr4a1 promoter requires the Ca(2+) pathway. Lastly, siRNA-mediated knockdown of CREB impaired NR4A1 expression and steroidogenesis. Together, our data indicate that the Ca(2+) signaling pathway increases Nr4a1 expression in MA-10 Leydig cells, at least in part, by enhancing the recruitment of coactivator most likely through the MEF2, AP1, and CREB transcription factors thus demonstrating an important interplay between the Ca(2+) and cAMP pathways in regulating Nr4a1 expression. PMID:26647388

  14. Baicalin induces NAD(P)H:quinone reductase through the transactivation of AP-1 and NF-kappaB in Hepa 1c1c7 cells.

    PubMed

    Park, H J; Lee, Y W; Lee, S K

    2004-12-01

    Baicalin (5,6,7-trihydroxyflavone-7-O-D-glucuronic acid, BA) is a flavone isolated from Scutellariae radix. In our previous report BA was a major active principle of NAD(P)H:quinone reductase (QR) induction mediated by Scutellariae radix extract and the induction was related to the transcriptional activation of the QR gene in Hepa 1c1c7 cells. The primary aim of the present study was to determine the molecular mechanism of QR gene expression by baicalin. The antioxidant or electrophile response element (ARE/EpRE) found at the 5'-flanking region of phase II genes may play an important role in mediating their induction by xenobiotics, including chemopreventive agents. In accordance, to study the molecular mechanisms of QR gene expression by BA, electrophoretic mobility shift assay (EMSA), using nuclear extracts of treated and untreated cells against ARE, activator protein-1 (AP-1) or nuclear factor-kappaB (NF-kappaB) binding sites, showed that BA increased the binding levels of the parameters in a dose-dependent manner. Further, Hepa 1c1c7 cells were transiently transfected with a plasmid containing three copies of the AP-1- or NF-kappaB-binding site linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Using the CAT reporter gene assay, a dose-dependent transactivation of AP-1- or NF-kappaB-mediated CAT expression was observed with the treatment of BA. These results clearly indicate that BA induces the QR gene expression and activity by transactivation of AP-1 and NF-kappaB, and thus BA may be considered as a potential cancer chemopreventive agent with the induction of phase II detoxification enzyme. PMID:15548947

  15. MEKK1-MKK4-JNK-AP1 Pathway Negatively Regulates Rgs4 Expression in Colonic Smooth Muscle Cells

    PubMed Central

    Zhang, Yonggang; Li, Fang; Liu, Shu; Wang, Hong; Mahavadi, Sunila; Murthy, Karnam S.; Khalili, Kamel; Hu, Wenhui

    2012-01-01

    Background Regulator of G-protein Signaling 4 (RGS4) plays an important role in regulating smooth muscle contraction, cardiac development, neural plasticity and psychiatric disorder. However, the underlying regulatory mechanisms remain elusive. Our recent studies have shown that upregulation of Rgs4 by interleukin (IL)-1β is mediated by the activation of NFκB signaling and modulated by extracellular signal-regulated kinases, p38 mitogen-activated protein kinase, and phosphoinositide-3 kinase. Here we investigate the effect of the c-Jun N-terminal kinase (JNK) pathway on Rgs4 expression in rabbit colonic smooth muscle cells. Methodology/Principal Findings Cultured cells at first passage were treated with or without IL-1β (10 ng/ml) in the presence or absence of the selective JNK inhibitor (SP600125) or JNK small hairpin RNA (shRNA). The expression levels of Rgs4 mRNA and protein were determined by real-time RT-PCR and Western blot respectively. SP600125 or JNK shRNA increased Rgs4 expression in the absence or presence of IL-1β stimulation. Overexpression of MEKK1, the key upstream kinase of JNK, inhibited Rgs4 expression, which was reversed by co-expression of JNK shRNA or dominant-negative mutants for MKK4 or JNK. Both constitutive and inducible upregulation of Rgs4 expression by SP600125 was significantly inhibited by pretreatment with the transcription inhibitor, actinomycin D. Dual reporter assay showed that pretreatment with SP600125 sensitized the promoter activity of Rgs4 in response to IL-1β. Mutation of the AP1-binding site within Rgs4 promoter increased the promoter activity. Western blot analysis confirmed that IL-1β treatment increased the phosphorylation of JNK, ATF-2 and c-Jun. Gel shift and chromatin immunoprecipitation assays validated that IL-1β increased the in vitro and ex vivo binding activities of AP1 within rabbit Rgs4 promoter. Conclusion/Significance Activation of MEKK1-MKK4-JNK-AP1 signal pathway plays a tonic inhibitory role in

  16. Guanylyl cyclase/natriuretic peptide receptor-A signaling antagonizes the vascular endothelial growth factor-stimulated MAPKs and downstream effectors AP-1 and CREB in mouse mesangial cells

    PubMed Central

    Tripathi, Satyabha; Pandey, Kailash N.

    2012-01-01

    Along with its natriuretic, diuretic, and vasodilatory properties, atrial natriuretic peptide (ANP) and its guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) exhibit an inhibitory effect on cell growth and proliferation. However, the signaling pathways mediating this inhibition are not well understood. The objective of this study was to determine the effect of ANP-NPRA system on mitogen-activated protein kinases (MAPKs) and the downstream proliferative transcription factors involving activating protein-1 (AP-1) and cAMP-response element binding protein (CREB) in agonist-stimulated mouse mesangial cells (MMCs). We found that ANP inhibited vascular endothelial growth factor (VEGF)-stimulated phosphorylation of MAPKs (Erk1, Erk2, JNK, and p38), to a greater extent in NPRA-transfected cells (50–60%) relative to vector-transfected cells (25–30%). The analyses of the phosphorylated transcription factors revealed that ANP inhibited VEGF-stimulated activation of CREB, and the AP-1 subunits (c-jun and c-fos). Gel shift assays demonstrated that ANP inhibited VEGF-stimulated AP-1 and CREB DNA-binding ability by 67 % and 62 %, respectively. The addition of the protein kinase G (PKG) inhibitor, KT-5823, restored the VEGF-stimulated activation of MAPKs, AP-1, and CREB, demonstrating the integral role of cGMP/PKG signaling in NPRA-mediated effects. Our results delineate the under lying mechanisms through which ANP-NPRA system exerts an inhibitory effect on MAPKs and down-stream effector molecules, AP-1 and CREB, critical for cell growth and proliferation. PMID:22610792

  17. Effects of Cigarette Smoke on the Activation of Oxidative Stress-Related Transcription Factors in Female A/J Mouse Lung

    PubMed Central

    Tharappel, Job C.; Cholewa, Jill; Espandiari, Parvaneh; Spear, Brett T.; Gairola, C. Gary; Glauert, Howard P.

    2010-01-01

    Cigarette smoke contains a high concentration of free radicals and induces oxidative stress in the lung and other tissues. Several transcription factors are known to be activated by oxidative stress, including nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and hypoxia-inducible factor (HIF). Studies were therefore undertaken to examine if cigarette smoke could activate these transcription factors, as well as other transcription factors that may be important in lung carcinogenesis. Female A/J mice were exposed to cigarette smoke for 2, 5, 10, 15, 20, 42, or 56 days (6 hr/day, 5 days/wk). Cigarette smoke did not increase NF-κB activation at any of these times, but NF-κB DNA binding activity was lower after 15 days and 56 days of smoke exposure. The DNA binding activity of AP-1 was lower after 10 days and 56 days but was not changed after 42 days of smoke exposure. The DNA binding activity of HIF was quantitatively increased after 42 days of smoke exposure but decreased after 56 days. Whether the activation of other transcription factors in the lung could be altered after exposure to cigarette smoke was subsequently examined. The DNA binding activities of FoxF2, myc-CF1, RORE, and p53 were examined after 10 days of smoke exposure. The DNA binding activities of FoxF2 and p53 were quantitatively increased, but those of myc-CF1 and RORE were unaffected. These studies show that cigarette smoke exposure leads to quantitative increases in DNA binding activities of FoxF2 and p53, while the activations of NF-κB, AP-1, and HIF are largely unaffected or reduced. PMID:20711931

  18. Divergent transcriptional activities determine limb identity

    PubMed Central

    Ouimette, Jean-François; Jolin, Marisol Lavertu; L'honoré, Aurore; Gifuni, Anthony; Drouin, Jacques

    2010-01-01

    Limbs develop using a common genetic programme despite widely differing morphologies. This programme is modulated by limb-restricted regulators such as hindlimb (HL) transcription factors Pitx1 and Tbx4 and the forelimb (FL) Tbx5. Both Tbx factors have been implicated in limb patterning and growth, but their relative activities and underlying mechanisms remain unclear. In this paper, we show that Tbx4 and Tbx5 harbour conserved and divergent transcriptional regulatory domains that account for their roles in limb development. In particular, both factors share an activator domain and the ability to stimulate limb growth. However, we find that Tbx4 is the primary effector of HL identity for both skeletal and muscle development; this activity relies on a repressor domain that is inactivated by a human TBX4 small-patella syndrome mutation. We propose that limb identity is largely achieved by default in FL, whereas a specific repressor activity unique to Tbx4 determines HL identity. PMID:20975709

  19. Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFβ expression.

    PubMed

    Bakiri, L; Macho-Maschler, S; Custic, I; Niemiec, J; Guío-Carrión, A; Hasenfuss, S C; Eger, A; Müller, M; Beug, H; Wagner, E F

    2015-02-01

    Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes

  20. Fra-1/AP-1 induces EMT in mammary epithelial cells by modulating Zeb1/2 and TGFβ expression

    PubMed Central

    Bakiri, L; Macho-Maschler, S; Custic, I; Niemiec, J; Guío-Carrión, A; Hasenfuss, S C; Eger, A; Müller, M; Beug, H; Wagner, E F

    2015-01-01

    Epithelial-to-mesenchymal transition (EMT) is essential for embryonic morphogenesis and wound healing and critical for tumour cell invasion and dissemination. The AP-1 transcription factor Fra-1 has been implicated in tumorigenesis and in tumour-associated EMT in human breast cancer. We observed a significant inverse correlation between Fra-1 mRNA expression and distant-metastasis-free survival in a large cohort of breast cancer patients derived from multiple array data sets. This unique correlation among Fos genes prompted us to assess the evolutionary conservation between Fra-1 functions in EMT of human and mouse cells. Ectopic expression of Fra-1 in fully polarized, non-tumourigenic, mouse mammary epithelial EpH4 cells induced a mesenchymal phenotype, characterized by a loss of epithelial and gain of mesenchymal markers. Proliferation, motility and invasiveness were also increased in the resulting EpFra1 cells, and the cells were tumourigenic and efficiently colonized the lung upon transplantation. Molecular analyses revealed increased expression of Tgfβ1 and the EMT-inducing transcription factors Zeb1, Zeb2 and Slug. Mechanistically, Fra-1 binds to the tgfb1 and zeb2 promoters and to an evolutionarily conserved region in the first intron of zeb1. Furthermore, increased activity of a zeb2 promoter reporter was detected in EpFra1 cells and shown to depend on AP-1-binding sites. Inhibiting TGFβ signalling in EpFra1 cells moderately increased the expression of epithelial markers, whereas silencing of zeb1 or zeb2 restored the epithelial phenotype and decreased migration in vitro and tumorigenesis in vivo. Thus Fra-1 induces changes in the expression of genes encoding EMT-related transcription factors leading to the acquisition of mesenchymal, invasive and tumorigenic capacities by epithelial cells. This study defines a novel function of Fra-1/AP-1 in modulating tgfb1, zeb1 and zeb2 expression through direct binding to genomic regulatory regions, which establishes

  1. Involvement of Blimp-1 and AP-1 dysregulation in the 2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated suppression of the IgM response by B cells.

    PubMed

    Schneider, Dina; Manzan, Maria A; Yoo, Byung Sun; Crawford, Robert B; Kaminski, Norbert

    2009-04-01

    B cell differentiation and humoral immune responses are markedly suppressed by the persistent environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The suppression of humoral immune responses by TCDD occurs by direct actions on the B cell and involves activation of the aryl hydrocarbon receptor. Transcriptional regulation of paired box gene 5 (Pax5), an important regulator of B cell differentiation, is altered by TCDD in concordance with the suppression of B cell differentiation and humoral immunoglobulin M response. We hypothesized that TCDD treatment leads to dysregulation of Pax5 transcription by interfering with the basic B cell differentiation mechanisms and aimed to determine the effects of TCDD on upstream regulators of Pax5. A critical regulator of B cell differentiation, B lymphocyte-induced maturation protein-1 (Blimp-1) acts as a transcriptional repressor of Pax5. In lipopolysaccharide (LPS)-activated murine B cell lymphoma, CH12.LX, Blimp-1 messenger RNA, and DNA-binding activity within the Pax5 promoter were suppressed by TCDD. Furthermore, LPS activation of CH12.LX cells upregulated DNA-binding activity of activator protein 1 (AP-1) at three responsive element-like motifs within the Blimp-1 promoter. TCDD treatment of LPS-activated CH12.LX cells suppressed AP-1 binding to these motifs between 24 and 72 h, in concordance with the suppression of Blimp-1 by TCDD. A more comprehensive analysis at 72 h demonstrated that the suppression of AP-1 binding within the Blimp-1 promoter by TCDD was concentration dependent. In summary, our findings link the TCDD-mediated suppression of Blimp-1 through AP-1 to the dysregulation of Pax5, which ultimately leads to the suppression of B cell differentiation and humoral immune responses. PMID:19237549

  2. Activating transcription factor 2 in mesenchymal tumors.

    PubMed

    Endo, Makoto; Su, Le; Nielsen, Torsten O

    2014-02-01

    Activating transcription factor 2 (ATF2) is a member of activator protein 1 superfamily, which can heterodimerize with other transcription factors regulating cell differentiation and survival. ATF2 assembles into a complex with the synovial sarcoma translocation, chromosome 18 (SS18)-synovial sarcoma, X breakpoint (SSX) fusion oncoprotein, and the transducin-like enhancer of split 1 (TLE1) corepressor, driving oncogenesis in synovial sarcoma. The fusion oncoproteins in many other translocation-associated sarcomas incorporate transcription factors from the ATF/cAMP response element binding or E26 families, which potentially form heterodimers with ATF2 to regulate transcription. ATF2 may therefore play an important role in the oncogenesis of many mesenchymal tumors, but as yet, little is known about its protein expression in patient specimens. Herein we perform immunohistochemical analyses using a validated specific antibody for ATF2 expression and intracellular localization on a cohort of 594 malignant and 207 benign mesenchymal tumors representing 47 diagnostic entities. Melanoma served as a positive control for nuclear and cytoplasmic staining. High nuclear ATF2 expression was mainly observed in translocation-associated and/or spindle cell sarcomas including synovial sarcoma, desmoplastic small round cell tumor, endometrial stromal sarcoma, gastrointestinal stromal tumor, malignant peripheral nerve sheath tumor, and solitary fibrous tumor. Cytoplasmic ATF2 expression was less frequently seen than nuclear expression in malignant mesenchymal tumors. Benign mesenchymal tumors mostly showed much lower nuclear and cytoplasmic ATF2 expression. PMID:24289970

  3. Inhibition of Phosphatase Activity Follows Decline in Sulfatase Activity and Leads to Transcriptional Effects through Sustained Phosphorylation of Transcription Factor MITF

    PubMed Central

    Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K.

    2016-01-01

    Arylsulfatase B (B-acetylgalactosamine 4-sulfatase; ARSB) is the enzyme that removes 4-sulfate groups from the non-reducing end of the glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate. Decline in ARSB has been shown in malignant prostate, colonic, and mammary cells and tissues, and decline in ARSB leads to transcriptional events mediated by galectin-3 with AP-1 and Sp1. Increased mRNA expression of GPNMB (transmembrane glycoprotein NMB) in HepG2 cells and in hepatic tissue from ARSB-deficient mice followed decline in expression of ARSB and was mediated by the microphthalmia-associated transcription factor (MITF), but was unaffected by silencing galectin-3. Since GPNMB is increased in multiple malignancies, studies were performed to determine how decline in ARSB increased GPNMB expression. The mechanism by which decline in ARSB increased nuclear phospho-MITF was due to reduced activity of SHP2, a protein tyrosine phosphatase with Src homology (SH2) domains that regulates multiple cellular processes. SHP2 activity declined due to increased binding with chondroitin 4-sulfate when ARSB was reduced. When SHP2 activity was inhibited, phosphorylations of p38 mitogen-associated phosphokinase (MAPK) and of MITF increased, leading to GPNMB promoter activation. A dominant negative SHP2 construct, the SHP2 inhibitor PHSP1, and silencing of ARSB increased phospho-p38, nuclear MITF, and GPNMB. In contrast, constitutively active SHP2 and overexpression of ARSB inhibited GPNMB expression. The interaction between chondroitin 4-sulfate and SHP2 is a novel intersection between sulfation and phosphorylation, by which decline in ARSB and increased chondroitin 4-sulfation can inhibit SHP2, thereby regulating downstream tyrosine phosphorylations by sustained phosphorylations with associated activation of signaling and transcriptional events. PMID:27078017

  4. Expression and purification of recombinant human c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro

    PubMed Central

    Ferguson, Heather A.; Goodrich, James A.

    2001-01-01

    c-Fos and c-Jun are members of the AP-1 family of transcriptional activators that regulate the expression of genes during cell proliferation. To facilitate in vitro studies of mechanisms of transcriptional activation by c-Jun and c-Fos we developed a method for obtaining recombinant c-Fos/c-Jun that is highly active in DNA binding and transcriptional activation in vitro. Full-length human c-Fos and c-Jun were expressed in Escherichia coli. The expression of c-Fos was dependent on a helper plasmid that encodes rare ArgtRNAs. Both over-expressed c-Fos and c-Jun were recovered from inclusion bodies. A c-Fos/c-Jun complex was generated by co-renaturation and purified via a His-tag on the full-length human c-Fos. The resulting c-Fos/c-Jun bound DNA with high affinity and specificity, and activated transcription in a reconstituted human RNA polymerase II transcription system. The availability of active recombinant human c-Fos/c-Jun will allow future biochemical studies of these important transcriptional activators. PMID:11600717

  5. Tungsten Carbide-Cobalt Nanoparticles Induce Reactive Oxygen Species, AKT, ERK, AP-1, NF-κB, VEGF, and Angiogenesis.

    PubMed

    Liu, Ling-Zhi; Ding, Min; Zheng, Jenny Z; Zhu, Yingxue; Fenderson, Bruce A; Li, Bingyun; Yu, Jing J; Jiang, Bing-Hua

    2015-07-01

    Powder mixtures of tungsten carbide and metallic cobalt (WC-Co) are widely used in various products. Nanoparticles are engineered structures with at least one dimension of 100 nm or smaller. WC-Co is known to be associated with lung injury and diseases. Angiogenesis is a key process during vasculature, carcinogenesis, recovery of injury, and inflammatory diseases. However, the cellular effects of WC-Co nanoparticles on angiogenesis remain to be elucidated. In this study, we investigated angiogenic response and relative mechanisms after exposure to WC-Co nanoparticles. Our results showed that WC-Co nanoparticles at 5 μg/cm(2) induced ROS production which activated AKT and ERK1/2 signaling pathways in lung epithelial cells by reactive oxygen species (ROS) staining and immunoblotting; WC-Co treatment also increased transcriptional activation of AP-1, NF-κB, and VEGF by reporter assay. Further studies demonstrated that ROS are upstream molecules of AKT and ERK signaling pathways; the activation of AP-1, NF-κB, and VEGF was through ROS generation, AKT and ERK1/2 activation. In addition, WC-Co nanoparticles affected the cells to induce angiogenesis by chicken chorioallantoic membrane (CAM) assay. These results illustrate that exposure to WC-Co nanoparticles induces angiogenic response by activating ROS, AKT, and ERK1/2 signaling pathways and the downstream molecules and elucidate the potential molecular mechanisms during this process. This information may be useful for preventing potential damage from nanoparticle exposure in the future. PMID:25893364

  6. Butyrate produced by commensal bacteria potentiates phorbol esters induced AP-1 response in human intestinal epithelial cells.

    PubMed

    Nepelska, Malgorzata; Cultrone, Antonietta; Béguet-Crespel, Fabienne; Le Roux, Karine; Doré, Joël; Arulampalam, Vermulugesan; Blottière, Hervé M

    2012-01-01

    The human intestine is a balanced ecosystem well suited for bacterial survival, colonization and growth, which has evolved to be beneficial both for the host and the commensal bacteria. Here, we investigated the effect of bacterial metabolites produced by commensal bacteria on AP-1 signaling pathway, which has a plethora of effects on host physiology. Using intestinal epithelial cell lines, HT-29 and Caco-2, stably transfected with AP-1-dependent luciferase reporter gene, we tested the effect of culture supernatant from 49 commensal strains. We observed that several bacteria were able to activate the AP-1 pathway and this was correlated to the amount of short chain fatty acids (SCFAs) produced. Besides being a major source of energy for epithelial cells, SCFAs have been shown to regulate several signaling pathways in these cells. We show that propionate and butyrate are potent activators of the AP-1 pathway, butyrate being the more efficient of the two. We also observed a strong synergistic activation of AP-1 pathway when using butyrate with PMA, a PKC activator. Moreover, butyrate enhanced the PMA-induced expression of c-fos and ERK1/2 phosphorylation, but not p38 and JNK. In conclusion, we showed that SCFAs especially butyrate regulate the AP-1 signaling pathway, a feature that may contribute to the physiological impact of the gut microbiota on the host. Our results provide support for the involvement of butyrate in modulating the action of PKC in colon cancer cells. PMID:23300800

  7. Dual role for mitogen-activated protein kinase (Erk) in insulin-dependent regulation of Fra-1 (fos-related antigen-1) transcription and phosphorylation.

    PubMed Central

    Hurd, Toby W; Culbert, Ainsley A; Webster, Kenneth J; Tavaré, Jeremy M

    2002-01-01

    Insulin regulates the activity of the AP-1 (activator protein-1) transcriptional complex in several cell types. One component of the AP-1 complex is the transcription factor Fra-1 (fos-related antigen-1), and we have demonstrated previously that insulin stimulates the expression of Fra-1 mRNA in CHO.T cells [Griffiths, Black, Culbert, Dickens, Shaw, Gillespie and Tavaré (1998) Biochem. J. 335, 19-26]. Here we demonstrate that insulin stimulates the activity of a fra-1 promoter linked to a luciferase reporter gene, indicating that the ability of insulin to induce expression of Fra-1 mRNA is due, at least in part, to an increase in gene transcription. Furthermore, we found that insulin induces the serine phosphorylation of Fra-1 and reduces its mobility during SDS/PAGE as a result of phosphorylation. The ability of insulin to induce the accumulation of Fra-1 mRNA, stimulate the fra-1 promoter and stimulate phosphorylation of Fra-1 all require the mitogen-activated protein (MAP) kinase cascade, which leads to the activation of extracellular-signal-regulated kinase (Erk) 1/2. Consequently, our results demonstrate that the Erk cascade plays a dual role in the co-ordinated regulation of the transcription and the phosphorylation of Fra-1 by insulin. PMID:12197835

  8. P4-ATPase Requirement for AP-1/Clathrin Function in Protein Transport from the trans-Golgi Network and Early Endosomes

    PubMed Central

    Liu, Ke; Surendhran, Kavitha; Nothwehr, Steven F.

    2008-01-01

    Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Δ cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN. PMID:18508916

  9. Radiolabeled anti-tissue factor antibody (AP-1) for imaging thrombotic disease by PET

    SciTech Connect

    Joshi, V.; Meinken, G.; Srivastava, S.

    1995-05-01

    The objective of this study was to develop and test radioimmunoconjugates of AP-1, an anti-tissue factor (TF) MAb, for PET imaging of vessel wall injury or associated thrombotic disease. Recently, anti rabbit MAb AP-1 was shown to prevent thrombosis following vascular injury in a rabbit model. In the represent study of AP-1 was conjugated with the conventional DTPA dianhydride (DTPA-DA) and with 4-isothiocyanato-cyclohexyl-EDTA (4-ICE) (2 to 2.5 ligands per MAb). Labeling with {sup 57}Co was done by adding {sup 57}CoCl{sub 2} in 0.1 N HCl to 500 {mu}g of conjugate in 0.1 M NaHCO{sub 3} containing 0.12 M acetate. The reaction mixture (pH {approximately} 5.5) was allowed to stand at room temperature for 8 h, and then purified by size exclusion HPLC following EDTA chase (10 {mu}l of 0.1 M EDTA, pH 7.0, 10 min). Labeling efficiencies were >90%. When incubated with mouse serum these conjugates showed similar stability ({approximately}3% activity loss for 4-ICE vs 6% for DTPA-DA at 24 h). The inhibition of tissue factor procoagulant activity was determined for the {sup 67}Co labeled conjugates using a two stage clotting assay. In the first stage, clotting times were determined using serial dilutions of reconstituted TF standards to check linearity. In the second stage, clotting times were determined for {sup 67}Co labeled AP-1 conjugates at various dilutions (1 ng to 1 {mu}g/mL) in presence of 150 ng/ml of TF. Results were compared with those obtained using unlabeled conjugates and the native AP-1. Neither conjugation with chelators nor radiolabelling affected the TF activity of AP-1. These conjugates labeled with {sup 66}Co (t l/1 17.5 h, {beta}{sup +} emission) should prove effective for PET imaging of vessel wall injury or thrombotic disease in our previously established rabbit model. Based on our previous data with other MAbs, the 4-ICE conjugate is expected to provide better biodistribution.

  10. The transcriptional corepressor DSP1 inhibits activated transcription by disrupting TFIIA-TBP complex formation.

    PubMed Central

    Kirov, N C; Lieberman, P M; Rushlow, C

    1996-01-01

    Transcriptional repression of eukaryotic genes is essential for many cellular and developmental processes, yet the precise mechanisms of repression remain poorly understood. The Dorsal Switch Protein (DSP1) was identified in a genetic screen for activities which convert Dorsal into a transcriptional repressor. DSP1 shares structural homology with the HMG-1/2 family and inhibits activation by the rel transcription factors Dorsal and NF-kappaB in transfection studies. Here we investigate the mechanism of transcriptional repression by DSP1. We found that DSP1 protein can act as a potent transcriptional repressor for multiple activator families in vitro and in transfection studies. DSP1 bound directly to the TATA binding protein (TBP), and formed a stable ternary complex with TBP bound to DNA. DSP1 preferentially disrupted the DNA binding of TBP complexes containing TFIIA and displaced TFIIA from binding to TBP. Consistent with the inhibition of TFIIA-bound complexes, DSP1 was shown to inhibit activated but not basal transcription reactions in vitro. The ability of DSP1 to interact with TBP and to repress transcription was mapped to the carboxy-terminal domain which contains two HMG boxes. Our results support the model that DSP1 represses activated transcription by interfering with the binding of TFIIA, a general transcription factor implicated in activated transcription pathways. Images PMID:9003783

  11. Methylation status and AP1 elements are involved in EBV-mediated miR-155 expression in EBV positive lymphoma cells.

    PubMed

    Yin, Qinyan; Wang, Xia; Roberts, Claire; Flemington, Erik K; Lasky, Joseph A

    2016-07-01

    The relationship between Epstein Barr Virus (EBV) and miR-155 is well established. EBV infection induces miR-155 expression, which is expressed at higher levels in EBV latency type III cells compared to EBV latency type I cells. However, the mechanism by which EBV latency genes activate miR-155 expression is still unclear. Here we present data showing that DNA methylation regulates miR-155 expression. We also provide evidence that the AP1 signaling pathway is involved in EBV-mediated miR-155 activation, and that Bay11 influences signaling of the miR-155 promoter AP1 element. Lastly, we show that LMP2A, LMP1 and EBNAs cannot activate miR-155 expression alone, indicating that the regulation of miR-155 by EBV is dependent on more than one EBV gene or cell signaling pathway. We conclude that the regulation of miR-155 in EBV-positive cells occurs through multiple cell signaling processes involving EBV-mediated chromatin remodeling, cell signaling regulation and transcription factor activation. PMID:27110708

  12. Pterostilbene Is Equally Potent as Resveratrol in Inhibiting 12-O-tetradecanoylphorbol-13-acetate Activated NFkappaB, AP-1, COX-2 and iNOS in Mouse Epidermis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Resveratrol, a phytoalexin present in grapes, has been reported to inhibit multistage mouse skin carcinogenesis. Recent studies showed that topically applied resveratrol significantly inhibited cyclooxygenase-2 (COX-2) expression and activation of nuclear factor-kB (NF-kB) induced by tumor promoter...

  13. SARS Coronavirus 3b Accessory Protein Modulates Transcriptional Activity of RUNX1b

    PubMed Central

    Varshney, Bhavna; Agnihotram, Sudhakar; Tan, Yee-Joo; Baric, Ralph; Lal, Sunil K.

    2012-01-01

    Background The causative agent of severe acute respiratory syndrome, SARS coronavirus (SARS-CoV) genome encodes several unique group specific accessory proteins with unknown functions. Among them, accessory protein 3b (also known as ORF4) was lately identified as one of the viral interferon antagonist. Recently our lab uncovered a new role for 3b in upregulation of AP-1 transcriptional activity and its downstream genes. Thus, we believe that 3b might play an important role in SARS-CoV pathogenesis and therefore is of considerable interest. The current study aims at identifying novel host cellular interactors of the 3b protein. Methodology/Principal Findings In this study, using yeast two-hybrid and co-immunoprecipitation techniques, we have identified a host transcription factor RUNX1b (Runt related transcription factor, isoform b) as a novel interacting partner for SARS-CoV 3b protein. Chromatin immunoprecipitaion (ChIP) and reporter gene assays in 3b expressing jurkat cells showed recruitment of 3b on the RUNX1 binding element that led to an increase in RUNX1b transactivation potential on the IL2 promoter. Kinase assay and pharmacological inhibitor treatment implied that 3b also affect RUNX1b transcriptional activity by regulating its ERK dependent phosphorylation levels. Additionally, mRNA levels of MIP-1α, a RUNX1b target gene upregulated in SARS-CoV infected monocyte-derived dendritic cells, were found to be elevated in 3b expressing U937 monocyte cells. Conclusions/Significance These results unveil a novel interaction of SARS-CoV 3b with the host factor, RUNX1b, and speculate its physiological relevance in upregulating cytokines and chemokine levels in state of SARS virus infection. PMID:22253733

  14. A Central Role for JNK/AP-1 Pathway in the Pro-Oxidant Effect of Pyrrolidine Dithiocarbamate through Superoxide Dismutase 1 Gene Repression and Reactive Oxygen Species Generation in Hematopoietic Human Cancer Cell Line U937

    PubMed Central

    Collin, Pascal; Lomri, Abderrahim

    2015-01-01

    Pyrrolidine dithiocarbamate (PDTC) known as antioxidant and specific inhibitor of NF-κB was also described as pro-oxidant by inducing cell death and reactive oxygen species (ROS) accumulation in cancer. However, the mechanism by which PDTC indices its pro-oxidant effect is unknown. Therefore, we aimed to evaluate the effect of PDTC on the human Cu/Zn superoxide dismutase 1 (SOD1) gene transcription in hematopoietic human cancer cell line U937. We herein show for the first time that PDTC decreases SOD1 transcripts, protein and promoter activity. Furthermore, SOD1 repression by PDTC was associated with an increase in oxidative stress as evidenced by ROS production. Electrophoretic mobility-shift assays (EMSA) show that PDTC increased binding of activating protein-1 (AP-1) in dose dependent-manner suggesting that the MAPkinase up-stream of AP-1 is involved. Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription. In contrast, in the presence of JNK inhibitor (SP600125), p65 induced a marked increase of SOD1 promoter, suggesting that JNK pathway is up-stream of NF-κB signaling and controls negatively its activity. Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production. Finally, PDTC represses SOD1 in U937 cells through JNK/c-Jun phosphorylation. Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation. PMID:25996379

  15. A position-dependent transcription-activating domain in TFIIIA.

    PubMed

    Mao, X; Darby, M K

    1993-12-01

    Transcription of the Xenopus 5S RNA gene by RNA polymerase III requires the gene-specific factor TFIIIA. To identify domains within TFIIIA that are essential for transcriptional activation, we have expressed C-terminal deletion, substitution, and insertion mutants of TFIIIA in bacteria as fusions with maltose-binding protein (MBP). The MBP-TFIIIA fusion protein specifically binds to the 5S RNA gene internal control region and complements transcription in a TFIIIA-depleted oocyte nuclear extract. Random, cassette-mediated mutagenesis of the carboxyl region of TFIIIA, which is not required for promoter binding, has defined a 14-amino-acid region that is critical for transcriptional activation. In contrast to activators of RNA polymerase II, the activity of the TFIIIA activation domain is strikingly sensitive to its position relative to the DNA-binding domain. When the eight amino acids that separate the transcription-activating domain from the last zinc finger are deleted, transcriptional activity is lost. Surprisingly, diverse amino acids can replace these eight amino acids with restoration of full transcriptional activity, suggesting that the length and not the sequence of this region is important. Insertion of amino acids between the zinc finger region and the transcription-activating domain causes a reduction in transcription proportional to the number of amino acids introduced. We propose that to function, the transcription-activating domain of TFIIIA must be correctly positioned at a minimum distance from the DNA-binding domain. PMID:8246967

  16. Induction of the Gene Encoding Macrophage Chemoattractant Protein 1 by Orientia tsutsugamushi in Human Endothelial Cells Involves Activation of Transcription Factor Activator Protein 1

    PubMed Central

    Cho, Nam-Hyuk; Seong, Seung-Yong; Huh, Myung-Sook; Kim, Na-Hyun; Choi, Myung-sik; Kim, Ik-sang

    2002-01-01

    Human macrophage chemoattractant protein 1 (MCP-1) is a potent mediator of macrophage migration and therefore plays an essential role in early events of inflammation. In endothelial cells, at least three independent pathways regulate MCP-1 expression by NF-κB and AP-1. Orientia tsutsugamushi causes vasculitis in humans by replicating inside macrophages and endothelial cells. In the present study, we investigated the cis-acting and trans-acting elements involved in O. tsutsugamushi-induced MCP-1 gene expression in human umbilical vein endothelial cells (HUVEC). Although NF-κB activation was observed in HUVEC infected with O. tsutsugamushi, inhibition of NF-κB activation did not affect the MCP-1 expression. However, treatment of HUVEC with extracellular signal-regulated kinase (ERK) kinase inhibitor or p38 mitogen-activated protein kinase (MAPK) inhibitor suppressed expression of MCP-1 mRNA concomitant with downregulation of activator protein 1 (AP-1) activation. Deletion of triphorbol acetate response elements (TRE) at position −69 to −63 of MCP-1 gene abolished inducible promoter activity. Deletion of TRE at position −69 to −63−96 to −90 or deletion of NF-κB-binding site at position −69 to −63−88 to −79 did not affect the inducibility of promoter. Site-directed mutagenesis of the NF-κB binding sites at positions −2640 to −2632, −2612 to −2603 in the enhancer region, or the AP-1 biding site at position −2276 to −2270 decreased the inducible activity of the promoter. Taken together, AP-1 activation by both the ERK pathway and the p38 MAPK pathway as well as their binding to TRE at position −69 to −63 in proximal promoter and TRE at position −2276 to −2270 in enhancer region is altogether essential in induction of MCP-1 mRNA in HUVEC infected with O. tsutsugamushi. Although NF-κB activation is not essential per se, the κB site in the enhancer region is important in MCP-1 induction of HUVEC. This discrepancy in the

  17. Bone development and inflammatory disease is regulated by AP-1 (Fos/Jun).

    PubMed

    Wagner, E F

    2010-01-01

    The Fos and Jun proteins are members of the AP-1 transcription factor complex, which is a central regulator for many cellular functions. This paper summarises the important functions of Fos proteins in bone development, with special emphasis on the Fos-related proteins Fra-1 and Fra-2. These factors determine the functions of osteoblasts and osteoclasts and regulate cytokine signalling during bone development. Likewise, the Jun proteins control the expression of cytokines and chemokines and are probably causally involved in inflammatory skin diseases such as psoriasis. Investigations into the molecular mechanisms responsible for skin inflammation have revealed that Jun proteins control cytokine expression, such as granulocyte colony-stimulating factor, IL-6 and tumour necrosis factor alpha by transcriptional and posttranscriptional pathways. Finally, the paper discusses the relevance of the Jun-dependent mouse model for psoriasis for preclinical studies in the field of anti-angiogenic therapies. PMID:19995753

  18. AP-1-mediated invasion requires increased expression of the hyaluronan receptor CD44.

    PubMed Central

    Lamb, R F; Hennigan, R F; Turnbull, K; Katsanakis, K D; MacKenzie, E D; Birnie, G D; Ozanne, B W

    1997-01-01

    Fibroblasts transformed by Fos oncogenes display increased expression of a number of genes implicated in tumor cell invasion and metastasis. In contrast to normal 208F rat fibroblasts, Fos-transformed 208F fibroblasts are growth factor independent for invasion. We demonstrate that invasion of v-Fos- or epidermal growth factor (EGF)-transformed cells requires AP-1 activity. v-Fos-transformed cell invasion is inhibited by c-jun antisense oligonucleotides and by expression of a c-jun dominant negative mutant, TAM-67. EGF-induced invasion is inhibited by both c-fos and c-jun antisense oligonucleotides. CD44s, the standard form of a transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. We demonstrate that increased expression of CD44 in Fos- and EGF-transformed cells is dependent upon AP-1. CD44 antisense oligonucleotides reduce expression of CD44 in v-Fos- or EGF-transformed cells and inhibit invasion but not migration. Expression of a fusion protein between human CD44s and Aequorea victoria green fluorescent protein (GFP) in 208F cells complements the inhibition of invasion by the rat-specific CD44 antisense oligonucleotide. We further show that both v-Fos and EGF transformations result in a concentration of endogenous CD44 or exogenous CD44-GFP at the ends of pseudopodial cell extensions. These results support the hypothesis that one role of AP-1 in transformation is to activate a multigenic invasion program. PMID:9001250

  19. Transcriptional activation of the human cytotoxic serine protease gene CSP-B in T lymphocytes.

    PubMed Central

    Hanson, R D; Ley, T J

    1990-01-01

    The cytotoxic serine protease B (CSP-B) gene is activated during cytotoxic T-lymphocyte maturation. In this report, we demonstrate that the PEER T-cell line (bearing gamma/delta T-cell receptors) accumulates CSP-B mRNA following exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA) and N6-2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (bt2cAMP) because of transcriptional activation of the CSP-B gene. TPA and bt2cAMP act synergistically to induce CSP-B expression, since neither agent alone causes activation of CSP-B transcription or mRNA accumulation. Chromatin upstream from the CSP-B gene is resistant to DNase I digestion in untreated PEER cells, but becomes sensitive following TPA-bt2cAMP treatment. Upon activation of PEER cells, a DNase I-hypersensitive site forms upstream from the CSP-B gene within a region that is highly conserved in the mouse. Transient transfection of CSP-B promoter constructs identified two regulatory regions in the CSP-B 5'-flanking sequence, located at positions -609 to -202 and positions -202 to -80. The region from -615 to -63 is sufficient to activate a heterologous promoter in activated PEER cells, but activation is orientation specific, suggesting that this region behaves as an upstream promoter element rather than a classical enhancer. Consensus AP-1, AP-2, and cAMP response elements are found upstream from the CSP-B gene (as are several T-cell-specific consensus elements), but the roles of these elements in CSP-B gene activation have yet to be determined. Images PMID:2233710

  20. Mechanisms of specificity in neuronal activity-regulated gene transcription

    PubMed Central

    Lyons, Michelle R.; West, Anne E.

    2011-01-01

    The brain is a highly adaptable organ that is capable of converting sensory information into changes in neuronal function. This plasticity allows behavior to be accommodated to the environment, providing an important evolutionary advantage. Neurons convert environmental stimuli into long-lasting changes in their physiology in part through the synaptic activity-regulated transcription of new gene products. Since the neurotransmitter-dependent regulation of Fos transcription was first discovered nearly 25 years ago, a wealth of studies have enriched our understanding of the molecular pathways that mediate activity-regulated changes in gene transcription. These findings show that a broad range of signaling pathways and transcriptional regulators can be engaged by neuronal activity to sculpt complex programs of stimulus-regulated gene transcription. However, the shear scope of the transcriptional pathways engaged by neuronal activity raises the question of how specificity in the nature of the transcriptional response is achieved in order to encode physiologically relevant responses to divergent stimuli. Here we summarize the general paradigms by which neuronal activity regulates transcription while focusing on the molecular mechanisms that confer differential stimulus-, cell-type-, and developmental-specificity upon activity-regulated programs of neuronal gene transcription. In addition, we preview some of the new technologies that will advance our future understanding of the mechanisms and consequences of activity-regulated gene transcription in the brain. PMID:21620929

  1. Constraints on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis.

    PubMed Central

    Almouzni, G; Wolffe, A P

    1995-01-01

    We have examined the cause of transcriptional quiescence prior to the mid-blastula transition (MBT) in Xenopus laevis. We have found distinct requirements for transcription of class II and class III genes. An artificial increase of the amount of DNA present within the embryo over that found at the MBT allows precocious transcription of tRNA genes, but not of the adenovirus E4 or human cytomegalovirus (CMV) promoters. Thus titration of an inhibitor by exogenous DNA determines class III but not class II gene activation. We demonstrate that the action of the inhibitor depends on the association of core histones with DNA. The addition of exogenous TBP, together with an increase in the amount of DNA within the embryo, allows significant basal transcription of class II genes prior to the MBT, whereas it does not increase transcription of tRNA genes. To examine the activation of transcription above basal levels, we used a defined minimal promoter containing five Gal4 binding sites and the activator Gal4-VP16. Precocious transcriptional activation is directed by Gal4-VP16 prior to the MBT, demonstrating that a functional transcriptional machinery exists at this early developmental stage. Furthermore, since this activation can occur in the absence of exogenous TBP or chromatin titration, a transcription factor that can penetrate chromatin is sufficient for recruitment of this machinery to a promoter. Our results support the hypothesis that the temporal regulation of transcription during early embryogenesis in Xenopus reflects not only a titration of inhibitors by DNA, but also a deficiency in the activity of transcriptional activators prior to the MBT. Images PMID:7737126

  2. TLR4-NOX4-AP-1 signaling mediates lipopolysaccharide-induced CXCR6 expression in human aortic smooth muscle cells

    SciTech Connect

    Patel, Devang N.; Bailey, Steven R.; Gresham, John K.; Schuchman, David B.; Shelhamer, James H.; Goldstein, Barry J.; Foxwell, Brian M.; Stemerman, Michael B.; Maranchie, Jodi K.; Valente, Anthony J.; Mummidi, Srinivas; Chandrasekar, Bysani . E-mail: chandraseka@uthscsa.edu

    2006-09-08

    CXCL16 is a transmembrane non-ELR CXC chemokine that signals via CXCR6 to induce aortic smooth muscle cell (ASMC) proliferation. While bacterial lipopolysaccharide (LPS) has been shown to stimulate CXCL16 expression in SMC, its effects on CXCR6 are not known. Here, we demonstrate that LPS upregulates CXCR6 mRNA, protein, and surface expression in human ASMC. Inhibition of TLR4 with neutralizing antibodies or specific siRNA interference blocked LPS-mediated CXCR6 expression. LPS stimulated both AP-1 (c-Fos, c-Jun) and NF-{kappa}B (p50 and p65) activation, but only inhibition of AP-1 attenuated LPS-induced CXCR6 expression. Using dominant negative expression vectors and siRNA interference, we demonstrate that LPS induces AP-1 activation via MyD88, TRAF6, ERK1/2, and JNK signaling pathways. Furthermore, the flavoprotein inhibitor diphenyleniodonium chloride significantly attenuated LPS-mediated AP-1-dependent CXCR6 expression, as did inhibition of NOX4 NADPH oxidase by siRNA. Finally, CXCR6 knockdown inhibited CXCL16-induced ASMC proliferation. These results demonstrate that LPS-TLR4-NOX4-AP-1 signaling can induce CXCR6 expression in ASMC, and suggest that the CXCL16-CXCR6 axis may be an important proinflammatory pathway in the pathogenesis of atherosclerosis.

  3. Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1

    PubMed Central

    2010-01-01

    Background Cinnamomum cassia bark is the outer skin of an evergreen tall tree belonging to the family Lauraceae containing several active components such as essential oils (cinnamic aldehyde and cinnamyl aldehyde), tannin, mucus and carbohydrate. They have various biological functions including anti-oxidant, anti-microbial, anti-inflammation, anti-diabetic and anti-tumor activity. Previously, we have reported that anti-cancer effect of cinnamon extracts is associated with modulation of angiogenesis and effector function of CD8+ T cells. In this study, we further identified that anti-tumor effect of cinnamon extracts is also link with enhanced pro-apoptotic activity by inhibiting the activities NFκB and AP1 in mouse melanoma model. Methods Water soluble cinnamon extract was obtained and quality of cinnamon extract was evaluated by HPLC (High Performance Liquid Chromatography) analysis. In this study, we tested anti-tumor activity and elucidated action mechanism of cinnamon extract using various types of tumor cell lines including lymphoma, melanoma, cervix cancer and colorectal cancer in vitro and in vivo mouse melanoma model. Results Cinnamon extract strongly inhibited tumor cell proliferation in vitro and induced active cell death of tumor cells by up-regulating pro-apoptotic molecules while inhibiting NFκB and AP1 activity and their target genes such as Bcl-2, BcL-xL and survivin. Oral administration of cinnamon extract in melanoma transplantation model significantly inhibited tumor growth with the same mechanism of action observed in vitro. Conclusion Our study suggests that anti-tumor effect of cinnamon extracts is directly linked with enhanced pro-apoptotic activity and inhibition of NFκB and AP1 activities and their target genes in vitro and in vivo mouse melanoma model. Hence, further elucidation of active components of cinnamon extract could lead to development of potent anti-tumor agent or complementary and alternative medicine for the treatment of

  4. DNA conformation driven by AP-1 triggers cell-specific expression via a strong epithelial enhancer.

    PubMed

    Virolle, T; Djabari, Z; Ortonne, J P; Aberdam, D

    2000-10-01

    We report here the characterization of the regulatory region of the human LAMA3 gene, coding for the alpha3A chain of laminin-5. A 202 bp fragment is sufficient to confer epithelial-specific expression to a thymidine kinase promoter through the cooperative effect of three AP-1 binding sites. Remarkably, removal of the sequences located between the AP-1 sites does not modify the promoter activity in keratinocytes but allows strong expression in fibroblasts. Replacement of the deleted sequences by non-homologous ones fully restores the restricted enhancement in keratinocytes. Functional analysis and mutagenesis experiments demonstrate that a minimal distance between the AP-1 sites is required for the enhancer DNA fragment to adopt a particular conformation driven by the binding of Jun-Fos heterodimers. In non-permissive cells, this conformation leads to the anchorage of non-DNA-binding fibroblastic cofactors to form an inhibitory ternary complex. Therefore, our results describe for the first time an unusual conformation-dependent epithelial-specific enhancer. PMID:11269498

  5. Mediator protein mutations that selectively abolish activated transcription.

    PubMed

    Myers, L C; Gustafsson, C M; Hayashibara, K C; Brown, P O; Kornberg, R D

    1999-01-01

    Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator. PMID:9874773

  6. The transcription factor NFAT promotes exhaustion of activated CD8⁺ T cells.

    PubMed

    Martinez, Gustavo J; Pereira, Renata M; Äijö, Tarmo; Kim, Edward Y; Marangoni, Francesco; Pipkin, Matthew E; Togher, Susan; Heissmeyer, Vigo; Zhang, Yi Chen; Crotty, Shane; Lamperti, Edward D; Ansel, K Mark; Mempel, Thorsten R; Lähdesmäki, Harri; Hogan, Patrick G; Rao, Anjana

    2015-02-17

    During persistent antigen stimulation, CD8(+) T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8(+) T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8(+) T cells and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8(+) T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1. PMID:25680272

  7. The Smad3 linker region contains a transcriptional activation domain.

    PubMed

    Wang, Guannan; Long, Jianyin; Matsuura, Isao; He, Dongming; Liu, Fang

    2005-02-15

    Transforming growth factor-beta (TGF-beta)/Smads regulate a wide variety of biological responses through transcriptional regulation of target genes. Smad3 plays a key role in TGF-beta/Smad-mediated transcriptional responses. Here, we show that the proline-rich linker region of Smad3 contains a transcriptional activation domain. When the linker region is fused to a heterologous DNA-binding domain, it activates transcription. We show that the linker region physically interacts with p300. The adenovirus E1a protein, which binds to p300, inhibits the transcriptional activity of the linker region, and overexpression of p300 can rescue the linker-mediated transcriptional activation. In contrast, an adenovirus E1a mutant, which cannot bind to p300, does not inhibit the linker-mediated transcription. The native Smad3 protein lacking the linker region is unable to mediate TGF-beta transcriptional activation responses, although it can be phosphorylated by the TGF-beta receptor at the C-terminal tail and has a significantly increased ability to form a heteromeric complex with Smad4. We show further that the linker region and the C-terminal domain of Smad3 synergize for transcriptional activation in the presence of TGF-beta. Thus our findings uncover an important function of the Smad3 linker region in Smad-mediated transcriptional control. PMID:15588252

  8. Anti-inflammatory effects of proanthocyanidin-rich red rice extract via suppression of MAPK, AP-1 and NF-κB pathways in Raw 264.7 macrophages

    PubMed Central

    Yodkeeree, Supachai; Pitchakarn, Pornsiri; Punfa, Wanisa

    2016-01-01

    BACKGROUND/OBJECTIVES Several pharmacological properties of red rice extract have been reported including anti-oxidant, anti-tumor, and reduced cancer cell invasion. This study was conducted to evaluate the anti-inflammatory effects of red rice extract on the production of inflammatory mediators in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages. MATERIALS/METHODS Pro-inflammatory cytokines including tumor necrosis factor-α and interleukin-6 were determined by ELISA and cyclooxygenase-2 and inducible nitric oxide synthase expression was evaluated using western blot analysis. In addition, the signaling pathway controlling the inflammatory cascade such as nuclear factor kappa B (NF-κB), activator proteins-1 (AP-1), and mitogen-activated protein kinase (MAPK) was determined. RESULTS Our results showed that red rice polar extract fraction (RR-P), but not non-polar extract fraction, inhibited interleukin-6, tumor necrosis factor-α, and nitric oxide production in LPS-induced Raw 264.7 cells. RR-P also reduced the expression of inflammatory enzymes, inducible nitric oxide synthase, and cyclooxygenase-2. In addition, activation of AP-1 and NF-κB transcription factor in the nucleus was abrogated by RR-P. RR-P inhibited the phosphorylation of extracellular signaling-regulated kinase 1/2, c-Jun NH2-terminal kinase, and p38 MAPK signaling responsible for the expression of inflammatory mediators in LPS-stimulated Raw 264.7 cells. Based on chemical analysis, high amounts of proanthocyanidin and catechins were detected in the RR-P fraction. However, only proanthocyanidin reduced NF-κB and AP-1 activation in LPS-activated Raw 264.7 cells. CONCLUSION These observations suggest that the anti-inflammatory properties of RR-P may stem from the inhibition of pro-inflammatory mediators via suppression of the AP-1, NF-κB, and MAPKs pathways. PMID:27247720

  9. RAD51AP2, a novel vertebrate- and meiotic-specific protein, sharesa conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    SciTech Connect

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-07-25

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1.

  10. Cooperative activation of Xenopus rhodopsin transcription by paired-like transcription factors

    PubMed Central

    2014-01-01

    Background In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking. Results We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element. Conclusions We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription. PMID:24499263

  11. Epidermal Growth Factor Receptor and PTEN Modulate Tissue Factor Expression in Glioblastoma through JunD/Activator Protein-1 Transcriptional Activity

    PubMed Central

    Rong, Yuan; Belozerov, Vladimir E.; Tucker-Burden, Carol; Chen, Gang; Durden, Donald L.; Olson, Jeffrey J.; Van Meir, Erwin G.; Mackman, Nigel; Brat, Daniel J.

    2009-01-01

    Hypoxia and necrosis are fundamental features of glioblastoma (GBM) and their emergence is critical for the rapid biological progression of this fatal tumor; yet, underlying mechanisms are poorly understood. We have suggested that vaso-occlusion following intravascular thrombosis could initiate or propagate hypoxia and necrosis in GBM. Tissue factor (TF), the main cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Epidermal growth factor receptor (EGFR) amplification and PTEN loss are two common genetic alterations seen in GBM but not in lower-grade astrocytomas that could be responsible for TF up-regulation. The most frequent EGFR mutation in GBM involves deletion of exons 2 to 7, resulting in the expression of a constitutively active receptor, EGFRvIII. Here, we show that overexpression of EGFR or EGFRvIII in human glioma cells causes increased basal TF expression and that stimulation of EGFR by its ligand, EGF, leads to a marked dose-dependent up-regulation of TF. In all cases, increased TF expression led to accelerated plasma coagulation in vitro. EGFR-mediated TF expression depended most strongly on activator protein-1 (AP-1) transcriptional activity and was associated with c-Jun NH2-terminal kinase (JNK) and JunD activation. Restoration of PTEN expression in PTEN-deficient GBM cells diminished EGFR-induced TF expression by inhibiting JunD/AP-1 transcriptional activity. PTEN mediated this effect by antagonizing phosphatidylinositol 3-kinase activity, which in turn attenuated both Akt and JNK activities. These mechanisms are likely at work in vivo, as EGFR expression was highly correlated with TF expression in human high-grade astrocytoma specimens. PMID:19276385

  12. The transcriptional activity of human Chromosome 22

    PubMed Central

    Rinn, John L.; Euskirchen, Ghia; Bertone, Paul; Martone, Rebecca; Luscombe, Nicholas M.; Hartman, Stephen; Harrison, Paul M.; Nelson, F. Kenneth; Miller, Perry; Gerstein, Mark; Weissman, Sherman; Snyder, Michael

    2003-01-01

    A DNA microarray representing nearly all of the unique sequences of human Chromosome 22 was constructed and used to measure global-transcriptional activity in placental poly(A)+ RNA. We found that many of the known, related and predicted genes are expressed. More importantly, our study reveals twice as many transcribed bases as have been reported previously. Many of the newly discovered expressed fragments were verified by RNA blot analysis and a novel technique called differential hybridization mapping (DHM). Interestingly, a significant fraction of these novel fragments are expressed antisense to previously annotated introns. The coding potential of these novel expressed regions is supported by their sequence conservation in the mouse genome. This study has greatly increased our understanding of the biological information encoded on a human chromosome. To facilitate the dissemination of these results to the scientific community, we have developed a comprehensive Web resource to present the findings of this study and other features of human Chromosome 22 at http://array.mbb.yale.edu/chr22. PMID:12600945

  13. Youngiasides A and C Isolated from Youngia denticulatum Inhibit UVB-Induced MMP Expression and Promote Type I Procollagen Production via Repression of MAPK/AP-1/NF-κB and Activation of AMPK/Nrf2 in HaCaT Cells and Human Dermal Fibroblasts.

    PubMed

    Kim, Myungsuk; Park, Young Gyun; Lee, Hee-Ju; Lim, Sue Ji; Nho, Chu Won

    2015-06-10

    This study investigated the effects of youngiaside A (YA), youngiaside C (YC), and Youngia denticulatum extract (YDE) on extrinsic aging and assessed its molecular mechanisms in UVB-irradiated HaCaT keratinocytes and human dermal fibroblasts (HDFs). The results showed that YA, YC, and YDE decreased matrix metalloproteinase (MMP) expression and production in HaCaT cell and HDFs and increased collagen expression and production in HDFs. In addition, YA, YC, and YDE significantly increased antioxidant enzyme expression, thereby down-regulating UVB-induced reactive oxygen species (ROS) production and ROS-induced mitogen-activated protein kinase (MAPK) and activator protein-1 (AP-1) signaling in HaCaT cells. Furthermore, YA, YC, and YDE reduced phosphorylation of IκBα and IKKα/β, blocked nuclear factor-κB (NF-κB) p65 nuclear translocation, and strongly suppressed pro-inflammatory mediators. Finally, YA, YC, and YDE augmented UVB-induced adenosine monophosphate activated protein kinase (AMPK) phosphorylation and YA and YC did not inhibit MMP-1 production in AMPK inhibitor or nuclear factor-erythroid 2-related factor-2 (Nrf2) siRNA-treated HaCaT cells. The results suggest that these compounds could be potential therapeutic agents for prevention and treatment of skin photoaging. PMID:25994852

  14. Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2

    PubMed Central

    Wurm, Stefanie; Zhang, Jisheng; Guinea-Viniegra, Juan; García, Fernando; Muñoz, Javier; Bakiri, Latifa; Ezhkova, Elena

    2015-01-01

    Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. PMID:25547114

  15. Terminal epidermal differentiation is regulated by the interaction of Fra-2/AP-1 with Ezh2 and ERK1/2.

    PubMed

    Wurm, Stefanie; Zhang, Jisheng; Guinea-Viniegra, Juan; García, Fernando; Muñoz, Javier; Bakiri, Latifa; Ezhkova, Elena; Wagner, Erwin F

    2015-01-15

    Altered epidermal differentiation characterizes numerous skin diseases affecting >25% of the human population. Here we identified Fra-2/AP-1 as a key regulator of terminal epidermal differentiation. Epithelial-restricted, ectopic expression of Fra-2 induced expression of epidermal differentiation genes located within the epidermal differentiation complex (EDC). Moreover, in a papilloma-prone background, a reduced tumor burden was observed due to precocious keratinocyte differentiation by Fra-2 expression. Importantly, loss of Fra-2 in suprabasal keratinocytes is sufficient to cause skin barrier defects due to reduced expression of differentiation genes. Mechanistically, Fra-2 binds and transcriptionally regulates EDC gene promoters, which are co-occupied by the transcriptional repressor Ezh2. Fra-2 remains transcriptionally inactive in nondifferentiated keratinocytes, where it was found monomethylated and dimethylated on Lys104 and interacted with Ezh2. Upon keratinocyte differentiation, Fra-2 is C-terminally phosphorylated on Ser320 and Thr322 by ERK1/2, leading to transcriptional activation. Thus, the induction of epidermal differentiation by Fra-2 is controlled by a dual mechanism involving Ezh2-dependent methylation and activation by ERK1/2-dependent phosphorylation. PMID:25547114

  16. Functional analysis of differences in transcriptional activity conferred by genetic variants in the 5' flanking region of the IL12RB2 gene.

    PubMed

    Kato-Kogoe, Nahoko; Ohyama, Hideki; Okano, Soichiro; Yamanegi, Koji; Yamada, Naoko; Hata, Masaki; Nishiura, Hiroshi; Abiko, Yoshimitsu; Terada, Nobuyuki; Nakasho, Keiji

    2016-01-01

    Interleukin 12 receptor β chain (IL12RB2) is a crucial regulatory factor involved in cell-mediated immune responses, and genetic variants of the gene encoding IL12RB2 are associated with susceptibility to various immune-related diseases. We previously demonstrated that haplotypes with single nucleotide polymorphisms (SNPs) in the 5' flanking region of IL12RB2, including -1035A>G (rs3762315) and -1023A>G (rs3762316), affect the expression of IL12RB2, thereby altering susceptibility to leprosy and periodontal diseases. In the present study, we identified transcription factors associated with the haplotype-specific transcriptional activity of IL12RB2 in T cells and NK cells. The -1023G polymorphism was found to create a consensus binding site for the transcription factor activating protein (AP)-1, and enzyme-linked immunosorbent assay (ELISA)-based binding assays showed that these SNPs enhanced AP-1 binding to this region. In reporter assays, suppression of JunB expression using siRNA eliminated differences in the -1035G/-1023G and -1035A/-1023A regions containing IL12RB2 promoter activity in Jurkat T cells and NK3.3 cells. These results suggested that the -1035/-1023 polymorphisms created differential binding affinities for JunB that could lead to differential IL12RB2 expression. Moreover, the -1035G and -1035A alleles formed binding sites for GATA-3 and myocyte enhancer factor-2 (MEF-2), respectively. Our data indicated that in addition to JunB, the SNP at -1035/-1023 influenced GATA-3 and MEF-2 binding affinity, potentially altering IL12RB2 transcriptional activity. These findings confirm the effects of rs3762315 and rs3762316 on IL12RB2 transcription. These genetic variants may alter cellular activation of T cells and NK cells and modify cell-mediated immune responses. PMID:26552659

  17. Wnt-11 signaling leads to down-regulation of the Wnt/{beta}-catenin, JNK/AP-1 and NF-{kappa}B pathways and promotes viability in the CHO-K1 cells

    SciTech Connect

    Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka Vainio, Seppo

    2008-08-01

    The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical {beta}-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical {beta}-catenin mediated Wnt signaling but also JNK/AP-1 and NF-{kappa}B signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-{kappa}B pathway. Consistent with the central role of Akt, JNK and NF-{kappa}B in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.

  18. Docosahexaenoic acid consumption inhibits deoxynivalenol-induced CREB/ATF1 activation and IL-6 gene transcription in mouse macrophages.

    PubMed

    Jia, Qunshan; Zhou, Hui-Ren; Shi, Yuhui; Pestka, James J

    2006-02-01

    The mycotoxin deoxynivalenol (DON) induces IgA nephropathy in mice by upregulating IL-6 expression, which is suppressed by (n-3) PUFA consumption. The purpose of this study was to test the hypothesis that consumption of the (n-3) PUFA docosahexaenoic acid (DHA) interferes with DON-induced transcriptional and post-transcriptional upregulation of IL-6 mRNA in murine macrophages. DON evoked expression of IL-6 mRNA and IL-6 heterogenous nuclear RNA (hnRNA), an indicator of ongoing IL-6 transcription, in macrophages elicited from mice fed control AIN-93G diet for 4 wk, whereas expression of both RNA species was suppressed in macrophages from mice fed AIN-93G modified to contain 30 g DHA/kg diet for the same time period. DON enhanced IL-6 mRNA stability similarly in macrophages from control and DHA-fed mice suggesting that (n-3) PUFA effects were not post-transcriptional. DON upregulated binding activity of cAMP response element binding protein (CREB) and activator protein (AP-1) to their respective consensus sequences in nuclear extracts from control-fed mice, whereas both activities were suppressed in nuclear extracts from DHA-fed mice. DON induced phosphorylation of CREB at Ser-133 and ATF1 at Ser-63 as well as intranuclear binding of phospho-CREB/ATF1 to the cis element of the IL-6 promoter in control macrophages, whereas both activities were inhibited in macrophages from DHA-fed mice. DHA consumption blocked DON-induced phosphorylation of the CREB kinase AKT. Inhibition of AKT suppressed both CREB/ATF1 phosphorylation and IL-6 transcription. These data suggest that DHA consumption suppresses DON-induced IL-6 transcription in macrophages in part by interfering with AKT-dependent phosphorylation and subsequent binding of CREB/ATF1 to the IL-6 promoter. PMID:16424113

  19. MCP-1 Upregulates Amylin Expression in Murine Pancreatic β Cells through ERK/JNK-AP1 and NF-κB Related Signaling Pathways Independent of CCR2

    PubMed Central

    Cai, Kun; Qi, Dongfei; Hou, Xinwei; Wang, Oumei; Chen, Juan; Deng, Bo; Qian, Lihua; Liu, Xiaolong; Le, Yingying

    2011-01-01

    Background Amylin is the most abundant component of islet amyloid implicated in the development of type 2 diabetes. Plasma amylin levels are elevated in individuals with obesity and insulin resistance. Monocyte chemoattractant protein-1 (MCP-1, CCL2) is involved in insulin resistance of obesity and type 2 diabetes. We investigated the effect of MCP-1 on amylin expression and the underlying mechanisms with murine pancreatic β-cell line MIN6 and pancreatic islets. Methodology/Principal Findings We found that MCP-1 induced amylin expression at transcriptional level and increased proamylin and intermediate forms of amylin at protein level in MIN6 cells and islets. However, MCP-1 had no effect on the expressions of proinsulin 1 and 2, as well as prohormone convertase (PC) 1/3 and PC2, suggesting that MCP-1 specifically induces amylin expression in β-cells. Mechanistic studies showed that although there is no detectable CCR2 mRNA in MIN6 cells and islets, pretreatment of MIN6 cells with pertussis toxin inhibited MCP-1 induced amylin expression, suggesting that alternative Gi-coupled receptor(s) mediates the inductive effect of MCP-1. MCP-1 rapidly induced ERK1/2 and JNK phosphorylation. Inhibitors for MEK1/2 (PD98059), JNK (SP600125) or AP1 (curcumin) significantly inhibited MCP-1-induced amylin mRNA expression. MCP-1 failed to induce amylin expression in pancreatic islets isolated from Fos knockout mice. EMSA showed that JNK and ERK1/2 were involved in MCP-1-induced AP1 activation. These results suggest that MCP-1 induces murine amylin expression through AP1 activation mediated by ERK1/2 or JNK. Further studies showed that treatment of MIN6 cells with NF-κB inhibitor or overexpression of IκBα dominant-negative construct in MIN6 cells significantly inhibited MCP-1-induced amylin expression, suggesting that NF-κB related signaling also participates in MCP-1-induced murine amylin expression. Conclusions/Significance MCP-1 induces amylin expression through ERK1/2/JNK-AP

  20. Activation domains of transcription factors mediate replication dependent transcription from a minimal HIV-1 promoter.

    PubMed Central

    Williams, R D; Lee, B A; Jackson, S P; Proudfoot, N J

    1996-01-01

    Transcription from a minimal HIV-1 promoter containing the three Sp1 binding sites and TATA box can be activated without Tat by template DNA replication. Here we show that this activation can also be mediated by recombinant GAL4 fusion proteins containing the activation domains of Sp1, VP16 or CTF (or by full-length GAL4) targeted to the HIV-1 promoter by replacing the Sp1 sites with five GAL4 binding sites. Thus Sp1 is not unique in its ability to mediate replication activated transcription, although the degree of processivity elicited by the different activators varied significantly from strongly processive (GAL4-VP16) to relatively non-processive (GAL4-Sp1 or -CTF). Processive GAL4-VP16-activated transcription, but not efficient initiation, required multiple GAL4 binding sites. In the presence of Tat, transcription with GAL4-SP1 and GAL4-CTF was further activated (principally at the level of processivity) but GAL4-VP16-potentiated transcription was only slightly stimulated. The Tat-dependent switch from non-processive to fully processive transcription was particularly marked for GAL4-Sp1, an effect which may be relevant to the selection of Sp1 binding sites by the HIV-1 promoter. PMID:8604293

  1. Biliverdin reductase, a novel regulator for induction of activating transcription factor-2 and heme oxygenase-1.

    PubMed

    Kravets, Anatoliy; Hu, Zhenbo; Miralem, Tihomir; Torno, Michael D; Maines, Mahin D

    2004-05-01

    Biliverdin IXalpha reductase (BVR) catalyzes reduction of the HO activity product, biliverdin, to bilirubin. hBVR is a serine/threonine kinase that contains a bZip domain. Presently, regulation of gene expression by hBVR was examined. 293A cells were infected with adenovirus-doxycycline (Ad-Dox)-inducible hBVR cDNA. High level expression of hBVR was determined at mRNA, protein, and activity levels 8 h after induction. Cell signal transduction microarray analysis of cells infected with expression or with the control Ad-inverted (INV)-hBVR vector identified ATF-2 among several up-regulated genes. ATF-2 is a bZip transcription factor for activation of cAMP response element (CRE) and a dimeric partner to c-jun in MAPK pathway that regulates the stress protein, HO-1, expression. Northern and Western blot analyses showed increases of approximately 10-fold in ATF-2 mRNA and protein at 16 and 24 h after Dox addition. Ad-INV-hBVR did not effect ATF-2 expression. In hBVR-infected cells, levels of HO-1 mRNA and protein were increased. In vitro translated hBVR and nuclear extract containing hBVR in gel mobility-shift assay bound to AP-1 sites in the ATF-2 promoter region and to an oligonucleotide containing the CRE site. Both bindings could be competed out by excess unlabeled probe; in the presence of hBVR antibody, they displayed shifted bands. Co-transfection of hBVR with ATF-2 or c-jun promoters caused a severalfold increase in luciferase activity. hBVR modulation of ATF-2 and HO-1 expression suggests it has a potential role in regulation of AP-1 and cAMP-regulated genes and a role in cell signaling. We propose that increased expression of the protein can be used to alter the gene expression profile in the cell. PMID:14988408

  2. α-Chaconine isolated from a Solanum tuberosum L. cv Jayoung suppresses lipopolysaccharide-induced pro-inflammatory mediators via AP-1 inactivation in RAW 264.7 macrophages and protects mice from endotoxin shock.

    PubMed

    Lee, Kyoung-Goo; Lee, Suel-Gie; Lee, Hwi-Ho; Lee, Hae Jun; Shin, Ji-Sun; Kim, Nan-Jung; An, Hyo-Jin; Nam, Jung-Hwan; Jang, Dae Sik; Lee, Kyung-Tae

    2015-06-25

    In this study, we investigated the molecular mechanisms underlying the anti-inflammatory effects of α-chaconine in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and in LPS-induced septic mice. α-Chaconine inhibited the expressions of cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) at the transcriptional level, and attenuated the transcriptional activity of activator protein-1 (AP-1) by reducing the translocation and phosphorylation of c-Jun. α-Chaconine also suppressed the phosphorylation of TGF-β-activated kinase-1 (TAK1), which lies upstream of mitogen-activated protein kinase kinase 7 (MKK7)/Jun N-terminal kinase (JNK) signaling. JNK knockdown using siRNA prevented the α-chaconine-mediated inhibition of pro-inflammatory mediators. In a sepsis model, pretreatment with α-chaconine reduced the LPS-induced lethality and the mRNA and production levels of pro-inflammatory mediators by inhibiting c-Jun activation. These results suggest that the anti-inflammatory effects of α-chaconine are associated with the suppression of AP-1, and support its possible therapeutic role for the treatment of sepsis. PMID:25913072

  3. MAPK/AP-1 signal pathway in tobacco smoke-induced cell proliferation and squamous metaplasia in the lungs of rats.

    PubMed

    Zhong, Cai-Yun; Zhou, Ya-Mei; Douglas, Gordon C; Witschi, Hanspeter; Pinkerton, Kent E

    2005-12-01

    Overwhelming evidence has demonstrated tobacco smoke (TS) is causally associated with various types of cancers, especially lung cancer. Sustained epithelial cell hyperplasia and squamous metaplasia are considered as preneoplastic lesions during the formation of lung cancer. The cellular and molecular mechanisms leading to lung cancer due to TS are not clear. Mitogen-activated protein kinases (MAPK)/activator protein-1 (AP-1) can be activated by various stimuli and play a critical role in the control of cell proliferation and differentiation. To date, information on the response of the MAPK/AP-1 pathway during hyperplasia and squamous metaplasia induced by TS is lacking. We therefore investigated the effects of TS on the development of epithelial hyperplasia and squamous metaplasia, regulation of MAPK/AP-1 activation, and expression of AP-1-regulated cell cycle proteins and differentiation markers in the lungs of rats. Exposure of rats to TS (30 mg/m(3) or 80 mg/m(3), 6 h/day, 3 days/week for 14 weeks) dramatically induced cell proliferation and squamous metaplasia in a dose-dependent manner, effects that paralleled the activation of AP-1-DNA binding activity. Phosphorylated ERK1/2, JNK, p38 and ERK5 were significantly increased by exposure to TS, indicating the activation of these MAPK pathways. Expression of Jun and Fos proteins were differentially regulated by TS. TS upregulated the expression of AP-1-dependent cell cycle proteins including cyclin D1 and proliferating cell nuclear antigen (PCNA). Among the AP-1-dependent cell differentiation markers, keratin 5 and 14 were upregulated, while loricrin, filaggrin and involucrin were downregulated following TS exposure. These findings suggest the important role of MAPK/AP-1 pathway in TS-induced pathogenesis, thus providing new insights into the molecular mechanisms of TS-associated lung diseases including lung cancers. PMID:16051644

  4. Activation of archaeal transcription mediated by recruitment of transcription factor B.

    PubMed

    Ochs, Simon M; Thumann, Sybille; Richau, Renate; Weirauch, Matt T; Lowe, Todd M; Thomm, Michael; Hausner, Winfried

    2012-05-25

    Archaeal promoters consist of a TATA box and a purine-rich adjacent upstream sequence (transcription factor B (TFB)-responsive element (BRE)), which are bound by the transcription factors TATA box-binding protein (TBP) and TFB. Currently, only a few activators of archaeal transcription have been experimentally characterized. The best studied activator, Ptr2, mediates activation by recruitment of TBP. Here, we present a detailed biochemical analysis of an archaeal transcriptional activator, PF1088, which was identified in Pyrococcus furiosus by a bioinformatic approach. Operon predictions suggested that an upstream gene, pf1089, is polycistronically transcribed with pf1088. We demonstrate that PF1088 stimulates in vitro transcription by up to 7-fold when the pf1089 promoter is used as a template. By DNase I and hydroxyl radical footprinting experiments, we show that the binding site of PF1088 is located directly upstream of the BRE of pf1089. Mutational analysis indicated that activation requires the presence of the binding site for PF1088. Furthermore, we show that activation of transcription by PF1088 is dependent upon the presence of an imperfect BRE and is abolished when the pf1089 BRE is replaced with a BRE from a strong archaeal promoter. Gel shift experiments showed that TFB recruitment to the pf1089 operon is stimulated by PF1088, and TFB seems to stabilize PF1088 operator binding even in the absence of TBP. Taken together, these results represent the first biochemical evidence for a transcriptional activator working as a TFB recruitment factor in Archaea, for which the designation TFB-RF1 is suggested. PMID:22496454

  5. Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CArG elements.

    PubMed

    Gius, D; Cao, X M; Rauscher, F J; Cohen, D R; Curran, T; Sukhatme, V P

    1990-08-01

    The Fos-Jun complex has been shown to activate transcription through the regulatory element known as the AP-1 binding site. We show that Fos down regulates several immediate-early genes (c-fos, Egr-1, and Egr-2) after mitogenic stimulation. Specifically, we demonstrate that the target for this repression is a sequence of the form CC(A/T)6GG, also known as a CArG box. Whereas Fos bound to the AP-1 site through a domain rich in basic amino acids and associated with Jun via a leucine zipper interaction, mutant Fos proteins lacking these structures were still capable of causing repression. Furthermore, Jun neither enhanced nor inhibited down regulation by Fos. Critical residues required for repression are located within the C-terminal 27 amino acids of c-Fos, since v-Fos and C-terminal truncations of c-Fos did not down regulate. In addition, transfer of 180 c-Fos C-terminal amino acids to Jun conferred upon it the ability to repress. Finally, Fra-1, a Fos-related protein which has striking similarity to Fos in its C-terminal 40 amino acids, also down regulated Egr-1 expression. Thus, Fos is a transcriptional regulator that can activate or repress gene expression by way of two separate functional domains that act on distinct regulatory elements. PMID:2115122

  6. Regulation of maternal transcript destabilization during egg activation in Drosophila.

    PubMed Central

    Tadros, Wael; Houston, Simon A; Bashirullah, Arash; Cooperstock, Ramona L; Semotok, Jennifer L; Reed, Bruce H; Lipshitz, Howard D

    2003-01-01

    In animals, the transfer of developmental control from maternal RNAs and proteins to zygotically derived products occurs at the midblastula transition. This is accompanied by the destabilization of a subset of maternal transcripts. In Drosophila, maternal transcript destabilization occurs in the absence of fertilization and requires specific cis-acting instability elements. We show here that egg activation is necessary and sufficient to trigger transcript destabilization. We have identified 13 maternal-effect lethal loci that, when mutated, result in failure of maternal transcript degradation. All mutants identified are defective in one or more additional processes associated with egg activation. These include vitelline membrane reorganization, cortical microtubule depolymerization, translation of maternal mRNA, completion of meiosis, and chromosome condensation (the S-to-M transition) after meiosis. The least pleiotropic class of transcript destabilization mutants consists of three genes: pan gu, plutonium, and giant nuclei. These three genes regulate the S-to-M transition at the end of meiosis and are thought to be required for the maintenance of cyclin-dependent kinase (CDK) activity during this cell cycle transition. Consistent with a possible functional connection between this S-to-M transition and transcript destabilization, we show that in vitro-activated eggs, which exhibit aberrant postmeiotic chromosome condensation, fail to initiate transcript degradation. Several genetic tests exclude the possibility that reduction of CDK/cyclin complex activity per se is responsible for the failure to trigger transcript destabilization in these mutants. We propose that the trigger for transcript destabilization occurs coincidently with the S-to-M transition at the end of meiosis and that pan gu, plutonium, and giant nuclei regulate maternal transcript destabilization independent of their role in cell cycle regulation. PMID:12871909

  7. Id-1 is induced in MDCK epithelial cells by activated Erk/MAPK pathway in response to expression of the Snail and E47 transcription factors

    SciTech Connect

    Jorda, Mireia; Vinyals, Antonia; Marazuela, Anna; Cubillo, Eva; Olmeda, David; Valero, Eva; Cano, Amparo; Fabra, Angels . E-mail: afabra@idibell.org

    2007-07-01

    Id-1, a member of the helix-loop-helix transcription factor family has been shown to be involved in cell proliferation, angiogenesis and invasion of many types of human cancers. We have previously shown that stable expression of E47 and Snail repressors of the E-cadherin promoter in MDCK epithelial cell line triggers epithelial mesenchymal transition (EMT) concomitantly with changes in gene expression. We show here that both factors activate the Id-1 gene promoter and induce Id-1 mRNA and protein. The upregulation of the Id-1 gene occurs through the transactivation of the promoter by the Erk/MAPK signaling pathway. Moreover, oncogenic Ras is also able to activate Id-1 promoter in MDCK cells in the absence of both E47 and Snail transcription factors. Several transcriptionally active regulatory elements have been identified in the proximal promoter, including AP-1, Sp1 and four putative E-boxes. By EMSA, we only detected an increased binding to Sp1 and AP-1 elements in E47- and Snail-expressing cells. Binding is affected by the treatment of cells with PD 98059 MEK inhibitor, suggesting that MAPK/Erk contributes to the recruitment or assembly of proteins to Id-1 promoter. Small interfering RNA directed against Sp1 reduced Id-1 expression and the upregulation of the promoter, indicating that Sp1 is required for Id-1 induction in E47- and Snail-expressing cells. Our results provide new insights into how some target genes are activated during and/or as a consequence of the EMT triggered by both E47 and Snail transcription factors.

  8. Proteins of the ETS family with transcriptional repressor activity.

    PubMed

    Mavrothalassitis, G; Ghysdael, J

    2000-12-18

    ETS proteins form one of the largest families of signal-dependent transcriptional regulators, mediating cellular proliferation, differentiation and tumorigenesis. Most of the known ETS proteins have been shown to activate transcription. However, four ETS proteins (YAN, ERF, NET and TEL) can act as transcriptional repressors. In three cases (ERF, NET and TEL) distinct repression domains have been identified and there are indications that NET and TEL may mediate transcription via Histone Deacetylase recruitment. All four proteins appear to be regulated by MAPKs, though for YAN and ERF this regulation seems to be restricted to ERKs. YAN, ERF and TEL have been implicated in cellular proliferation although there are indications suggesting a possible involvement of YAN and TEL in differentiation as well. Other ETS-domain proteins have been shown to repress transcription in a context specific manner, and there are suggestions that the ETS DNA-binding domain may act as a transcriptional repressor. Transcriptional repression by ETS domain proteins adds an other level in the orchestrated regulation by this diverse family of transcription factors that often recognize similar if not identical binding sites on DNA and are believed to regulate critical genes in a variety of biological processes. Definitive assessment of the importance of this novel regulatory level will require the identification of ETS proteins target genes and the further analysis of transcriptional control and biological function of these proteins in defined pathways. PMID:11175368

  9. NF-Y activates mouse tryptophan hydroxylase transcription.

    PubMed

    Reed, G E; Kirchner, J E; Carr, L G

    1995-06-01

    Tryptophan hydroxylase catalyses the rate-limiting step in the biosynthesis of serotonin, a neurotransmitter which has been implicated in the etiologies of clinically important psychiatric illnesses. Tryptophan hydroxylase is expressed in a tissue-specific manner, but little is known about its transcriptional regulation. By analysing transcriptional activities of a set 5'-deletion constructs of promoter-reporter plasmids in P815-HTR mastocytoma cells, we found that transcription was activated by sequences between nucleotides -343 and -21. DNase I footprint analysis, using nuclear protein extracts from P815-HTR cells, revealed a protein-DNA interaction between nucleotides -77 and -46. A double stranded oligonucleotide, representing this binding site, specifically bound nuclear protein in a gel shift assay. Methylation interference analysis of this complex revealed that nuclear protein interacted with an inverted GGCCAAT element, which is a high-affinity binding motif for the transcription factor NF-Y (also known as CP1 or CBF). An NF-Y specific antibody abolished protein binding in a gel shift assay. Mutagenesis of specific base pairs abolished protein binding in vitro, and mutagenesis of the same base pairs in a reporter gene construct resulted in a 65% decrease in transcriptional activity. Our results suggest that the transcription factor NF-Y binds to a GGCCAAT motif in the tph proximal promoter and activates transcription. PMID:7552299

  10. Selective Activation of Transcription by a Novel CCAAT Binding Factor

    NASA Astrophysics Data System (ADS)

    Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

    1988-07-01

    A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

  11. Trim69 regulates zebrafish brain development by ap-1 pathway

    PubMed Central

    Han, Ruiqin; Wang, Renxian; Zhao, Qing; Han, Yongqing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

    2016-01-01

    Proteins belonging to the TRIM family have been implicated in a variety of cellular processes such as apoptosis, differentiation, neurogenesis, muscular physiology and innate immune responses. Trim69, previously identified as a novel gene cloned from a human testis cDNA library, has a homologous gene in zebrafish and this study focused on investigating the function of trim69 in zebrafish neurogenesis. Trim69 was found to be expressed in zebrafish embryo brain at the early stages. Knockdown of trim69 led to deformed brain development, obvious signs of apoptosis present in the head, and decreased expression of neuronal differentiation and stem cell markers. This phenotype was rescued upon co-injection of human mRNA together along with the trim69 knockdown. Results of this study also showed an interaction between TRIM69 and c-Jun in human cells, and upon TRIM69 knock down c-Jun expression subsequently increased, whereas the over-expression of TRIM69 led to the down-regulation of c-Jun. Additionally, knockdown both c-Jun and trim69 can rescue the deformed brain, evident cellular apoptosis in the head and decreased expression of neuronal differentiation and stem cell markers. Overall, our results support a role for trim69 in the development of the zebrafish brain through ap-1 pathway. PMID:27050765

  12. RKIP Inhibits Local Breast Cancer Invasion by Antagonizing the Transcriptional Activation of MMP13

    PubMed Central

    Qiu, Xiaoliang; Lewandowski, John; Yeung, Miranda; Ren, Gang; Aras, Shweta; Al-Mulla, Fahd; Cui, Hongjuan; Trumbly, Robert; Arudra, Sri Krishna Chaitanya; De Las Casas, Luis E.; de la Serna, Ivana; Bitar, Milad S.; Yeung, Kam C.

    2015-01-01

    Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a

  13. Molecular Mechanism of Transcriptional Regulation of Matrix Metalloproteinase-9 in Diabetic Retinopathy.

    PubMed

    Mishra, Manish; Flaga, Jadwiga; Kowluru, Renu A

    2016-08-01

    Increase in matrix metalloproteinase-9 (MMP-9) is implicated in retinal capillary cell apoptosis, a phenomenon which precedes the development of diabetic retinopathy. MMP-9 promoter has multiple sites for binding the transcriptional factors, including two for activator protein 1 (AP-1). The binding of AP-1, a heterodimer of c-Jun and c-Fos, is regulated by posttranslational modifications, and in diabetes, deacetylating enzyme, Sirt1, is inhibited. Our aim, is to investigate the molecular mechanism of MMP-9 transcriptional regulation in diabetes. Binding of AP-1 (c-Jun, c-Fos) at the MMP-9 promoter, and AP-1 acetylation were analyzed in retinal endothelial cells incubated in normal or high glucose by chromatin-immunoprecipitation and co-immunoprecipitation respectively. Role of AP-1 in MMP-9 regulation was confirmed by c-Jun or c-Fos siRNAs, and that of its acetylation, by Sirt1 overexpression. In vitro results were validated in the retina from diabetic mice overexpressing Sirt1, and in the retinal microvessels from human donors with diabetic retinopathy. In experimental models, AP-1 binding was increased at the proximal and distal sites of the MMP-9 promoter, and similar phenomenon was confirmed in the retinal microvessels from human donors with diabetic retinopathy. Silencing of AP-1, or overexpression of Sirt1 ameliorated glucose-induced increase in MMP-9 expression and cell apoptosis. Thus, in diabetes, due to Sirt1 inhibition, AP-1 is hyperacetylated, which increases its binding at MMP-9 promoter, and hence, activation of Sirt1 could inhibit the development of diabetic retinopathy by impeding MMP-9-mediated mitochondrial damage. J. Cell. Physiol. 231: 1709-1718, 2016. © 2015 Wiley Periodicals, Inc. PMID:26599598

  14. 5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells.

    PubMed

    Wu, Lei; Li, Xueqin; Wu, Haifeng; Long, Wei; Jiang, Xiaojian; Shen, Ting; Qiang, Qian; Si, Chuanling; Wang, Xinfeng; Jiang, Yunyao; Hu, Weicheng

    2016-01-01

    For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E₂ (PGE₂), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases. PMID:26938526

  15. 5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells

    PubMed Central

    Wu, Lei; Li, Xueqin; Wu, Haifeng; Long, Wei; Jiang, Xiaojian; Shen, Ting; Qiang, Qian; Si, Chuanling; Wang, Xinfeng; Jiang, Yunyao; Hu, Weicheng

    2016-01-01

    For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases. PMID:26938526

  16. Enhancement of Flow-Induced AP-1 Gene Expression by Cyclosporin A Requires NFAT-Independent Signaling in Bone Cells

    PubMed Central

    WORTON, LEAH E.; KWON, RONALD Y.; GARDINER, EDITH M.; GROSS, TED S.; SRINIVASAN, SUNDAR

    2014-01-01

    Growing evidence suggests that aging compromises the ability of the skeleton to respond to anabolic mechanical stimuli. Recently, we reported that treating senescent mice with Cyclosporin A (CsA) rescued aging-related deficits in loading-induced bone formation. Given that the actions of CsA are often attributed to inhibition of the calcineurin/NFAT axis, we hypothesized that CsA enhances gene expression in bone cells exposed to fluid flow, by inhibiting nuclear NFATc1 accumulation. When exposed to flow, MC3T3-E1 osteoblastic cells exhibited rapid nuclear accumulation of NFATc1 that was abolished by CsA treatment. Under differentiation conditions, intermittent CsA treatment enhanced gene expression of late osteoblastic differentiation markers and activator protein 1 (AP-1) family members. Superimposing flow upon CsA further enhanced expression of the AP-1 members Fra-1 and c-Jun. To delineate the contribution of NFAT in this response, cells were treated with VIVIT, a specific inhibitor of the calcineurin/NFAT interaction. Treatment with VIVIT blocked flow-induced nuclear NFATc1 accumulation but did not recapitulate the CsA-mediated enhancement of flow-induced AP-1 component gene expression. Taken together, our study is the first to demonstrate that CsA enhances mechanically-induced gene expression of AP-1 components in bone cells, and suggests that this response requires calcineurin-dependent mechanisms that are independent of inhibiting NFATc1 nuclear accumulation. PMID:25484988

  17. MafG Sumoylation Is Required for Active Transcriptional Repression

    PubMed Central

    Motohashi, Hozumi; Katsuoka, Fumiki; Miyoshi, Chika; Uchimura, Yasuhiro; Saitoh, Hisato; Francastel, Claire; Engel, James Douglas; Yamamoto, Masayuki

    2006-01-01

    A straightforward mechanism for eliciting transcriptional repression would be to simply block the DNA binding site for activators. Such passive repression is often mediated by transcription factors that lack an intrinsic repressor activity. MafG is a bidirectional regulator of transcription, a repressor in its homodimeric state but an activator when heterodimerized with p45. Here, we report that MafG is conjugated to SUMO-2/3 in vivo. To clarify the possible physiological role(s) for sumoylation in regulating MafG activity, we evaluated mutant and wild-type MafG in transgenic mice and cultured cells. Whereas sumoylation-deficient MafG activated p45-dependent transcription normally and did not affect heterodimer activity, repression by the sumoylation-deficient MafG mutant was severely compromised in vivo. Furthermore, the SUMO-dependent repression activity of MafG was sensitive to histone deacetylase inhibition. Thus, repression by MafG is not achieved through simple passive repression by competing for the activator binding site but requires sumoylation, which then mediates transcriptional repression through recruitment of a repressor complex containing histone deacetylase activity. PMID:16738329

  18. Potential Role of Activating Transcription Factor 5 during Osteogenesis.

    PubMed

    Vicari, Luisa; Calabrese, Giovanna; Forte, Stefano; Giuffrida, Raffaella; Colarossi, Cristina; Parrinello, Nunziatina Laura; Memeo, Lorenzo

    2016-01-01

    Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB) family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2), encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology. PMID:26770207

  19. p12CDK2-AP1 interacts with CD82 to regulate the proliferation and survival of human oral squamous cell carcinoma cells.

    PubMed

    Chai, Juan; Ju, Jun; Zhang, Shao-Wu; Shen, Zhi-Yuan; Liang, Liang; Yang, Xiang-Ming; Ma, Chao; Ni, Qian-Wei; Sun, Mo-Yi

    2016-08-01

    p12 cyclin-dependent kinase 2 (CDK2)-associating protein 1 (p12CDK2-AP1) has been demonstrated to negatively regulate the activity of CDK2. However, the underlying molecular mechanism remains largely unknown. We aimed to determine the potential binding proteins of p12CDK2-AP1 and to elucidate the role of p12CDK2-AP1 in the regulation of the proliferation, invasion, apoptosis, and in vivo growth of human oral squamous cell carcinoma cells. The protein-protein interaction was predicted using computational decision templates. The predicted p12CDK2‑AP1 interacting proteins were overexpressed in human oral squamous cell carcinoma OSCC-15 cells, and the protein binding was examined using co-precipitation (Co-IP). Cell proliferation and invasion were determined via MTT assay and Transwell system, respectively. Cell apoptosis was evaluated using Annexin V-FITC/PI double staining followed by flow cytometric analysis. The in vivo growth of OSCC-15 cells was examined in nude mouse tumor xenografts. We found that overexpression of either p12CDK2-AP1 or CD82 significantly suppressed the proliferation and invasion but promoted the apoptosis of OSCC-15 cells (P<0.05). Importantly, combined overexpression of p12CDK2-AP1 and CD82 showed synergistic antitumor activity compared with the overexpression of a single protein alone (P<0.05). Additionally, the simultaneous overexpression of p12CDK2-AP1 and CD82 significantly suppressed the in vivo tumor growth of OSCC-15 cells in nude mice compared with the negative control (P<0.05). Our findings indicate that p12CDK2-AP1 interacts with CD82 to play a functional role in suppressing the in vitro and in vivo growth of OSCC-15 cells. PMID:27349208

  20. Theory on the dynamic memory in the transcription-factor-mediated transcription activation

    NASA Astrophysics Data System (ADS)

    Murugan, R.

    2011-04-01

    We develop a theory to explain the origin of the static and dynamical memory effects in transcription-factor-mediated transcription activation. Our results suggest that the following inequality conditions should be satisfied to observe such memory effects: (a) τL≫max(τR,τE), (b) τLT≫τT, and (c) τI⩾(τEL+τTR) where τL is the average time required for the looping-mediated spatial interactions of enhancer—transcription-factor complex with the corresponding promoter—RNA-polymerase or eukaryotic RNA polymerase type II (PolII in eukaryotes) complex that is located L base pairs away from the cis-acting element, (τR,τE) are respectively the search times required for the site-specific binding of the RNA polymerase and the transcription factor with the respective promoter and the cis-regulatory module, τLT is the time associated with the relaxation of the looped-out segment of DNA that connects the cis-acting site and promoter, τT is the time required to generate a complete transcript, τI is the transcription initiation time, τEL is the elongation time, and τTR is the termination time. We have theoretically derived the expressions for the various searching, looping, and loop-relaxation time components. Using the experimentally determined values of various time components we further show that the dynamical memory effects cannot be experimentally observed whenever the segment of DNA that connects the cis-regulatory element with the promoter is not loaded with bulky histone bodies. Our analysis suggests that the presence of histone-mediated compaction of the connecting segment of DNA can result in higher values of looping and loop-relaxation times, which is the origin of the static memory in the transcription activation that is mediated by the memory gene loops in eukaryotes.

  1. Targeting Gli Transcription Activation by Small Molecule Suppresses Tumor Growth

    PubMed Central

    Bosco-Clément, Geneviève; Zhang, Fang; Chen, Zhao; Zhou, Hai-Meng; Li, Hui; Mikami, Iwao; Hirata, Tomomi; Yagui-Beltran, Adam; Lui, Natalie; Do, Hanh T.; Cheng, Tiffany; Tseng, Hsin-Hui; Choi, Helen; Fang, Li-Tai; Kim, Il-Jin; Yue, Dongsheng; Wang, Changli; Zheng, Qingfeng; Fujii, Naoaki; Mann, Michael; Jablons, David M.; He, Biao

    2014-01-01

    Targeted inhibition of Hedgehog signaling at the cell membrane has been associated with anti-cancer activity in preclinical and early clinical studies. Hedgehog signaling involves activation of Gli transcription factors that can also be induced by alternative pathways. In this study we identified an interaction between Gli proteins and a transcription co-activator TAF9, and validated its functional relevance in regulating Gli transactivation. We also describe a novel, synthetic small molecule, FN1-8, that efficiently interferes with Gli/TAF9 interaction and down-regulate Gli/TAF9 dependent transcriptional activity. More importantly, FN1-8 suppresses cancer cell proliferation in vitro and inhibits tumor growth in vivo. Our results suggest that blocking Gli transactivation, a key control point of multiple oncogenic pathways, may be an effective anti-cancer strategy. PMID:23686308

  2. Oxytocin-Stimulated NFAT Transcriptional Activation in Human Myometrial Cells

    PubMed Central

    McArdle, Craig A.; López Bernal, Andrés

    2012-01-01

    Oxytocin (OXT) is a peptide hormone that binds the OXT receptor on myometrial cells, initiating an intracellular signaling cascade, resulting in accumulation of intracellular calcium and smooth muscle contraction. In other systems, an elevation of intracellular Ca2+ stimulates nuclear translocation of the transcription factor, nuclear factor of activated T cells (NFAT), which is transcriptionally active in arterial and ileal smooth muscle. Here we have investigated the role of NFAT in the mechanism of action of OXT. Human myometrial cells expressed all five NFAT isoforms (NFATC1–C4 and -5). Myometrial cells were transduced with a recombinant adenovirus expressing a NFATC1-EFP reporter, and a semi-automated imaging system was used to monitor effects of OXT on reporter localization in live cells. OXT induced a concentration-dependent nuclear translocation of NFATC1-EFP in a reversible manner, which was inhibited by OXT antagonists and calcineurin inhibitors. Pulsatile stimulation with OXT caused intermittent, pulse-frequency-dependent, nuclear translocation of NFATC1-EFP, which was more efficient than sustained stimulation. OXT induced nuclear translocation of endogenous NFAT that was transcriptionally active, because OXT stimulated activity of a NFAT-response element-luciferase reporter and induced calcineurin-NFAT dependent expression of RGS2, RCAN1, and PTGS2 (COX2) mRNA. Furthermore, OXT-dependent transcription was dependent on protein neosynthesis; cycloheximide abolished RGS2 transcription but augmented RCAN1 and COX2 transcriptional readouts. This study identifies a novel signaling mechanism within the myometrium, whereby calcineurin-NFAT signaling mediates OXT-induced transcriptional activity. Furthermore, we show NFATC1-EFP is responsive to pulses of OXT, a mechanism by which myometrial cells could decode OXT pulse frequency. PMID:22902539

  3. p53-independent activation of the hdm2-P2 promoter through multiple transcription factor response elements results in elevated hdm2 expression in estrogen receptor alpha-positive breast cancer cells.

    PubMed

    Phelps, Monika; Darley, Matthew; Primrose, John N; Blaydes, Jeremy P

    2003-05-15

    The negative-regulatory feedback loop between p53 and hdm2 forms part of a finely balanced regulatory network of proteins that controls cell cycle progression and commitment to apoptosis. Expression of hdm2, and its mouse orthologue mdm2, is known to be induced by p53, but recent evidence has demonstrated mdm2 expression can also be regulated via p53-independent pathways. However the p53 independent mechanisms that control transcription of the human hdm2 gene have not been studied. Differential levels of hdm2 mRNA and protein expression have been reported in several types of human malignancy, including breast cancers in which hdm2 expression correlates with positive estrogen receptor alpha (ERalpha) status. Experimental models have demonstrated that hdm2 overexpression can promote breast cancer development. Here, we show that the elevated level of hdm2 protein in ERalpha(+ve) breast cancer cell lines such as MCF-7 and T47D is because of transcription from the p53-inducible P2 promoter of hdm2. The P2 promoter is inactive in ERalpha(-ve) cell lines such as SKBr3. Hdm2-P2 promoter activity in T47D cells is independent of p53, as well as of known regulators of the mouse mdm2-P2 promoter, including ERalpha and ras-raf-mitogen-activated protein/extracellular signal-regulated kinase (MEK) mitogen-activated protein kinase (MAPK) signaling. We show that hdm2-P2 activity in T47D cells is dependent on the integrity of both an evolutionarily conserved composite binding site for AP1 and ETS family transcription factors (AP1-ETS) and a nonconserved upstream (nnGGGGC)(5) repeat sequence. Lack of hdm2-P2 activity in ERalpha(-ve) cells is shown to be a consequence of reduced transcriptional activation through the AP1-ETS element. Overexpression of ETS2 in SKBr3 cells reconstitutes AP1-ETS element-dependent hdm2-P2 promoter activity, resulting in increased levels of hdm2 protein in the cells. Our findings support the hypothesis that the elevated levels of hdm2 expression reported

  4. The CREB Transcription Factor Controls Transcriptional Activity of the Human RIC8B Gene.

    PubMed

    Maureira, Alejandro; Sánchez, Rodolfo; Valenzuela, Nicole; Torrejón, Marcela; Hinrichs, María V; Olate, Juan; Gutiérrez, José L

    2016-08-01

    Proper regulation of gene expression is essential for normal development, cellular growth, and differentiation. Differential expression profiles of mRNA coding for vertebrate Ric-8B during embryo and adult stages have been observed. In addition, Ric-8B is expressed in few cerebral nuclei subareas. These facts point to a dynamic control of RIC8B gene expression. In order to understand the transcriptional regulation of this gene, we searched for cis-elements in the sequence of the human RIC8B promoter region, identifying binding sites for the basic/leucine zipper (bZip) CREB transcription factor family (CRE sites) and C/EBP transcription factor family (C/EBP sites). CRE sites were found clustered near the transcription start site, while the C/EBP sites were found clustered at around 300 bp upstream the CRE sites. Here, we demonstrate the ability of CREB1 and C/EBPβ to bind their respective elements identified in the RIC8B promoter. Comparative protein-DNA interaction analyses revealed only the proximal elements as high affinity sites for CREB1 and only the distal elements as high affinity sites for C/EBPβ. Chromatin immunoprecipitation analyses, carried out using a human neuroblastoma cell line, confirmed the preferential association of CREB to the proximal region of the RIC8B promoter. By performing luciferase reporter assays, we found the CRE sites as the most relevant elements for its transcriptional activity. Taken together, these data show the existence of functional CREB and C/EBP binding sites in the human RIC8B gene promoter, a particular distribution of these sites and demonstrate a relevant role of CREB in stimulating transcriptional activity of this gene. J. Cell. Biochem. 117: 1797-1805, 2016. © 2016 Wiley Periodicals, Inc. PMID:26729411

  5. Modulation of Cytokine Production and Transcription Factors Activities in Human Jurkat T Cells by Thymol and Carvacrol

    PubMed Central

    Gholijani, Nasser; Gharagozloo, Marjan; Kalantar, Fathollah; Ramezani, Amin; Amirghofran, Zahra

    2015-01-01

    Purpose: Thymol and carvacrol, two main components of thyme, have shown anti-inflammatory effects. The aim of this study was to assess the effects of these components on Jurkat leukemia cells as an in vitro T cell model and their molecular mechanisms of activity. Methods: Cells were cultured in the presence of components and subsequently stimulated with phorbol-12-myristate-13-acetate (PMA)/calcium ionophore for evaluating interleukin (IL)-2 and interferon (IFN)-γ production. The activation of T cell transcription factors that included nuclear factors of activated T cells (NFATs), activator protein-1 (AP-1; c-Jun/c-Fos), and nuclear factor (NF)-κB were examined by Western blot analysis. Results: Thymol and carvacrol at 25 µg/ml significantly reduced IL-2 levels from 119.4 ± 8pg/ml in control cells treated only with PMA/Calcium ionophore and the solvent to 66.9 ± 6.4pg/ml (thymol) and 32.3 ± 3.6pg/ml (carvacrol) and IFN-γ from 423.7 ± 19.7pg/ml in control cells to 311.9 ± 11.6pg/ml (thymol) and 293.5 ± 16.7pg/ml (carvacrol). Western blot analyses of nuclear extracts showed that the same concentrations of components significantly reduced NFAT-2 to 44.2 ± 2.7% (thymol) and 91.4 ± 2.3% (carvacrol) of the control (p<0.05), and c-Fos to 31.2 ± 6.2% (thymol) and 27.6 ± 3.1% (carvacrol) of the control (p<0.01). No effects on NFAT-1, c-Jun and phospho-NF-κBp65 levels were observed. Conclusion: Thymol and carvacrol could contribute to modulation of T cell activity by reducing IL-2 and IFN-γ production possibly through down regulation of AP-1 and NFAT-2 transcription factors suggesting their potential usefulness for reduction of T cell overactivity in immune-mediated diseases. PMID:26793612

  6. Sug1 modulates yeast transcription activation by Cdc68.

    PubMed Central

    Xu, Q; Singer, R A; Johnston, G C

    1995-01-01

    The Cdc68 protein is required for the transcription of a variety of genes in the yeast Saccharomyces cerevisiae. In a search for proteins involved in the activity of the Cdc68 protein, we identified four suppressor genes in which mutations reverse the temperature sensitivity caused by the cdc68-1 allele. We report here the molecular characterization of mutations in one suppressor gene, the previously identified SUG1 gene. The Sug1 protein has been implicated in both transcriptional regulation and proteolysis. sug1 suppressor alleles reversed most aspects of the cdc68-1 mutant phenotype but did not suppress the lethality of a cdc68 null allele, indicating that sug1 suppression is by restoration of Cdc68 activity. Our evidence suggests that suppression by sug1 is unlikely to be due to increased stability of mutant Cdc68 protein, despite the observation that Sug1 affected proteolysis of mutant Cdc68. We report here that attenuated Sug1 activity strengthens mutant Cdc68 activity, whereas increased Sug1 activity further inhibits enfeebled Cdc68 activity, suggesting that Sug1 antagonizes the activator function of Cdc68 for transcription. Consistent with this hypothesis, we find that Sug1 represses transcription in vivo. PMID:7565755

  7. An arcane role of DNA in transcription activation.

    PubMed Central

    Ryu, S; Garges, S; Adhya, S

    1994-01-01

    The mechanism by which the cAMP receptor protein (CRP) activates transcription has been investigated using the lac promoter of Escherichia coli. For transcription activation, an interaction between DNA-bound CRP and RNA polymerase is not sufficient. CRP must bind to a site in the same DNA and close to the promoter. CRP action requires an intact spacer DNA to provide a rigid support in building a CRP-RNA polymerase protein bridge or to allow a conformational change in the DNA to be transmitted to the lac promoter using the protein bridge as a structural support. Images PMID:7811325

  8. Nucleosomes unfold completely at a transcriptionally active promoter.

    PubMed

    Boeger, Hinrich; Griesenbeck, Joachim; Strattan, J Seth; Kornberg, Roger D

    2003-06-01

    It has long been known that promoter DNA is converted to a nuclease-sensitive state upon transcriptional activation. Recent findings have raised the possibility that this conversion reflects only a partial unfolding or other perturbation of nucleosomal structure, rather than the loss of nucleosomes. We report topological, sedimentation, nuclease digestion, and ChIP analyses, which demonstrate the complete unfolding of nucleosomes at the transcriptionally active PHO5 promoter of the yeast Saccharomyces cerevisiae. Although nucleosome loss occurs at all promoter sites, it is not complete at any of them, suggesting the existence of an equilibrium between the removal of nucleosomes and their reformation. PMID:12820971

  9. Evidence of a retinoid signaling alteration involving the activator protein 1 complex in tumorigenic human bronchial epithelial cells and non-small cell lung cancer cells.

    PubMed

    Lee, H Y; Dawson, M I; Claret, F X; Chen, J D; Walsh, G L; Hong, W K; Kurie, J M

    1997-03-01

    Retinoids, including retinol and retinoic acid derivatives, inhibit the growth of normal human bronchial epithelial (HBE) cells. Using a lung carcinogenesis model consisting of normal, immortalized, and tumorigenic HBE cells, we showed previously that, compared to normal HBE cells, the tumorigenic HBE cell line 11701 is resistant to the growth-inhibitory effects of all-trans-retinoic acid (t-RA). Retinoid receptor function is preserved in tumorigenic 11701 cells, suggesting that other retinoid signaling components are altered. The activator protein 1 (AP-1) complex is a component of the retinoid signaling pathway and has demonstrated importance in cellular growth and differentiation. Therefore, we investigated whether AP-1 is involved in a retinoid signaling defect in tumorigenic 11701 cells and in retinoid-resistant non-small cell lung cancer (NSCLC) cell lines. We found that t-RA treatment inhibited AP-1 transcriptional activity in normal HBE cells but not in tumorigenic 11701 cells nor in the NSCLC cell lines Calu-1, Calu-6, SKMES-1, and ChaGo K1. We sought mechanisms for this retinoid signaling alteration involving AP-1 in tumorigenic 11701 cells. Basal AP-1 transcriptional activity; AP-1 DNA-binding activity; and the mRNA levels of c-fos, the AP-1 coactivator Jun activation domain-binding protein 1, and the retinoid receptor corepressor, the silencing mediator for retinoid and thyroid hormone receptors (SMRT), were lower in tumorigenic 11701 cells than in normal HBE cells. Transient transfection of tumorigenic 11701 cells with c-fos or CREB binding protein, which is a coactivator of AP-1 and retinoid receptors, enhanced basal AP-1 transcriptional activity but did not alter the effects of t-RA on AP-1 transcriptional activity. These findings provide evidence of a retinoid signaling alteration involving AP-1 in tumorigenic 11701 and NSCLC cells. Furthermore, the inhibitory effect of t-RA on AP-1 transcriptional activity was not restored in tumorigenic 11701

  10. NF-κB/AP-1-targeted inhibition of macrophage-mediated inflammatory responses by depigmenting compound AP736 derived from natural 1,3-diphenylpropane skeleton.

    PubMed

    Ha, Van Thai; Beak, Heung Soo; Kim, Eunji; Baek, Kwang-Soo; Hossen, Muhammad Jahangir; Yang, Woo Seok; Kim, Yong; Kim, Jun Ho; Yang, Sungjae; Kim, Jeong-Hwan; Joo, Yung Hyup; Lee, Chang Seok; Choi, Joonho; Shin, Hong-Ju; Hong, Sungyoul; Shin, Song Seok; Cho, Jae Youl

    2014-01-01

    AP736 was identified as an antimelanogenic drug that can be used for the prevention of melasma, freckles, and dark spots in skin by acting as a suppressor of melanin synthesis and tyrosinase expression. Since macrophage-mediated inflammatory responses are critical for skin health, here we investigated the potential anti-inflammatory activity of AP736. The effects of AP736 on various inflammatory events such as nitric oxide (NO)/prostaglandin (PG) E2 production, inflammatory gene expression, phagocytic uptake, and morphological changes were examined in RAW264.7 cells. AP736 was found to strongly inhibit the production of both NO and PGE2 in lipopolysaccharide- (LPS-) treated RAW264.7 cells. In addition, AP736 strongly inhibited both LPS-induced morphological changes and FITC-dextran-induced phagocytic uptake. Furthermore, AP736 also downregulated the expression of multiple inflammatory genes, such as inducible NO synthase (iNOS), cyclooxygenase- (COX-) 2, and interleukin- (IL-) 1β in LPS-treated RAW264.7 cells. Transcription factor analysis, including upstream signalling events, revealed that both NF-κB and AP-1 were targeted by AP736 via inhibition of the IKK/IκBα and IRAK1/TAK1 pathways. Therefore, our results strongly suggest that AP736 is a potential anti-inflammatory drug due to its suppression of NF-κB-IKK/IκBα and AP-1-IRAK1/TAK1 signalling, which may make AP736 useful for the treatment of macrophage-mediated skin inflammation. PMID:25386046

  11. PKG-1α mediates GATA4 transcriptional activity.

    PubMed

    Ma, Yanlin; Wang, Jun; Yu, Yanhong; Schwartz, Robert J

    2016-06-01

    GATA4, a zinc-finger transcription factor, is central for cardiac development and diseases. Here we show that GATA4 transcriptional activity is mediated by cell signaling via cGMP dependent PKG-1α activity. Protein kinase G (PKG), a serine/tyrosine specific kinase is the major effector of cGMP signaling. We observed enhanced transcriptional activity elicited by co-expressed GATA4 and PKG-1α. Phosphorylation of GATA4 by PKG-1α was detected on serine 261 (S261), while the C-terminal activation domain of GATA4 associated with PKG-1α. GATA4's DNA binding activity was enhanced by PKG-1α via by both phosphorylation and physical association. More importantly, a number of human disease-linked GATA4 mutants exhibited impaired S261 phosphorylation, pointing to defective S261 phosphorylation in the elaboration of human heart diseases. We showed S261 phosphorylation was favored by PKG-1α but not by PKA, and several other kinase signaling pathways such as MAPK and PKC. Our observations demonstrate that cGMP-PKG signaling mediates transcriptional activity of GATA4 and links defective GATA4 and PKG-1α mutations to the development of human heart disease. PMID:26946174

  12. Involvement of the AP-1 Adaptor Complex in Early Steps of Phagocytosis and Macropinocytosis

    PubMed Central

    Lefkir, Yaya; Malbouyres, Marilyne; Gotthardt, Daniel; Ozinsky, Adrian; Cornillon, Sophie; Bruckert, Franz; Aderem, Alan A.; Soldati, Thierry; Cosson, Pierre; Letourneur, François

    2004-01-01

    The best described function of the adaptor complex-1 (AP-1) is to participate in the budding of clathrin-coated vesicles from the trans-Golgi network and endosomes. Here, we show that AP-1 is also localized to phagocytic cups in murine macrophages as well as in Dictyostelium amoebae. AP-1 is recruited to phagosomal membranes at this early stage of phagosome formation and rapidly dissociates from maturing phagosomes. To establish the role of AP-1 in phagocytosis, we made used of Dictyostelium mutant cells (apm1- cells) disrupted for AP-1 medium chain. In this mutant, phagocytosis drops by 60%, indicating that AP-1 is necessary for efficient phagocytosis. Furthermore, phagocytosis in apm1- cells is more affected for large rather than small particles, and cells exhibiting incomplete engulfment are then often observed. This suggests that AP-1 could participate in the extension of the phagocytic cup. Interestingly, macropinocytosis, a process dedicated to fluid-phase endocytosis and related to phagocytosis, is also impaired in apm1- cells. In summary, our data suggest a new role of AP-1 at an early stage of phagosome and macropinosome formation. PMID:14617812

  13. Ubiquitously expressed transcript is a novel interacting protein of protein inhibitor of activated signal transducer and activator of transcription 2

    PubMed Central

    KONG, XIANG; MA, SHIKUN; GUO, JIAQIAN; MA, YAN; HU, YANQIU; WANG, JIANJUN; ZHENG, YING

    2015-01-01

    Protein inhibitor of activated signal transducer and activator of transcription 2 (PIAS2) is a member of the PIAS protein family. This protein family modulates the activity of several transcription factors and acts as an E3 ubiquitin ligase in the sumoylation pathway. To improve understanding of the physiological roles of PIAS2, the current study used a yeast two-hybrid system to screen mouse stem cell cDNA libraries for proteins that interact with PIAS2. The screening identified an interaction between PIAS2 and ubiquitously expressed transcript (UXT). UXT, also termed androgen receptor trapped clone-27, is an α-class prefoldin-type chaperone that acts as a coregulator for various transcription factors, including nuclear factor-κB and androgen receptor (AR). A direct interaction between PIAS2 and UXT was confirmed by direct yeast two-hybrid analysis. In vitro evidence of the association of UXT with PIAS2 was obtained by co-immunoprecipitation. Colocalization between PIAS2 and UXT was identified in the nucleus and cytoplasm of HEK 293T and human cervical carcinoma HeLa cells. The results of the current study suggested that UXT is a binding protein of PIAS2, and interaction between PIAS2 and UXT may be important for the transcriptional activation of AR. PMID:25434787

  14. AP-1 Is a Key Regulator of Proinflammatory Cytokine TNFα-mediated Triple-negative Breast Cancer Progression.

    PubMed

    Qiao, Yichun; He, Huan; Jonsson, Philip; Sinha, Indranil; Zhao, Chunyan; Dahlman-Wright, Karin

    2016-03-01

    Triple-negative breast cancer (TNBC) represents a highly aggressive form of breast cancer with limited treatment options. Proinflammatory cytokines such as TNFα can facilitate tumor progression and metastasis. However, the mechanistic aspects of inflammation mediated TNBC progression remain unclear. Using ChIP-seq, we demonstrate that the cistrome for the AP-1 transcription factor c-Jun is comprised of 13,800 binding regions in TNFα-stimulated TNBC cells. In addition, we show that c-Jun regulates nearly a third of the TNFα-regulated transcriptome. Interestingly, high expression level of the c-Jun-regulated pro-invasion gene program is associated with poor clinical outcome in TNBCs. We further demonstrate that c-Jun drives TNFα-mediated increase of malignant characteristics of TNBC cells by transcriptional regulation of Ninj1. As exemplified by the CXC chemokine genes clustered on chromosome 4, we demonstrate that NF-κB might be a pioneer factor required for the regulation of TNFα-inducible inflammatory genes, whereas c-Jun has little effect. Together, our results uncover AP-1 as an important determinant for inflammation-induced cancer progression, rather than inflammatory response. PMID:26792858

  15. Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC.

    PubMed

    Yang, Hongchun; Howard, Martin; Dean, Caroline

    2016-08-16

    Establishment and maintenance of gene expression states is central to development and differentiation. Transcriptional and epigenetic mechanisms interconnect in poorly understood ways to determine these states. We explore these mechanisms through dissection of the regulation of Arabidopsis thaliana FLOWERING LOCUS C (FLC). FLC can be present in a transcriptionally active state marked by H3K36me3 or a silent state marked by H3K27me3. Here, we investigate the trans factors modifying these opposing histone states and find a physical coupling in vivo between the H3K36 methyltransferase, SDG8, and the H3K27me3 demethylase, ELF6. Previous modeling has predicted this coupling would exist as it facilitates bistability of opposing histone states. We also find association of SDG8 with the transcription machinery, namely RNA polymerase II and the PAF1 complex. Delivery of the active histone modifications is therefore likely to be through transcription at the locus. SDG8 and ELF6 were found to influence the localization of each other on FLC chromatin, showing the functional importance of the interaction. In addition, both influenced accumulation of the associated H3K27me3 and H3K36me3 histone modifications at FLC We propose the physical coupling of activation and derepression activities coordinates transcriptional activity and prevents ectopic silencing. PMID:27482092

  16. Physical coupling of activation and derepression activities to maintain an active transcriptional state at FLC

    PubMed Central

    Yang, Hongchun; Howard, Martin; Dean, Caroline

    2016-01-01

    Establishment and maintenance of gene expression states is central to development and differentiation. Transcriptional and epigenetic mechanisms interconnect in poorly understood ways to determine these states. We explore these mechanisms through dissection of the regulation of Arabidopsis thaliana FLOWERING LOCUS C (FLC). FLC can be present in a transcriptionally active state marked by H3K36me3 or a silent state marked by H3K27me3. Here, we investigate the trans factors modifying these opposing histone states and find a physical coupling in vivo between the H3K36 methyltransferase, SDG8, and the H3K27me3 demethylase, ELF6. Previous modeling has predicted this coupling would exist as it facilitates bistability of opposing histone states. We also find association of SDG8 with the transcription machinery, namely RNA polymerase II and the PAF1 complex. Delivery of the active histone modifications is therefore likely to be through transcription at the locus. SDG8 and ELF6 were found to influence the localization of each other on FLC chromatin, showing the functional importance of the interaction. In addition, both influenced accumulation of the associated H3K27me3 and H3K36me3 histone modifications at FLC. We propose the physical coupling of activation and derepression activities coordinates transcriptional activity and prevents ectopic silencing. PMID:27482092

  17. Aerobic glycolysis tunes YAP/TAZ transcriptional activity

    PubMed Central

    Enzo, Elena; Santinon, Giulia; Pocaterra, Arianna; Aragona, Mariaceleste; Bresolin, Silvia; Forcato, Mattia; Grifoni, Daniela; Pession, Annalisa; Zanconato, Francesca; Guzzo, Giulia; Bicciato, Silvio; Dupont, Sirio

    2015-01-01

    Increased glucose metabolism and reprogramming toward aerobic glycolysis are a hallmark of cancer cells, meeting their metabolic needs for sustained cell proliferation. Metabolic reprogramming is usually considered as a downstream consequence of tumor development and oncogene activation; growing evidence indicates, however, that metabolism on its turn can support oncogenic signaling to foster tumor malignancy. Here, we explored how glucose metabolism regulates gene transcription and found an unexpected link with YAP/TAZ, key transcription factors regulating organ growth, tumor cell proliferation and aggressiveness. When cells actively incorporate glucose and route it through glycolysis, YAP/TAZ are fully active; when glucose metabolism is blocked, or glycolysis is reduced, YAP/TAZ transcriptional activity is decreased. Accordingly, glycolysis is required to sustain YAP/TAZ pro-tumorigenic functions, and YAP/TAZ are required for the full deployment of glucose growth-promoting activity. Mechanistically we found that phosphofructokinase (PFK1), the enzyme regulating the first committed step of glycolysis, binds the YAP/TAZ transcriptional cofactors TEADs and promotes their functional and biochemical cooperation with YAP/TAZ. Strikingly, this regulation is conserved in Drosophila, where phosphofructokinase is required for tissue overgrowth promoted by Yki, the fly homologue of YAP. Moreover, gene expression regulated by glucose metabolism in breast cancer cells is strongly associated in a large dataset of primary human mammary tumors with YAP/TAZ activation and with the progression toward more advanced and malignant stages. These findings suggest that aerobic glycolysis endows cancer cells with particular metabolic properties and at the same time sustains transcription factors with potent pro-tumorigenic activities such as YAP/TAZ. PMID:25796446

  18. TBP domain symmetry in basal and activated archaeal transcription.

    PubMed

    Ouhammouch, Mohamed; Hausner, Winfried; Geiduschek, E Peter

    2009-01-01

    The TATA box binding protein (TBP) is the platform for assembly of archaeal and eukaryotic transcription preinitiation complexes. Ancestral gene duplication and fusion events have produced the saddle-shaped TBP molecule, with its two direct-repeat subdomains and pseudo-two-fold symmetry. Collectively, eukaryotic TBPs have diverged from their present-day archaeal counterparts, which remain highly symmetrical. The similarity of the N- and C-halves of archaeal TBPs is especially pronounced in the Methanococcales and Thermoplasmatales, including complete conservation of their N- and C-terminal stirrups; along with helix H'1, the C-terminal stirrup of TBP forms the main interface with TFB/TFIIB. Here, we show that, in stark contrast to its eukaryotic counterparts, multiple substitutions in the C-terminal stirrup of Methanocaldococcus jannaschii (Mja) TBP do not completely abrogate basal transcription. Using DNA affinity cleavage, we show that, by assembling TFB through its conserved N-terminal stirrup, Mja TBP is in effect ambidextrous with regard to basal transcription. In contrast, substitutions in either its N- or the C-terminal stirrup abrogate activated transcription in response to the Lrp-family transcriptional activator Ptr2. PMID:19007415

  19. Eupafolin inhibits PGE2 production and COX2 expression in LPS-stimulated human dermal fibroblasts by blocking JNK/AP-1 and Nox2/p47{sup phox} pathway

    SciTech Connect

    Tsai, Ming-Horng; Liang, Chan-Jung; Yen, Feng-Lin; Chiang, Yao-Chang; Lee, Chiang-Wen

    2014-09-01

    Eupafolin, a major active component found in the methanol extracts of Phyla nodiflora, has been used to treat inflammation of skin. We examined its effects on cyclooxygenase-2 (COX-2) expression in LPS-treated human dermal fibroblasts. Lipopolysaccharide (LPS) significantly increased prostaglandin-E2 (PGE2) production associated with increased COX-2 expression in Hs68 cells. This effect was blocked by eupafolin, TLR-4 antibody, antioxidants (APO and NAC), as well as inhibitors, including U0126 (ERK1/2), SB202190 (p38), SP600125 (JNK1/2), and Tanshinone IIA (AP-1). In gene regulation level, qPCR and promoter assays revealed that COX-2 expression was attenuated by eupafolin. In addition, eupafolin also ameliorated LPS-induced p47 phox activation and decreased reactive oxygen species (ROS) generation and NADPH oxidase (Nox) activity. Moreover, pretreatment with eupafolin and APO led to reduced LPS-induced phosphorylation of ERK1/2, JNK, and p38. Further, eupafolin attenuated LPS-induced increase in AP-1 transcription factor binding activity as well as the increase in the phosphorylation of c-Jun and c-Fos. In vivo studies have shown that in dermal fibroblasts of LPS treated mice, eupafolin exerted anti-inflammation effects by decreasing COX-2 protein levels. Our results reveal a novel mechanism for anti-inflammatory and anti-oxidative effects of eupafolin that involved inhibition of LPS-induced ROS generation, suppression of MAPK phosphorylation, diminished DNA binding activity of AP-1 and attenuated COX-2 expression leading to reduced production of prostaglandin E2 (PGE2). Our results demonstrate that eupafolin may be used to treat inflammatory responses associated with dermatologic diseases. - Highlights: • LPS activates the Nox2/p47{sup phox}/JNK/AP-1 and induces COX2 expression in Hs68 cells. • Eupafolin inhibits LPS-induced COX-2 expression via Nox2/p47{sup phox} inhibition. • Eupafolin may be used in the treatment of skin diseases involving inflammation.

  20. Combinatorial influence of environmental parameters on transcription factor activity

    PubMed Central

    Knijnenburg, T.A.; Wessels, L.F.A.; Reinders, M.J.T.

    2008-01-01

    Motivation: Cells receive a wide variety of environmental signals, which are often processed combinatorially to generate specific genetic responses. Changes in transcript levels, as observed across different environmental conditions, can, to a large extent, be attributed to changes in the activity of transcription factors (TFs). However, in unraveling these transcription regulation networks, the actual environmental signals are often not incorporated into the model, simply because they have not been measured. The unquantified heterogeneity of the environmental parameters across microarray experiments frustrates regulatory network inference. Results: We propose an inference algorithm that models the influence of environmental parameters on gene expression. The approach is based on a yeast microarray compendium of chemostat steady-state experiments. Chemostat cultivation enables the accurate control and measurement of many of the key cultivation parameters, such as nutrient concentrations, growth rate and temperature. The observed transcript levels are explained by inferring the activity of TFs in response to combinations of cultivation parameters. The interplay between activated enhancers and repressors that bind a gene promoter determine the possible up- or downregulation of the gene. The model is translated into a linear integer optimization problem. The resulting regulatory network identifies the combinatorial effects of environmental parameters on TF activity and gene expression. Availability: The Matlab code is available from the authors upon request. Contact: t.a.knijnenburg@tudelft.nl Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18586711

  1. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme

    PubMed Central

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  2. Moonlighting transcriptional activation function of a fungal sulfur metabolism enzyme.

    PubMed

    Levati, Elisabetta; Sartini, Sara; Bolchi, Angelo; Ottonello, Simone; Montanini, Barbara

    2016-01-01

    Moonlighting proteins, including metabolic enzymes acting as transcription factors (TF), are present in a variety of organisms but have not been described in higher fungi so far. In a previous genome-wide analysis of the TF repertoire of the plant-symbiotic fungus Tuber melanosporum, we identified various enzymes, including the sulfur-assimilation enzyme phosphoadenosine-phosphosulfate reductase (PAPS-red), as potential transcriptional activators. A functional analysis performed in the yeast Saccharomyces cerevisiae, now demonstrates that a specific variant of this enzyme, PAPS-red A, localizes to the nucleus and is capable of transcriptional activation. TF moonlighting, which is not present in the other enzyme variant (PAPS-red B) encoded by the T. melanosporum genome, relies on a transplantable C-terminal polypeptide containing an alternating hydrophobic/hydrophilic amino acid motif. A similar moonlighting activity was demonstrated for six additional proteins, suggesting that multitasking is a relatively frequent event. PAPS-red A is sulfur-state-responsive and highly expressed, especially in fruitbodies, and likely acts as a recruiter of transcription components involved in S-metabolism gene network activation. PAPS-red B, instead, is expressed at low levels and localizes to a highly methylated and silenced region of the genome, hinting at an evolutionary mechanism based on gene duplication, followed by epigenetic silencing of this non-moonlighting gene variant. PMID:27121330

  3. Transcriptional Activation of the Cyclin A Gene by the Architectural Transcription Factor HMGA2

    PubMed Central

    Tessari, Michela A.; Gostissa, Monica; Altamura, Sandro; Sgarra, Riccardo; Rustighi, Alessandra; Salvagno, Clio; Caretti, Giuseppina; Imbriano, Carol; Mantovani, Roberto; Del Sal, Giannino; Giancotti, Vincenzo; Manfioletti, Guidalberto

    2003-01-01

    The HMGA2 protein belongs to the HMGA family of architectural transcription factors, which play an important role in chromatin organization. HMGA proteins are overexpressed in several experimental and human tumors and have been implicated in the process of neoplastic transformation. Hmga2 knockout results in the pygmy phenotype in mice and in a decreased growth rate of embryonic fibroblasts, thus indicating a role for HMGA2 in cell proliferation. Here we show that HMGA2 associates with the E1A-regulated transcriptional repressor p120E4F, interfering with p120E4F binding to the cyclin A promoter. Ectopic expression of HMGA2 results in the activation of the cyclin A promoter and induction of the endogenous cyclin A gene. In addition, chromatin immunoprecipitation experiments show that HMGA2 associates with the cyclin A promoter only when the gene is transcriptionally activated. These data identify the cyclin A gene as a cellular target for HMGA2 and, for the first time, suggest a mechanism for HMGA2-dependent cell cycle regulation. PMID:14645522

  4. An Essential Viral Transcription Activator Modulates Chromatin Dynamics

    PubMed Central

    Gibeault, Rebecca L.; Bildersheim, Michael D.

    2016-01-01

    Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes. PMID:27575707

  5. In Vitro Activation of the Transcription of araBAD Operon by araC Activator

    PubMed Central

    Lee, Nancy; Wilcox, Gary; Gielow, William; Arnold, John; Cleary, Paul; Englesberg, Ellis

    1974-01-01

    The transcription of araBAD operon requires araC activator and cyclic AMP. D-Fucose inhibits ara mRNA synthesis. Our results indicate that the positive control by araC activator is exerted at the level of transcription. PMID:4362626

  6. Signal Transducer and Activator of Transcription-3, Inflammation, and Cancer

    PubMed Central

    Aggarwal, Bharat B.; Kunnumakkara, Ajaikumar B.; Harikumar, Kuzhuvelil B.; Gupta, Shan R.; Tharakan, Sheeja T.; Koca, Cemile; Dey, Sanjit; Sung, Bokyung

    2011-01-01

    Signal transducer and activator of transcription-3 (STAT-3) is one of six members of a family of transcription factors. It was discovered almost 15 years ago as an acute-phase response factor. This factor has now been associated with inflammation, cellular transformation, survival, proliferation, invasion, angiogenesis, and metastasis of cancer. Various types of carcinogens, radiation, viruses, growth factors, oncogenes, and inflammatory cytokines have been found to activate STAT-3. STAT-3 is constitutively active in most tumor cells but not in normal cells. Phosphorylation of STAT-3 at tyrosine 705 leads to its dimerization, nuclear translocation, DNA binding, and gene transcription. The phosphorylation of STAT-3 at serine 727 may regulate its activity negatively or positively. STAT-3 regulates the expression of genes that mediate survival (survivin, bcl-xl, mcl-1, cellular FLICE-like inhibitory protein), proliferation (c-fos, c-myc, cyclin D1), invasion (matrix metalloproteinase-2), and angiogenesis (vascular endothelial growth factor). STAT-3 activation has also been associated with both chemoresistance and radioresistance. STAT-3 mediates these effects through its collaboration with various other transcription factors, including nuclear factor-κB, hypoxia-inducible factor-1, and peroxisome proliferator activated receptor-γ. Because of its critical role in tumorigenesis, inhibitors of this factor’s activation are being sought for both prevention and therapy of cancer. This has led to identification of small peptides, oligonucleotides, and small molecules as potential STAT-3 inhibitors. Several of these small molecules are chemo-preventive agents derived from plants. This review discusses the intimate relationship between STAT-3, inflammation, and cancer in more detail. PMID:19723038

  7. Isolated HIV-1 core is active for reverse transcription.

    PubMed

    Warrilow, David; Stenzel, Deborah; Harrich, David

    2007-01-01

    Whether purified HIV-1 virion cores are capable of reverse transcription or require uncoating to be activated is currently controversial. To address this question we purified cores from a virus culture and tested for the ability to generate authentic reverse transcription products. A dense fraction (approximately 1.28 g/ml) prepared without detergent, possibly derived from disrupted virions, was found to naturally occur as a minor sub-fraction in our preparations. Core-like particles were identified in this active fraction by electron microscopy. We are the first to report the detection of authentic strong-stop, first-strand transfer and full-length minus strand products in this core fraction without requirement for an uncoating activity. PMID:17956635

  8. The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis.

    PubMed

    Hai, T; Hartman, M G

    2001-07-25

    The mammalian ATF/CREB family of transcription factors represents a large group of basic region-leucine zipper (bZip) proteins which was originally defined in the late 1980s by their ability to bind to the consensus ATF/CRE site 'TGACGTCA'. Over the past decade, cDNA clones encoding identical or homologous proteins have been isolated by different laboratories and given different names. These proteins can be grouped into subgroups according to their amino acid similarity. In this review, we will briefly describe the classification of these proteins with a historical perspective of their nomenclature. We will then review three members of the ATF/CREB family of proteins: ATF3, ATF4 and ATF6. We will address four issues for each protein: (a) homologous proteins and alternative names, (b) dimer formation with other bZip proteins, (c) transcriptional activity, and (d) potential physiological functions. Although the name Activating Transcription Factor (ATF) implies that they are transcriptional activators, some of these proteins are transcriptional repressors. ATF3 homodimer is a transcriptional repressor and ATF4 has been reported to be either an activator or a repressor. We will review the reports on the transcriptional activities of ATF4, and propose potential explanations for the discrepancy. Although the physiological functions of these proteins are not well understood, some clues can be gained from studies with different approaches. When the data are available, we will address the following questions. (a) How is the expression (at the mRNA level or protein level) regulated? (b) How are the transcriptional activities regulated? (c) What are the interacting proteins (other than bZip partners)? (d) What are the consequences of ectopically expressing the gene (gain-of-function) or deleting the gene (loss-of-function)? Although answers to these questions are far from being complete, together they provide clues to the functions of these ATF proteins. Despite the

  9. RAD51AP2, a novel vertebrate- and meiotic-specific protein, shares a conserved RAD51-interacting C-terminal domain with RAD51AP1/PIR51

    PubMed Central

    Kovalenko, Oleg V.; Wiese, Claudia; Schild, David

    2006-01-01

    Many interacting proteins regulate and/or assist the activities of RAD51, a recombinase which plays a critical role in both DNA repair and meiotic recombination. Yeast two-hybrid screening of a human testis cDNA library revealed a new protein, RAD51AP2 (RAD51 Associated Protein 2), that interacts strongly with RAD51. A full-length cDNA clone predicts a novel vertebrate-specific protein of 1159 residues, and the RAD51AP2 transcript was observed only in meiotic tissue (i.e. adult testis and fetal ovary), suggesting a meiotic-specific function for RAD51AP2. In HEK293 cells the interaction of RAD51 with an ectopically-expressed recombinant large fragment of RAD51AP2 requires the C-terminal 57 residues of RAD51AP2. This RAD51-binding region shows 81% homology to the C-terminus of RAD51AP1/PIR51, an otherwise totally unrelated RAD51-binding partner that is ubiquitously expressed. Analyses using truncations and point mutations in both RAD51AP1 and RAD51AP2 demonstrate that these proteins use the same structural motif for RAD51 binding. RAD54 shares some homology with this RAD51-binding motif, but this homologous region plays only an accessory role to the adjacent main RAD51-interacting region, which has been narrowed here to 40 amino acids. A novel protein, RAD51AP2, has been discovered that interacts with RAD51 through a C-terminal motif also present in RAD51AP1. PMID:16990250

  10. Potentiation of glucocorticoid receptor transcriptional activity by sumoylation.

    PubMed

    Le Drean, Yves; Mincheneau, Nathalie; Le Goff, Pascale; Michel, Denis

    2002-09-01

    The glucocorticoid receptor (GR) is a transcription factor, subject to several types of posttranslational modifications including phosphorylation and ubiquitination. We showed that the GR is covalently modified by the small ubiquitin-related modifier-1 (SUMO-1) peptide in mammalian cells. We demonstrated that GR sumoylation is not dependent on the presence of the ligand and regulates the stability of the protein as well as its transcriptional activity. SUMO-1 overexpression induces dramatic GR degradation, abolished by proteasome inhibition. We also found that SUMO-1 stimulates the transactivation capacity of GRs to an extent largely exceeding those observed so far for other sumoylated transcription factors. Overexpression of SUMO-1 specifically enhances the ligand-induced transactivation of GR up to 8-fold. However, this hyperactivation occurs only in the context of a synergy between multiple molecules of GRs. It requires more than one receptor DNA-binding site in promoter and becomes more prominent as the number of sites increases. Interestingly, these observations may be related to the transcriptional properties of the synergy control region of GRs, which precisely contains two evolutionary conserved sumoylation sites. We propose a model in which SUMO-1 regulates the synergy control function of GR and serves as a unique signal for activation and destruction. PMID:12193561

  11. The transcriptional activities and cellular localization of the human estrogen receptor alpha are affected by the synonymous Ala87 mutation.

    PubMed

    Fernández-Calero, Tamara; Astrada, Soledad; Alberti, Alvaro; Horjales, Sofía; Arnal, Jean Francois; Rovira, Carlos; Bollati-Fogolín, Mariela; Flouriot, Gilles; Marin, Mónica

    2014-09-01

    Until recently, synonymous mutations (which do not change amino acids) have been much neglected. Some evidence suggests that this kind of mutations could affect mRNA secondary structure or stability, translation kinetics and protein structure. To explore deeper the role of synonymous mutations, we studied their consequence on the functional activity of the estrogen receptor alpha (ERα). The ERα is a ligand-inducible transcription factor that orchestrates pleiotropic cellular effects, at both genomic and non-genomic levels in response to estrogens. In this work we analyzed in transient transfection experiments, the activity of ERα carrying the synonymous mutation Ala87, a polymorphism involving about 5-10% of the population. In comparison to the wild type receptor, our results show that ERαA87 mutation reduces the transactivation efficiency of ERα on an ERE reporter gene while its expression level remains similar. This mutation enhances 4-OHT-induced transactivation of ERα on an AP1 reporter gene. Finally, the mutation affects the subcellular localization of ERα in a cell type specific manner. It enhances the cytoplasmic location of ERα without significant changes in non-genomic effects of E2. The functional alteration of the ERαA87 determined in this work highlights the relevance of synonymous mutations for biomedical and pharmacological points of view. PMID:24607813

  12. Ectopic expression of Jatropha curcas APETALA1 (JcAP1) caused early flowering in Arabidopsis, but not in Jatropha.

    PubMed

    Tang, Mingyong; Tao, Yan-Bin; Xu, Zeng-Fu

    2016-01-01

    Jatropha curcas is a promising feedstock for biofuel production because Jatropha oil is highly suitable for the production of biodiesel and bio-jet fuels. However, Jatropha exhibits a low seed yield as a result of unreliable and poor flowering. APETALA1 (AP1) is a floral meristem and organ identity gene in higher plants. The flower meristem identity genes of Jatropha have not yet been identified or characterized. To better understand the genetic control of flowering in Jatropha, an AP1 homolog (JcAP1) was isolated from Jatropha. An amino acid sequence analysis of JcAP1 revealed a high similarity to the AP1 proteins of other perennial plants. JcAP1 was expressed in inflorescence buds, flower buds, sepals and petals. The highest expression level was observed during the early developmental stage of the flower buds. The overexpression of JcAP1 using the cauliflower mosaic virus (CaMV) 35S promoter resulted in extremely early flowering and abnormal flowers in transgenic Arabidopsis plants. Several flowering genes downstream of AP1 were up-regulated in the JcAP1-overexpressing transgenic plant lines. Furthermore, JcAP1 overexpression rescued the phenotype caused by the Arabidopsis AP1 loss-of-function mutant ap1-11. Therefore, JcAP1 is an ortholog of AtAP1, which plays a similar role in the regulation of flowering in Arabidopsis. However, the overexpression of JcAP1 in Jatropha using the same promoter resulted in little variation in the flowering time and floral organs, indicating that JcAP1 may be insufficient to regulate flowering by itself in Jatropha. This study helps to elucidate the function of JcAP1 and contributes to the understanding of the molecular mechanisms of flower development in Jatropha. PMID:27168978

  13. Ectopic expression of Jatropha curcas APETALA1 (JcAP1) caused early flowering in Arabidopsis, but not in Jatropha

    PubMed Central

    Tang, Mingyong; Tao, Yan-Bin

    2016-01-01

    Jatropha curcas is a promising feedstock for biofuel production because Jatropha oil is highly suitable for the production of biodiesel and bio-jet fuels. However, Jatropha exhibits a low seed yield as a result of unreliable and poor flowering. APETALA1 (AP1) is a floral meristem and organ identity gene in higher plants. The flower meristem identity genes of Jatropha have not yet been identified or characterized. To better understand the genetic control of flowering in Jatropha, an AP1 homolog (JcAP1) was isolated from Jatropha. An amino acid sequence analysis of JcAP1 revealed a high similarity to the AP1 proteins of other perennial plants. JcAP1 was expressed in inflorescence buds, flower buds, sepals and petals. The highest expression level was observed during the early developmental stage of the flower buds. The overexpression of JcAP1 using the cauliflower mosaic virus (CaMV) 35S promoter resulted in extremely early flowering and abnormal flowers in transgenic Arabidopsis plants. Several flowering genes downstream of AP1 were up-regulated in the JcAP1-overexpressing transgenic plant lines. Furthermore, JcAP1 overexpression rescued the phenotype caused by the Arabidopsis AP1 loss-of-function mutant ap1-11. Therefore, JcAP1 is an ortholog of AtAP1, which plays a similar role in the regulation of flowering in Arabidopsis. However, the overexpression of JcAP1 in Jatropha using the same promoter resulted in little variation in the flowering time and floral organs, indicating that JcAP1 may be insufficient to regulate flowering by itself in Jatropha. This study helps to elucidate the function of JcAP1 and contributes to the understanding of the molecular mechanisms of flower development in Jatropha. PMID:27168978

  14. Truncated and Helix-Constrained Peptides with High Affinity and Specificity for the cFos Coiled-Coil of AP-1

    PubMed Central

    Rao, Tara; Ruiz-Gómez, Gloria; Hill, Timothy A.; Hoang, Huy N.; Fairlie, David P.; Mason, Jody M.

    2013-01-01

    Protein-based therapeutics feature large interacting surfaces. Protein folding endows structural stability to localised surface epitopes, imparting high affinity and target specificity upon interactions with binding partners. However, short synthetic peptides with sequences corresponding to such protein epitopes are unstructured in water and promiscuously bind to proteins with low affinity and specificity. Here we combine structural stability and target specificity of proteins, with low cost and rapid synthesis of small molecules, towards meeting the significant challenge of binding coiled coil proteins in transcriptional regulation. By iteratively truncating a Jun-based peptide from 37 to 22 residues, strategically incorporating i→i+4 helix-inducing constraints, and positioning unnatural amino acids, we have produced short, water-stable, α-helical peptides that bind cFos. A three-dimensional NMR-derived structure for one peptide (24) confirmed a highly stable α-helix which was resistant to proteolytic degradation in serum. These short structured peptides are entropically pre-organized for binding with high affinity and specificity to cFos, a key component of the oncogenic transcriptional regulator Activator Protein-1 (AP-1). They competitively antagonized the cJun–cFos coiled-coil interaction. Truncating a Jun-based peptide from 37 to 22 residues decreased the binding enthalpy for cJun by ∼9 kcal/mol, but this was compensated by increased conformational entropy (TΔS ≤7.5 kcal/mol). This study demonstrates that rational design of short peptides constrained by α-helical cyclic pentapeptide modules is able to retain parental high helicity, as well as high affinity and specificity for cFos. These are important steps towards small antagonists of the cJun-cFos interaction that mediates gene transcription in cancer and inflammatory diseases. PMID:23544065

  15. Suppression of acetylpolyamine oxidase by selected AP-1 members regulates DNp73 abundance: mechanistic insights for overcoming DNp73-mediated resistance to chemotherapeutic drugs.

    PubMed

    Bunjobpol, W; Dulloo, I; Igarashi, K; Concin, N; Matsuo, K; Sabapathy, K

    2014-08-01

    Enhanced resistance to chemotherapy has been correlated with high levels of Delta-Np73 (DNp73), an anti-apoptotic protein of the p53 tumor-suppressor family which inhibits the pro-apoptotic members such as p53 and TAp73. Although genotoxic drugs have been shown to induce DNp73 degradation, lack of mechanistic understanding of this process precludes strategies to enhance the targeting of DNp73 and improve treatment outcomes. Antizyme (Az) is a mediator of ubiquitin-independent protein degradation regulated by the polyamine biosynthesis pathway. We show here that acetylpolyamine oxidase (PAOX), a catabolic enzyme of this pathway, upregulates DNp73 levels by suppressing its degradation via the Az pathway. Conversely, downregulation of PAOX activity by siRNA-mediated knockdown or chemical inhibition leads to DNp73 degradation in an Az-dependent manner. PAOX expression is suppressed by several genotoxic drugs, via selected members of the activator protein-1 (AP-1) transcription factors, namely c-Jun, JunB and FosB, which are required for stress-mediated DNp73 degradation. Finally, chemical- and siRNA-mediated inhibition of PAOX significantly reversed the resistant phenotype of DNp73-overexpressing cancer cells to genotoxic drugs. Together, these data define a critical mechanism for the regulation of DNp73 abundance, and reveal that inhibition of PAOX could widen the therapeutic index of cytotoxic drugs and overcome DNp73-mediated chemoresistance in tumors. PMID:24722210

  16. Stromelysin-3 induction and interstitial collagenase repression by retinoic acid. Therapeutical implication of receptor-selective retinoids dissociating transactivation and AP-1-mediated transrepression.

    PubMed

    Guérin, E; Ludwig, M G; Basset, P; Anglard, P

    1997-04-25

    Human stromelysin-3 and interstitial collagenase are matrix metalloproteinases whose expression by stromal cells in several types of carcinomas has been associated with cancer progression. We compared here the regulation of the expression of both proteinases by retinoids in human fibroblasts. Physiological concentrations of retinoic acid were found to simultaneously induce stromelysin-3 and repress interstitial collagenase. In both cases, the involvement of a transcriptional mechanism was supported by run-on assays. Furthermore, in transient transfection experiments, the activity of the stromelysin-3 promoter was induced by retinoic acid through endogenous receptors acting on a DR1 retinoic acid-responsive element. The ligand-dependent activation of the receptors was also investigated by using selective synthetic retinoids, and we demonstrated that retinoic acid-retinoid X receptor heterodimers were the most potent functional units controlling both stromelysin-3 induction and interstitial collagenase repression. However, specific retinoids dissociating the transactivation and the AP-1-mediated transrepression functions of the receptors were found to repress interstitial collagenase without inducing stromelysin-3. These findings indicate that such retinoids may represent efficient inhibitors of matrix metalloproteinase expression in the treatment of human carcinomas. PMID:9111003

  17. The Positive Transcription Elongation Factor b Is an Essential Cofactor for the Activation of Transcription by Myocyte Enhancer Factor 2

    PubMed Central

    Nojima, Masanori; Huang, Yehong; Tyagi, Mudit; Kao, Hung-Ying; Fujinaga, Koh

    2014-01-01

    The positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 and cyclin T1, stimulates the elongation of transcription by hyperphosphorylating the C-terminal region of RNA polymerase II. Aberrant activation of P-TEFb results in manifestations of cardiac hypertrophy in mice, suggesting that P-TEFb is an essential factor for cardiac myocyte function and development. Here, we present evidence that P-TEFb selectively activates transcription mediated by the myocyte enhancer factor 2 (MEF2) family of transcription factors, key regulatory factors for myocyte development. Knockdown of endogenous cyclin T1 in murine C2C12 cells abolishes MEF2-dependent reporter gene expression as well as transcription of endogenous MEF2 target genes, whereas overexpression of P-TEFb enhances MEF2-dependent transcription. P-TEFb interacts with MEF2 both in vitro and in vivo. Activation of MEF2-dependent transcription induced by serum starvation is mediated by a rapid dissociation of P-TEFb from its inhibitory subunit, HEXIM1, and a subsequent recruitment of P-TEFb to MEF2 binding sites in the promoter region of MEF2 target genes. These results indicate that recruitment of P-TEFb is a critical step for stimulation of MEF2-dependent transcription, therefore providing a fundamentally important regulatory mechanism underlying the transcriptional program in muscle cells. PMID:18662700

  18. AP1S3 is required for hepatitis C virus infection by stabilizing E2 protein.

    PubMed

    Li, Xiang; Niu, Yuqiang; Cheng, Min; Chi, Xiaojing; Liu, Xiuying; Yang, Wei

    2016-07-01

    Hepatitis C virus (HCV) infects 130 million people worldwide and is a leading cause of liver cirrhosis, end-stage liver disease and hepatocellular carcinoma. The interactions between viral elements and host factors play critical role on HCV invade, replication and release. Here, we identified adaptor protein complex 1 sigma 3 subunit (AP1S3) as a dependency factor for the efficient HCV infection in hepatoma cells. AP1S3 silencing in cultivated Huh7.5.1 cells significantly reduced the production of HCV progeny particles. Immunoprecipitation analysis revealed that AP1S3 interacted with the HCV E2 protein. With this interaction, AP1S3 could protect HCV E2 from ubiquitin-mediated proteasomal degradation. Using in vivo ubiquitylation assay, we identified that E6-Associated Protein (E6AP) was associated with HCV E2. In addition, treatment with synthetic peptide that contains the AP1S3-recognized motif inhibited HCV infection in Huh7.5.1 cells. Our data reveal AP1 as a novel host network that is required by viruses during infection and provides a potential target for developing broad-spectrum anti-virus strategies. PMID:27079945

  19. The Role of the Clathrin Adaptor AP-1: Polarized Sorting and Beyond

    PubMed Central

    Nakatsu, Fubito; Hase, Koji; Ohno, Hiroshi

    2014-01-01

    The selective transport of proteins or lipids by vesicular transport is a fundamental process supporting cellular physiology. The budding process involves cargo sorting and vesicle formation at the donor membrane and constitutes an important process in vesicular transport. This process is particularly important for the polarized sorting in epithelial cells, in which the cargo molecules need to be selectively sorted and transported to two distinct destinations, the apical or basolateral plasma membrane. Adaptor protein (AP)-1, a member of the AP complex family, which includes the ubiquitously expressed AP-1A and the epithelium-specific AP-1B, regulates polarized sorting at the trans-Golgi network and/or at the recycling endosomes. A growing body of evidence, especially from studies using model organisms and animals, demonstrates that the AP-1-mediated polarized sorting supports the development and physiology of multi-cellular units as functional organs and tissues (e.g., cell fate determination, inflammation and gut immune homeostasis). Furthermore, a possible involvement of AP-1B in the pathogenesis of human diseases, such as Crohn’s disease and cancer, is now becoming evident. These data highlight the significant contribution of AP-1 complexes to the physiology of multicellular organisms, as master regulators of polarized sorting in epithelial cells. PMID:25387275

  20. CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection

    PubMed Central

    Pérez-Núñez, Daniel; García-Urdiales, Eduardo; Martínez-Bonet, Marta; Nogal, María L.; Barroso, Susana; Revilla, Yolanda; Madrid, Ricardo

    2015-01-01

    African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity. PMID:25915900

  1. Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling.

    PubMed

    Kwon, Gyoo Taik; Cho, Han Jin; Chung, Won-Yoon; Park, Kwang-Kyun; Moon, Aree; Park, Jung Han Yoon

    2009-09-01

    Isoliquiritigenin (ISL, 4,2',4'-trihydroxychalcone), which is found in licorice, shallot and bean sprouts, is a potent antioxidant with anti-inflammatory and anti-carcinogenic effects. The purpose of this study was to investigate the effects of ISL treatment on the migration, invasion and adhesion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0-20 micromol/L ISL with or without 10 microg/L epidermal growth factor (EGF). ISL inhibited basal and EGF-induced cell migration, invasion and adhesion dose dependently. ISL decreased EGF-induced secretion of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF), but increased TIMP-2 secretion in a concentration-dependent manner. In addition, ISL decreased the protein levels of integrin-alpha2, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), and mRNA levels of uPA, MMP-9, VEGF, ICAM and integrin-alpha2. Furthermore, basal and EGF-induced activator protein (AP)-1 binding activity and phosphorylation of Jun N-terminal kinase (JNK), c-Jun and Akt were decreased after ISL treatment. However, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase was not altered. The JNK inhibitor SP600125 inhibited basal and EGF-induced secretion of uPA, VEGF, MMP-9 and TIMP-1, as well as AP-1 DNA binding activity and cell migration. These results provide evidence for the role of ISL as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of prostate cancer cells. The inhibition of JNK/AP-1 signaling may be one of the mechanisms by which ISL inhibits cancer cell invasion and migration. PMID:18824345

  2. Negative transcriptional regulation of the interferon-gamma promoter by glucocorticoids and dominant negative mutants of c-Jun.

    PubMed

    Cippitelli, M; Sica, A; Viggiano, V; Ye, J; Ghosh, P; Birrer, M J; Young, H A

    1995-05-26

    Interferon-gamma (IFN-gamma) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN-gamma gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN-gamma promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN-gamma promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN-gamma promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1 CREB-ATF factors were able to block the glucocorticoid effect. These results suggest a model involving the inhibition of IFN-gamma AP-1 CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN-gamma gene expression and support the relevance of AP-1 CREB-ATF binding factors during the transcriptional activation of the IFN-gamma promoter in T cells. PMID:7759501

  3. Status of APS 1-Mwe Parabolic Trough Project

    SciTech Connect

    Canada, S.; Brosseau, D.; Kolb, G.; Moore, L.; Cable, R.; Price, H.

    2005-11-01

    Arizona Public Service (APS) is currently installing new power facilities to generate a portion of its electricity from solar resources that will satisfy its obligation under the Arizona Environmental Portfolio Standard (EPS). During FY04, APS began construction on a 1-MWe parabolic trough concentrating solar power plant. This plant represents the first parabolic trough plant to begin construction since 1991. Site preparation and construction activities continued throughout much of FY05, and startup activities are planned for Fall 2005 (with completion early in FY06). The plant will be the first commercial deployment of the Solargenix parabolic trough collector technology developed under contract to the National Renewable Energy Laboratory. The plant will use an organic Rankine cycle (ORC) power plant, provided by Ormat. The ORC power plant is much simpler than the conventional steam Rankine cycle plant and allows unattended operation of the facility.

  4. Regulation of human alcohol dehydrogenase gene ADH7: importance of an AP-1 site.

    PubMed

    Kotagiri, S; Edenberg, H J

    1998-07-01

    The structure and function of the human alcohol dehydrogenase 7 (ADH7) promoter were analyzed. A promoter fragment extending to bp -232 functioned well in H4IIE-C3, CV-1, and HeLa cells, whereas the region extending further upstream to bp -799 had no significant effect on activity. We identified cis-acting elements in the proximal 232 bp and examined their effect on promoter activity. Mutation of site A, where c-Jun bound, caused a drastic decrease in the promoter activity in H4IIE-C3 and CV-1 cells, suggesting that AP-1 plays an important role in the regulation of ADH7. Mutation of site B also caused a large drop in promoter activity in both cell lines; C/EBPalpha can bind to this site, but because the site affects activity approximately equally in CV-1 cells that lack C/EBPalpha and in H4IIE-C3 cells that contain low levels, other proteins are likely to play the major roles in vivo. Mutation of site C, where C/EBP bound and c-Jun bound weakly, had different effects in the two cell lines: in H4IIE-C3 cells, the site C mutation did not significantly increase promoter activity, whereas in CV-1 cells, which lack C/EBPalpha, it led to a doubling of activity. Surprisingly, cotransfection of the wild-type promoter with C/EBPa or C/EBPbeta led to a decrease in promoter activity, which might in part explain the lack of activity of ADH7 in adult liver. PMID:9703017

  5. Protein-DNA array-based identification of transcription factor activities differentially regulated in skeletal muscle of normal and dystrophin-deficient mdx mice.

    PubMed

    Dogra, Charu; Srivastava, Daya Shankar; Kumar, Ashok

    2008-05-01

    Inactivation of dystrophin gene is the primary cause of Duchenne muscular dystrophy (DMD) in humans and mdx mice. However, the underpinning mechanisms, which govern the pathogenesis of dystrophin-deficient skeletal muscle, remain poorly understood. We have previously reported activation of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-kappaB), and phosphatidyl-inositol 3-kinase/Akt (PI3K/Akt) signaling pathways in diaphragm muscle of mdx mice. In this study, using a protein-DNA array-based approach, we have investigated the activation of 345 transcription factors in diaphragm muscle of 6-week old normal and dystrophin-deficient mdx mice. Our data demonstrate increased activation of a number nuclear transcription factors including AP1, HFH-3, PPARalpha, c.myb BP, ETF, Fra-1/JUN, kBF-A, N-rasBP, lactoferrin BP, Myb(2), EBP40_45, EKLF(1), p53(2), TFEB, Myc-Max; c-Rel; E2, ISRE; NF-kB; Stat1 p84/p91, Antioxidant RE, EVI-1, Stat3, AP3, p53, Stat4, AP4, HFH-1, FAST-1, Pax-5, and Beta-RE in the diaphragm muscle of mdx mice compared to corresponding normal mice. The level of activation for p53 was highest among all the transcription factors studied. Furthermore, higher activation of p53 in diaphragm muscle of mdx mice was associated with its increased phosphorylation and nuclear translocation. Collectively, our data suggest that the primary deficiency of dystrophin leads to the aberrant activation of nuclear transcription factors which might further contribute to muscle pathogenesis in mdx mice. PMID:18278580

  6. A new type of NtrC transcriptional activator.

    PubMed Central

    Foster-Hartnett, D; Cullen, P J; Monika, E M; Kranz, R G

    1994-01-01

    The enteric NtrC (NRI) protein has been the paradigm for a class of bacterial enhancer-binding proteins (EBPs) that activate transcription of RNA polymerase containing the sigma 54 factor. Activators in the NtrC class are characterized by essentially three properties: (i) they bind to sites distant from the promoters that they activate (> 100 bp upstream of the transcriptional start site), (ii) they contain a conserved nucleotide-binding fold and exhibit ATPase activity that is required for activation, and (iii) they activate the sigma 54 RNA polymerase. We have characterized the NtrC protein from a photosynthetic bacterium, Rhodobacter capsulatus, which represents a metabolically versatile group of bacteria found in aquatic environments. We have shown that the R. capsulatus NtrC protein (RcNtrC) binds to two tandem sites that are distant from promoters that it activates, nifA1 and nifA2. These tandem binding sites are shown to be important for RcNtrC-dependent nitrogen regulation in vivo. Moreover, the conserved nucleotide-binding fold of RcNtrC is required to activate nifA1 and nifA2 but is not required for DNA binding of RcNtrC to upstream activation sequences. However, nifA1 and nifA2 genes do not require the sigma 54 for activation and do not contain the highly conserved nucleotides that are present in all sigma 54-type, EBP-activated promoters. Thus, the NtrC from this photosynthetic bacterium represents a novel member of the class of bacterial EBPs. It is probable that this class of EBPs is more versatile in prokaryotes than previously envisioned. Images PMID:7928986

  7. Promoter occlusion: transcription through a promoter may inhibit its activity.

    PubMed

    Adhya, S; Gottesman, M

    1982-07-01

    Induction of prophage lambda inhibits the expression of the gal operon from its cognate promoters. The effect is observed only in cis, and is due to frequent transcription of the gal promoter region by RNA polymerase molecules initiating upstream at the prophage PL promoter. The frequency of transcription initiation at PL is some 30 times greater than that at the gal promoter, Pg1. PL is one of the strongest procaryotic promoters. This "promoter occlusion" is essentially complete when the distance between gal and PL is small (less than or equal to 10 kb); and when PL is fully active (that is, in the absence of the cl or cro repressors). We discuss the possibility that promoter occlusion at two lambda promoters, Pint and PR', might play a role in the sequential expression of viral functions. PMID:6217898

  8. Identification of novel transcriptional regulators involved in macrophage differentiation and activation in U937 cells

    PubMed Central

    Baek, Young-Sook; Haas, Stefan; Hackstein, Holger; Bein, Gregor; Hernandez-Santana, Maria; Lehrach, Hans; Sauer, Sascha; Seitz, Harald

    2009-01-01

    Background Monocytes and macrophages play essential role in innate immunity. Understanding the underlying mechanism of macrophage differentiation and the identification of regulatory mechanisms will help to find new strategies to prevent their harmful effects in chronic inflammatory diseases and sepsis. Results Maturation of blood monocytes into tissue macrophages and subsequent inflammatory response was mimicked in U937 cells of human histocytic lymphoma origin. Whole genome array analysis was employed to evaluate gene expression profile to identify underlying transcriptional networks implicated during the processes of differentiation and inflammation. In addition to already known transcription factors (i.e. MAFB, EGR, IRF, BCL6, NFkB, AP1, Nur77), gene expression analysis further revealed novel genes (i.e. MEF2, BRI, HLX, HDAC5, H2AV, TCF7L2, NFIL3) previously uncharacterized to be involved in the differentiation process. A total of 58 selected genes representing cytokines, chemokines, surface antigens, signaling molecules and transcription factors were validated by real time PCR and compared to primary monocyte-derived macrophages. Beside the verification of several new genes, the comparison reveals individual heterogeneity of blood donors. Conclusion Up regulation of MEF2 family, HDACs, and H2AV during cell differentiation and inflammation sheds new lights onto regulation events on transcriptional and epigenetic level controlling these processes. Data generated will serve as a source for further investigation of macrophages differentiation pathways and related biological responses. PMID:19341462

  9. Genome-wide activities of Polycomb complexes control pervasive transcription.

    PubMed

    Lee, Hun-Goo; Kahn, Tatyana G; Simcox, Amanda; Schwartz, Yuri B; Pirrotta, Vincenzo

    2015-08-01

    Polycomb group (PcG) complexes PRC1 and PRC2 are well known for silencing specific developmental genes. PRC2 is a methyltransferase targeting histone H3K27 and producing H3K27me3, essential for stable silencing. Less well known but quantitatively much more important is the genome-wide role of PRC2 that dimethylates ∼70% of total H3K27. We show that H3K27me2 occurs in inverse proportion to transcriptional activity in most non-PcG target genes and intergenic regions and is governed by opposing roaming activities of PRC2 and complexes containing the H3K27 demethylase UTX. Surprisingly, loss of H3K27me2 results in global transcriptional derepression proportionally greatest in silent or weakly transcribed intergenic and genic regions and accompanied by an increase of H3K27ac and H3K4me1. H3K27me2 therefore sets a threshold that prevents random, unscheduled transcription all over the genome and even limits the activity of highly transcribed genes. PRC1-type complexes also have global roles. Unexpectedly, we find a pervasive distribution of histone H2A ubiquitylated at lysine 118 (H2AK118ub) outside of canonical PcG target regions, dependent on the RING/Sce subunit of PRC1-type complexes. We show, however, that H2AK118ub does not mediate the global PRC2 activity or the global repression and is predominantly produced by a new complex involving L(3)73Ah, a homolog of mammalian PCGF3. PMID:25986499

  10. Human T-cell leukemia virus type 1 Tax activates transcription of the human fra-1 gene through multiple cis elements responsive to transmembrane signals.

    PubMed Central

    Tsuchiya, H; Fujii, M; Niki, T; Tokuhara, M; Matsui, M; Seiki, M

    1993-01-01

    We have shown that Tax1 of human T-cell leukemia virus type 1 stimulates the expression of several cellular immediate-early genes (M. Fujii, T. Niki, T. Mori, T. Matsuda, M. Matsui, N. Nomura, and M. Seiki, Oncogene 6:1023-1029, 1991). In this study, the 5'-flanking region of the human fra-1 gene, which is a Tax1-inducible fos-related gene, was isolated and Tax1 or serum-responsive cis elements were analyzed to obtain further insight into the mechanism of Tax1 action. The 62-bp sequence starting 46 nucleotides upstream from the translation initiation site showed 71% homology with the sequence surrounding the TATA box of the c-fos promoter. Regulatory motifs identified in the c-fos promoter, such as an Ets-binding site, E boxes, a CArG box, c-fos AP-1 sites, and two retinoblastoma control elements, were also found upstream of the c-fos homology region. A 502-bp fragment containing these motifs mediated transcriptional activation by Tax1 or by serum in a transient transfection assay. Three independent Tax1-responsive regions (TRRs) were identified, and mutations in each revealed that one of the retinoblastoma control elements in TRR1 and the c-fos AP-1 sites in TRR2 and TRR3 were essential for the activation. Although TRR2 contains a CArG box-like sequence, it was a weak binding site for p67SRF, if it bound at all, and was not required for activation. All three TRRs could also mediate the signals stimulated by serum. Thus, Tax1 appears to activate fra-1 gene expression by means of a part of the cellular machinery similar to that which mediates growth signals. Images PMID:8230424

  11. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

    PubMed Central

    Naranjo, José R.; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M.; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C.; Arrabal, María D.; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-01-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  12. Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease.

    PubMed

    Naranjo, José R; Zhang, Hongyu; Villar, Diego; González, Paz; Dopazo, Xose M; Morón-Oset, Javier; Higueras, Elena; Oliveros, Juan C; Arrabal, María D; Prieto, Angela; Cercós, Pilar; González, Teresa; De la Cruz, Alicia; Casado-Vela, Juan; Rábano, Alberto; Valenzuela, Carmen; Gutierrez-Rodriguez, Marta; Li, Jia-Yi; Mellström, Britt

    2016-02-01

    Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HD. PMID:26752648

  13. Visualization of Estrogen Receptor Transcriptional Activation in Zebrafish

    PubMed Central

    Halpern, Marnie E.

    2011-01-01

    Estrogens regulate a diverse range of physiological processes and affect multiple tissues. Estrogen receptors (ERs) regulate transcription by binding to DNA at conserved estrogen response elements, and such elements have been used to report ER activity in cultured cells and in transgenic mice. We generated stable, transgenic zebrafish containing five consecutive elements upstream of a c-fos minimal promoter and green fluorescent protein (GFP) to visualize and quantify transcriptional activation in live larvae. Transgenic larvae show robust, dose-dependent estrogen-dependent fluorescent labeling in the liver, consistent with er gene expression, whereas ER antagonists inhibit GFP expression. The nonestrogenic steroids dexamethasone and progesterone fail to activate GFP, confirming ER selectivity. Natural and synthetic estrogens activated the transgene with varying potency, and two chemicals, genistein and bisphenol A, preferentially induce GFP expression in the heart. In adult fish, fluorescence was observed in estrogenic tissues such as the liver, ovary, pituitary gland, and brain. Individual estrogen-responsive neurons and their projections were visualized in the adult brain, and GFP-positive neurons increased in number after 17β-estradiol exposure. The transgenic estrogen-responsive zebrafish allow ER signaling to be monitored visually and serve as in vivo sentinels for detection of estrogenic compounds. PMID:21540282

  14. Transcription activities of human papillomavirus type 11 E6 promoter-proximal elements in raft and submerged cultures of foreskin keratinocytes.

    PubMed Central

    Zhao, W; Chow, L T; Broker, T R

    1997-01-01

    Human papillomaviruses (HPVs) replicate only in differentiated squamous epithelia in warts and in epithelial raft cultures grown at the medium-air interface. Virus-encoded and host transcription factors are thought to be responsible for repressing the viral enhancer and promoter located within the upstream regulatory region (URR) in the undifferentiated basal and parabasal cells while up-regulating their activities in the differentiated spinous cells. Using recombinant retroviruses, we acutely transduced neonatal foreskin keratinocytes (PHKs) with a lacZ reporter gene driven by the wild-type URR of the low-risk HPV type 11 or by a URR with individual mutations in seven promoter-proximal elements, some of which have not been characterized previously. Beta-galactosidase activities were detected in the submerged, proliferating PHKs and also in the differentiated spinous cells, but not in the steady-state proliferating basal cells, of stratified raft cultures. In particular, mutation of an Oct1, an Sp1, or a previously unknown promoter-proximal AP1 site severely reduced the reporter activity, whereas mutation of either of two NF1 sites flanking the Oct1 site had no effect. These results demonstrate changes in cellular transcription factor profiles under different culture conditions and begin to characterize the naturally differentiation-dependent activation of the URR. They provide one molecular explanation for the patterns of HPV expression in warts and help validate epithelial raft cultures as an important experimental system for genetic dissection of HPV regulatory elements. PMID:9343243

  15. Clinical application of transcriptional activators of bile salt transporters☆

    PubMed Central

    Baghdasaryan, Anna; Chiba, Peter; Trauner, Michael

    2014-01-01

    Hepatobiliary bile salt (BS) transporters are critical determinants of BS homeostasis controlling intracellular concentrations of BSs and their enterohepatic circulation. Genetic or acquired dysfunction of specific transport systems causes intrahepatic and systemic retention of potentially cytotoxic BSs, which, in high concentrations, may disturb integrity of cell membranes and subcellular organelles resulting in cell death, inflammation and fibrosis. Transcriptional regulation of canalicular BS efflux through bile salt export pump (BSEP), basolateral elimination through organic solute transporters alpha and beta (OSTα/OSTβ) as well as inhibition of hepatocellular BS uptake through basolateral Na+-taurocholate cotransporting polypeptide (NTCP) represent critical steps in protection from hepatocellular BS overload and can be targeted therapeutically. In this article, we review the potential clinical implications of the major BS transporters BSEP, OSTα/OSTβ and NTCP in the pathogenesis of hereditary and acquired cholestatic syndromes, provide an overview on transcriptional control of these transporters by the key regulatory nuclear receptors and discuss the potential therapeutic role of novel transcriptional activators of BS transporters in cholestasis. PMID:24333169

  16. Regulating the regulators: modulators of transcription factor activity.

    PubMed

    Everett, Logan; Hansen, Matthew; Hannenhalli, Sridhar

    2010-01-01

    Gene transcription is largely regulated by DNA-binding transcription factors (TFs). However, the TF activity itself is modulated via, among other things, post-translational modifications (PTMs) by specific modification enzymes in response to cellular stimuli. TF-PTMs thus serve as "molecular switchboards" that map upstream signaling events to the downstream transcriptional events. An important long-term goal is to obtain a genome-wide map of "regulatory triplets" consisting of a TF, target gene, and a modulator gene that specifically modulates the regulation of the target gene by the TF. A variety of genome-wide data sets can be exploited by computational methods to obtain a rough map of regulatory triplets, which can guide directed experiments. However, a prerequisite to developing such computational tools is a systematic catalog of known instances of regulatory triplets. We first describe PTM-Switchboard, a recent database that stores triplets of genes such that the ability of one gene (the TF) to regulate a target gene is dependent on one or more PTMs catalyzed by a third gene, the modifying enzyme. We also review current computational approaches to infer regulatory triplets from genome-wide data sets and conclude with a discussion of potential future research. PTM-Switchboard is accessible at http://cagr.pcbi.upenn.edu/PTMswitchboard / PMID:20827600

  17. Deletion mutants of AP-1 adaptin subunits display distinct phenotypes in fission yeast.

    PubMed

    Ma, Yan; Takeuchi, Mai; Sugiura, Reiko; Sio, Susie O; Kuno, Takayoshi

    2009-08-01

    Adaptins are subunits of the heterotetrameric (beta/mu/gamma/sigma) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (beta), Apl4 (gamma), Apm1 (mu) and Aps1 (sigma)] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the Deltaapm1 and Deltaapl2 mutants displayed similar but more severe phenotypes than those of Deltaaps1 or Deltaapl4 mutants. Furthermore, the Deltaapl2Deltaaps1 and Deltaapl2Deltaapl4 double mutants displayed synthetic growth defects, whereas the Deltaaps1Deltaapl4 and Deltaapl2Deltaapm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes. PMID:19624755

  18. A Temporal Gate for Viral Enhancers to Co-opt Toll-Like-Receptor Transcriptional Activation Pathways upon Acute Infection

    PubMed Central

    Kropp, Kai A.; Hsieh, Wei Yuan; Isern, Elena; Forster, Thorsten; Krause, Eva; Brune, Wolfram; Angulo, Ana; Ghazal, Peter

    2015-01-01

    series of pharmacologic, siRNA and genetic loss-of-function experiments we determined that signalling mediated by the TLR-adaptor protein MyD88 plays a vital role for governing the inflammatory activation of the CMV enhancer in macrophages. Downstream TLR-regulated transcription factor binding motif disruption for NFκB, AP1 and CREB/ATF in the CMV enhancer demonstrated the requirement of these inflammatory signal-regulated elements in driving viral gene expression and growth in cells as well as in primary infection of neonatal mice. Thus, this study shows that the prototypical CMV enhancer, in a restricted time-gated manner, co-opts through DNA regulatory mimicry elements, innate-immune transcription factors to drive viral expression and replication in the face of on-going pro-inflammatory antiviral responses in vitro and in vivo and; suggests an unexpected role for inflammation in promoting acute infection and has important future implications for regulating latency. PMID:25856589

  19. Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1.

    PubMed

    Weekes, D; Kashima, T G; Zandueta, C; Perurena, N; Thomas, D P; Sunters, A; Vuillier, C; Bozec, A; El-Emir, E; Miletich, I; Patiño-Garcia, A; Lecanda, F; Grigoriadis, A E

    2016-06-01

    Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant owing to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signalling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 (fibroblast growth factor receptor 1) was identified as a novel c-Fos/activator protein-1(AP-1)-regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of mitogen-activated protein kinases (MAPKs), morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1-silenced osteosarcoma cells caused a marked twofold to fivefold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small-molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus deregulated FGFR signalling has an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy. PMID:26387545

  20. Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

    PubMed Central

    Cerqueira, Gustavo M.; Souza, Natalie M.; Araújo, Eduardo R.; Barros, Aline T.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Nascimento, Ana L. T. O.

    2011-01-01

    Background Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. Methodology and Principal Findings A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. Conclusions The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa. PMID:21445252

  1. Distinct TFIID complexes mediate the effect of different transcriptional activators.

    PubMed Central

    Brou, C; Chaudhary, S; Davidson, I; Lutz, Y; Wu, J; Egly, J M; Tora, L; Chambon, P

    1993-01-01

    Multiple chromatographically separable complexes containing the TATA binding protein (TBP), which exhibit different functional properties, exist in HeLa cells. At least three distinct subpopulations of such complexes can be functionally defined as TFIID since they function with RNA polymerase II. Using a partially reconstituted HeLa cell in vitro transcription system and immunoprecipitation with a monoclonal antibody directed against TBP, we show that stimulation of transcription by the chimeric activators GAL-VP16, GAL-TEF-1 and GAL-ER(EF) requires the presence of factors which are tightly associated with these TFIID complexes. Moreover, the activity of GAL-TEF-1 appears to be mediated by at least two chromatographically distinct populations of TFIID. The factor(s) associated with one of these populations is also required for the activity of GAL-ER (EF) and GAL-VP16, while the factor(s) associated with the other population functions selectively with GAL-TEF-1. These two TFIID populations are composed of both common and unique TBP associated factors (TAFs). Images PMID:8440239

  2. The lipopolysaccharide-binding protein is a secretory class 1 acute-phase protein whose gene is transcriptionally activated by APRF/STAT/3 and other cytokine-inducible nuclear proteins.

    PubMed Central

    Schumann, R R; Kirschning, C J; Unbehaun, A; Aberle, H P; Knope, H P; Lamping, N; Ulevitch, R J; Herrmann, F

    1996-01-01

    Acute-phase reactants (APRs) are proteins synthesized in the liver following induction by interleukin-1 (IL-1), IL-6, and glucocorticoids, involving transcriptional gene activation. Lipopolysaccharide-binding protein (LBP) is a recently identified hepatic secretory protein potentially involved in the pathogenesis of sepsis, capable of binding the bacterial cell wall product endotoxin and directing it to its cellular receptor, CD14. In order to examine the transcriptional induction mechanisms by which the LBP gene is activated, we have investigated the regulation of expression of its mRNA in vitro and in vivo as well as the organization of 5' upstream regulatory DNA sequences. We show that induction of LBP expression is transcriptionally regulated and is dependent on stimulation with IL-1beta, IL-6, and dexamethasone. By definition, LBP thus has to be viewed as a class 1 acute-phase protein and represents the first APR identified which is capable of detecting pathogenic bacteria. Furthermore, cloning of the LBP promoter revealed the presence of regulatory elements, including the common APR promoter motif APRE/STAT-3 (acute-phase response element/signal transducer and activator of transcription 3). Luciferase reporter gene assays utilizing LBP promoter truncation and point mutation variants indicated that transcriptional activation of the LBP gene required a functional APRE/STAT-3 binding site downstream of the transcription start site, as well as an AP-1 and a C/EBP (CCAAT enhancer-binding protein) binding site. Gel retardation and supershift assays confirmed that upon cytokine stimulation APRF/STAT-3 binds to its recognition site, leading to strong activation of the LBP gene. Unraveling of the mechanism of transcriptional activation of the LBP gene, involving three known transcription factors, may contribute to our understanding of the acute-phase response and the pathophysiology of sepsis and septic shock. PMID:8668165

  3. Anti-Inflammatory Effect of Apigenin on LPS-Induced Pro-Inflammatory Mediators and AP-1 Factors in Human Lung Epithelial Cells.

    PubMed

    Patil, Rajeshwari H; Babu, R L; Naveen Kumar, M; Kiran Kumar, K M; Hegde, Shubha M; Nagesh, Rashmi; Ramesh, Govindarajan T; Sharma, S Chidananda

    2016-02-01

    Apigenin is one of the plant flavonoids present in fruits and vegetables, acting as an important nutraceutical component. It is recognized as a potential antioxidant, antimicrobial, and anti-inflammatory molecule. In the present study, the mechanism of anti-inflammatory action of apigenin on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and activator protein-1 (AP-1) factors in human lung A549 cells was investigated. The anti-inflammatory activity of apigenin on LPS-induced inflammation was determined by analyzing the expression of pro-inflammatory cytokines, nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and different AP-1 factors. Apigenin significantly inhibited the LPS-induced expression of iNOS, COX-2, expression of pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IL-8, and TNF-α), and AP-1 proteins (c-Jun, c-Fos, and JunB) including nitric oxide production. Study confirms the anti-inflammatory effect of apigenin by inhibiting the expression of inflammatory mediators and AP-1 factors involved in the inflammation and its importance in the treatment of lung inflammatory diseases. PMID:26276128

  4. Dynamic Mechanism for the Transcription Apparatus Orchestrating Reliable Responses to Activators

    NASA Astrophysics Data System (ADS)

    Wang, Yaolai; Liu, Feng; Wang, Wei

    2012-05-01

    The transcription apparatus (TA) is a huge molecular machine. It detects the time-varying concentrations of transcriptional activators and initiates mRNA transcripts at appropriate rates. Based on the general structural organizations of the TA, we propose how the TA dynamically orchestrates transcriptional responses. The activators rapidly cycle in and out of a clamp-like space temporarily formed between the enhancer and the Mediator, with the concentration of activators encoded as their temporal occupancy rate (RTOR) within the space. The entry of activators into this space induces allostery in the Mediator, resulting in a facilitated circumstance for transcriptional reinitiation. The reinitiation rate is much larger than the cycling rate of activators, thereby RTOR guiding the amount of transcripts. Based on this mechanism, stochastic simulations can qualitatively reproduce and interpret multiple features of gene expression, e.g., transcriptional bursting is not mere noise as traditionally believed, but rather the basis of reliable transcriptional responses.

  5. Diallyl disulfide and diallyl trisulfide up-regulate the expression of the pi class of glutathione S-transferase via an AP-1-dependent pathway.

    PubMed

    Tsai, Chia-Wen; Chen, Haw-Wen; Yang, Jaw-Ji; Sheen, Lee-Yan; Lii, Chong-Kuei

    2007-02-01

    Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I. PMID:17263507

  6. The Unfolded Protein Response and the Phosphorylations of Activating Transcription Factor 2 in the trans-Activation of il23a Promoter Produced by β-Glucans*

    PubMed Central

    Rodríguez, Mario; Domingo, Esther; Alonso, Sara; Frade, Javier García; Eiros, José; Crespo, Mariano Sánchez; Fernández, Nieves

    2014-01-01

    Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP), X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities. PMID:24982422

  7. Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1

    PubMed Central

    Weekes, Daniel; Zandueta, Carolina; Perurena, Naiara; Thomas, David P; Sunters, Andrew; Vuillier, Céline; Bozec, Aline; El-Emir, Ethaar; Miletich, Isabelle; Patiño-Garcia, Ana; Lecanda, Fernando; Grigoriadis, Agamemnon E

    2015-01-01

    Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant due to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signaling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 was identified as a novel c-Fos/AP-1 regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of MAPKs, morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1 silenced osteosarcoma cells caused a marked 2- to 5-fold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus, deregulated FGFR signalling plays an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy. PMID:26387545

  8. Macrophage-derived BAFF induces AID expression through the p38MAPK/CREB and JNK/AP-1 pathways.

    PubMed

    Kim, Hyun-A; Seo, Goo-Young; Kim, Pyeung-Hyeun

    2011-03-01

    BAFF is expressed primarily by macrophages and DCs. BAFF stimulates the differentiation and survival of B cells and induces Ig production. We have demonstrated previously that murine macrophages treated with TGF-β1 or IFN-γ express membrane-bound and soluble forms of BAFF. The ability of these two forms of BAFF to induce expression of AID, which plays a critical role in Ig CSR in B cells, was investigated. Both forms of BAFF, derived from macrophages activated by IFN-γ or TGF-β1, can increase AID expression. Subsequent analysis of BAFF signaling suggested that BAFF induces AID through BCMA, a BAFF-receptor, and p38MAPK and CREB act as intermediates in AID expression. In addition, JNK and AP-1 have similar activities. Our findings suggest that macrophage-derived BAFF stimulates B cells to express AID through BCMA and at least two different pathways, including the p38MAPK/CREB and the JNK/AP-1 pathways. PMID:21169521

  9. Curcumin Nanoparticles Ameliorate ICAM-1 Expression in TNF-α-Treated Lung Epithelial Cells through p47 phox and MAPKs/AP-1 Pathways

    PubMed Central

    Yang, Chuen-Mao; Liang, Chan-Jung; Lin, Chun-Ching; Chiang, Yao-Chang; Lee, Hui-Chun; Ko, Horng-Huey; Lee, Chiang-Wen

    2013-01-01

    Upregulation of intercellular adhesion molecule-1 (ICAM-1) involves adhesions between both circulating and resident leukocytes and the human lung epithelial cells during lung inflammatory reactions. We have previously demonstrated that curcumin-loaded polyvinylpyrrolidone nanoparticles (CURN) improve the anti-inflammatory and anti-oxidative properties of curcumin in hepatocytes. In this study, we focused on the effects of CURN on the expression of ICAM-1 in TNF-α-treated lung epithelial cells and compared these to the effects of curcumin water preparation (CURH). TNF-αinduced ICAM-1 expression, ROS production, and cell-cell adhesion were significantly attenuated by the pretreatment with antioxidants (DPI, APO, or NAC) and CURN, but not by CURH, as revealed by western blot analysis, RT-PCR, promoter assay, and ROS detection and adhesion assay. In addition, treatment of TNF-α-treated cells with CURN and antioxidants also resulted in an inhibition of activation of p47 phox and phosphorylation of MAPKs, as compared to that using CURH. Our findings also suggest that phosphorylation of MAPKs may eventually lead to the activation of transcription factors. We also observed that the effects of TNF-α treatment for 30 min, which includes a significant increase in the binding activity of AP-1 and phosphorylation of c-jun and c-fos genes, were reduced by CURN treatment. In vivo studies have revealed that CURN improved the anti-inflammation activities of CURH in the lung epithelial cells of TNF-α-treated mice. Our results indicate that curcumin-loaded polyvinylpyrrolidone nanoparticles may potentially serve as an anti-inflammatory drug for the treatment of respiratory diseases. PMID:23671702

  10. The metabolic activator FOXO1 binds hepatitis B virus DNA and activates its transcription

    SciTech Connect

    Shlomai, Amir; Shaul, Yosef

    2009-04-17

    Hepatitis B virus (HBV) is a small DNA virus that targets the liver and infects humans worldwide. Recently we have shown that the metabolic regulator PGC-1{alpha} coactivates HBV transcription thereby rendering the virus susceptible to fluctuations in the nutritional status of the liver. PGC-1{alpha} coactivation of HBV is mediated through the liver-enriched nuclear receptor HNF4{alpha} and through another yet unknown transcription factor(s). Here we show that the forkhead transcription factor FOXO1, a known target for PGC-1{alpha} coactivation and a central mediator of glucose metabolism in the liver, binds HBV core promoter and activates its transcription. This activation is further enhanced in the presence of PGC-1{alpha}, implying that FOXO1 is a target for PGC-1{alpha} coactivation of HBV transcription. Thus, our results identify another key metabolic regulator as an activator of HBV transcription, thereby supporting the principle that HBV gene expression is regulated in a similar way to key hepatic metabolic genes.

  11. A Role for APETALA1/FRUITFULL Transcription Factors in Tomato Leaf Development[C][W

    PubMed Central

    Burko, Yogev; Shleizer-Burko, Sharona; Yanai, Osnat; Shwartz, Ido; Zelnik, Iris Daphne; Jacob-Hirsch, Jasmine; Kela, Itai; Eshed-Williams, Leor; Ori, Naomi

    2013-01-01

    Flexible maturation rates underlie part of the diversity of leaf shape, and tomato (Solanum lycopersicum) leaves are compound due to prolonged organogenic activity of the leaf margin. The CINCINNATA -TEOSINTE BRANCHED1, CYCLOIDEA, PCF (CIN-TCP) transcription factor LANCEOLATE (LA) restricts this organogenic activity and promotes maturation. Here, we show that tomato APETALA1/FRUITFULL (AP1/FUL) MADS box genes are involved in tomato leaf development and are repressed by LA. AP1/FUL expression is correlated negatively with LA activity and positively with the organogenic activity of the leaf margin. LA binds to the promoters of the AP1/FUL genes MBP20 and TM4. Overexpression of MBP20 suppressed the simple-leaf phenotype resulting from upregulation of LA activity or from downregulation of class I knotted like homeobox (KNOXI) activity. Overexpression of a dominant-negative form of MBP20 led to leaf simplification and partly suppressed the increased leaf complexity of plants with reduced LA activity or increased KNOXI activity. Tomato plants overexpressing miR319, a negative regulator of several CIN-TCP genes including LA, flower with fewer leaves via an SFT-dependent pathway, suggesting that miR319-sensitive CIN-TCPs delay flowering in tomato. These results identify a role for AP1/FUL genes in vegetative development and show that leaf and plant maturation are regulated via partially independent mechanisms. PMID:23771895

  12. In vitro transcriptional activation by a metabolic intermediate: activation by Leu3 depends on alpha-isopropylmalate.

    PubMed

    Sze, J Y; Woontner, M; Jaehning, J A; Kohlhaw, G B

    1992-11-13

    In the absence of the leucine biosynthetic precursor alpha-isopropylmalate (alpha-IPM), the yeast LEU3 protein (Leu3p) binds DNA and acts as a transcriptional repressor in an in vitro extract. Addition of alpha-IPM resulted in a dramatic increase in Leu3p-dependent transcription. The presence of alpha-IPM was also required for Leu3p to compete effectively with another transcriptional activator, GAL4/VP16, for limiting transcription factors. Therefore, the addition of alpha-IPM appears to convert a transcriptional repressor into an activator. This represents an example in eukaryotes of direct transcriptional regulation by a small effector molecule. PMID:1439822

  13. Transcriptional activation by simian virus 40 large T antigen: interactions with multiple components of the transcription complex.

    PubMed Central

    Gruda, M C; Zabolotny, J M; Xiao, J H; Davidson, I; Alwine, J C

    1993-01-01

    Simian virus 40 (SV40) large T antigen is a potent transcriptional activator of both viral and cellular promoters. Within the SV40 late promoter, a specific upstream element necessary for T-antigen transcriptional activation is the binding site for transcription-enhancing factor 1 (TEF-1). The promoter structure necessary for T-antigen-mediated transcriptional activation appears to be simple. For example, a promoter consisting of upstream TEF-1 binding sites (or other factor-binding sites) and a downstream TATA or initiator element is efficiently activated. It has been demonstrated that transcriptional activation by T antigen does not require direct binding to the DNA; thus, the most direct effect that T antigen could have on these simple promoters would be through protein-protein interactions with either upstream-bound transcription factors, the basal transcription complex, or both. To determine whether such interactions occur, full-length T antigen or segments of it was fused to the glutathione-binding site (GST fusions) or to the Gal4 DNA-binding domain (amino acids 1 to 147) (Gal4 fusions). With the GST fusions, it was found that TEF-1 and the TATA-binding protein (TBP) bound different regions of T antigen. A GST fusion containing amino acids 5 to 172 (region T1) efficiently bound TBP. TEF-1 bound neither region T1 nor a region between amino acids 168 and 373 (region T2); however, it bound efficiently to the combined region (T5) containing amino acids 5 to 383.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:8423815

  14. Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis

    PubMed Central

    Zhang, Hongjie; Kim, Ahlee; Abraham, Nessy; Khan, Liakot A.; Hall, David H.; Fleming, John T.; Gobel, Verena

    2012-01-01

    Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C. elegans intestine. We show that CHC-1/AP-1-mediated polarized transport intersects with a sphingolipid-dependent apical sorting process. Depleting each presumed trafficking component mislocalizes the same set of apical membrane molecules basolaterally, including the polarity regulator PAR-6, and generates ectopic lateral lumens. GFP::CHC-1 and BODIPY-ceramide vesicles associate perinuclearly and assemble asymmetrically at polarized plasma membrane domains in a co-dependent and AP-1-dependent manner. Based on these findings, we propose a trafficking pathway for apical membrane polarity and lumen morphogenesis that implies: (1) a clathrin/AP-1 function on an apically directed transport route; and (2) the convergence of this route with a sphingolipid-dependent apical trafficking path. PMID:22535410

  15. Necrotic cells influence migration and invasion of glioblastoma via NF-κB/AP-1-mediated IL-8 regulation

    PubMed Central

    Ahn, So-Hee; Park, Hyunju; Ahn, Young-Ho; Kim, Sewha; Cho, Min-Sun; Kang, Jihee Lee; Choi, Youn-Hee

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. Diffuse infiltration into normal brain parenchyma, rapid growth, and the presence of necrosis are remarkable hallmarks of GBM. However, the effect of necrotic cells on GBM growth and metastasis is poorly understood at present. In this study, we examined the biological significance of necrotic tissues by exploring the molecular mechanisms underlying the signaling network between necrotic tissues and GBM cells. The migration and invasion of the GBM cell line CRT-MG was significantly enhanced by treatment with necrotic cells, as shown by assays for scratch wound healing and spheroid invasion. Incubation with necrotic cells induced IL-8 secretion in CRT-MG cells in a dose-dependent manner. In human GBM tissues, IL-8 positive cells were mainly distributed in the perinecrotic region, as seen in immunohistochemistry and immunofluorescence analysis. Necrotic cells induced NF-κB and AP-1 activation and their binding to the IL-8 promoter, leading to enhanced IL-8 production and secretion in GBM cells. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the migration and invasion of GBM cells are enhanced and facilitated via NF-κB/AP-1 mediated IL-8 upregulation. PMID:27076368

  16. Necrotic cells influence migration and invasion of glioblastoma via NF-κB/AP-1-mediated IL-8 regulation.

    PubMed

    Ahn, So-Hee; Park, Hyunju; Ahn, Young-Ho; Kim, Sewha; Cho, Min-Sun; Kang, Jihee Lee; Choi, Youn-Hee

    2016-01-01

    Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. Diffuse infiltration into normal brain parenchyma, rapid growth, and the presence of necrosis are remarkable hallmarks of GBM. However, the effect of necrotic cells on GBM growth and metastasis is poorly understood at present. In this study, we examined the biological significance of necrotic tissues by exploring the molecular mechanisms underlying the signaling network between necrotic tissues and GBM cells. The migration and invasion of the GBM cell line CRT-MG was significantly enhanced by treatment with necrotic cells, as shown by assays for scratch wound healing and spheroid invasion. Incubation with necrotic cells induced IL-8 secretion in CRT-MG cells in a dose-dependent manner. In human GBM tissues, IL-8 positive cells were mainly distributed in the perinecrotic region, as seen in immunohistochemistry and immunofluorescence analysis. Necrotic cells induced NF-κB and AP-1 activation and their binding to the IL-8 promoter, leading to enhanced IL-8 production and secretion in GBM cells. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the migration and invasion of GBM cells are enhanced and facilitated via NF-κB/AP-1 mediated IL-8 upregulation. PMID:27076368

  17. Post-transcriptional gene silencing activity of human GIGYF2.

    PubMed

    Kryszke, Marie-Hélène; Adjeriou, Badia; Liang, Feifei; Chen, Hong; Dautry, François

    2016-07-01

    In mammalian post-transcriptional gene silencing, the Argonaute protein AGO2 indirectly recruits translation inhibitors, deadenylase complexes, and decapping factors to microRNA-targeted mRNAs, thereby repressing mRNA translation and accelerating mRNA decay. However, the exact composition and assembly pathway of the microRNA-induced silencing complex are not completely elucidated. As the GYF domain of human GIGYF2 was shown to bind AGO2 in pulldown experiments, we wondered whether GIGYF2 could be a novel protein component of the microRNA-induced silencing complex. Here we show that full-length GIGYF2 coimmunoprecipitates with AGO2 in human cells, and demonstrate that, upon tethering to a reporter mRNA, GIGYF2 exhibits strong, dose-dependent silencing activity, involving both mRNA destabilization and translational repression. PMID:27157137

  18. The Regulatory Role of Activating Transcription Factor 2 in Inflammation

    PubMed Central

    Yu, Tao; Li, Yong Jun; Bian, Ai Hong; Zuo, Hui Bin; Zhu, Ti Wen; Ji, Sheng Xiang; Kong, Fanming; Yin, De Qing; Wang, Chuan Bao; Wang, Zi Fu; Wang, Hong Qun; Yang, Yanyan; Yoo, Byong Chul

    2014-01-01

    Activating transcription factor 2 (ATF2) is a member of the leucine zipper family of DNA-binding proteins and is widely distributed in tissues including the liver, lung, spleen, and kidney. Like c-Jun and c-Fos, ATF2 responds to stress-related stimuli and may thereby influence cell proliferation, inflammation, apoptosis, oncogenesis, neurological development and function, and skeletal remodeling. Recent studies clarify the regulatory role of ATF2 in inflammation and describe potential inhibitors of this protein. In this paper, we summarize the properties and functions of ATF2 and explore potential applications of ATF2 inhibitors as tools for research and for the development of immunosuppressive and anti-inflammatory drugs. PMID:25049453

  19. AP-1/Fos-TGase2 Axis Mediates Wounding-induced Plasmodium falciparum Killing in Anopheles gambiae*

    PubMed Central

    Nsango, Sandrine E.; Pompon, Julien; Xie, Ting; Rademacher, Annika; Fraiture, Malou; Thoma, Martine; Awono-Ambene, Parfait H.; Moyou, Roger S.; Morlais, Isabelle; Levashina, Elena A.

    2013-01-01

    Anopheline mosquitoes are the only vectors of human malaria worldwide. It is now widely accepted that mosquito immune responses play a crucial role in restricting Plasmodium development within the vector; therefore, further dissection of the molecular mechanisms underlying these processes should inform new vector control strategies urgently needed to roll back the disease. Here, using genome-wide transcriptional profiling, bioinformatics, and functional gene analysis, we identify a new axis of mosquito resistance to monoclonal Plasmodium falciparum infections that includes the AP-1 transcription factor Fos and the transglutaminase 2 (TGase2), a cross-linking enzyme with known roles in wound responses. We demonstrate that Fos regulates induction of TGase2 expression after wounding but does not affect expression of the components of the well characterized complement-like system. Silencing of Fos or of TGase2 aborts the wounding-induced mosquito killing of P. falciparum. These results reveal multiple signaling pathways that are required for efficient Plasmodium killing in Anopheles gambiae. PMID:23592781

  20. Transcription factors of Lotus: regulation of isoflavonoid biosynthesis requires coordinated changes in transcription factor activity.

    PubMed

    Shelton, Dale; Stranne, Maria; Mikkelsen, Lisbeth; Pakseresht, Nima; Welham, Tracey; Hiraka, Hideki; Tabata, Satoshi; Sato, Shusei; Paquette, Suzanne; Wang, Trevor L; Martin, Cathie; Bailey, Paul

    2012-06-01

    Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors. PMID:22529285

  1. AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking.

    PubMed

    Setta-Kaffetzi, Niovi; Simpson, Michael A; Navarini, Alexander A; Patel, Varsha M; Lu, Hui-Chun; Allen, Michael H; Duckworth, Michael; Bachelez, Hervé; Burden, A David; Choon, Siew-Eng; Griffiths, Christopher E M; Kirby, Brian; Kolios, Antonios; Seyger, Marieke M B; Prins, Christa; Smahi, Asma; Trembath, Richard C; Fraternali, Franca; Smith, Catherine H; Barker, Jonathan N; Capon, Francesca

    2014-05-01

    Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has the unique potential to deliver insights into gene function. Here, we report two founder mutations (c.11T>G [p.Phe4Cys] and c.97C>T [p.Arg33Trp]) in AP1S3, the gene encoding AP-1 complex subunit σ1C, in 15 unrelated individuals with a severe autoinflammatory skin disorder known as pustular psoriasis. Because the variants are predicted to destabilize the 3D structure of the AP-1 complex, we generated AP1S3-knockdown cell lines to investigate the consequences of AP-1 deficiency in skin keratinocytes. We found that AP1S3 silencing disrupted the endosomal translocation of the innate pattern-recognition receptor TLR-3 (Toll-like receptor 3) and resulted in a marked inhibition of downstream signaling. These findings identify pustular psoriasis as an autoinflammatory phenotype caused by defects in vesicular trafficking and demonstrate a requirement of AP-1 for Toll-like receptor homeostasis. PMID:24791904

  2. Transcriptional activation of Brassica napus β-ketoacyl-ACP synthase II with an engineered zinc finger protein transcription factor.

    PubMed

    Gupta, Manju; DeKelver, Russell C; Palta, Asha; Clifford, Carla; Gopalan, Sunita; Miller, Jeffrey C; Novak, Stephen; Desloover, Daniel; Gachotte, Daniel; Connell, James; Flook, Josh; Patterson, Thomas; Robbins, Kelly; Rebar, Edward J; Gregory, Philip D; Urnov, Fyodor D; Petolino, Joseph F

    2012-09-01

    Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. Canola seed oil composition is dictated largely by the expression of genes encoding enzymes in the fatty acid biosynthetic pathway. In the present study, zinc finger proteins (ZFPs) were designed to bind DNA sequences common to two canola β-ketoacyl-ACP Synthase II (KASII) genes downstream of their transcription start site. Transcriptional activators (ZFP-TFs) were constructed by fusing these ZFP DNA-binding domains to the VP16 transcriptional activation domain. Following transformation using Agrobacterium, transgenic events expressing ZFP-TFs were generated and shown to have elevated KASII transcript levels in the leaves of transgenic T(0) plants when compared to 'selectable marker only' controls as well as of T(1) progeny plants when compared to null segregants. In addition, leaves of ZFP-TF-expressing T(1) plants contained statistically significant decreases in palmitic acid (consistent with increased KASII activity) and increased total C18. Similarly, T(2) seed displayed statistically significant decreases in palmitic acid, increased total C18 and reduced total saturated fatty acid contents. These results demonstrate that designed ZFP-TFs can be used to regulate the expression of endogenous genes to elicit specific phenotypic modifications of agronomically relevant traits in a crop species. PMID:22520333

  3. E proteins are required to activate germline transcription of the TCR Vbeta8.2 gene.

    PubMed

    Jia, Jingquan; Dai, Meifang; Zhuang, Yuan

    2008-10-01

    Each TCR Vbeta gene is regulated by an individual Vbeta promoter, which becomes active prior to V(D) J recombination and drives germline transcription. It has been shown that Vbeta gene locus activation and recombination are dependent on the Vbeta promoter. However, transcription factors that regulate Vbeta germline transcription remain largely undefined. A major challenge in studying Vbeta gene germline transcription is the quantitative assessment of relatively low-level transcripts in T-cell progenitors. Here we used the established Vbeta8.2(CD2) knock-in mouse model to assess functions of E-protein transcription factors in Vbeta8.2 germline transcription. We show that E proteins are required for the activation but not the maintenance of the Vbeta8.2 germline transcription during thymocyte development. The activation of Vbeta8.2 germline transcription depends more on the E proteins encoded by the E2A gene than by the HEB gene. We further show that IL-7 receptor (IL-7R)-mediated signals are essential for Vbeta8.2 germline transcription. We provide evidence that IL-7R expression is only partially controlled by E2A, suggesting a role for E2A in driving Vbeta8.2 germline transcription independent of IL-7R activation. PMID:18958875

  4. Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells.

    PubMed

    Teng, Yun; Radde, Brandie N; Litchfield, Lacey M; Ivanova, Margarita M; Prough, Russell A; Clark, Barbara J; Doll, Mark A; Hein, David W; Klinge, Carolyn M

    2015-06-19

    Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3-12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction. PMID:25969534

  5. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions.

    PubMed

    Scholes, Natalie S; Weinzierl, Robert O J

    2016-05-01

    Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators. PMID:27175900

  6. Molecular Dynamics of "Fuzzy" Transcriptional Activator-Coactivator Interactions

    PubMed Central

    Scholes, Natalie S.; Weinzierl, Robert O. J.

    2016-01-01

    Transcriptional activation domains (ADs) are generally thought to be intrinsically unstructured, but capable of adopting limited secondary structure upon interaction with a coactivator surface. The indeterminate nature of this interface made it hitherto difficult to study structure/function relationships of such contacts. Here we used atomistic accelerated molecular dynamics (aMD) simulations to study the conformational changes of the GCN4 AD and variants thereof, either free in solution, or bound to the GAL11 coactivator surface. We show that the AD-coactivator interactions are highly dynamic while obeying distinct rules. The data provide insights into the constant and variable aspects of orientation of ADs relative to the coactivator, changes in secondary structure and energetic contributions stabilizing the various conformers at different time points. We also demonstrate that a prediction of α-helical propensity correlates directly with the experimentally measured transactivation potential of a large set of mutagenized ADs. The link between α-helical propensity and the stimulatory activity of ADs has fundamental practical and theoretical implications concerning the recruitment of ADs to coactivators. PMID:27175900

  7. Up-Regulation of Human Inducible Nitric Oxide Synthase by p300 Transcriptional Complex.

    PubMed

    Guo, Zhong; Zheng, Liang; Liao, Xinghua; Geller, David

    2016-01-01

    p300, a ubiquitous transcription coactivator, plays an important role in gene activation. Our previous work demonstrated that human inducible nitric oxide synthase (hiNOS) expression can be highly induced with the cytokine mixture (CM) of TNF-α + IL-1β + IFN-γ. In this study, we investigated the functional role of p300 in the regulation of hiNOS gene expression. Our initial data showed that overexpression of p300 significantly increased the basal and cytokine-induced hiNOS promoter activities in A549 cells. Interestingly, p300 activated cytokine-induced hiNOS transcriptional activity was completely abrogated by deleting the upstream hiNOS enhancer at -5 kb to -6 kb in the promoter. Furthermore, p300 over-expression increased cytokine-induced transcriptional activity on a heterologous minimal TK promoter with the same hiNOS enhancer. Site-directed mutagenesis of the hiNOS AP-1 motifs revealed that an intact upstream (-5.3 kb) AP-1 binding site was critical for p300 mediated cytokine-induced hiNOS transcription. Furthermore, our ChIP analysis demonstrated that p300 was binding to Jun D and Fra-2 proteins at -5.3 kb AP-1 binding site in vivo. Lastly, our 3C assay was able to detect a long DNA loop between the hiNOS enhancer and core promoter site, and ChIP loop assay confirmed that p300 binds to AP-1 and RNA pol II proteins. Overall, our results suggest that coactivator p300 mediates cytokine-induced hiNOS transactivation by forming a distant DNA loop between its enhancer and core promoter region. PMID:26751080

  8. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity

    PubMed Central

    Ahmed, Afsar U.; Williams, Bryan R. G.; Hannigan, Gregory E.

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  9. Transcriptional Activation of Inflammatory Genes: Mechanistic Insight into Selectivity and Diversity.

    PubMed

    Ahmed, Afsar U; Williams, Bryan R G; Hannigan, Gregory E

    2015-01-01

    Acute inflammation, an integral part of host defence and immunity, is a highly conserved cellular response to pathogens and other harmful stimuli. An inflammatory stimulation triggers transcriptional activation of selective pro-inflammatory genes that carry out specific functions such as anti-microbial activity or tissue healing. Based on the nature of inflammatory stimuli, an extensive exploitation of selective transcriptional activations of pro-inflammatory genes is performed by the host to ensure a defined inflammatory response. Inflammatory signal transductions are initiated by the recognition of inflammatory stimuli by transmembrane receptors, followed by the transmission of the signals to the nucleus for differential gene activations. The differential transcriptional activation of pro-inflammatory genes is precisely controlled by the selective binding of transcription factors to the promoters of these genes. Among a number of transcription factors identified to date, NF-κB still remains the most prominent and studied factor for its diverse range of selective transcriptional activities. Differential transcriptional activities of NF-κB are dictated by post-translational modifications, specificities in dimer formation, and variability in activation kinetics. Apart from the differential functions of transcription factors, the transcriptional activation of selective pro-inflammatory genes is also governed by chromatin structures, epigenetic markers, and other regulators as the field is continuously expanding. PMID:26569329

  10. Cell cycle-dependent regulation of RNA polymerase II basal transcription activity.

    PubMed Central

    Yonaha, M; Chibazakura, T; Kitajima, S; Yasukochi, Y

    1995-01-01

    Regulation of transcription by RNA polymerase II (pol II) in eukaryotic cells requires both basal and regulatory transcription factors. In this report we have investigated in vitro pol II basal transcription activity during the cell cycle by using nuclear extracts from synchronized HeLa cells. It is shown that pol II basal transcription activity is low in the S and G2 phases and high in early G1 phase and TFIID is the rate limiting component of pol II basal transcription activity during the cell cycle. Further analyses reveal that TFIID exists as a less active form in the S and G2 phases and nuclear extracts from S and G2 phase cells contain a heat-sensitive repressor(s) of TATA box binding protein (TBP). These results suggest that pol II basal transcription activity is regulated by a qualitative change in the TFIID complex, which could involve repression of TBP, during the cell cycle. Images PMID:7479063

  11. Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation

    NASA Technical Reports Server (NTRS)

    D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Karsenty, Gerard; Partridge, Nicola C.

    2002-01-01

    Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

  12. Long-Range Enhancer Associated with Chromatin Looping Allows AP-1 Regulation of the Peptidylarginine Deiminase 3 Gene in Differentiated Keratinocyte

    PubMed Central

    Chavanas, Stéphane; Adoue, Véronique; Méchin, Marie-Claire; Ying, Shibo; Dong, Sijun; Duplan, Hélène; Charveron, Marie; Takahara, Hidenari; Serre, Guy; Simon, Michel

    2008-01-01

    Transcription control at a distance is a critical mechanism, particularly for contiguous genes. The peptidylarginine deiminases (PADs) catalyse the conversion of protein-bound arginine into citrulline (deimination), a critical reaction in the pathophysiology of multiple sclerosis, Alzheimer's disease and rheumatoid arthritis, and in the metabolism of the major epidermal barrier protein filaggrin, a strong predisposing factor for atopic dermatitis. PADs are encoded by 5 clustered PADI genes (1p35-6). Unclear are the mechanisms controlling the expression of the gene PADI3 encoding the PAD3 isoform, a strong candidate for the deimination of filaggrin in the terminally differentiating epidermal keratinocyte. We describe the first PAD Intergenic Enhancer (PIE), an evolutionary conserved non coding segment located 86-kb from the PADI3 promoter. PIE is a strong enhancer of the PADI3 promoter in Ca2+-differentiated epidermal keratinocytes, and requires bound AP-1 factors, namely c-Jun and c-Fos. As compared to proliferative keratinocytes, calcium stimulation specifically associates with increased local DNase I hypersensitivity around PIE, and increased physical proximity of PIE and PADI3 as assessed by Chromosome Conformation Capture. The specific AP-1 inhibitor nordihydroguaiaretic acid suppresses the calcium-induced increase of PADI3 mRNA levels in keratinocytes. Our findings pave the way to the exploration of deimination control during tumorigenesis and wound healing, two conditions for which AP-1 factors are critical, and disclose that long-range transcription control has a role in the regulation of the gene PADI3. Since invalidation of distant regulators causes a variety of human diseases, PIE results to be a plausible candidate in association studies on deimination-related disorders or atopic disease. PMID:18923650

  13. Adenovirus E1A downregulates cJun- and JunB-mediated transcription by targeting their coactivator p300.

    PubMed Central

    Lee, J S; See, R H; Deng, T; Shi, Y

    1996-01-01

    Transcription factors and cofactors play critical roles in cell growth and differentiation. Alterations of their activities either through genetic mutations or by viral oncoproteins often result in aberrant cell growth and tumorigenesis. The transcriptional cofactor p300 has recently been shown to be complexed with transcription factors YY1 and CREB. Adenovirus E1A oncoproteins target these transcription complexes via physical interactions with p300, resulting in alterations of transcription mediated by these transcription factors. Here we show that p300 is also critical for repression by E1A of the activities of cJun and JunB, two members of the AP-1 transcriptional complexes. This repressive effect of E1A is dependent on the p300-binding domain of E1A and can be relieved by overexpression of p300. These results suggest that p300 serves as a mediator protein for downregulation of AP-1 activity by E1A. This hypothesis was further supported by the following observations: (i) in the absence of E1A, overexpression of p300 stimulated transcription both through an AP-1 site present in the collagenase promoter and through Jun proteins in GAL4 fusion protein-based assays; and (ii) overexpression of a mutant p300 lacking the E1A-interacting domain reduced the responsiveness of Jun-dependent transcription to E1A repression. As predicted from the functional results, p300 physically interacted with the Jun proteins. These findings thus established that p300 is a cofactor for cJun and JunB. We propose that p300 is a common mediator protein through which E1A gains control over multiple transcriptional regulatory pathways in the host cells. PMID:8754832

  14. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase

    PubMed Central

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2016-01-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5′- and 3′-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

  15. Inhibition of human insulin gene transcription and MafA transcriptional activity by the dual leucine zipper kinase.

    PubMed

    Stahnke, Marie-Jeannette; Dickel, Corinna; Schröder, Sabine; Kaiser, Diana; Blume, Roland; Stein, Roland; Pouponnot, Celio; Oetjen, Elke

    2014-09-01

    Insulin biosynthesis is an essential β-cell function and inappropriate insulin secretion and biosynthesis contribute to the pathogenesis of diabetes mellitus type 2. Previous studies showed that the dual leucine zipper kinase (DLK) induces β-cell apoptosis. Since β-cell dysfunction precedes β-cell loss, in the present study the effect of DLK on insulin gene transcription was investigated in the HIT-T15 β-cell line. Downregulation of endogenous DLK increased whereas overexpression of DLK decreased human insulin gene transcription. 5'- and 3'-deletion human insulin promoter analyses resulted in the identification of a DLK responsive element that mapped to the DNA binding-site for the β-cell specific transcription factor MafA. Overexpression of DLK wild-type but not its kinase-dead mutant inhibited MafA transcriptional activity conferred by its transactivation domain. Furthermore, in the non-β-cell line JEG DLK inhibited MafA overexpression-induced human insulin promoter activity. Overexpression of MafA and DLK or its kinase-dead mutant into JEG cells revealed that DLK but not its mutant reduced MafA protein content. Inhibition of the down-stream DLK kinase c-Jun N-terminal kinase (JNK) by SP600125 attenuated DLK-induced MafA loss. Furthermore, mutation of the serine 65 to alanine, shown to confer MafA protein stability, increased MafA-dependent insulin gene transcription and prevented DLK-induced MafA loss in JEG cells. These data suggest that DLK by activating JNK triggers the phosphorylation and degradation of MafA thereby attenuating insulin gene transcription. Given the importance of MafA for β-cell function, the inhibition of DLK might preserve β-cell function and ultimately retard the development of diabetes mellitus type 2. PMID:24726898

  16. Decreased expression of hepatocyte nuclear factor 3 alpha during the acute-phase response influences transthyretin gene transcription.

    PubMed Central

    Qian, X; Samadani, U; Porcella, A; Costa, R H

    1995-01-01

    Three distinct hepatocyte nuclear factor 3 (HNF-3) proteins (alpha, beta, and gamma) are known to regulate the transcription of numerous liver-specific genes. The HNF-3 proteins bind to DNA as monomers through a winged-helix motif, which is also utilized by a number of developmental regulators, including the Drosophila homeotic fork head (fkh) protein. We have previously characterized a strong-affinity HNF-3S site in the transthyretin (TTR) promoter region which is essential for expression in human hepatoma (HepG2) cells. In the current study, we identify an activating protein 1 (AP-1) site which partially overlaps the HNF-3S sequence in the TTR promoter. We show that in HepG2 cells the AP-1 sequence confers 12-O-tetradecanoylphorbol-13-acetate inducibility to the TTR promoter and contributes to normal TTR transcriptional activity. We also demonstrate that the HNF-3 proteins and AP-1 bind independently to the TTR AP-1-HNF-3 site, and cotransfection experiments suggest that they do not cooperate to activate an AP-1-HNF-3 reporter construct. In addition, 12-O-tetradecanoylphorbol-13-acetate exposure of HepG2 cells results in a reciprocal decrease in HNF-3 alpha and -3 gamma expression which may facilitate interaction of AP-1 with the TTR AP-1-HNF-3 site. In order to explore the role of HNF-3 in the liver, we have examined expression patterns of TTR and HNF-3 during the acute-phase response and liver regeneration. Partial hepatectomy produced minimal fluctuation in HNF-3 and TTR expression, suggesting that HNF-3 expression is not influenced by proliferative signals induced during liver regeneration. In acute-phase livers, we observed a dramatic reduction in HNF-3 alpha expression which correlates with a decrease in the expression of its target gene, the TTR gene. Furthermore, consistent with previous studies, the acute-phase livers are induced for c-jun but not c-fos expression. We propose that the reduction in TTR gene expression during the acute phase is likely due

  17. Mutations in ap1b1 Cause Mistargeting of the Na+/K+-ATPase Pump in Sensory Hair Cells

    PubMed Central

    Clemens Grisham, Rachel; Kindt, Katie; Finger-Baier, Karin; Schmid, Bettina; Nicolson, Teresa

    2013-01-01

    The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1) gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na+/K+-ATPase pump (NKA) was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na+ levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells. PMID:23593334

  18. Mutations in ap1b1 cause mistargeting of the Na(+)/K(+)-ATPase pump in sensory hair cells.

    PubMed

    Clemens Grisham, Rachel; Kindt, Katie; Finger-Baier, Karin; Schmid, Bettina; Nicolson, Teresa

    2013-01-01

    The hair cells of the inner ear are polarized epithelial cells with a specialized structure at the apical surface, the mechanosensitive hair bundle. Mechanotransduction occurs within the hair bundle, whereas synaptic transmission takes place at the basolateral membrane. The molecular basis of the development and maintenance of the apical and basal compartments in sensory hair cells is poorly understood. Here we describe auditory/vestibular mutants isolated from forward genetic screens in zebrafish with lesions in the adaptor protein 1 beta subunit 1 (ap1b1) gene. Ap1b1 is a subunit of the adaptor complex AP-1, which has been implicated in the targeting of basolateral membrane proteins. In ap1b1 mutants we observed that although the overall development of the inner ear and lateral-line organ appeared normal, the sensory epithelium showed progressive signs of degeneration. Mechanically-evoked calcium transients were reduced in mutant hair cells, indicating that mechanotransduction was also compromised. To gain insight into the cellular and molecular defects in ap1b1 mutants, we examined the localization of basolateral membrane proteins in hair cells. We observed that the Na(+)/K(+)-ATPase pump (NKA) was less abundant in the basolateral membrane and was mislocalized to apical bundles in ap1b1 mutant hair cells. Accordingly, intracellular Na(+) levels were increased in ap1b1 mutant hair cells. Our results suggest that Ap1b1 is essential for maintaining integrity and ion homeostasis in hair cells. PMID:23593334

  19. Novel and recurrent mutations in the AIRE gene of autoimmune polyendocrinopathy syndrome type 1 (APS1) patients.

    PubMed

    Faiyaz-Ul-Haque, M; Bin-Abbas, B; Al-Abdullatif, A; Abdullah Abalkhail, H; Toulimat, M; Al-Gazlan, S; Almutawa, A M; Al-Sagheir, A; Peltekova, I; Al-Dayel, F; Zaidi, S H E

    2009-11-01

    Autoimmune polyendocrinopathy syndrome type 1 (APS1) is characterized by the presence of at least two out of three clinical features, which include Addison's disease, hypoparathyroidism, and chronic mucocutaneous candidiasis. This disorder is caused by mutations in the AIRE (autoimmune regulator) gene. While several AIRE mutations have been described in APS1 patients of various ethnic origins, the genetic cause of APS1 in Arab patients requires further investigation. This study describes seven Arab families, in which 18 patients had APS1. In addition to the cardinal features of APS1, some patients exhibited alopecia, diabetes mellitus, nephrocalcinosis and other phenotypes associated with APS1. DNA sequencing of the AIRE gene of patients from this study identified four novel and one recurrent mutation. These mutations likely result in loss of AIRE function in the patients. In addition, it was noted that the non-pathogenic c.834C> G mutation (rs1800520, encoding for p.Ser278Arg) occurs with high incidence in the AIRE gene of Arab individuals. Furthermore, this investigation demonstrates inflammation of the hair follicles in APS1 patients with alopecia universalis. We conclude that Arab APS1 patients carry novel and recurrent mutations in the AIRE gene. PMID:19758376

  20. N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

    PubMed Central

    Chen, Kai; Deng, Xin; Yu, Miao; Han, Dali; Hao, Ziyang; Liu, Jianzhao; Lu, Xingyu; Dore, Louis C; Weng, Xiaocheng; Ji, Quanjiang; Mets, Laurens; He, Chuan

    2015-01-01

    SUMMARY N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. PMID:25936837

  1. Transcription factor PIF4 controls the thermosensory activation of flowering.

    PubMed

    Kumar, S Vinod; Lucyshyn, Doris; Jaeger, Katja E; Alós, Enriqueta; Alvey, Elizabeth; Harberd, Nicholas P; Wigge, Philip A

    2012-04-12

    Plant growth and development are strongly affected by small differences in temperature. Current climate change has already altered global plant phenology and distribution, and projected increases in temperature pose a significant challenge to agriculture. Despite the important role of temperature on plant development, the underlying pathways are unknown. It has previously been shown that thermal acceleration of flowering is dependent on the florigen, FLOWERING LOCUS T (FT). How this occurs is, however, not understood, because the major pathway known to upregulate FT, the photoperiod pathway, is not required for thermal acceleration of flowering. Here we demonstrate a direct mechanism by which increasing temperature causes the bHLH transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4) to activate FT. Our findings provide a new understanding of how plants control their timing of reproduction in response to temperature. Flowering time is an important trait in crops as well as affecting the life cycles of pollinator species. A molecular understanding of how temperature affects flowering will be important for mitigating the effects of climate change. PMID:22437497

  2. Transcription factors as targets of the anti-inflammatory treatment. A cell culture study with extracts from some Mediterranean diet plants.

    PubMed

    Stalińska, K; Guzdek, A; Rokicki, M; Koj, A

    2005-03-01

    During the inflammatory response at least 2 transcription factors, NF-kappaB and AP-1, are involved in the altered profile of gene expression. We used human hepatoma (HepG2) and human umbilical vein endothelial cells (HUVEC) as a model system: NF-kappaB and AP-1 were activated by the proinflammatory cytokine IL-1 in the absence or presence of 21 selected plant extracts and the effect was evaluated by the electrophoretic mobility shift assay (EMSA). In both types of cells activation of NF-kappaB by IL-1 was significantly inhibited by extracts from Scandix australis and Artemisia alba, whereas extracts from Amaranthus sp., Eryngium campestre, Thymus pulegioides and Reichardia picroides elicited cell-type dependent response. The IL-1-induced AP-1 activation was diminished by extracts from Scandix australis, Amaranthus sp. and Artemisia alba more potently in HUVEC, while extracts from Urospermum picroides and Scandix pecten-veneris in HepG2 cells. Inhibitory activities of plant extracts towards cytokine activated NF-kappaB and AP-1 depend to some extent on the order of addition of IL-1 and plant extract to the cell culture, but the mechanism of action of extract components is not clear: although plant polyphenols may participate they are unlikely to be the only mediators, and MAP kinases were found generally not involved in down-regulation of transcription factors activity by plant extracts. PMID:15800392

  3. TRIC: Capturing the direct cellular targets of promoter-bound transcriptional activators.

    PubMed

    Dugan, Amanda; Pricer, Rachel; Katz, Micah; Mapp, Anna K

    2016-08-01

    Transcriptional activators coordinate the dynamic assembly of multiprotein coactivator complexes required for gene expression to occur. Here we combine the power of in vivo covalent chemical capture with p-benzoyl-L-phenylalanine (Bpa), a genetically incorporated photo-crosslinking amino acid, and chromatin immunoprecipitation (ChIP) to capture the direct protein interactions of the transcriptional activator VP16 with the general transcription factor TBP at the GAL1 promoter in live yeast. PMID:27213278

  4. Single molecule microscopy reveals mechanistic insight into RNA polymerase II preinitiation complex assembly and transcriptional activity

    PubMed Central

    Horn, Abigail E.; Kugel, Jennifer F.; Goodrich, James A.

    2016-01-01

    Transcription by RNA polymerase II (Pol II) is a complex process that requires general transcription factors and Pol II to assemble on DNA into preinitiation complexes that can begin RNA synthesis upon binding of NTPs (nucleoside triphosphate). The pathways by which preinitiation complexes form, and how this impacts transcriptional activity are not completely clear. To address these issues, we developed a single molecule system using TIRF (total internal reflection fluorescence) microscopy and purified human transcription factors, which allows us to visualize transcriptional activity at individual template molecules. We see that stable interactions between polymerase II (Pol II) and a heteroduplex DNA template do not depend on general transcription factors; however, transcriptional activity is highly dependent upon TATA-binding protein, TFIIB and TFIIF. We also found that subsets of general transcription factors and Pol II can form stable complexes that are precursors for functional transcription complexes upon addition of the remaining factors and DNA. Ultimately we found that Pol II, TATA-binding protein, TFIIB and TFIIF can form a quaternary complex in the absence of promoter DNA, indicating that a stable network of interactions exists between these proteins independent of promoter DNA. Single molecule studies can be used to learn how different modes of preinitiation complex assembly impact transcriptional activity. PMID:27112574

  5. SUMO-activating SAE1 transcription is positively regulated by Myc

    PubMed Central

    Amente, Stefano; Lavadera, Miriam Lubrano; Palo, Giacomo Di; Majello, Barbara

    2012-01-01

    Myc protein plays a fundamental role in regulation of cell cycle, proliferation, differentiation and apoptosis by modulating the expression of a large number of targets. Here we report the transactivation ability of the human Myc protein to activate the SUMO-activating enzyme SAE1 transcription. We found that Myc activates SAE1 transcription via direct binding to canonical E-Boxes sequences located close to the SAE1 transcription start site. A recent report has highlighted the crucial role of the SAE gene expression in Myc mediated oncogenesis. Our study adds new insight in this context since we show here that Myc directly activates SAE1 transcription, suggesting that Myc oncogenic activity which depends on SAE1 is ensured by Myc itself through direct binding and transcriptional activation of SAE1 expression. PMID:22679563

  6. Transcription activation at class II CRP-dependent promoters: the role of different activating regions.

    PubMed Central

    Rhodius, V A; West, D M; Webster, C L; Busby, S J; Savery, N J

    1997-01-01

    Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at Class II promoters is dependent on direct interactions between two surface-exposed activating regions (AR1 and AR2) and two contact sites in RNA polymerase. The effects on transcription activation of disrupting either AR1 or AR2 have been measured at different Class II promoters. AR2 but not AR1 is essential for activation at all the Class II promoters that were tested. The effects of single positive control substitutions in AR1 and AR2 vary from one promoter to another: the effects of the different substitutions are contingent on the -35 hexamer sequence. Abortive initiation assays have been used to quantify the effects of positive control substitutions in each activating region on the kinetics of transcription initiation at the Class II CRP- dependent promoter pmelRcon. At this promoter, the HL159 substitution in AR1 results in a defect in the initial binding of RNA polymerase whilst the KE101 substitution in AR2 reduces the rate of isomerization from the closed to the open complex. PMID:9016561

  7. Improving fold activation of small transcription activating RNAs (STARs) with rational RNA engineering strategies.

    PubMed

    Meyer, Sarai; Chappell, James; Sankar, Sitara; Chew, Rebecca; Lucks, Julius B

    2016-01-01

    Regulatory RNAs have become integral components of the synthetic biology and bioengineering toolbox for controlling gene expression. We recently expanded this toolbox by creating small transcription activating RNAs (STARs) that act by disrupting the formation of a target transcriptional terminator hairpin placed upstream of a gene. While STARs are a promising addition to the repertoire of RNA regulators, much work remains to be done to optimize the fold activation of these systems. Here we apply rational RNA engineering strategies to improve the fold activation of two STAR regulators. We demonstrate that a combination of promoter strength tuning and multiple RNA engineering strategies can improve fold activation from 5.4-fold to 13.4-fold for a STAR regulator derived from the pbuE riboswitch terminator. We then validate the generality of our approach and show that these same strategies improve fold activation from 2.1-fold to 14.6-fold for an unrelated STAR regulator, opening the door to creating a range of additional STARs to use in a broad array of biotechnologies. We also establish that the optimizations preserve the orthogonality of these STARs between themselves and a set of RNA transcriptional repressors, enabling these optimized STARs to be used in sophisticated circuits. PMID:26134708

  8. Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator.

    PubMed Central

    Kato, N; Lan, K H; Ono-Nita, S K; Shiratori, Y; Omata, M

    1997-01-01

    The hepatitis C virus (HCV) nonstructural region 5A (NS5A) protein, without its 146 amino-terminal amino acids and fused to the DNA-binding domain of GAL4, strongly activates transcription in yeast and human hepatoma cells. Transcriptional activation by the HCV NS5A protein may play a role in viral replication and hepatocarcinogenesis. PMID:9343247

  9. PU.1 can participate in an active enhancer complex without its transcriptional activation domain

    PubMed Central

    Pongubala, Jagan M. R.; Atchison, Michael L.

    1997-01-01

    The transcription factor PU.1 is necessary for the development of multiple hematopoietic lineages and contributes to the activity of the immunoglobulin κ 3′ enhancer. A variety of proteins bind to the 3′ enhancer (PU.1, PIP, ATF1, CREM, c-Fos, c-Jun, and E2A), but the mechanism of 3′-enhancer activity and the proteins necessary for its activity are presently unclear. We show here that PU.1 participates with other transcription factors in forming a higher-order complex with 3′-enhancer DNA sequences. Each protein is necessary for formation of this complex. Individually, transcription factors that bind to the 3′ enhancer do not appreciably stimulate transcription in a cell type in which the 3′ enhancer is normally silent (NIH 3T3). However, mixture of multiple transcription factors (PU.1, PIP, c-Fos, and c-Jun) can greatly activate the enhancer. PU.1 is necessary for maximal enhancer activity, but mutants of PU.1 that lack the transcriptional activation domain are nearly as efficient at stimulating enhancer activity as the wild-type PU.1 protein. PU.1 apparently can activate transcription by playing an architectural role in interactions with other transcription factors. PMID:8990172

  10. A modified reverse one-hybrid screen identifies transcriptional activation in Phyochrome-Interacting Factor 3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptional activation domains (TAD) are difficult to predict and identify, since they are not conserved and have little consensus. Here, we describe a yeast-based screening method that is able to identify individual amino acid residues involved in transcriptional activation in a high throughput...

  11. AP1- and NF-kappaB-binding sites conserved among mammalian WNT10B orthologs elucidate the TNFalpha-WNT10B signaling loop implicated in carcinogenesis and adipogenesis.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2007-04-01

    WNT signals are context-dependently transduced to canonical and non-canonical signaling cascades. We cloned and characterized wild-type human WNT10B, while another group cloned aberrant human WNT10B with Gly60Asp amino-acid substitution. Proto-oncogene WNT10B is expressed in gastric cancer, pancreatic cancer, breast cancer, esophageal cancer, and cervical cancer. Because WNT10B blocks adipocyte differentiation, coding SNP of WNT10B gene is associated with familial obesity. In 2001, we reported WNT10B upregulation by TNFalpha. Here, comparative integromics analyses on WNT10B orthologs were performed to elucidate the transcriptional mechanism of WNT10B. Chimpanzee WNT10B and cow Wnt10b genes were identified within NW_001223159.1 and AC150975.2 genome sequences, respectively, by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee WNT10B and cow Wnt10b showed 98.7% and 95.1% total-amino-acid identity with human WNT10B, respectively. N-terminal signal peptide, 24 Cys residues, two Asn-linked glycosylation sites, and Gly60 of human WNT10B were conserved among mammalian WNT10B orthologs. Transcription start site of human WNT10B gene was 106-bp upstream of NM_003394.2 RefSeq 5'-end. Number of GC di-nucleotide repeats just down-stream of WNT10B transcription start site varied among primates and human population. Comparative genomics analyses revealed that double AP1-binding sites in the 5'-flanking promoter region and NF-kappaB-binding site in intron 3 were conserved among human, chimpanzee, cow, mouse, and rat WNT10B orthologs. Because TNFalpha signaling through TNFR1 and TRADD/RIP/TRAF2 complex activates JUN kinase (JNK) and IkappaB kinase (IKK) signaling cascades, conserved AP1- and NF-kappaB-binding sites explain the mechanism of TNFalpha-induced WNT10B upregulation. TNFalpha-WNT10B signaling loop is the negative feedback mechanism of adipogenesis to prevent obesity and metabolic syndrome. On the other hand, TNFalpha-WNT10B signaling loop is

  12. c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

    SciTech Connect

    Zhang Dongyun; Li Jingxia; Gao Jimin; Huang Chuanshu

    2009-02-15

    Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.

  13. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  14. Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

    PubMed Central

    Woontner, M; Wade, P A; Bonner, J; Jaehning, J A

    1991-01-01

    We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor. Images PMID:1875938

  15. Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

    PubMed

    Woontner, M; Wade, P A; Bonner, J; Jaehning, J A

    1991-09-01

    We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor. PMID:1875938

  16. Prediction of Pathway Activation by Xenobiotic-Responsive Transcription Factors in the Mouse Liver

    EPA Science Inventory

    Many drugs and environmentally-relevant chemicals activate xenobioticresponsive transcription factors (TF). Identification of target genes of these factors would be useful in predicting pathway activation in in vitro chemical screening. Starting with a large compendium of Affymet...

  17. The Nuclear Matrix Protein, NRP/B, Acts as a Transcriptional Repressor of E2F-mediated Transcriptional Activity

    PubMed Central

    Choi, Jina; Yang, Eun Sung; Cha, Kiweon; Whang, John; Choi, Woo-Jung; Avraham, Shalom; Kim, Tae-Aug

    2014-01-01

    Background: NRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated. Methods: Three-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity. Results: Ectopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex). Conclusions: The present study shows that NRP/B acts as a

  18. Active transcription and essential role of RNA polymerase II at the centromere during mitosis

    PubMed Central

    Chan, F. Lyn; Marshall, Owen J.; Saffery, Richard; Won Kim, Bo; Earle, Elizabeth; Choo, K. H. Andy; Wong, Lee H.

    2012-01-01

    Transcription of the centromeric regions has been reported to occur in G1 and S phase in different species. Here, we investigate whether transcription also occurs and plays a functional role at the mammalian centromere during mitosis. We show the presence of actively transcribing RNA polymerase II (RNAPII) and its associated transcription factors, coupled with the production of centromere satellite transcripts at the mitotic kinetochore. Specific inhibition of RNAPII activity during mitosis leads to a decrease in centromeric α-satellite transcription and a concomitant increase in anaphase-lagging cells, with the lagging chromosomes showing reduced centromere protein C binding. These findings demonstrate an essential role of RNAPII in the transcription of α-satellite DNA, binding of centromere protein C, and the proper functioning of the mitotic kinetochore. PMID:22308327

  19. Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

    PubMed Central

    Wiese, Claudia; Dray, Eloïse; Groesser, Torsten; Filippo, Joseph San; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams, Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-01-01

    Summary Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds both dsDNA and a D-loop structure, and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement. PMID:17996711

  20. The kinesin KIF16B mediates apical transcytosis of transferrin receptor in AP-1B-deficient epithelia

    PubMed Central

    Perez Bay, Andres E; Schreiner, Ryan; Mazzoni, Francesca; Carvajal-Gonzalez, Jose M; Gravotta, Diego; Perret, Emilie; Lehmann Mantaras, Gullermo; Zhu, Yuan-Shan; Rodriguez-Boulan, Enrique J

    2013-01-01

    Polarized epithelial cells take up nutrients from the blood through receptors that are endocytosed and recycle back to the basolateral plasma membrane (PM) utilizing the epithelial-specific clathrin adaptor AP-1B. Some native epithelia lack AP-1B and therefore recycle cognate basolateral receptors to the apical PM, where they carry out important functions for the host organ. Here, we report a novel transcytotic pathway employed by AP-1B-deficient epithelia to relocate AP-1B cargo, such as transferrin receptor (TfR), to the apical PM. Lack of AP-1B inhibited basolateral recycling of TfR from common recycling endosomes (CRE), the site of function of AP-1B, and promoted its transfer to apical recycling endosomes (ARE) mediated by the plus-end kinesin KIF16B and non-centrosomal microtubules, and its delivery to the apical membrane mediated by the small GTPase rab11a. Hence, our experiments suggest that the apical recycling pathway of epithelial cells is functionally equivalent to the rab11a-dependent TfR recycling pathway of non-polarized cells. They define a transcytotic pathway important for the physiology of native AP-1B-deficient epithelia and report the first microtubule motor involved in transcytosis. PMID:23749212

  1. EGF activates TTP expression by activation of ELK-1 and EGR-1 transcription factors

    PubMed Central

    2012-01-01

    Background Tristetraprolin (TTP) is a key mediator of processes such as inflammation resolution, the inhibition of autoimmunity and in cancer. It carries out this role by the binding and degradation of mRNA transcripts, thereby decreasing their half-life. Transcripts modulated by TTP encode proteins such as cytokines, pro-inflammatory agents and immediate-early response proteins. TTP can also modulate neoplastic phenotypes in many cancers. TTP is induced and functionally regulated by a spectrum of both pro- and anti-inflammatory cytokines, mitogens and drugs in a MAPK-dependent manner. So far the contribution of p38 MAPK to the regulation of TTP expression and function has been best described. Results Our results demonstrate the induction of the gene coding TTP (ZFP36) by EGF through the ERK1/2-dependent pathway and implicates the transcription factor ELK-1 in this process. We show that ELK-1 regulates ZFP36 expression by two mechanisms: by binding the ZFP36 promoter directly through ETS-binding site (+ 883 to +905 bp) and by inducing expression of EGR-1, which in turn increases ZFP36 expression through sequences located between -111 and -103 bp. Conclusions EGF activates TTP expression via ELK-1 and EGR-1 transcription factors. PMID:22433566

  2. The nuclear factor SPBP contains different functional domains and stimulates the activity of various transcriptional activators.

    PubMed

    Rekdal, C; Sjøttem, E; Johansen, T

    2000-12-22

    SPBP (stromelysin-1 platelet-derived growth factor-responsive element binding protein) was originally cloned from a cDNA expression library by virtue of its ability to bind to a platelet-derived growth factor-responsive element in the human stromelysin-1 promoter. A 937-amino acid-long protein was deduced from a 3995-nucleotide murine cDNA sequence. By analyses of both human and murine cDNAs, we now show that SPBP is twice as large as originally found. The human SPBP gene contains six exons and is located on chromosome 22q13.1-13.3. Two isoforms differing in their C termini are expressed due to alternative splicing. PCR analyses of multitissue cDNA panels showed that SPBP is expressed in most tissues except for ovary and prostate. Functional mapping revealed that SPBP is a nuclear, multidomain protein containing an N-terminal region with transactivating ability, a novel type of DNA-binding domain containing an AT hook motif, and a bipartite nuclear localization signal as well as a C-terminal zinc finger domain. This type of zinc finger domain is also found in the trithorax family of chromatin-based transcriptional regulator proteins. Using cotransfection experiments, we find that SPBP enhances the transcriptional activity of various transcription factors such as c-Jun, Ets1, Sp1, and Pax6. Hence, SPBP seems to act as a transcriptional coactivator. PMID:10995766

  3. Transcriptional activation upon pheromone stimulation mediated by a small domain of Saccharomyces cerevisiae Ste12p.

    PubMed Central

    Pi, H; Chien, C T; Fields, S

    1997-01-01

    In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element. We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins. A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity. Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation. By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction. This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone. By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines. The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain. These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain. PMID:9343403

  4. Oleic acid-induced ADRP expression requires both AP-1 and PPAR response elements, and is reduced by Pycnogenol through mRNA degradation in NMuLi liver cells.

    PubMed

    Fan, Bin; Ikuyama, Shoichiro; Gu, Jian-Qiu; Wei, Ping; Oyama, Jun-ichi; Inoguchi, Toyoshi; Nishimura, Junji

    2009-07-01

    Fatty acids stimulate lipid accumulation in parallel with increased expression of adipose differentiation-related protein (ADRP) in liver cells. Although it is generally considered that the fatty acid effect on ADRP expression is mediated by peroxisome proliferator-activated receptors (PPARs), we identified here an additional molecular mechanism using the NMuLi mouse liver nonparenchymal cell line, which expresses PPARgamma and delta but not alpha. Oleic acid (OA) and specific ligands for PPARgamma and -delta stimulated ADRP expression as well as the -2,090-bp ADRP promoter activity which encompasses the PPAR response element (PPRE) adjacent to an Ets/activator protein (AP)-1 site. When the AP-1 site was mutated, OA failed to stimulate the activity despite the presence of the PPRE, whereas ligands for PPARgamma and -delta did stimulate it and so did a PPARalpha ligand under the coexpression of PPARalpha. DNA binding of AP-1 was stimulated by OA but not by PPAR ligands. Because we previously demonstrated that Pycnogenol (PYC), a French maritime pine bark extract, suppressed ADRP expression in macrophages partly by suppression of AP-1 activity, we tested the effect of PYC on NMuLi cells. PYC reduced the OA-induced ADRP expression along with suppression of lipid droplet formation. However, PYC neither suppressed the OA-stimulated ADRP promoter activity nor DNA binding of AP-1 but, instead, reduced the ADRP mRNA half-life. All these results indicate that the effect of OA on ADRP expression requires AP-1 as well as PPRE, and PYC suppresses the ADRP expression in part by facilitating mRNA degradation. PYC, a widely used dietary supplement, could be beneficial for the prevention of excessive lipid accumulation such as hepatic steatosis. PMID:19383873

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

    PubMed Central

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

    2013-01-01

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

  6. Multiple steps in the regulation of transcription-factor level and activity.

    PubMed Central

    Calkhoven, C F; Ab, G

    1996-01-01

    This review focuses on the regulation of transcription factors, many of which are DNA-binding proteins that recognize cis-regulatory elements of target genes and are the most direct regulators of gene transcription. Transcription factors serve as integration centres of the different signal-transduction pathways affecting a given gene. It is obvious that the regulation of these regulators themselves is of crucial importance for differential gene expression during development and in terminally differentiated cells. Transcription factors can be regulated at two, principally different, levels, namely concentration and activity, each of which can be modulated in a variety of ways. The concentrations of transcription factors, as of intracellular proteins in general, may be regulated at any of the steps leading from DNA to protein, including transcription, RNA processing, mRNA degradation and translation. The activity of a transcription factor is often regulated by (de) phosphorylation, which may affect different functions, e.g. nuclear localization DNA binding and trans-activation. Ligand binding is another mode of transcription-factor activation. It is typical for the large super-family of nuclear hormone receptors. Heterodimerization between transcription factors adds another dimension to the regulatory diversity and signal integration. Finally, non-DNA-binding (accessory) factors may mediate a diverse range of functions, e.g. serving as a bridge between the transcription factor and the basal transcription machinery, stabilizing the DNA-binding complex or changing the specificity of the target sequence recognition. The present review presents an overview of different modes of transcription-factor regulation, each illustrated by typical examples. PMID:8713055

  7. O-GlcNAc modification of Sp3 and Sp4 transcription factors negatively regulates their transcriptional activities.

    PubMed

    Ha, Changhoon; Lim, Kihong

    2015-11-13

    The addition of O-linked N-acetylglucosamine (O-GlcNAc) on serine or threonine modifies a myriad of proteins and regulates their function, stability and localization. O-GlcNAc modification is common among chromosome-associated proteins, such as transcription factors, suggesting its extensive involvement in gene expression regulation. In this study, we demonstrate the O-GlcNAc status of the Sp family members of transcription factors and the functional impact on their transcriptional activities. We highlight the presence of O-GlcNAc residues in Sp3 and Sp4, but not Sp2, as demonstrated by their enrichment in GlcNAc positive protein fractions and by detection of O-GlcNAc residues on Sp3 and Sp4 co-expressed in Escherichia coli together with O-GlcNAc transferase (OGT) using an O-GlcNAc-specific antibody. Deletion mutants of Sp3 and Sp4 indicate that the majority of O-GlcNAc sites reside in their N-terminal transactivation domain. Overall, using reporter gene assays and co-immunoprecipitations, we demonstrate a functional inhibitory role of O-GlcNAc modifications in Sp3 and Sp4 transcription factors. Thereby, our study strengthens the current notion that O-GlcNAc modification is an important regulator of protein interactome. PMID:26431879

  8. Distance and Helical Phase Dependence of Synergistic Transcription Activation in cis-Regulatory Module

    PubMed Central

    Huang, Qilai; Gong, Chenguang; Li, Jiahuang; Zhuo, Zhu; Chen, Yuan; Wang, Jin; Hua, Zi-Chun

    2012-01-01

    Deciphering of the spatial and stereospecific constraints on synergistic transcription activation mediated between activators bound to cis-regulatory elements is important for understanding gene regulation and remains largely unknown. It has been commonly believed that two activators will activate transcription most effectively when they are bound on the same face of DNA double helix and within a boundary distance from the transcription initiation complex attached to the TATA box. In this work, we studied the spatial and stereospecific constraints on activation by multiple copies of bound model activators using a series of engineered relative distances and stereospecific orientations. We observed that multiple copies of the activators GAL4-VP16 and ZEBRA bound to engineered promoters activated transcription more effectively when bound on opposite faces of the DNA double helix. This phenomenon was not affected by the spatial relationship between the proximal activator and initiation complex. To explain these results, we proposed the novel concentration field model, which posits the effective concentration of bound activators, and therefore the transcription activation potential, is affected by their stereospecific positioning. These results could be used to understand synergistic transcription activation anew and to aid the development of predictive models for the identification of cis-regulatory elements. PMID:22299056

  9. Selective activation of human heat shock gene transcription by nitrosourea antitumor drugs mediated by isocyanate-induced damage and activation of heat shock transcription factor

    SciTech Connect

    Kroes, R.A. Northwestern Univ., Evanston, IL ); Abravaya, K.; Morimoto, R.I. ); Seidenfeld, J. )

    1991-06-01

    Treatment of cultured human tumor cells with the chloroethylnitrosourea antitumor drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) selectively induces transcription and protein synthesis of a subset of the human heat shock or stress-induced genes (HSP90 and HSP70) with little effect on other stress genes or on expression of the c-fos, c-myc, or {beta}-actin genes. The active component of BCNU and related compounds appears to be the isocyanate moiety that causes carbamoylation of proteins and nucleic acids. Transcriptional activation of the human HSP70 gene by BCNU is dependent on the heat shock element and correlates with the level of heat shock transcription factor and its binding to the heat shock element in vivo. Unlike activation by heat or heavy metals, BCNU-mediated activation is strongly dependent upon new protein synthesis. This suggests that BCNU-induced, isocyanate-mediated damage to newly synthesized protein(s) may be responsible for activation of the heat shock transcription factor and increased transcription of the HSP90 and HSP70 genes.

  10. Transcriptional trans activators of human and simian foamy viruses contain a small, highly conserved activation domain.

    PubMed Central

    Garrett, E D; He, F; Bogerd, H P; Cullen, B R

    1993-01-01

    The Bel-1 protein of human foamy virus is a potent transcriptional trans activator of its homologous long terminal repeat promoter element. Here, we demonstrate that Bel-1 can also efficiently activate gene expression when targeted to a heterologous promoter by fusion to the DNA-binding motif of the yeast GAL4 protein. Analysis of a series of deletion mutants of Bel-1 generated in this hybrid protein context suggests the presence of a single transcription activation domain that is fully contained within a discrete, approximately 30-amino-acid segment located proximal to the Bel-1 carboxy terminus. Although this short motif can be shown to function effectively in eukaryotic cells of mammalian, avian, and fungal origin, it does not bear any evident sequence homology to the known classes of eukaryotic activation domain. However, this Bel-1 activation domain was found to be fully conserved, in terms of both biological activity and location, in the distantly related Taf trans activator of simian foamy virus type 1. Images PMID:8411385

  11. The cellular bromodomain protein Brd4 has multiple functions in E2-mediated papillomavirus transcription activation.

    PubMed

    Helfer, Christine M; Yan, Junpeng; You, Jianxin

    2014-08-01

    The cellular bromodomain protein Brd4 functions in multiple processes of the papillomavirus life cycle, including viral replication, genome maintenance, and gene transcription through its interaction with the viral protein, E2. However, the mechanisms by which E2 and Brd4 activate viral transcription are still not completely understood. In this study, we show that recruitment of positive transcription elongation factor b (P-TEFb), a functional interaction partner of Brd4 in transcription activation, is important for E2's transcription activation activity. Furthermore, chromatin immunoprecipitation (ChIP) analyses demonstrate that P-TEFb is recruited to the actual papillomavirus episomes. We also show that E2's interaction with cellular chromatin through Brd4 correlates with its papillomavirus transcription activation function since JQ1(+), a bromodomain inhibitor that efficiently dissociates E2-Brd4 complexes from chromatin, potently reduces papillomavirus transcription. Our study identifies a specific function of Brd4 in papillomavirus gene transcription and highlights the potential use of bromodomain inhibitors as a method to disrupt the human papillomavirus (HPV) life cycle. PMID:25140737

  12. Fur-mediated activation of gene transcription in the human pathogen Neisseria gonorrhoeae.

    PubMed

    Yu, Chunxiao; Genco, Caroline Attardo

    2012-04-01

    It is well established that the ferric uptake regulatory protein (Fur) functions as a transcriptional repressor in diverse microorganisms. Recent studies demonstrated that Fur also functions as a transcriptional activator. In this study we defined Fur-mediated activation of gene transcription in the sexually transmitted disease pathogen Neisseria gonorrhoeae. Analysis of 37 genes which were previously determined to be iron induced and which contained putative Fur boxes revealed that only 30 of these genes exhibited reduced transcription in a gonococcal fur mutant strain. Fur-mediated activation was established by examining binding of Fur to the putative promoter regions of 16 Fur-activated genes with variable binding affinities observed. Only ∼50% of the newly identified Fur-regulated genes bound Fur in vitro, suggesting that additional regulatory circuits exist which may function through a Fur-mediated indirect mechanism. The gonococcal Fur-activated genes displayed variable transcription patterns in a fur mutant strain, which correlated with the position of the Fur box in each (promoter) region. These results suggest that Fur-mediated direct transcriptional activation is fulfilled by multiple mechanisms involving either competing with a repressor or recruiting RNA polymerase. Collectively, our studies have established that gonococcal Fur functions as an activator of gene transcription through both direct and indirect mechanisms. PMID:22287521

  13. Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts

    PubMed Central

    Pandian, Ganesh N.; Taniguchi, Junichi; Junetha, Syed; Sato, Shinsuke; Han, Le; Saha, Abhijit; AnandhaKumar, Chandran; Bando, Toshikazu; Nagase, Hiroki; Vaijayanthi, Thangavel; Taylor, Rhys D.; Sugiyama, Hiroshi

    2014-01-01

    The influential role of the epigenome in orchestrating genome-wide transcriptional activation instigates the demand for the artificial genetic switches with distinct DNA sequence recognition. Recently, we developed a novel class of epigenetically active small molecules called SAHA-PIPs by conjugating selective DNA binding pyrrole-imidazole polyamides (PIPs) with the histone deacetylase inhibitor SAHA. Screening studies revealed that certain SAHA-PIPs trigger targeted transcriptional activation of pluripotency and germ cell genes in mouse and human fibroblasts, respectively. Through microarray studies and functional analysis, here we demonstrate for the first time the remarkable ability of thirty-two different SAHA-PIPs to trigger the transcriptional activation of exclusive clusters of genes and noncoding RNAs. QRT-PCR validated the microarray data, and some SAHA-PIPs activated therapeutically significant genes like KSR2. Based on the aforementioned results, we propose the potential use of SAHA-PIPs as reagents capable of targeted transcriptional activation. PMID:24457603

  14. Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors

    PubMed Central

    Morbitzer, Robert; Römer, Patrick; Boch, Jens; Lahaye, Thomas

    2010-01-01

    Proteins that can be tailored to bind desired DNA sequences are key tools for molecular biology. Previous studies suggested that DNA-binding specificity of transcription activator-like effectors (TALEs) from the bacterial genus Xanthomonas is defined by repeat-variable diresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units. We have studied chimeras of two TALEs differing in RVDs and non-RVDs and found that, in contrast to the critical contributions by RVDs, non-RVDs had no major effect on the DNA-binding specificity of the chimeras. This finding suggests that one needs only to modify the RVDs to generate designer TALEs (dTALEs) to activate transcription of user-defined target genes. We used the scaffold of the TALE AvrBs3 and changed its RVDs to match either the tomato Bs4, the Arabidopsis EGL3, or the Arabidopsis KNAT1 promoter. All three dTALEs transcriptionally activated the desired promoters in a sequence-specific manner as mutations within the targeted DNA sequences abolished promoter activation. This study is unique in showing that chromosomal loci can be targeted specifically by dTALEs. We also engineered two AvrBs3 derivatives with four additional repeat units activating specifically either the pepper Bs3 or UPA20 promoter. Because AvrBs3 activates both promoters, our data show that addition of repeat units facilitates TALE-specificity fine-tuning. Finally, we demonstrate that the RVD NK mediates specific interaction with G nucleotides that thus far could not be targeted specifically by any known RVD type. In summary, our data demonstrate that the TALE scaffold can be tailored to target user-defined DNA sequences in whole genomes. PMID:21106758

  15. Overexpression of Two PsnAP1 Genes from Populus simonii × P. nigra Causes Early Flowering in Transgenic Tobacco and Arabidopsis

    PubMed Central

    Zheng, Tangchun; Li, Shuang; Zang, Lina; Dai, Lijuan; Yang, Chuanping; Qu, Guan-Zheng

    2014-01-01

    In Arabidopsis, AP1 is a floral meristem identity gene and plays an important role in floral organ development. In this study, PsnAP1-1 and PsnAP1-2 were isolated from the male reproductive buds of poplar (Populus simonii × P. nigra), which are the orthologs of AP1 in Arabidopsis, by sequence analysis. Northern blot and qRT-PCR analysis showed that PsnAP1-1 and PsnAP1-2 exhibited high expression level in early inflorescence development of poplar. Subcellular localization showed the PsnAP1-1 and PsnAP1-2 proteins are localized in the nucleus. Overexpression of PsnAP1-1 and PsnAP1-2 in tobacco under the control of a CaMV 35S promoter significantly enhanced early flowering. These transgenic plants also showed much earlier stem initiation and higher rates of photosynthesis than did wild-type tobacco. qRT-PCR analysis further indicated that overexpression of PsnAP1-1 and PsnAP1-2 resulted in up-regulation of genes related to flowering, such as NtMADS4, NtMADS5 and NtMADS11. Overexpression of PsnAP1-1 and PsnAP1-2 in Arabidopsis also induced early flowering, but did not complement the ap1-10 floral morphology to any noticeable extent. This study indicates that PsnAP1-1 and PsnAP1-2 play a role in floral transition of poplar. PMID:25360739

  16. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    SciTech Connect

    Kasim, Vivi; Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia; Yang, Li; Miyagishi, Makoto; Wu, Shourong

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  17. An Asp7Gly substitution in PPARG is associated with decreased transcriptional activation activity.

    PubMed

    Hua, Liushuai; Wang, Jing; Li, Mingxun; Sun, Xiaomei; Zhang, Liangzhi; Lei, Chuzhao; Lan, Xianyong; Fang, Xingtang; Zhao, Xin; Chen, Hong

    2014-01-01

    As the master regulator of adipogenesis, peroxisome proliferator-activated receptor gamma (PPARG) is required for the accumulation of adipose tissue and hence contributes to obesity. A previous study showed that the substitution of +20A>G in PPARG changed the 7(th) amino acid from Asp to Gly, creating a mutant referred to as PPARG Asp7Gly. In this study, association analysis indicated that PPARG Asp7Gly was associated with lower body height, body weight and heart girth in cattle (P<0.05). Overexpression of PPARG in NIH3T3-L1 cells showed that the Asp7Gly substitution may cause a decrease in its adipogenic ability and the mRNA levels of CIDEC (cell death-inducing DFFA-like effector c) and aP2, which are all transcriptionally activated by PPARG during adipocyte differentiation. A dual-luciferase reporter assay was used to analyze the promoter activity of CIDEC. The results confirmed that the mutant PPARG exhibited weaker transcriptional activation activity than the wild type (P<0.05). These findings likely explain the associations between the Asp7Gly substitution and the body measurements. Additionally, the Asp7Gly mutation may be used in molecular marker assisted selection (MAS) of cattle breeding in the future. PMID:24466299

  18. The clathrin adaptor AP-1 complex and Arf1 regulate planar cell polarity in vivo

    PubMed Central

    Mendoza, Meg; Dussert, Aurore; Collu, Giovanna; Roman, Angel-Carlos; Weber, Ursula; Ciruna, Brian; Mlodzik, Marek

    2015-01-01

    A key step in generating planar cell polarity (PCP) is the formation of restricted junctional domains containing Frizzled/Dishevelled/Diego (Fz/Dsh/Dgo) or Van Gogh/Prickle (Vang/Pk) complexes within the same cell, stabilized via Flamingo (Fmi) across cell membranes. Although models have been proposed for how these complexes acquire and maintain their polarized localization, the machinery involved in moving core PCP proteins around cells remains unknown. We describe the AP-1 adaptor complex and Arf1 as major regulators of PCP protein trafficking in vivo. AP-1 and Arf1 disruption affects the accumulation of Fz/Fmi and Vang/Fmi complexes in the proximo–distal axis, producing severe PCP phenotypes. Using novel tools, we demonstrate a direct and specific Arf1 involvement in Fz trafficking in vivo. Moreover, we uncover a conserved Arf1 PCP function in vertebrates. Our data support a model whereby the trafficking machinery plays an important part during PCP establishment, promoting formation of polarized PCP-core complexes in vivo. PMID:25849195

  19. HDAC inhibitors suppress c-Jun/Fra-1-mediated proliferation through transcriptionally downregulating MKK7 and Raf1 in neuroblastoma cells

    PubMed Central

    Tang, Xiaomei; Xia, Yong; He, Guozhen; Min, Zhiqun; Li, Chun; Xiong, Shiqiu; Shi, Zhi; Lu, Yongjian; Yuan, Zhongmin

    2016-01-01

    Activator protein 1 (AP-1) is a transcriptional factor composed of the dimeric members of bZIP proteins, which are frequently deregulated in human cancer cells. In this study, we aimed to identify an oncogenic AP-1 dimer critical for the proliferation of neuroblastoma cells and to investigate whether histone deacetylase inhibitors (HDACIs), a new generation of anticancer agents, could target the AP-1 dimer. We report here that HDACIs including trichostatin A, suberoylanilidehydroxamic acid, valproic acid and M344 can transcriptionally suppress both c-Jun and Fra-1, preceding their inhibition of cell growth. c-Jun preferentially interacting with Fra-1 as a heterodimer is responsible for AP-1 activity and critical for cell growth. Mechanistically, HDACIs suppress Fra-1 expression through transcriptionally downregulating Raf1 and subsequently decreasing MEK1/2-ERK1/2 activity. Unexpectedly, HDACI treatment caused MKK7 downregulation at both the protein and mRNA levels. Deletion analysis of the 5′-flanking sequence of the MKK7 gene revealed that a major element responsible for the downregulation by HDACI is located at −149 to −3 relative to the transcriptional start site. Knockdown of MKK7 but not MKK4 remarkably decreased JNK/c-Jun activity and proliferation, whereas ectopic MKK7-JNK1 reversed HDACI-induced c-Jun suppression. Furthermore, suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings demonstrated that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1. PMID:26734995

  20. HMGA proteins as modulators of chromatin structure during transcriptional activation

    PubMed Central

    Ozturk, Nihan; Singh, Indrabahadur; Mehta, Aditi; Braun, Thomas; Barreto, Guillermo

    2013-01-01

    High mobility group (HMG) proteins are the most abundant non-histone chromatin associated proteins. HMG proteins bind to DNA and nucleosome and alter the structure of chromatin locally and globally. Accessibility to DNA within chromatin is a central factor that affects DNA-dependent nuclear processes, such as transcription, replication, recombination, and repair. HMG proteins associate with different multi-protein complexes to regulate these processes by mediating accessibility to DNA. HMG proteins can be subdivided into three families: HMGA, HMGB, and HMGN. In this review, we will focus on recent advances in understanding the function of HMGA family members, specifically their role in gene transcription regulation during development and cancer. PMID:25364713

  1. Chromatin dynamics associated with HIV-1 Tat activated transcription

    PubMed Central

    Easley, Rebecca; Van Duyne, Rachel; Coley, Will; Guendel, Irene; Dadgar, Sherry; Kehn-Hall, Kylene; Kashanchi, Fatah

    2009-01-01

    Summary Chromatin remodeling is an essential event for HIV-1 transcription. Over the last two decades this field of research has come to the forefront, as silencing of the HIV-1 provirus through chromatin modifications has been linked to latency. Here, we focus on chromatin remodeling, especially in relation to the transactivator Tat, and review the most important and newly emerging studies that investigate remodeling mechanisms. We begin by discussing covalent modifications that can alter chromatin structure including acetylation, deacetylation, and methylation, as well as topics addressing the interplay between chromatin remodeling and splicing. Next, we focus on complexes that use the energy of ATP to remove or secure nucleosomes and can additionally act to control HIV-1 transcription. Finally, we cover recent literature on viral microRNAs which have been shown to alter chromatin structure by inducing methylation or even by remodeling nucleosomes. PMID:19716452

  2. On the way of revealing coactivator complexes cross-talk during transcriptional activation.

    PubMed

    Krasnov, Aleksey N; Mazina, Marina Yu; Nikolenko, Julia V; Vorobyeva, Nadezhda E

    2016-01-01

    Transcriptional activation is a complex, multistage process implemented by hundreds of proteins. Many transcriptional proteins are organized into coactivator complexes, which participate in transcription regulation at numerous genes and are a driver of this process. The molecular action mechanisms of coactivator complexes remain largely understudied. Relevant publications usually deal with the involvement of these complexes in the entire process of transcription, and only a few studies are aimed to elucidate their functions at its particular stages. This review summarizes available information on the participation of key coactivator complexes in transcriptional activation. The timing of coactivator complex binding/removal has been used for restructuring previously described information about the transcriptional process. Several major stages of transcriptional activation have been distinguished based on the presence of covalent histone modifications and general transcriptional factors, and the recruitment and/or removal phases have been determined for each coactivator included in analysis. Recruitment of Mediator, SWItch/Sucrose Non-Fermentable and NUcleosome Remodeling Factor complexes during transcription activation has been investigated thoroughly; CHD and INOsitol auxotrophy 80 families are less well studied. In most cases, the molecular mechanisms responsible for the removal of certain coactivator complexes after the termination of their functions at the promoters are still not understood. On the basis of the summarized information, we propose a scheme that illustrates the involvement of coactivator complexes in different stages of the transcription activation process. This scheme may help to gain a deeper insight into the molecular mechanism of functioning of coactivator complexes, find novel participants of the process, and reveal novel structural or functional connections between different coactivators. PMID:26913181

  3. Sip1, a Conserved AP-1 Accessory Protein, Is Important for Golgi/Endosome Trafficking in Fission Yeast

    PubMed Central

    Yu, Yang; Kita, Ayako; Udo, Masako; Katayama, Yuta; Shintani, Mami; Park, Kwihwa; Hagihara, Kanako; Umeda, Nanae; Sugiura, Reiko

    2012-01-01

    We had previously identified the mutant allele of apm1+ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4+/sip1+, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1-deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl− and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking. PMID:23028933

  4. AaeAP1 and AaeAP2: novel antimicrobial peptides from the venom of the scorpion, Androctonus aeneas: structural characterisation, molecular cloning of biosynthetic precursor-encoding cDNAs and engineering of analogues with enhanced antimicrobial and anticancer activities.

    PubMed

    Du, Qiang; Hou, Xiaojuan; Wang, Lei; Zhang, Yingqi; Xi, Xinping; Wang, Hui; Zhou, Mei; Duan, Jinao; Wei, Minjie; Chen, Tianbao; Shaw, Chris

    2015-02-01

    The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation. PMID:25626077

  5. AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion, Androctonus aeneas: Structural Characterisation, Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities

    PubMed Central

    Du, Qiang; Hou, Xiaojuan; Wang, Lei; Zhang, Yingqi; Xi, Xinping; Wang, Hui; Zhou, Mei; Duan, Jinao; Wei, Minjie; Chen, Tianbao; Shaw, Chris

    2015-01-01

    The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3'- and 5'-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation. PMID:25626077

  6. Manganese peroxidase gene transcription in Phanerochaete chrysosporium: Activation by manganese

    SciTech Connect

    Brown, J.A.; Alic, M. Gold, M.H. )

    1991-07-01

    The expression of manganese peroxidase in nitrogen-limited cultures of Phanerochaete chrysosporium is dependent on Mn, and initial work suggested that Mn regulates transcription of the mnp gene. In this study, using Northern (RNA) blot analysis of kinetic, dose-response, and inhibitor experiments, the authors demonstrate unequivocally that Mn regulates mnp gene transcription. The amount of mnp mRNA in cells of 4-day-old nitrogen-limited cultures is a direct function of the concentration of Mn in the culture medium up to a maximum of 180 {mu}M. Addition of Mn to nitrogen-limited Mn-deficient secondary metabolic (4-, 5-, and 6-day-old) cultures results in the appearance of mnp mRNA within 40 min. The appearance of this message is completely inhibited by the RNA synthesis inhibitor dactinomycin but not by the protein synthesis inhibitor cycloheximide. Furthermore, the amount of mnp mRNA produced is a direct function of the concentration of added Mn. In contrast, addition of Mn to low-nitrogen Mn-deficient 2- or 3-day-old cultures does not result in the appearance of mnp mRNA. Manganese peroxidase protein is detected by specific immunoprecipitation of the in vitro translation products of poly(A) RNA isolated from Mn-supplemented (but nor from Mn-deficient) cells. All of these results demonstrate that Mn, the substrate for the enzyme, regulates mnp gene transcription via a growth-stage-specific and concentration-dependent mechanism.

  7. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes

    SciTech Connect

    Youn, Gi Soo; Kwon, Dong-Joo; Ju, Sung Mi; Rhim, Hyangshuk; Bae, Yong Soo; Choi, Soo Young; Park, Jinseu

    2014-10-01

    HIV-1 Tat causes extensive neuroinflammation that may progress to AIDS-related encephalitis and dementia. Celastrol possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we investigated the modulatory effects of celastrol on HIV-1 Tat-induced inflammatory responses and the molecular mechanisms underlying its action in astrocytes. Pre-treatment of CRT-MG human astroglioma cells with celastrol significantly inhibited HIV-1 Tat-induced expression of ICAM-1/VCAM-1 and subsequent monocyte adhesiveness in CRT-MG cells. In addition, celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory chemokines, such as CXCL10, IL-8, and MCP-1. Celastrol decreased HIV-1 Tat-induced activation of JNK MAPK, AP-1, and NF-κB. Furthermore, celastrol induced mRNA and protein expression of HO-1 as well as Nrf2 activation. Blockage of HO-1 expression using siRNA reversed the inhibitory effect of celastrol on HIV-1 Tat-induced inflammatory responses. These results suggest that celastrol has regulatory effects on HIV-1 Tat-induced inflammatory responses by blocking the JNK MAPK-AP-1/NF-κB signaling pathways and inducing HO-1 expression in astrocytes. - Highlights: • Celastrol suppressed HIV-1 Tat-induced expression of pro-inflammatory genes. • Celastrol inhibited HIV-1 Tat -induced activation of JNK MAPK. • Celastrol inhibited HIV-1 Tat-induced activation of both NF-κB and AP-1. • Celastrol inhibited HIV-1 Tat-induced inflammatory responses via HO-1 induction.

  8. TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation.

    PubMed Central

    Bohjanen, P R; Liu, Y; Garcia-Blanco, M A

    1997-01-01

    The ability of the HIV-1 Tat protein to trans -activate HIV-1 transcription in vitro is specifically inhibited by a circular TAR RNA decoy. This inhibition is not overcome by adding an excess of Tat to the reaction but is partially overcome by adding Tat in combination with nuclear extract, suggesting that TAR RNA might function by interacting with a complex containing Tat and cellular factor(s). A cell-free transcription system involving immobilized DNA templates was used to further define the factor(s) that interact with TAR RNA. Preinitiation complexes formed in the presence or absence of Tat were purified on immobilized templates containing the HIV-1 promoter. After washing, nucleotides and radiolabelled UTP were added and transcription was measured. The presence of Tat during preinitiation complex formation resulted in an increase in the level of full-length HIV-1 transcripts. This Tat-activated increase in HIV-1 transcription was not inhibited by circular TAR decoys added during preinitiation complex formation but was inhibited by circular TAR decoys subsequently added during the transcription reaction. These results suggest that TAR decoys inhibit Tat-activated HIV-1 transcription after preinitiation complex formation, perhaps by interacting with components of transcription complexes. PMID:9358155

  9. The yeast Hot1 transcription factor is critical for activating a single target gene, STL1

    PubMed Central

    Bai, Chen; Tesker, Masha; Engelberg, David

    2015-01-01

    Transcription factors are commonly activated by signal transduction cascades and induce expression of many genes. They therefore play critical roles in determining the cell's fate. The yeast Hog1 MAP kinase pathway is believed to control the transcription of hundreds of genes via several transcription factors. To identify the bona fide target genes of Hog1, we inducibly expressed the spontaneously active variant Hog1D170A+F318L in cells lacking the Hog1 activator Pbs2. This system allowed monitoring the effects of Hog1 by itself. Expression of Hog1D170A+F318L in pbs2∆ cells imposed induction of just 105 and suppression of only 26 transcripts by at least twofold. We looked for the Hog1-responsive element within the promoter of the most highly induced gene, STL1 (88-fold). A novel Hog1 responsive element (HoRE) was identified and shown to be the direct target of the transcription factor Hot1. Unexpectedly, we could not find this HoRE in any other yeast promoter. In addition, the only gene whose expression was abolished in hot1∆ cells was STL1. Thus Hot1 is essential for transcription of just one gene, STL1. Hot1 may represent a class of transcription factors that are essential for transcription of a very few genes or even just one. PMID:25904326

  10. GW8510 Increases Insulin Expression in Pancreatic Alpha Cells through Activation of p53 Transcriptional Activity

    PubMed Central

    Fomina-Yadlin, Dina; Kubicek, Stefan; Vetere, Amedeo; He, Kaihui Hu; Schreiber, Stuart L.; Wagner, Bridget K.

    2012-01-01

    Background Expression of insulin in terminally differentiated non-beta cell types in the pancreas could be important to treating type-1 diabetes. Previous findings led us to hypothesize involvement of kinase inhibition in induction of insulin expression in pancreatic alpha cells. Methodology/Principal Findings Alpha (αTC1.6) cells and human islets were treated with GW8510 and other small-molecule inhibitors for up to 5 days. Alpha cells were assessed for gene- and protein-expression levels, cell-cycle status, promoter occupancy status by chromatin immunoprecipitation (ChIP), and p53-dependent transcriptional activity. GW8510, a putative CDK2 inhibitor, up-regulated insulin expression in mouse alpha cells and enhanced insulin secretion in dissociated human islets. Gene-expression profiling and gene-set enrichment analysis of GW8510-treated alpha cells suggested up-regulation of the p53 pathway. Accordingly, the compound increased p53 transcriptional activity and expression levels of p53 transcriptional targets. A predicted p53 response element in the promoter region of the mouse Ins2 gene was verified by chromatin immunoprecipitation (ChIP). Further, inhibition of Jun N-terminal kinase (JNK) and p38 kinase activities suppressed insulin induction by GW8510. Conclusions/Significance The induction of Ins2 by GW8510 occurred through p53 in a JNK- and p38-dependent manner. These results implicate p53 activity in modulation of Ins2 expression levels in pancreatic alpha cells, and point to a potential approach toward using small molecules to generate insulin in an alternative cell type. PMID:22242153

  11. Helix-loop-helix transcription factors mediate activation and repression of the p75LNGFR gene.

    PubMed Central

    Chiaramello, A; Neuman, K; Palm, K; Metsis, M; Neuman, T

    1995-01-01

    Sequence analysis of rat and human low-affinity nerve growth factor receptor p75LNGFR gene promoter regions revealed a single E-box cis-acting element, located upstream of the major transcription start sites. Deletion analysis of the E-box sequence demonstrated that it significantly contributes to p75LNGFR promoter activity. This E box has a dual function; it mediates either activation or repression of the p75LNGFR promoter activity, depending on the interacting transcription factors. We showed that the two isoforms of the class A basic helix-loop-helix (bHLH) transcription factor ME1 (ME1a and ME1b), the murine homolog of the human HEB transcription factor, specifically repress p75LNGFR promoter activity. This repression can be released by coexpression of the HLH Id2 transcriptional regulator. In vitro analyses demonstrated that ME1a forms a stable complex with the p75LNGFR E box and likely competes with activating E-box-binding proteins. By using ME1a-overexpressing PC12 cells, we showed that the endogenous p75LNGFR gene is a target of ME1a repression. Together, these data demonstrate that the p75LNGFR E box and the interacting bHLH transcription factors are involved in the regulation of p75LNGFR gene expression. These results also show that class A bHLH transcription factors can repress and Id-like negative regulators can stimulate gene expression. PMID:7565756

  12. Transcriptional Control by A-Factor of strR, the Pathway-Specific Transcriptional Activator for Streptomycin Biosynthesis in Streptomyces griseus

    PubMed Central

    Tomono, Ayami; Tsai, Yisan; Yamazaki, Haruka; Ohnishi, Yasuo; Horinouchi, Sueharu

    2005-01-01

    A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) triggers streptomycin production by inducing the transcription of strR, encoding the pathway-specific transcriptional activator, through signal transduction in the A-factor regulatory cascade in Streptomyces griseus. AdpA, one of the key transcriptional activators in the cascade, bound two upstream activation sites, approximately at nucleotide positions −270 and −50 with respect to the transcriptional start point of strR, as determined by gel mobility shift assays and DNase I footprinting. Transcriptional analysis of the strR promoter with mutated AdpA-binding sites showed that both sites were required for full transcriptional activation of strR by AdpA. Potassium permanganate footprinting showed that AdpA assisted RNA polymerase in forming an open complex at an appropriate position for transcriptional initiation of strR. Nine transcriptional units within the streptomycin biosynthesis gene cluster, including the strR-aphD operon, depended on StrR, indicating that StrR is the pathway-specific transcriptional activator for the whole gene cluster. Consistent with this, expression of strR under the control of a constitutively expressed promoter in an adpA null mutant caused the host to produce streptomycin. PMID:16077104

  13. The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

    PubMed Central

    Rudd, M D; Izban, M G; Luse, D S

    1994-01-01

    RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active. Images PMID:8058756

  14. Characteristics of Antisense Transcript Promoters and the Regulation of Their Activity

    PubMed Central

    Lin, Shudai; Zhang, Li; Luo, Wen; Zhang, Xiquan

    2015-01-01

    Recently, an increasing number of studies on natural antisense transcripts have been reported, especially regarding their classification, temporal and spatial expression patterns, regulatory functions and mechanisms. It is well established that natural antisense transcripts are produced from the strand opposite to the strand encoding a protein. Despite the pivotal roles of natural antisense transcripts in regulating the expression of target genes, the transcriptional mechanisms initiated by antisense promoters (ASPs) remain unknown. To date, nearly all of the studies conducted on this topic have focused on the ASP of a single gene of interest, whereas no study has systematically analyzed the locations of ASPs in the genome, ASP activity, or factors influencing this activity. This review focuses on elaborating on and summarizing the characteristics of ASPs to extend our knowledge about the mechanisms of antisense transcript initiation. PMID:26703594

  15. Promotion of Homologous Recombination and Genomic Stability byRAD51AP1 via RAD51 Recombinase Enhancement

    SciTech Connect

    Wiese, Claudia; Dray, Eloise; Groesser, Torsten; San Filippo,Joseph; Shi, Idina; Collins, David W.; Tsai, Miaw-Sheue; Williams,Gareth; Rydberg, Bjorn; Sung, Patrick; Schild, David

    2007-04-11

    Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand pairing step in HR. RAD51AP1 (RAD51 Associated Protein 1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA damaging treatment. Purified RAD51AP1 binds dsDNA and RAD51, and it greatly stimulates the RAD51-mediated D-loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide the first evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

  16. v-SNARE cellubrevin is required for basolateral sorting of AP-1B–dependent cargo in polarized epithelial cells

    PubMed Central

    Fields, Ian C.; Shteyn, Elina; Pypaert, Marc; Proux-Gillardeaux, Véronique; Kang, Richard S.; Galli, Thierry; Fölsch, Heike

    2007-01-01

    The epithelial cell–specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B–dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking. PMID:17485489

  17. Promotion of RAD51-Mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex.

    PubMed

    Liang, Fengshan; Longerich, Simonne; Miller, Adam S; Tang, Caroline; Buzovetsky, Olga; Xiong, Yong; Maranon, David G; Wiese, Claudia; Kupfer, Gary M; Sung, Patrick

    2016-06-01

    The UAF1-USP1 complex deubiquitinates FANCD2 during execution of the Fanconi anemia DNA damage response pathway. As such, UAF1 depletion results in persistent FANCD2 ubiquitination and DNA damage hypersensitivity. UAF1-deficient cells are also impaired for DNA repair by homologous recombination. Herein, we show that UAF1 binds DNA and forms a dimeric complex with RAD51AP1, an accessory factor of the RAD51 recombinase, and a trimeric complex with RAD51 through RAD51AP1. Two small ubiquitin-like modifier (SUMO)-like domains in UAF1 and a SUMO-interacting motif in RAD51AP1 mediate complex formation. Importantly, UAF1 enhances RAD51-mediated homologous DNA pairing in a manner that is dependent on complex formation with RAD51AP1 but independent of USP1. Mechanistically, RAD51AP1-UAF1 co-operates with RAD51 to assemble the synaptic complex, a critical nucleoprotein intermediate in homologous recombination, and cellular studies reveal the biological significance of the RAD51AP1-UAF1 protein complex. Our findings provide insights into an apparently USP1-independent role of UAF1 in genome maintenance. PMID:27239033

  18. An AGEF-1/Arf GTPase/AP-1 Ensemble Antagonizes LET-23 EGFR Basolateral Localization and Signaling during C. elegans Vulva Induction

    PubMed Central

    Skorobogata, Olga; Escobar-Restrepo, Juan M.; Rocheleau, Christian E.

    2014-01-01

    LET-23 Epidermal Growth Factor Receptor (EGFR) signaling specifies the vulval cell fates during C. elegans larval development. LET-23 EGFR localization on the basolateral membrane of the vulval precursor cells (VPCs) is required to engage the LIN-3 EGF-like inductive signal. The LIN-2 Cask/LIN-7 Veli/LIN-10 Mint (LIN-2/7/10) complex binds LET-23 EGFR, is required for its basolateral membrane localization, and therefore, vulva induction. Besides the LIN-2/7/10 complex, the trafficking pathways that regulate LET-23 EGFR localization have not been defined. Here we identify vh4, a hypomorphic allele of agef-1, as a strong suppressor of the lin-2 mutant Vulvaless (Vul) phenotype. AGEF-1 is homologous to the mammalian BIG1 and BIG2 Arf GTPase guanine nucleotide exchange factors (GEFs), which regulate secretory traffic between the Trans-Golgi network, endosomes and the plasma membrane via activation of Arf GTPases and recruitment of the AP-1 clathrin adaptor complex. Consistent with a role in trafficking we show that AGEF-1 is required for protein secretion and that AGEF-1 and the AP-1 complex regulate endosome size in coelomocytes. The AP-1 complex has previously been implicated in negative regulation of LET-23 EGFR, however the mechanism was not known. Our genetic data indicate that AGEF-1 is a strong negative regulator of LET-23 EGFR signaling that functions in the VPCs at the level of the receptor. In line with AGEF-1 being an Arf GEF, we identify the ARF-1.2 and ARF-3 GTPases as also negatively regulating signaling. We find that the agef-1(vh4) mutation results in increased LET-23 EGFR on the basolateral membrane in both wild-type and lin-2 mutant animals. Furthermore, unc-101(RNAi), a component of the AP-1 complex, increased LET-23 EGFR on the basolateral membrane in lin-2 and agef-1(vh4); lin-2 mutant animals. Thus, an AGEF-1/Arf GTPase/AP-1 ensemble functions opposite the LIN-2/7/10 complex to antagonize LET-23 EGFR basolateral membrane localization and signaling

  19. Biomechanical Signals Suppress TAK1 Activation to Inhibit NF-κB Transcriptional Activation in Fibrochondrocytes

    PubMed Central

    Madhavan, Shashi; Anghelina, Mirela; Sjostrom, Danen; Dossumbekova, Anar; Guttridge, Denis C.; Agarwal, Sudha

    2016-01-01

    Exercise/joint mobilization is therapeutic for inflammatory joint diseases like rheumatoid and osteoarthritis, but the mechanisms underlying its actions remain poorly understood. We report that biomechanical signals at low/physiological magnitudes are potent inhibitors of inflammation induced by diverse proinflammatory activators like IL-1β, TNF-α, and lipopolysaccharides, in fibrochondrocytes. These signals exert their anti-inflammatory effects by inhibiting phosphorylation of TAK1, a critical point where signals generated by IL-1β, TNF-α, and LPS converge to initiate NF-κB signaling cascade and proinflammatory gene induction. Additionally, biomechanical signals inhibit multiple steps in the IL-1β-induced proinflammatory cascade downstream of IκB kinase activation to regulate IκBα and IκBβ degradation and synthesis, and promote IκBα shuttling to export nuclear NF-κB and terminate its transcriptional activity. The findings demonstrate that biomechanical forces are but another important signal that uses NF-κB pathway to regulate inflammation by switching the molecular activation of discrete molecules involved in proinflammatory gene transcription. PMID:17947700

  20. Proapoptotic RYBP interacts with FANK1 and induces tumor cell apoptosis through the AP-1 signaling pathway.

    PubMed

    Ma, Wen; Zhang, Xuan; Li, Meng; Ma, Xiaoli; Huang, Bingren; Chen, Hong; Chen, Deng

    2016-08-01

    Ring1 and YY1 Binding Protein (RYBP) induces tumor-specific cell apoptosis, but the underlying molecular mechanism has not been fully understood. Here we conducted a yeast two hybrid screen and identified FANK1 (Fibronectin type III and ankyrin repeat domains 1) as a novel RYBP-interacting protein. This interaction was confirmed by coimmunoprecipitation, GST pulldown and immunofluorescence assays. We mapped that the FNIII domain at the N-terminal of FANK1 binds to the Serine/Threonine-rich region at the C-terminal of RYBP. Further studies showed that overexpression of RYBP stabilized, whereas knockdown of RYBP by its specific shRNAs reduced, the expression of FANK1. Mechanistic studies revealed that RYBP inhibited the proteasome degradation of polyubiquitinated FANK1, thus prolonging the half-life of FANK1 protein. Functional studies indicated that RYBP activates FANK1-mediated activator protein 1 (AP-1) signaling pathway which contributes to tumor cell apoptosis. Taken together, our current study uncovered a new mechanism which RYBP utilizes to exert its pro-apoptotic activity in human tumor cells. PMID:27060496