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Sample records for mapk-mediated ap-1 activation

  1. 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

  2. Small molecule inhibitors targeting activator protein 1 (AP-1).

    PubMed

    Ye, Na; Ding, Ye; Wild, Christopher; Shen, Qiang; Zhou, Jia

    2014-08-28

    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.

  3. Antioxidants and AP-1 activation: a brief overview.

    PubMed

    Gomez del Arco, P; Martínez-Martínez, S; Calvo, V; Armesilla, A L; Redondo, J M

    1997-12-01

    Activity of the transcription factor AP-1 is controlled by different MAPK cascades that regulate the different AP-1 components at the transcriptional and posttranscriptional level. Recently, AP-1 has been shown to behave as a redox-sensitive transcription factor that can be induced under both pro-oxidative and antioxidative conditions. In this overview we summarize the signaling pathways that converge on the activation of AP-1 and the components of these pathways that have been shown to be targets of antioxidants. The activation of AP-1 by antioxidants may account for the expression of a number of genes that mediate important functions under physiological conditions.

  4. Modulation of AP-1 activity by nitric oxide (NO) in vitro: NO-mediated modulation of AP-1.

    PubMed

    Tabuchi, A; Sano, K; Oh, E; Tsuchiya, T; Tsuda, M

    1994-08-29

    To understand the role of nitric oxide (NO) in controlling the specific DNA-binding activities of transcriptional factors, we investigated the in vitro effect of the NO-donor sodium nitroprusside (SNP) on the AP-1 activity of cultured mouse cerebellar granule cells. A gel-mobility assay showed that SNP inhibited AP-1 activity in the presence, but not the absence of dithiothreitol (DTT). This DTT-dependent inhibition of AP-1 activity by SNP corresponded with the activation of the chemical reactivity of SNP with DTT, which can be monitored by the production of nitrite (NO2-). In contrast, diamide, a typical sulfhydryl oxidizing agent, inhibited AP-1 activity in the absence of DTT and its inhibitory effect was reversed competitively by DTT. Studies using structurally or functionally related analogues of SNP demonstrated that S-nitrosylation of the AP-1 moiety mediated by some NO-carriers but not by free NO, which can be produced by the chemical reaction of SNP with DTT, was responsible for the inhibition of AP-1 activity, suggesting NO-mediated regulation of the AP-1 transcriptional factor.

  5. Activation of transcriptional activities of AP-1 and SRE by a new zinc-finger protein ZNF641

    SciTech Connect

    Qi Xingzhu; Li Yongqing; Xiao Jing; Yuan Wuzhou; Yan Yan; Wang Yuequn; Liang Shuyuan; Zhu Chuanbing; Chen Yingduan; Liu Mingyao . E-mail: mliu@ibt.tamhsc.edu; Wu Xiushan

    2006-01-27

    Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that zinc-finger proteins are involved in the regulation of the MAPK signaling pathways. Here, we report the identification and characterization of a novel human zinc-finger protein, ZNF641. The cDNA of ZNF641 is 4.9 kb, encoding 438 amino acids in the nucleus. The protein is highly conserved in evolution across different vertebrate species from mouse to human. Northern blot analysis indicates that ZNF641 is expressed in most of the examined human tissues, with a high level in skeletal muscle. Overexpression of pCMV-Tag2B-ZNF641 in the COS-7 cells activates the transcriptional activities of AP-1 and SRE. Deletion analysis indicates that the linker between KRAB box and C{sub 2}H{sub 2}-type zinc-fingers represents the basal activation domain. These results suggest that ZNF641 may be a positive regulator in MAPK-mediated signaling pathways that lead to the activation of AP-1 and SRE.

  6. 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.

  7. 7-O-Geranylquercetin induces apoptosis in gastric cancer cells via ROS-MAPK mediated mitochondrial signaling pathway activation.

    PubMed

    Zhu, Yanyan; Jiang, Yameng; Shi, Lei; Du, Linying; Xu, Xiaodong; Wang, Enxia; Sun, Yong; Guo, Xin; Zou, Boyang; Wang, Huaxin; Wang, Changyuan; Sun, Lidan; Zhen, Yuhong

    2017-03-01

    7-O-Geranylquercetin (GQ) is a novel O-alkylated derivate of quercetin. In this study, we evaluated its apoptosis induction effects in human gastric cancer cell lines SGC-7901 and MGC-803 and explored the potential molecular mechanisms. The results demonstrated that GQ lowered viability of SGC-7901 and MGC-803 cells in a dose- and time-dependent manner without apparent cytotoxicity to human gastric epithelial cell line GES-1. GQ could induce apoptosis in SGC-7901 and MGC-803cells, and arrest the gastric cancer cells at G2/M phase. Mechanism study showed that GQ triggered generation of reactive oxygen species (ROS), then activated p38 and JNK signaling pathways, subsequently led to mitochondrial impairment by regulating the expression of Bcl-2, Bcl-xl and Bax, and finally promoted the release of cytochrome c and the activation of caspases to induce apoptosis. In addition, Z-VAD-FMK (caspase inhibitor) could reverse GQ-induced apoptosis. SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) could rescue GQ-induced cell death and attenuate mitochondrial signal pathway activation. Furthermore, NAC (ROS inhibitor) could rescue GQ-induced cell death, reduce ROS generation, decrease the phosphorylation of p38 and JNK, and then attenuate the activation of mitochondrial signal pathway. Taken together, GQ induces caspase-dependent apoptosis in gastric cancer cells through activating ROS-MAPK mediated mitochondrial signal pathway. This study highlights the potential use of GQ as a gastric cancer therapeutic agent.

  8. 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.

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

    PubMed

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

    2015-09-25

    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.

  10. Mineralocorticoid Receptor (MR) trans-Activation of Inflammatory AP-1 Signaling: DEPENDENCE ON DNA SEQUENCE, MR CONFORMATION, AND AP-1 FAMILY MEMBER EXPRESSION.

    PubMed

    Dougherty, Edward J; Elinoff, Jason M; Ferreyra, Gabriela A; Hou, Angela; Cai, Rongman; Sun, Junfeng; Blaine, Kevin P; Wang, Shuibang; Danner, Robert L

    2016-11-04

    Glucocorticoids are commonly used to treat inflammatory disorders. The glucocorticoid receptor (GR) can tether to inflammatory transcription factor complexes, such as NFκB and AP-1, and trans-repress the transcription of cytokines, chemokines, and adhesion molecules. In contrast, aldosterone and the mineralocorticoid receptor (MR) primarily promote cardiovascular inflammation by incompletely understood mechanisms. Although MR has been shown to weakly repress NFκB, its role in modulating AP-1 has not been established. Here, the effects of GR and MR on NFκB and AP-1 signaling were directly compared using a variety of ligands, two different AP-1 consensus sequences, GR and MR DNA-binding domain mutants, and siRNA knockdown or overexpression of core AP-1 family members. Both GR and MR repressed an NFκB reporter without influencing p65 or p50 binding to DNA. Likewise, neither GR nor MR affected AP-1 binding, but repression or activation of AP-1 reporters occurred in a ligand-, AP-1 consensus sequence-, and AP-1 family member-specific manner. Notably, aldosterone interactions with both GR and MR demonstrated a potential to activate AP-1. DNA-binding domain mutations that eliminated the ability of GR and MR to cis-activate a hormone response element-driven reporter variably affected the strength and polarity of these responses. Importantly, MR modulation of NFκB and AP-1 signaling was consistent with a trans-mechanism, and AP-1 effects were confirmed for specific gene targets in primary human cells. Steroid nuclear receptor trans-effects on inflammatory signaling are context-dependent and influenced by nuclear receptor conformation, DNA sequence, and the expression of heterologous binding partners. Aldosterone activation of AP-1 may contribute to its proinflammatory effects in the vasculature.

  11. Interplay between TAp73 Protein and Selected Activator Protein-1 (AP-1) Family Members Promotes AP-1 Target Gene Activation and Cellular Growth.

    PubMed

    Subramanian, Deepa; Bunjobpol, Wilawan; Sabapathy, Kanaga

    2015-07-24

    Unlike p53, which is mutated at a high rate in human cancers, its homologue p73 is not mutated but is often overexpressed, suggesting a possible context-dependent role in growth promotion. Previously, we have shown that co-expression of TAp73 with the proto-oncogene c-Jun can augment cellular growth and potentiate transactivation of activator protein (AP)-1 target genes such as cyclin D1. Here, we provide further mechanistic insights into the cooperative activity between these two transcription factors. Our data show that TAp73-mediated AP-1 target gene transactivation relies on c-Jun dimerization and requires the canonical AP-1 sites on target gene promoters. Interestingly, only selected members of the Fos family of proteins such as c-Fos and Fra1 were found to cooperate with TAp73 in a c-Jun-dependent manner to transactivate AP-1 target promoters. Inducible expression of TAp73 led to the recruitment of these Fos family members to the AP-1 target promoters on which TAp73 was found to be bound near the AP-1 site. Consistent with the binding of TAp73 and AP-1 members on the target promoters in a c-Jun-dependent manner, TAp73 was observed to physically interact with c-Jun specifically at the chromatin via its carboxyl-terminal region. Furthermore, co-expression of c-Fos or Fra1 was able to cooperate with TAp73 in potentiating cellular growth, similarly to c-Jun. These data together suggest that TAp73 plays a vital role in activation of AP-1 target genes via direct binding to c-Jun at the target promoters, leading to enhanced loading of other AP-1 family members, thereby leading to cellular growth.

  12. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal-regulated kinase and CBP recruitment to an AP1-responsive promoter.

    PubMed

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-07-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPK(ERK)) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters.

  13. Retinoic Acid Receptors Inhibit AP1 Activation by Regulating Extracellular Signal-Regulated Kinase and CBP Recruitment to an AP1-Responsive Promoter

    PubMed Central

    Benkoussa, Madjid; Brand, Céline; Delmotte, Marie-Hélène; Formstecher, Pierre; Lefebvre, Philippe

    2002-01-01

    Retinoids exhibit antineoplastic activities that may be linked to retinoid receptor-mediated transrepression of activating protein 1 (AP1), a heterodimeric transcription factor composed of fos- and jun-related proteins. Here we show that transcriptional activation of an AP1-regulated gene through the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway (MAPKERK) is characterized, in intact cells, by a switch from a fra2-junD dimer to a junD-fosB dimer loading on its promoter and by simultaneous recruitment of ERKs, CREB-binding protein (CBP), and RNA polymerase II. All-trans-retinoic acid (atRA) receptor (RAR) was tethered constitutively to the AP1 promoter. AP1 transrepression by retinoic acid was concomitant to glycogen synthase kinase 3 activation, negative regulation of junD hyperphosphorylation, and to decreased RNA polymerase II recruitment. Under these conditions, fra1 loading to the AP1 response element was strongly increased. Importantly, CBP and ERKs were excluded from the promoter in the presence of atRA. AP1 transrepression by retinoids was RAR and ligand dependent, but none of the functions required for RAR-mediated transactivation was necessary for AP1 transrepression. These results indicate that transrepressive effects of retinoids are mediated through a mechanism unrelated to transcriptional activation, involving the RAR-dependent control of transcription factors and cofactor assembly on AP1-regulated promoters. PMID:12052862

  14. Structural basis for recruitment and activation of the AP-1 clathrin adaptor complex by Arf1.

    PubMed

    Ren, Xuefeng; Farías, Ginny G; Canagarajah, Bertram J; Bonifacino, Juan S; Hurley, James H

    2013-02-14

    AP-1 is a clathrin adaptor complex that sorts cargo between the trans-Golgi network and endosomes. AP-1 recruitment to these compartments requires Arf1-GTP. The crystal structure of the tetrameric core of AP-1 in complex with Arf1-GTP, together with biochemical analyses, shows that Arf1 activates cargo binding by unlocking AP-1. Unlocking is driven by two molecules of Arf1 that bridge two copies of AP-1 at two interaction sites. The GTP-dependent switch I and II regions of Arf1 bind to the N terminus of the β1 subunit of one AP-1 complex, while the back side of Arf1 binds to the central part of the γ subunit trunk of a second AP-1 complex. A third Arf1 interaction site near the N terminus of the γ subunit is important for recruitment, but not activation. These observations lead to a model for the recruitment and activation of AP-1 by Arf1.

  15. Multiple Bcl-2 family immunomodulators from vaccinia virus regulate MAPK/AP-1 activation.

    PubMed

    Torres, Alice A; Albarnaz, Jonas D; Bonjardim, Cláudio A; Smith, Geoffrey L

    2016-09-01

    Vaccinia virus (VACV) is a poxvirus and encodes many proteins that modify the host cell metabolism or inhibit the host response to infection. For instance, it is known that VACV infection can activate the mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) pathway and inhibit activation of the pro-inflammatory transcription factor NF-κB. Since NF-κB and MAPK/AP-1 share common upstream activators we investigated whether six different VACV Bcl-2-like NF-κB inhibitors can also influence MAPK/AP-1 activation. Data presented show that proteins A52, B14 and K7 each contribute to AP-1 activation during VACV infection, and when expressed individually outwith infection. B14 induced the greatest stimulation of AP-1 and further investigation showed B14 activated mainly the MAPKs ERK (extracellular signal-regulated kinase) and JNK (Jun N-terminal kinase), and their substrate c-Jun (a component of AP-1). These data indicate that the same viral protein can have different effects on distinct signalling pathways, in blocking NF-κB activation whilst leading to MAPK/AP-1 activation.

  16. Activator protein-1 (AP-1) and response to pathogen infection in the Hong Kong oyster (Crassostrea hongkongensis).

    PubMed

    Xiang, Zhiming; Qu, Fufa; Li, Jun; Qi, Lin; Yang, Zhang; Kong, Xiaoyu; Yu, Ziniu

    2014-01-01

    Growing evidence suggests that the transcription factor activator protein-1 (AP-1), a downstream target of mitogen-activated protein kinase (MAPK) signaling, plays a major role in stimulating the synthesis of immune effector molecules during innate immune responses. We have characterized ChAP-1, an AP-1-like protein in Crassostrea hongkongensis that is a member of the AP-1 family of proteins. ChAP-1 is composed of 290 amino acid residues with a Jun and bZIP domain at the N- and C-termini, respectively, a structure similar to that of known Ap-1 proteins. ChAP-1 mRNA is expressed in several tissues analyzed, with highest expression in the mantle. Expression of ChAP-1 increases in response to Vibrio alginolyticus, Salmo haemolyticus or Salmo cerevisiae infection and, despite the location of GFP-tagged full-length ChAP-1 protein in the cytoplasm, ChAP-1 activates the transcription of an L8G5-luc reporter gene, and its over-expression can also activate the AP-1-Luc reporter gene in HEK293T cells.

  17. AMP-activated protein kinase α1-sensitive activation of AP-1 in cardiomyocytes.

    PubMed

    Voelkl, Jakob; Alesutan, Ioana; Primessnig, Uwe; Feger, Martina; Mia, Sobuj; Jungmann, Andreas; Castor, Tatsiana; Viereck, Robert; Stöckigt, Florian; Borst, Oliver; Gawaz, Meinrad; Schrickel, Jan Wilko; Metzler, Bernhard; Katus, Hugo A; Müller, Oliver J; Pieske, Burkert; Heinzel, Frank R; Lang, Florian

    2016-08-01

    AMP-activated protein kinase (Ampk) regulates myocardial energy metabolism and plays a crucial role in the response to cell stress. In the failing heart, an isoform shift of the predominant Ampkα2 to the Ampkα1 was observed. The present study explored possible isoform specific effects of Ampkα1 in cardiomyocytes. To this end, experiments were performed in HL-1 cardiomyocytes, as well as in Ampkα1-deficient and corresponding wild-type mice and mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1. As a result, in HL-1 cardiomyocytes, overexpression of constitutively active Ampkα1 increased the phosphorylation of Pkcζ. Constitutively active Ampkα1 further increased AP-1-dependent transcriptional activity and mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1, effects blunted by Pkcζ silencing. In HL-1 cardiomyocytes, angiotensin-II activated AP-1, an effect blunted by silencing of Ampkα1 and Pkcζ, but not of Ampkα2. In wild-type mice, angiotensin-II infusion increased cardiac Ampkα1 and cardiac Pkcζ protein levels, as well as c-Fos, Il6 and Ncx1 mRNA expression, effects blunted in Ampkα1-deficient mice. Pressure overload by transverse aortic constriction (TAC) similarly increased cardiac Ampkα1 and Pkcζ abundance as well as c-Fos, Il6 and Ncx1 mRNA expression, effects again blunted in Ampkα1-deficient mice. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 increased Pkcζ protein abundance and the mRNA expression of c-Fos, Il6 and Ncx1 in cardiac tissue. In conclusion, Ampkα1 promotes myocardial AP-1 activation in a Pkcζ-dependent manner and thus contributes to cardiac stress signaling.

  18. 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...

  19. Human ZCCHC12 activates AP-1 and CREB signaling as a transcriptional co-activator.

    PubMed

    Li, Hong; Liu, Qian; Hu, Xiang; Feng, Du; Xiang, Shuanglin; He, Zhicheng; Hu, Xingwang; Zhou, Jianlin; Ding, Xiaofeng; Zhou, Chang; Zhang, Jian

    2009-07-01

    Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) signaling, and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR). However, the details of how human ZCCHC12 involve in the NS-XLMR still remain unclear. In this study, we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization. Multiple-tissue northern blot analysis indicated that ZCCHC12 is highly expressed in human brain. Furthermore, in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain, midbrain, and diencephalon regions of mouse E10.5 embryos. Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcriptional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a coactivator. In conclusion, we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.

  20. Specific activation of human interleukin-5 depends on de novo synthesis of an AP-1 complex.

    PubMed

    Schwenger, Gretchen T F; Kok, Chee Choy; Arthaningtyas, Estri; Thomas, Marc A; Sanderson, Colin J; Mordvinov, Viatcheslav A

    2002-12-06

    It is clear from the biology of eosinophilia that a specific regulatory mechanism must exist. Because interleukin-5 (IL5) is the key regulatory cytokine, it follows that a gene-specific control of IL5 expression must exist that differs even from closely related cytokines such as IL4. Two features of IL5 induction make it unique compared with other cytokines; first, induction by cyclic adenosine monophosphate (cAMP), which inhibits other T-cell-derived cytokines, and second, sensitivity to protein synthesis inhibitors, which have no effect on other cytokines. This study has utilized the activation of different transcription factors by different stimuli in a human T-cell line to study the role of conserved lymphokine element 0 (CLE0) in the specific induction of IL5. In unstimulated cells the ubiquitous Oct-1 binds to CLE0. Stimulation induces de novo synthesis of the AP-1 members JunD and Fra-2, which bind to CLE0. The amount of IL5 produced correlates with the production of the AP-1 complex, suggesting a key role in IL5 expression. The formation of the AP-1 complex is essential, but the rate-limiting step is the synthesis of AP-1, especially Fra-2. This provides an explanation for the sensitivity of IL5 to protein synthesis inhibitors and a mechanism for the specific induction of IL5 compared with other cytokines.

  1. Transcriptional upregulation of DDR2 by ATF4 facilitates osteoblastic differentiation through p38 MAPK-mediated Runx2 activation.

    PubMed

    Lin, Kuan-Liang; Chou, Ching-Heng; Hsieh, Shu-Chen; Hwa, Su-Yang; Lee, Ming-Ta; Wang, Fung-Fang

    2010-11-01

    Deficiency of the collagen receptor discoidin domain receptor tyrosine kinase (DDR2) in mice and humans results in dwarfism and short limbs, of which the mechanism remains unknown. Here we report that DDR2 is a key regulator of osteoblast differentiation. DDR2 mRNA expression was increased at an early stage of induced osteoblast differentiation. In the subchondral bone of human osteoarthritic knee, DDR2 was detected in osteoblastic cells. In mouse embryos, DDR2 expression was found from E11 to E15, preceding osteocalcin (OCN) and coinciding with Runx2 expression. Activating transcription factor 4 (ATF4) enhanced DDR2 mRNA expression, and knockdown of ATF4 expression delayed DDR2 induction during osteoblast differentiation. A CCAAT/enhancer binding protein (C/EBP) binding site at -1150 bp in the DDR2 promoter was required for ATF4-mediated DDR2 activation. C/EBPβ bound to and cooperated with ATF4 in stimulating DDR2 transcription; accordingly, the ATF4 mutants deficient of C/EBPβ binding were incapable of transactivating DDR2. Overexpression of DDR2 increased osteoblast-specific gene expression. Conversely, knockdown of DDR2 suppressed osteogenic marker gene expression and matrix mineralization during the induced osteogenesis. The stimulation of p38 MAPK by DDR2 was required for DDR2-induced activation of Runx2 and OCN promoters. Together our findings uncover a pathway in which ATF4, by binding to C/EBPβ transcriptionally upregulates DDR2 expression, and DDR2, in turn, activates Runx2 through p38 MAPK to promote osteoblast differentiation.

  2. Palmitoleic acid prevents palmitic acid-induced macrophage activation and consequent p38 MAPK-mediated skeletal muscle insulin resistance.

    PubMed

    Talbot, Nicola A; Wheeler-Jones, Caroline P; Cleasby, Mark E

    2014-08-05

    Obesity and saturated fatty acid (SFA) treatment are both associated with skeletal muscle insulin resistance (IR) and increased macrophage infiltration. However, the relative effects of SFA and unsaturated fatty acid (UFA)-activated macrophages on muscle are unknown. Here, macrophages were treated with palmitic acid, palmitoleic acid or both and the effects of the conditioned medium (CM) on C2C12 myotubes investigated. CM from palmitic acid-treated J774s (palm-mac-CM) impaired insulin signalling and insulin-stimulated glycogen synthesis, reduced Inhibitor κBα and increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase in myotubes. p38 MAPK inhibition or siRNA partially ameliorated these defects, as did addition of tumour necrosis factor-α blocking antibody to the CM. Macrophages incubated with both FAs generated CM that did not induce IR, while palmitoleic acid-mac-CM alone was insulin sensitising. Thus UFAs may improve muscle insulin sensitivity and counteract SFA-mediated IR through an effect on macrophage activation.

  3. Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway

    PubMed Central

    Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

    2015-01-01

    Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue. PMID:26412401

  4. 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

  5. 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

  6. Thioredoxin reductase regulates AP-1 activity as well as thioredoxin nuclear localization via active cysteines in response to ionizing radiation.

    PubMed

    Karimpour, Shervin; Lou, Junyang; Lin, Lilie L; Rene, Luis M; Lagunas, Lucio; Ma, Xinrong; Karra, Sreenivasu; Bradbury, C Matthew; Markovina, Stephanie; Goswami, Prabhat C; Spitz, Douglas R; Hirota, Kiichi; Kalvakolanu, Dhananjaya V; Yodoi, Junji; Gius, David

    2002-09-12

    A recently identified class of signaling factors uses critical cysteine motif(s) that act as redox-sensitive 'sulfhydryl switches' to reversibly modulate specific signal transduction cascades regulating downstream proteins with similar redox-sensitive sites. For example, signaling factors such as redox factor-1 (Ref-1) and transcription factors such as the AP-1 complex both contain redox-sensitive cysteine motifs that regulate activity in response to oxidative stress. The mammalian thioredoxin reductase-1 (TR) is an oxidoreductase selenocysteine-containing flavoprotein that also appears to regulate multiple downstream intracellular redox-sensitive proteins. Since ionizing radiation (IR) induces oxidative stress as well as increases AP-1 DNA-binding activity via the activation of Ref-1, the potential roles of TR and thioredoxin (TRX) in the regulation of AP-1 activity in response to IR were investigated. Permanently transfected cell lines that overexpress wild type TR demonstrated constitutive increases in AP-1 DNA-binding activity as well as AP-1-dependent reporter gene expression, relative to vector control cells. In contrast, permanently transfected cell lines expressing a TR gene with the active site cysteine motif deleted were unable to induce AP-1 activity or reporter gene expression in response to IR. Transient genetic overexpression of either the TR wild type or dominant-negative genes demonstrated similar results using a transient assay system. One mechanism through which TR regulates AP-1 activity appears to involve TRX sub-cellular localization, with no change in the total TRX content of the cell. These results identify a novel function of the TR enzyme as a signaling factor in the regulation of AP-1 activity via a cysteine motif located in the protein.

  7. The novel secretory protein CGREF1 inhibits the activation of AP-1 transcriptional activity and cell proliferation.

    PubMed

    Deng, Weiwei; Wang, Lan; Xiong, Ying; Li, Jing; Wang, Ying; Shi, Taiping; Ma, Dalong

    2015-08-01

    The transcription factor AP-1 plays an important role in inflammation and cell survival. Using a dual-luciferase reporter assay system and a library of 940 candidate human secretory protein cDNA clones, we identified that CGREF1 can inhibit the transcriptional activity of AP-1. We demonstrated that CGREF1 is secreted via the classical secretory pathway through the ER-to-Golgi apparatus. Functional investigations revealed that overexpression of CGREF1 can significantly inhibit the phosphorylation of ERK and p38 MAPK, and suppress the proliferation of HEK293T and HCT116 cells. Conversely, specific siRNAs against CGREF1 can increase the transcriptional activity of AP-1. These results clearly indicated that CGREF1 is a novel secretory protein, and plays an important role in regulation of AP-1 transcriptional activity and cell proliferation.

  8. E1A dependent up-regulation of c-jun/AP-1 activity.

    PubMed Central

    Kitabayashi, I; Chiu, R; Gachelin, G; Yokoyama, K

    1991-01-01

    E1A, the early region 1A transcription unit of human adenovirus, exhibits multiple functions that regulate the expression of some cellular genes and promote cell growth and division. We found that E1A stimulated c-jun gene expression at least fifty-fold in rat 3Y1 cells in a serum-independent manner, concomitantly with E1A down-regulation of jun B expression. The E1A-dependent induction of c-jun transcription resulted in increase amount of cJun/AP1. This induction was mediated by the enhancement of the binding activity of the transcription factor cJun/AP1 to an AP1 binding site in the c-jun promoter. Additionally, this induction can be repressed by introducing junB into the cells. Taken collectively, these results suggest that the differential expression of two closely related proteins greatly expands their cellular regulation. Induction of c-jun expression by E1A as well as c-jun autoregulation may amplify the action of E1A during adenovirus infection. Therefore, some of the biological effects of E1A may include mediating the constitutive activation of c-jun, which is important in transcriptional regulation and oncogenic transformation. Images PMID:1826351

  9. Impaired dynamin 2 function leads to increased AP-1 transcriptional activity through the JNK/c-Jun pathway.

    PubMed

    Szymanska, Ewelina; Skowronek, Agnieszka; Miaczynska, Marta

    2016-01-01

    Activation of AP-1 transcription factors, composed of the Jun and Fos proteins, regulates cellular fates, such as proliferation, differentiation or apoptosis. Among other stimuli, the AP-1 pathway can be initiated by extracellular ligands, such as growth factors or cytokines, which undergo internalization in complex with their receptors. Endocytosis has been implicated in the regulation of several signaling pathways; however its possible impact on AP-1 signaling remains unknown. Here we show that inhibition of dynamin 2 (Dyn2), a major regulator of endocytic internalization, strongly stimulates the AP-1 pathway. Specifically, expression of a dominant-negative Dyn2 K44A mutant increases the total levels of c-Jun, its phosphorylation on Ser63/73 and transcription of AP-1 target genes. Interestingly, DNM2 mutations implicated in human neurological disorders exhibit similar effects on AP-1 signaling. Mechanistically, Dyn2 K44A induces AP-1 by increasing phosphorylation of several receptor tyrosine kinases. Their activation is required to initiate a Src- and JNK-dependent signaling cascade converging on c-Jun and stimulating expression of AP-1 target genes. Cumulatively, our data uncover a link between the Dyn2 function and JNK signaling which leads to AP-1 induction.

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

    PubMed

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

    2013-02-01

    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.

  11. Chloroquine inhibits human CD4+ T-cell activation by AP-1 signaling modulation

    PubMed Central

    Schmidt, Ralf L. J.; Jutz, Sabrina; Goldhahn, Katrin; Witzeneder, Nadine; Gerner, Marlene C.; Trapin, Doris; Greiner, Georg; Hoermann, Gregor; Steiner, Guenter; Pickl, Winfried F.; Burgmann, Heinz; Steinberger, Peter; Ratzinger, Franz; Schmetterer, Klaus G.

    2017-01-01

    Chloroquine (CQ) is widely used as an anti-inflammatory therapeutic for rheumatic diseases. Although its modes of action on the innate immune system are well described, there is still insufficient knowledge about its direct effects on the adaptive immune system. Thus, we evaluated the influence of CQ on activation parameters of human CD4+ T-cells. CQ directly suppressed proliferation, metabolic activity and cytokine secretion of T-cells following anti-CD3/anti-CD28 activation. In contrast, CQ showed no effect on up-regulation of T-cell activation markers. CQ inhibited activation of all T helper cell subsets, although IL-4 and IL-13 secretion by Th2 cells were less influenced compared to other Th-specific cytokines. Up to 10 μM, CQ did not reduce cell viability, suggesting specific suppressive effects on T-cells. These properties of CQ were fully reversible in re-stimulation experiments. Analyses of intracellular signaling showed that CQ specifically inhibited autophagic flux and additionally activation of AP-1 by reducing phosphorylation of c-JUN. This effect was mediated by inhibition of JNK catalytic activity. In summary, we characterized selective and reversible immunomodulatory effects of CQ on human CD4+ T-cells. These findings provide new insights into the biological actions of JNK/AP-1 signaling in T-cells and may help to expand the therapeutic spectrum of CQ. PMID:28169350

  12. Inhibiting AP-1 activity alters cocaine induced gene expression and potentiates sensitization

    PubMed Central

    Paletzki, Ronald F.; Myakishev, Max V.; Polesskaya, Oksana; Orosz, Andras; Hyman, Steven E.; Vinson, Charles

    2008-01-01

    We have expressed A-FOS, an inhibitor of AP-1 DNA binding, in adult mouse striatal neurons. We observe normal behavior including locomotion and exploratory activities. Following a single injection of cocaine, locomotion increased similarly in both the A-FOS expressing and littermate controls. However, following repeated injections of cocaine, the A-FOS expressing mice showed increased locomotion relative to littermate controls, an increase that persisted following a week of withdrawal and subsequent cocaine administration. These results indicate that AP-1 suppresses this behavioral responses to cocaine. We analyzed mRNA from the striatum before and 4 and 24 hours after a single cocaine injection in both A-FOS and control striata using Affymetrix microarrays (430 2.0 Array) to identify genes mis-regulated by A-FOS that may mediate the increased locomotor sensitization to cocaine. A-FOS expression did not change gene expression in the basal state or 4 hours following cocaine treatment relative to controls. However, 24 hours after an acute cocaine treatment, 84 genes were identified that were differentially expressed between the A-FOS and control mice. 56 gene are down regulated while 28 genes are up regulated including previously identified candidates for addiction including BDNF and Per1. Using a random sample of identified genes, quantitative PCR was used to verify the microarray studies. The chromosomal location of these 84 genes was compared to human genome scans of addiction to identify potential genes in humans that are involved in addiction. PMID:18355967

  13. Activator protein-1 (AP-1) signalling in human atherosclerosis: results of a systematic evaluation and intervention study.

    PubMed

    Meijer, C Arnoud; Le Haen, Pum A A; van Dijk, Rogier A; Hira, Mitsuhisa; Hamming, Jaap F; van Bockel, J Hajo; Lindeman, Jan H

    2012-05-01

    Animal studies implicate the AP-1 (activator protein-1) pro-inflammatory pathway as a promising target in the treatment of atherosclerotic disease. It is, however, unclear whether these observations apply to human atherosclerosis. Therefore we evaluated the profile of AP-1 activation through histological analysis and tested the potential benefit of AP-1 inhibition in a clinical trial. AP-1 activation was quantified by phospho-c-Jun nuclear translocation (immunohistochemistry) on a biobank of aortic wall samples from organ donors. The effect of AP-1 inhibition on vascular parameters was tested through a double blind placebo-controlled cross-over study of 28 days doxycycline or placebo in patients with symptomatic peripheral artery disease. Vascular function was assessed by brachial dilation as well as by plasma samples analysed for hs-CRP (high-sensitivity C-reactive protein), IL-6 (interleukin-6), IL-8, ICAM-1 (intercellular adhesion molecule-1), vWF (von Willebrand factor), MCP-1 (monocyte chemoattractant protein-1), PAI-1 (plasminogen activator inhibitor-1) and fibrinogen. Histological evaluation of human atherosclerosis showed minimal AP-1 activation in non-diseased arterial wall (i.e. vessel wall without any signs of atherosclerotic disease). A gradual increase of AP-1 activation was found in non-progressive and progressive phases of atherosclerosis respectively (P<0.044). No significant difference was found between progressive and vulnerable lesions. The expression of phospho-c-Jun diminished as the lesion stabilized (P<0.016) and does not significantly differ from the normal aortic wall (P<0.33). Evaluation of the doxycycline intervention only revealed a borderline-significant reduction of circulating hs-CRP levels (-0.51 μg/ml, P=0.05) and did not affect any of the other markers of systemic inflammation and vascular function. Our studies do not characterize AP-1 as a therapeutic target for progressive human atherosclerotic disease.

  14. MAPK/AP-1-Targeted Anti-Inflammatory Activities of Xanthium strumarium.

    PubMed

    Hossen, Muhammad Jahangir; Kim, Mi-Yeon; Cho, Jae Youl

    2016-01-01

    Xanthium strumarium L. (Asteraceae), a traditional Chinese medicine, is prescribed to treat arthritis, bronchitis, and rhinitis. Although the plant has been used for many years, the mechanism by which it ameliorates various inflammatory diseases is not yet fully understood. To explore the anti-inflammatory mechanism of methanol extracts of X. strumarium (Xs-ME) and its therapeutic potential, we used lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells and human monocyte-like U937 cells as well as a LPS/D-galactosamine (GalN)-induced acute hepatitis mouse model. To find the target inflammatory pathway, we used holistic immunoblotting analysis, reporter gene assays, and mRNA analysis. Xs-ME significantly suppressed the up-regulation of both the activator protein (AP)-1-mediated luciferase activity and the production of LPS-induced proinflammatory cytokines, including interleukin (IL)-1[Formula: see text], IL-6, and tumor necrosis factor (TNF)-[Formula: see text]. Moreover, Xs-ME strongly inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) in LPS-stimulated RAW264.7 and U937 cells. Additionally, these results highlighted the hepatoprotective and curative effects of Xs-ME in a mouse model of LPS/D-GalN-induced acute liver injury, as assessed by elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and histological damage. Therefore, our results strongly suggest that the ethnopharmacological roles of Xs-ME in hepatitis and other inflammatory diseases might result from its inhibitory activities on the inflammatory signaling of MAPK and AP-1.

  15. Cloning and expression of a transcription factor activator protein-1 (AP-1) member identified from manila clam Venerupis philippinarum.

    PubMed

    Wu, Luning; Zhang, Lei; Zhao, Jianmin; Ning, Xuanxuan; Mu, Changkao; Wang, Chunlin

    2015-02-15

    The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

  16. Interleukin-35 Inhibits Endothelial Cell Activation by Suppressing MAPK-AP-1 Pathway.

    PubMed

    Sha, Xiaojin; Meng, Shu; Li, Xinyuan; Xi, Hang; Maddaloni, Massimo; Pascual, David W; Shan, Huimin; Jiang, Xiaohua; Wang, Hong; Yang, Xiao-feng

    2015-07-31

    Vascular response is an essential pathological mechanism underlying various inflammatory diseases. This study determines whether IL-35, a novel responsive anti-inflammatory cytokine, inhibits vascular response in acute inflammation. Using a mouse model of LPS-induced acute inflammation and plasma samples from sepsis patients, we found that IL-35 was induced in the plasma of mice after LPS injection as well as in the plasma of sepsis patients. In addition, IL-35 decreased LPS-induced proinflammatory cytokines and chemokines in the plasma of mice. Furthermore, IL-35 inhibited leukocyte adhesion to the endothelium in the vessels of lung and cremaster muscle and decreased the numbers of inflammatory cells in bronchoalveolar lavage fluid. Mechanistically, IL-35 inhibited the LPS-induced up-regulation of endothelial cell (EC) adhesion molecule VCAM-1 through IL-35 receptors gp130 and IL-12Rβ2 via inhibition of the MAPK-activator protein-1 (AP-1) signaling pathway. We also found that IL-27, which shares the EBI3 subunit with IL-35, promoted LPS-induced VCAM-1 in human aortic ECs and that EBI3-deficient mice had similar vascular response to LPS when compared with that of WT mice. These results demonstrated for the first time that inflammation-induced IL-35 inhibits LPS-induced EC activation by suppressing MAPK-AP1-mediated VCAM-1 expression and attenuates LPS-induced secretion of proinflammatory cytokines/chemokines. Our results provide insight into the control of vascular inflammation by IL-35 and suggest that IL-35 is an attractive novel therapeutic reagent for sepsis and cardiovascular diseases.

  17. 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.

  18. Ror2/Frizzled Complex Mediates Wnt5a-Induced AP-1 Activation by Regulating Dishevelled Polymerization▿ †

    PubMed Central

    Nishita, Michiru; Itsukushima, Sumiyo; Nomachi, Akira; Endo, Mitsuharu; Wang, ZhiChao; Inaba, Daisuke; Qiao, Sen; Takada, Shinji; Kikuchi, Akira; Minami, Yasuhiro

    2010-01-01

    The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the β-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl. PMID:20457807

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

    PubMed

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

    2015-12-28

    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.

  20. SARM inhibits both TRIF- and MyD88-mediated AP-1 activation.

    PubMed

    Peng, Jun; Yuan, Quan; Lin, Bin; Panneerselvam, Porkodi; Wang, Xiaowei; Luan, Xiao Lei; Lim, Soon Kok; Leung, Bernard P; Ho, Bow; Ding, Jeak Ling

    2010-06-01

    SARM (sterile alpha- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-beta)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF- and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.

  1. 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

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

    PubMed Central

    Shen, Ting; Yang, Woo Seok; 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) IκB kinase ε (IKKε)/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

  3. Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1.

    PubMed

    Castellanos, M C; Muñoz, C; Montoya, M C; Lara-Pezzi, E; López-Cabrera, M; de Landázuri, M O

    1997-12-01

    The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1.

  4. TLR7 and CD40 cooperate in IL-6 production via enhanced JNK and AP-1 activation.

    PubMed

    Vanden Bush, Tony J; Bishop, Gail A

    2008-02-01

    During vaccination or infection, adaptive and innate immune receptors of B cells are engaged by microbial antigens/ligands. A better understanding of how innate and adaptive signaling pathways interact could enlighten B lymphocyte biology as well as aid immunotherapy strategies and vaccine design. To address this goal, we examined the effects of TLR stimulation on BCR and CD40-induced B cell activation. Synergistic production of IL-6 was observed in both human and mouse primary B cells stimulated through B cell antigen receptors, CD40 and TLR7, and these two receptors also cooperated independently of BCR signals. The enhanced IL-6 production was dependent upon the activity of c-Jun kinase (JNK) and cFos. Dual stimulation through CD40 and TLR7 markedly enhanced JNK activity. The increased level of active JNK in dual-stimulated cells was accompanied by an increase in the level of active AP-1 monomers cJun and cFos. The stimulation of B cells through both CD40 and TLR7 therefore enhanced the production of cytokines through increased JNK signaling and AP-1 activity. In addition, the dual stimulation increased cFos/AP-1 species in stimulated cells, effectively expanding the repertoire of AP-1 dimers as compared to singly stimulated B cells.

  5. 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

  6. First molluscan transcription factor activator protein-1 (Ap-1) member from disk abalone and its expression profiling against immune challenge and tissue injury.

    PubMed

    De Zoysa, Mahanama; Nikapitiya, Chamilani; Lee, Youngdeuk; Lee, Sukkyoung; Oh, Chulhong; Whang, Ilson; Yeo, Sang-Yeop; Choi, Cheol Young; Lee, Jehee

    2010-12-01

    The regulation of transcriptional activation is an essential and critical point in gene expression. In this study, we describe a novel transcription factor activator protein-1 (Ap-1) gene from disk abalone Haliotis discus discus (AbAp-1) for the first time in mollusk. It was identified by homology screening of an abalone normalized cDNA library. The cloned AbAp-1 consists of a 945 bp coding region that encodes a putative protein containing 315 amino acids. The AbAp-1 gene is composed of a characteristic Jun transcription factor domain and a highly conserved basic leucine zipper (bZIP) signature similar to known Ap-1 genes. The AbAp-1 shares 46, 43 and, 40% amino acid identities with fish (Takifugu rubripes), human and insect (Ixodes scapularis) Ap-1, respectively. Quantitative real time RT-PCR analysis confirmed that AbAp-1 gene expression is constitutive in all selected tissues. AbAp-1 was upregulated in gills after bacteria (Vibrio alginolyticus, Vibrio parahemolyticus and Lysteria monocytogenes) challenge; and, upregulated in hemocytes and gills by viral hemorrhagic septicemia virus (VHSV) challenge. Shell damage and tissue injury also increased the transcriptional level of Ap-1 in mantle together with other transcription factors (NF-kB, LITAF) and pro-inflammatory TNF-α. All results considered, identification and gene expression data demonstrate that abalone Ap-1 is an important regulator in innate immune response against bacteria and virus, as well as in the inflammatory response during tissue injury. In addition, stimulation of Ap-1 under different external stimuli could be useful to understand the Ap-1 biology and its downstream target genes, especially in abalone-like mollusks.

  7. AP-1 mediated relief of repressive activity of the CD30 promoter microsatellite in Hodgkin and Reed-Sternberg cells.

    PubMed

    Watanabe, Mariko; Ogawa, Yuji; Ito, Kinji; Higashihara, Masaaki; Kadin, Marshall E; Abraham, Lawrence J; Watanabe, Toshiki; Horie, Ryouichi

    2003-08-01

    Overexpression of CD30 is the hallmark of Hodgkin and Reed-Sternberg (H-RS) cells and drives constitutive nuclear factor-kappaB activation that is the molecular basis for the pathophysiology of Hodgkin's lymphoma. Transcription of the CD30 gene is controlled by the core promoter that is driven by Sp-1 and the microsatellite sequences (MSs) that represses core promoter activity. To understand the mechanism(s) of CD30 overexpression in H-RS cells, we structurally and functionally characterized the CD30 MSs. Although the CD30 MS of H-RS cell lines was polymorphic, it was not truncated compared with that of control cells. A strong core promoter activity and constitutive Sp-1 binding were revealed in all cell lines examined irrespective of the levels of CD30 expression. In transient reporter gene assays, all MS clones derived from H-RS cell lines repressed the core promoter activity in unrelated cell lines, but not in the H-RS cell lines. An AP-1-binding site was found in the MS at nucleotide position of -377 to -371, the presence of which was found to relieve repression of the core promoter in H-RS cell lines but not in other tumor cell lines. H-RS cell lines showed constitutive and strong AP-1-binding activity, but other cell lines did not. The AP-1 complex contained JunB, whose overexpression activated reporter constructs driven by the CD30 promoter including the MSs, and was dependent on the AP-1 site. JunB expression was detected in H-RS cells in vitro and in vivo, but not in reactive cells or tumor cells of non-Hodgkin's lymphoma of diffuse large B-cell type. Transduction of JunB small interfering RNAs suppressed CD30 promoter activity in L428 cells but not in control cells. Taken together, overexpression and binding of JunB to the AP-1 site appear to relieve the repression of the core promoter by the CD30 MS in H-RS cells, which provide one basis for the constitutive overexpression of CD30 in Hodgkin's lymphoma.

  8. Pseudoephedrine inhibits T-cell activation by targeting NF-κB, NFAT and AP-1 signaling pathways.

    PubMed

    Fiebich, Bernd L; Collado, Juan A; Stratz, Cristian; Valina, Christian; Hochholzer, Willibald; Muñoz, Eduardo; Bellido, Luz M

    2012-02-01

    Pseudoephedrine (PSE) is a stereoisomer of ephedrine that is commonly used as a nasal decongestant in combination with other anti-inflammatory drugs for the symptomatic treatment of some common pathologies such as common cold. Herein, we describe for the first time the effects of PSE on T-cell activation events. We found that PSE inhibits interleukin-2 (IL-2) and tumor necrosis factor (TNF) alpha-gene transcription in stimulated Jurkat cells, a human T-cell leukemia cell line. To further characterize the inhibitory mechanisms of PSE at the transcriptional level, we examined the transcriptional activities of nuclear factor kappa B (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1) transcription factors and found that PSE inhibited NF-κB-dependent transcriptional activity without affecting either the phosphorylation, the degradation of the cytoplasmic NF-κB inhibitory protein, IκBα or the DNA-binding activity. However, phosphorylation of the p65/RelA subunit was clearly inhibited by PSE in stimulated cells. In addition, PSE inhibited the transcriptional activity of NFAT without interfering with the calcium-induced NFAT dephosphorylation event, which represents the major signaling pathway for its activation. NFAT cooperates with c-Jun, a compound of the AP-1 complex, to activate target genes, and we also found that PSE inhibited both JNK activation and AP-1 transcriptional activity. These findings provide new mechanistic insights into the potential immunomodulatory activities of PSE and highlight their potential in designing novel therapeutic strategies to manage inflammatory diseases.

  9. 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.

  10. Anti-inflammatory activities of Physalis alkekengi var. franchetii extract through the inhibition of MMP-9 and AP-1 activation.

    PubMed

    Hong, Ju-Mi; Kwon, Ok-Kyoung; Shin, In-Sik; Song, Hyuck-Hwan; Shin, Na-Rae; Jeon, Chan-Mi; Oh, Sei-Ryang; Han, Sang-Bae; Ahn, Kyung-Seop

    2015-01-01

    Physalis alkekengi has been traditionally used for the treatment of coughs, middle ear infections, and sore throats in Korea, Europe, and China. It exhibits a variety of pharmacological activities such as anti-inflammatory, anti-oxidant, and anti-cancer effects. The anti-inflammatory effects of the P. alkekengi methanol extract (PA) and its molecular mechanisms have not yet been fully investigated. In the present study, the chromatogram of PA was established by UPLC analysis. The anti-inflammatory effects of PA were also investigated using murine microphage cell lines, RAW 264.7 cells, and a murine model of OVA induced asthma. In LPS-stimulated RAW264.7 cells, PA reduced the MMP-9 expression with decreases in the production of nitric oxide, inteleukin-6, and tumor necrosis factor-α. Furthermore, PA suppressed the phosphorylation of MAPKs, which resulted in the inhibition of AP-1 activation. These effects of PA were consistent with the results of the in vivo experiment. PA-treated mice significantly inhibited inflammatory cell counts and cytokine production in bronchoalveolar lavage fluids and airway-hyperresponsiveness in OVA-induced asthmatic mice. PA treated mice also showed a marked inhibition of inducible nitric oxide synthase and MMP-9 expression. In conclusion, our results suggest that PA may be a valuable therapeutic material in treating various inflammatory diseases, including allergic asthma.

  11. Cobalt chloride induces PC12 cells apoptosis through reactive oxygen species and accompanied by AP-1 activation.

    PubMed

    Zou, W; Yan, M; Xu, W; Huo, H; Sun, L; Zheng, Z; Liu, X

    2001-06-15

    Reactive oxygen species (ROS) are supposed to play an important role in hypoxia- and ischemia/reperfusion-mediated neuronal injury with the characteristics of apoptosis. There are many reports showing that cobalt chloride (CoCl(2)) could mimic the hypoxic responses in some aspects including production of ROS in cultured cells. The cytotoxicity of CoCl(2) and its molecular mechanisms have yet to be elucidated. We report that CoCl(2) triggered neuronal PC12 cells apoptosis in a dose- and time-dependent manner. Apoptosis was demonstrated by morphological changes and DNA fragmentation, and was dependent on macromolecular synthesis. Apoptosis was also confirmed by the decrease of the expression of Bcl-X(L). To our knowledge, this is the first documentation of the apoptotic induction of CoCl(2) on PC12 cells. Furthermore, ROS production in PC12 cells was increased during CoCl(2) treatment. Antioxidants, which could inhibit ROS production, significantly blocked CoCl(2)-induced apoptosis, suggesting that apoptosis is mediated by ROS production. We also observed a significant increase of the DNA-binding activity of AP-1 in response to CoCl(2) and this increase was blocked by antioxidants, showing that CoCl(2)-induced apoptosis is accompanied by ROS-activated AP-1. CoCl(2)-treated PC12 cells may serve as an in vitro model for studies of molecular mechanisms in ROS-linked neuronal disorders.

  12. Anticancer activity of Phyllanthus emblica Linn. (Indian gooseberry): inhibition of transcription factor AP-1 and HPV gene expression in cervical cancer cells.

    PubMed

    Mahata, Sutapa; Pandey, Arvind; Shukla, Shirish; Tyagi, Abhishek; Husain, Syed Akhtar; Das, Bhudev Chandra; Bharti, Alok Chandra

    2013-01-01

    Plant products of Phyllanthus emblica Linn. are traditionally consumed for its immense nutritive and medicinal values. However, the molecular mechanism(s) by which it exerts it effects is less understood. In this study, we investigated mechanism of action of P. emblica fruit extract (PE) by studying its effect on activator protein-1 (AP-1) activity and human papillomavirus (HPV) transcription that are essential for tumorigenicity of cervical cancer cells. PE resulted in a dose-and time-dependent inhibition of DNA binding activity of constitutively active AP-1 in both HPV16-positive (SiHa) and HPV18-positive (HeLa) cervical cancer cells. PE-induced AP-1 inhibition was found mediated through downregulation of constituent AP-1 proteins, c-Jun, JunB, JunD, and c-Fos; however, the kinetics of their inhibition varied in both the cell types. Inhibition of AP-1 by PE was accompanied by suppression of viral transcription that resulted in growth inhibition of cervical cancer cells. Growth inhibitory activity of PE was primarily manifested through induction of apoptotic cell death. These results suggest that P. emblica exhibits its anticancer activities through inhibition of AP-1 and targets transcription of viral oncogenes responsible for development and progression of cervical cancer thus indicating its possible utility for treatment of HPV-induced cervical cancers.

  13. Glucocorticoid receptor-mediated suppression of the interleukin 2 gene expression through impairment of the cooperativity between nuclear factor of activated T cells and AP-1 enhancer elements

    PubMed Central

    1992-01-01

    The immunosuppressant hormone dexamethasone (Dex) interferes with T cell-specific signals activating the enhancer sequences directing interleukin 2 (IL-2) transcription. We report that the Dex-dependent downregulation of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and calcium ionophore-induced activity of the IL-2 enhancer are mediated by glucocorticoid receptor (GR) via a process that requires intact NH2- and COOH-terminal and DNA-binding domains. Functional analysis of chloramphenicol acetyltransferase (CAT) vectors containing internal deletions of the -317 to +47 bp IL-2 enhancer showed that the GR- responsive elements mapped to regions containing nuclear factor of activated T cells protein (NFAT) (-279 to -263 bp) and AP-1 (-160 to - 150 bp) motifs. The AP-1 motif binds TPA and calcium ionophore-induced nuclear factor(s) containing fos protein. TPA and calcium ionophore- induced transcriptional activation of homo-oligomers of the NFAT element were not inhibited by Dex, while AP-1 motif concatemers were not stimulated by TPA and calcium ionophore. When combined, NFAT and AP- 1 motifs significantly synergized in directing CAT transcription. Such a synergism was impaired by specific mutations affecting the trans- acting factor binding to either NFAT or AP-1 motifs. In spite of the lack of hormone regulation of isolated cis elements, TPA/calcium ionophore-mediated activation of CAT vectors containing a combination of the NFAT and the AP-1 motifs became suppressible by Dex. Our results show that the IL-2-AP-1 motif confers GR sensitivity to a flanking region containing a NFAT element and suggest that synergistic cooperativity between the NFAT and AP-1 sites allows GR to mediate the Dex inhibition of IL-2 gene transcription. Therefore, a Dex-modulated second level of IL-2 enhancer regulation, based on a combinatorial modular interplay, appears to be present. PMID:1740658

  14. ETV6-NTRK3 fusion oncogene initiates breast cancer from committed mammary progenitors via activation of AP1 complex

    PubMed Central

    Li, Zhe; Tognon, Cristina E.; Godinho, Frank J.; Yasaitis, Laura; Hock, Hanno; Herschkowitz, Jason I.; Lannon, Chris L.; Cho, Eunah; Kim, Seong-Jin; Bronson, Roderick T.; Perou, Charles M.; Sorensen, Poul H.; Orkin, Stuart H.

    2007-01-01

    SUMMARY To better understand the cellular origin of breast cancer, we developed a mouse model that recapitulates expression of the ETV6-NTRK3 (EN) fusion oncoprotein, the product of the t(12;15)(p13;q25) translocation characteristic of human secretory breast carcinoma. Activation of EN expression in mammary tissues by Wap-Cre leads to fully penetrant, multifocal malignant breast cancer with short latency. We provide genetic evidence that in nulliparous Wap-Cre;EN females, committed alveolar bipotent or CD61+ luminal progenitors, are targets of tumorigenesis. Furthermore, EN transforms these otherwise transient progenitors through activation of the AP1 complex. Given increasing relevance of chromosomal translocations in epithelial cancers, such mice serve as a paradigm for the study of their genetic pathogenesis and cellular origins, and generation of novel preclinical models. SIGNIFICANCE For the largest class of human tumors, those of epithelial origin, little is known about their initiating genetic hits or cells of origin. Whether tissue stem cells or more committed progenitors are targets for transformation is uncertain. We developed a system in which epithelial tumorigenesis can be assessed from the initial event to frank malignancy. In this breast cancer model based on chromosomal translocation, we show through genetic marking that committed mammary progenitors, rather than mammary stem cells, are direct targets of transformation. We show that activation of the AP1 complex represents a critical downstream event of the ETV6-NTRK3 translocation. Further focus on this transcriptional complex as a target in human breast cancer is warranted. PMID:18068631

  15. AP-1-Targeted Anti-Inflammatory Activities of the Nanostructured, Self-Assembling S5 Peptide

    PubMed Central

    Yang, Woo Seok; Son, Young-Jin; Kim, Mi-Yeon; Kim, Soochan; Kim, Jong-Hoon

    2015-01-01

    Peptide-based therapeutics have received increasing attention in medical research. However, the local delivery of such therapeutics poses unique challenges. Self-assembling peptides that use decorated nanofibers are one approach by which these therapeutics may be delivered. We previously found that the self-assembling K5 peptide affects the anti-inflammatory response. The aim of the present study was to investigate another self-assembling peptide, S5. Unlike the K5 peptide which has a positive charge, the S5 peptide has a free hydroxyl (-OH) group. We first examined whether the S5 peptide regulates the inflammatory response in primary cells and found that the S5 peptide reduced the production of prostaglandin E2 (PGE2) and tumor necrosis factor (TNF)-α in lipopolysaccharide- (LPS-) treated bone marrow-derived macrophages. Moreover, the S5 peptide significantly downregulated cyclooxygenase- (COX-) 2, TNF-α, and interleukin- (IL-) 1β expression by blocking the nuclear translocation of c-Jun. Consistent with this finding, the S5 peptide diminished the activation of inflammatory signaling enzymes related to p38. The S5 peptide also inhibited the formation of the p38/c-Jun signaling complex in RAW264.7 cells. Similarly, p38 and MKK3/6 were inhibited by the S5 peptide in LPS-activated peritoneal macrophages. Taken together, these results strongly suggest that the S5 peptide could exert anti-inflammatory effects by inhibiting the c-Jun/p38 signaling pathway. PMID:26074678

  16. Clinical Light Exposure, Photoreceptor Degeneration, and AP-1 Activation: A Cell Death or Cell Survival Signal in the Rhodopsin Mutant Retina?

    PubMed Central

    Gu, Danian; Beltran, William A.; Li, Zexiao; Acland, Gregory M.; Aguirre, Gustavo D.

    2008-01-01

    Purpose The T4R RHO mutant dog retina shows retinal degeneration with exposures to light comparable to those used in clinical eye examinations of patients. To define the molecular mechanisms of the degeneration, AP-1 DNA-binding activity, composition, posttranslational modification of the protein complex, and modulation of ERK/MAPK signaling pathways were examined in light-exposed mutant retinas. Methods Dark-adapted retinas were exposed to short-duration light flashes from a retinal camera used clinically for retinal photography and were collected at different time points after exposure. Electrophoretic mobility shift assay (EMSA), super-shift EMSA, Western blot analysis, and immunocytochemistry were used to examine AP-1 signaling. Results Exposure to light of mutant retinas significantly increased AP-1 DNA-binding activity by 1 hour after exposure, and levels remained elevated for 6 hours. Shielded mutant retinas had similar AP-1 levels to shielded or exposed wild-type retinas. The parallel phosphorylation of c-Fos and activation of ERK1/2 was detected only in exposed mutant retinas. Exposure to light changed the composition of the AP-1 protein complex in the mutant retina from c-Jun/Fra-1/c-Fos to JunB/c-Fos. Immunohistochemistry showed that the components of activated AP-1 (JunB, and phosphorylated c-Fos, and phosphorylated ERK1/2 isoforms) were localized in Müller cells. Conclusions The inner nuclear layer/Müller cell localization of the key proteins induced by light exposure raises the question of the direct involvement of AP-1 in mediating photoreceptor cell death in this model of autosomal dominant retinitis pigmentosa. PMID:17962438

  17. Megakaryocytic Maturation in Response to Shear Flow Is Mediated by the Activator Protein 1 (AP-1) Transcription Factor via Mitogen-activated Protein Kinase (MAPK) Mechanotransduction.

    PubMed

    Luff, Stephanie A; Papoutsakis, Eleftherios T

    2016-04-08

    Megakaryocytes (MKs) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation to release pro/preplatelets into circulating blood. Shear forces promote DNA synthesis, polyploidization, and maturation in MKs, and platelet biogenesis. To investigate mechanisms underlying these MK responses to shear, we carried out transcriptional analysis on immature and mature stem cell-derived MKs exposed to physiological shear. In immature (day (d)9) MKs, shear exposure up-regulated genes related to growth and MK maturation, whereas in mature (d12) MKs, it up-regulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, six activator protein 1 (AP-1) transcripts (ATF4,JUNB,JUN,FOSB,FOS, andJUND) were up-regulated at d9 and two AP-1 proteins (JunD and c-Fos) were up-regulated both at d9 and d12. We show that mitogen-activated protein kinase (MAPK) signaling is linked to both the shear stress response and AP-1 up-regulation. c-Jun N-terminal kinase (JNK) phosphorylation increased significantly following shear stimulation, whereas JNK inhibition reduced shear-induced JunD expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos expression. JNK inhibition reduced fibrinogen binding and P-selectin expression of d12 platelet-like particles (PLPs), whereas p38 inhibition reduced fibrinogen binding of d12 PLPs. AP-1 expression correlated with increased MK DNA synthesis and polyploidization, which might explain the observed impact of shear on MKs. To summarize, we show that MK exposure to shear forces results in JNK activation, AP-1 up-regulation, and downstream transcriptional changes that promote maturation of immature MKs and platelet biogenesis in mature MKs.

  18. Arctigenin, a phenylpropanoid dibenzylbutyrolactone lignan, inhibits MAP kinases and AP-1 activation via potent MKK inhibition: the role in TNF-alpha inhibition.

    PubMed

    Cho, Min Kyung; Jang, Young Pyo; Kim, Young Choong; Kim, Sang Geon

    2004-10-01

    Arctigenin, naturally occurring in Bardanae fructus, Saussurea medusa, Arctium lappa L., Torreya nucifera and Ipomea cairica, is a phenylpropanoid dibenzylbutyrolactone lignan with antioxidant and anti-inflammatory activities. Previously, we showed that arctigenin potently inhibited the induction of nitric oxide synthase (iNOS) by lipopolysaccharide (LPS), which involved suppression of NF-kappaB activation. In the present study, we examined the effects of arctigenin on mitogen-activated protein (MAP) kinase activation in Raw264.7 cells and MAP kinase kinase (MKK) activity. The effect of arctigenin on activator protein-1 (AP-1) activation was also studied in association with tumor necrosis factor-alpha (TNF-alpha) expression. Immunoblot analysis showed that arctigenin inhibited phosphorylation of MAP kinases ERK1/2, p38 kinase and JNK and their activities in Raw264.7 cells treated with LPS. Arctigenin potently inhibited the activity of MKK1 in vitro with the IC(50) value of 1 nM. Gel shift and reporter gene analyses revealed that arctigenin inhibited LPS-inducible AP-1 binding to the AP-1 consensus oligonucleotide and AP-1-mediated reporter gene expression. In view of the potential role of AP-1 in the induction of TNF-alpha, we next examined the inhibitory effects of arctigenin on the expression of TNF-alpha. Arctigenin blocked TNF-alpha production and decreased the level of TNF-alpha mRNA in the cells exposed to LPS. These results showed that arctigenin inhibited activation of MAP kinases including ERK1/2, p38 kinase and JNK through the inhibition of MKK activities, leading to AP-1 inactivation, which might, at least in part, contribute to the inhibition of TNF-alpha production.

  19. 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

  20. Costunolide inhibits interleukin-1beta expression by down-regulation of AP-1 and MAPK activity in LPS-stimulated RAW 264.7 cells.

    PubMed

    Kang, Jong Soon; Yoon, Yeo Dae; Lee, Ki Hoon; Park, Song-Kyu; Kim, Hwan Mook

    2004-01-02

    Costunolide, a sesquiterpene lactone isolated from the root of Saussurea lappa Clarke, is known to have a variety of biological activities, including anti-carcinogenic and anti-fungal activities. Here, we demonstrated the inhibitory effect of costunolide on the protein and mRNA expression of interleukin-1beta (IL-1beta) in LPS-stimulated RAW 264.7 cells. We also showed that costunolide suppressed the transcriptional activity of the IL-1beta promoter. Moreover, costunolide inhibited the activity of AP-1 transcription factor, and the phosphorylation of MAPKs, including SAPK/JNK and p38 MAP kinase. The inhibitory effect of costunolide on AP-1 activity was also confirmed by an electrophoretic mobility shift assay. Additionally, specific inhibitors of SAPK/JNK and p38 MAP kinase, SP600125 and SB203580, also suppressed LPS-induced increase in IL-1beta gene expression and AP-1 DNA binding. Taken together, these results demonstrate that costunolide inhibits IL-1beta gene expression by blocking the activation of MAPKs and DNA binding of AP-1 in LPS-stimulated RAW 264.7 cells.

  1. Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope

    PubMed Central

    González, José María; Navarro-Puche, Ana; Casar, Berta; Crespo, Piero; Andrés, Vicente

    2008-01-01

    Sequestration of c-Fos at the nuclear envelope (NE) through interaction with A-type lamins suppresses AP-1–dependent transcription. We show here that c-Fos accumulation within the extraction-resistant nuclear fraction (ERNF) and its interaction with lamin A are reduced and enhanced by gain-of and loss-of ERK1/2 activity, respectively. Moreover, hindering ERK1/2-dependent phosphorylation of c-Fos attenuates its release from the ERNF induced by serum and promotes its interaction with lamin A. Accordingly, serum stimulation rapidly releases preexisting c-Fos from the NE via ERK1/2-dependent phosphorylation, leading to a fast activation of AP-1 before de novo c-Fos synthesis. Moreover, lamin A–null cells exhibit increased AP-1 activity and reduced levels of c-Fos phosphorylation. We also find that active ERK1/2 interacts with lamin A and colocalizes with c-Fos and A-type lamins at the NE. Thus, NE-bound ERK1/2 functions as a molecular switch for rapid mitogen-dependent AP-1 activation through phosphorylation-induced release of preexisting c-Fos from its inhibitory interaction with lamin A/C. PMID:19015316

  2. C3 toxin activates the stress signaling pathways, JNK and p38, but antagonizes the activation of AP-1 in rat-1 cells.

    PubMed

    Beltman, J; Erickson, J R; Martin, G A; Lyons, J F; Cook, S J

    1999-02-05

    Lysophosphatidic acid (LPA) stimulates the c-Fos serum response element (SRE) by activating two distinct signal pathways regulated by the small GTPases, Ras and RhoA. Ras activates the ERK cascade leading to phosphorylation of the transcription factors Elk-1 and Sap1a at the Ets/TCF site. RhoA regulates an undefined pathway required for the activation of the SRF/CArG site. Here we have examined the role of the Ras and RhoA pathways in activation of the SRE and c-Fos expression in Rat-1 cells. Pertussis toxin and PD98059 strongly inhibited LPA-stimulated c-Fos expression and activation of a SRE:Luc reporter. C3 toxin completely inhibited RhoA function, partially inhibited SRE:Luc activity, but had no effect on LPA-stimulated c-Fos expression. Thus, in a physiological context the Ras-Raf-MEK-ERK pathway, but not RhoA, is required for LPA-stimulated c-Fos expression in Rat-1 cells. C3 toxin stimulated the stress-activated protein kinases JNK and p38 and potentiated c-Jun expression and phosphorylation; these properties were shared by another cellular stress agonist the protein kinase C inhibitor Ro-31-8220. However, C3 toxin alone or in combination with growth factors did not stimulate AP-1:Luc activity and actually antagonized the synergistic activation of AP-1:Luc observed in response to co-stimulation with growth factors and Ro-31-8220. These data indicate that C3 toxin is a cellular stress which antagonizes activation of AP-1 at a point downstream of stress-activated kinase activation or immediate-early gene induction.

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

    PubMed

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

    2016-04-07

    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.

  4. 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

  5. Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia.

    PubMed Central

    Yao, K S; Xanthoudakis, S; Curran, T; O'Dwyer, P J

    1994-01-01

    Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells

  6. 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.

  7. IL-1β and IL-6 activate inflammatory responses of astrocytes against Naegleria fowleri infection via the modulation of MAPKs and AP-1.

    PubMed

    Kim, J-H; Song, A-R; Sohn, H-J; Lee, J; Yoo, J-K; Kwon, D; Shin, H-J

    2013-01-01

    Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

  8. Molecular targets of the antiinflammatory Harpagophytum procumbens (devil's claw): inhibition of TNFα and COX-2 gene expression by preventing activation of AP-1.

    PubMed

    Fiebich, Bernd L; Muñoz, Eduardo; Rose, Thorsten; Weiss, Gabriele; McGregor, Gerard P

    2012-06-01

    Harpagophytum procumbens (Hp) is often used in the supportive treatment of inflammatory and degenerative diseases of the skeletal system. Although the clinical efficacy in osteoarthritis has been demonstrated in clinical trials, the molecular target(s) of Hp are unclear. This study quantified the effects of the ethanol Hp extract (60% v/v ethanol, sole active ingredient of Pascoe®-Agil), on the expression and release of the major pro-inflammatory mediators in LPS-stimulated human monocytes and the intracellular signalling pathways involved in inflammation. The Hp extract dose-dependently inhibited the release of TNFα as well as that of interleukin (IL)-6, IL-1β and prostaglandin E₂ (PGE₂). The Hp prevented TNFα and IL-6 mRNA expression in human monocytes and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. Furthermore, the Hp extract inhibited LPS-stimulated AP-1-mediated gene transcription activity and binding to the AP-1 response elements. The extract had no effect on the LPS-induced binding of nuclear factor-κB in RAW 264.7 cells, on LPS-induced degradation of IκBα or on LPS-induced activation of mitogen-activated protein kinases (MAPK), p38MAPK and JNK in human monocytes. The data indicate that a standardized ethanol Hp extract inhibits induction of pro-inflammatory gene expression, possibly by blocking the AP-1 pathway. This is novel evidence of a possible mechanism of action of this antiinflammatory drug.

  9. 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.

  10. Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1.

    PubMed

    Yang, Xinyu; Li, Lin; Liu, Jin; Lv, Ben; Chen, Fangping

    2016-01-01

    Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1.

  11. Ultraviolet irradiation induces CYR61/CCN1, a mediator of collagen homeostasis, through activation of transcription factor AP-1 in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Xu, Yiru; He, Tianyuan; Kang, Sewon; Voorhees, John J; Fisher, Gary J

    2010-06-01

    UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

  12. 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.

  13. Activation of Nrf2 Reduces UVA-Mediated MMP-1 Upregulation via MAPK/AP-1 Signaling Cascades: The Photoprotective Effects of Sulforaphane and Hispidulin

    PubMed Central

    Chaiprasongsuk, Anyamanee; Lohakul, Jinaphat; Soontrapa, Kitipong; Sampattavanich, Somponnat; Akarasereenont, Pravit

    2017-01-01

    UVA irradiation plays a role in premature aging of the skin through triggering oxidative stress-associated stimulation of matrix metalloproteinase-1 (MMP-1) responsible for collagen degradation, a hallmark of photoaged skin. Compounds that can activate nuclear factor E2-related factor 2 (Nrf2), a transcription factor regulating antioxidant gene expression, should therefore serve as effective antiphotoaging agents. We investigated whether genetic silencing of Nrf2 could relieve UVA-mediated MMP-1 upregulation via activation of mitogen-activated protein kinase (MAPK)/activator protein 1 (AP-1) signaling using human keratinocyte cell line (HaCaT). Antiphotoaging effects of hispidulin (HPD) and sulforaphane (SFN) were assessed on their abilities to activate Nrf2 in controlling MMP-1 and collagen expressions in association with phosphorylation of MAPKs (extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38), c-Jun, and c-Fos, using the skin of BALB/c mice subjected to repetitive UVA irradiation. Our findings suggested that depletion of Nrf2 promoted both mRNA expression and activity of MMP-1 in the UVA-irradiated HaCaT cells. Treatment of Nrf2 knocked-down HaCaT cells with MAPK inhibitors significantly suppressed UVA-induced MMP-1 and AP-1 activities. Moreover, pretreatment of the mouse skin with HPD and SFN, which could activate Nrf2, provided protective effects against UVA-mediated MMP-1 induction and collagen depletion in correlation with the decreased levels of phosphorylated MAPKs, c-Jun, and c-Fos in the mouse skin. In conclusion, Nrf2 could influence UVA-mediated MMP-1 upregulation through the MAPK/AP-1 signaling cascades. HPD and SFN may therefore represent promising antiphotoaging candidates. PMID:28011874

  14. 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...

  15. 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.

  16. Low doses of LPS and minimally oxidized LDL cooperatively activate macrophages via NF-kappaB and AP-1: Possible mechanism for acceleration of atherosclerosis by subclinical endotoxemia

    PubMed Central

    Wiesner, Philipp; Choi, Soo-Ho; Almazan, Felicidad; Benner, Christopher; Huang, Wendy; Diehl, Cody J.; Gonen, Ayelet; Butler, Susan; Witztum, Joseph L.; Glass, Christopher K.; Miller, Yury I.

    2010-01-01

    Rationale Oxidized low-density lipoprotein (LDL) is an important determinant of inflammation in atherosclerotic lesions. It has also been documented that certain chronic infectious diseases, such as periodontitis and chlamydial infection, exacerbate clinical manifestations of atherosclerosis. In addition, low-level but persistent metabolic endotoxemia is often found in diabetic and obese subjects and is induced in mice fed a high-fat diet. Objective In this study, we examined cooperative macrophage activation by low levels of bacterial LPS and by minimally oxidized LDL (mmLDL), as a model for subclinical endotoxemia-complicated atherosclerosis. Methods and Results We found that both in vitro and in vivo, mmLDL and LPS (Kdo2-LipidA) cooperatively activated macrophages to express pro-inflammatory cytokines Cxcl2 (MIP-2), Ccl3 (MIP-1alpha), and Ccl4 (MIP-1beta). Importantly, the mmLDL and LPS cooperative effects were evident at a threshold LPS concentration (1 ng/ml) at which LPS alone induced only a limited macrophage response. Analyzing microarray data with a de novo motif discovery algorithm, we found that genes transcribed by promoters containing an AP-1 binding site were significantly upregulated by co-stimulation with mmLDL and LPS. In a nuclear factor-DNA binding assay, the cooperative effect of mmLDL and LPS co-stimulation on c-Jun and c-Fos DNA binding, but not on p65 or p50, was dependent on mmLDL-induced activation of ERK1/2. In addition, mmLDL induced JNK-dependent derepression of AP-1 by removing the corepressor NCoR from the chemokine promoters. Conclusions The cooperative engagement of AP-1 and NF-kappaB by mmLDL and LPS may constitute a mechanism of increased transcription of inflammatory cytokines within atherosclerotic lesions. PMID:20489162

  17. A wogonin-rich-fraction of Scutellaria baicalensis root extract exerts chondroprotective effects by suppressing IL-1β-induced activation of AP-1 in human OA chondrocytes

    PubMed Central

    Khan, Nazir M.; Haseeb, Abdul; Ansari, Mohammad Y.; Haqqi, Tariq M.

    2017-01-01

    Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1β-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1β showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1β-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1β-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions. PMID:28256567

  18. 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

  19. Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells.

    PubMed

    Stapleton, Genevieve; Malliri, Angeliki; Ozanne, Bradford W

    2002-07-01

    Progression to an invasive, metastatic tumour requires the coordinated expression and function of a number of gene products, as well as their regulation in the context of invasion. The transcription factor AP-1 regulates expression of many of those genes necessary for implementation of the invasion programme. Two such gene products, CD44 and ezrin, are both upregulated in fibroblasts transformed by v-fos and are commonly implicated in cell motility and invasion. Here we report that CD44 and ezrin colocalise to membrane ruffles and microvilli of A431 cells after treatment with EGF. However, A431 cells expressing dominant-negative c-Jun (TAM67), and which as a consequence fail to invade in response to EGF, also fail to correctly localise CD44 and ezrin. CD44 and ezrin are both substrates for Protein Kinase C, and we show that their EGF-dependent colocalisation requires Protein Kinase C activity. Associated with TAM67 expression and disrupted CD44 and ezrin colocalisation is the increased expression and activation of the novel PKC theta isoform. Expression of PKC theta in A431 cells results in the inhibition of cell motility and disrupted localisation of CD44 and ezrin. We propose that AP-1 regulates the integrity of Protein Kinase C signalling and identifies PKC theta as a potential suppressor of the invasion programme.

  20. Transcriptional Activation of Metalloid Tolerance Genes in Saccharomyces cerevisiae Requires the AP-1–like Proteins Yap1p and Yap8p

    PubMed Central

    Wysocki, Robert; Fortier, Pierre-Karl; Maciaszczyk, Ewa; Thorsen, Michael; Leduc, Anick; Odhagen, Åsa; Owsianik, Grzegorz; Ulaszewski, Stanislaw; Ramotar, Dindial; Tamás, Markus J.

    2004-01-01

    All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1–like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H2O2 treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1–like proteins respond to metalloids through distinct mechanisms. PMID:14978214

  1. Tamoxifen increases nuclear respiratory factor 1 transcription by activating estrogen receptor beta and AP-1 recruitment to adjacent promoter binding sites.

    PubMed

    Ivanova, Margarita M; Luken, Kristen H; Zimmer, Amber S; Lenzo, Felicia L; Smith, Ryan J; Arteel, Maia W; Kollenberg, Tara J; Mattingly, Kathleen A; Klinge, Carolyn M

    2011-04-01

    Little is known about endogenous estrogen receptor β (ERβ) gene targets in human breast cancer. We reported that estradiol (E(2)) induces nuclear respiratory factor-1 (NRF-1) transcription through ERα in MCF-7 breast cancer cells. Here we report that 4-hydroxytamoxifen (4-OHT), with an EC(50) of ~1.7 nM, increases NRF-1 expression by recruiting ERβ, cJun, cFos, CBP, and RNA polymerase II to and dismissing NCoR from the NRF1 promoter. Promoter deletion and transient transfection studies showed that the estrogen response element (ERE) is essential and that an adjacent AP-1 site contributes to maximal 4-OHT-induced NRF-1 transcription. siRNA knockdown of ERβ revealed that ERβ inhibits basal NRF-1 expression and is required for 4-OHT-induced NRF-1 transcription. An AP-1 inhibitor blocked 4-OHT-induced NRF-1 expression. The 4-OHT-induced increase in NRF-1 resulted in increased transcription of NRF-1 target CAPNS1 but not CYC1, CYC2, or TFAM despite increased NRF-1 coactivator PGC-1α protein. The absence of TFAM induction corresponds to a lack of Akt-dependent phosphorylation of NRF-1 with 4-OHT treatment. Overexpression of NRF-1 inhibited 4-OHT-induced apoptosis and siRNA knockdown of NRF-1 increased apoptosis, indicating an antiapoptotic role for NRF-1. Overall, NRF-1 expression and activity is regulated by 4-OHT via endogenous ERβ in MCF-7 cells.

  2. Cobalt chloride induces delayed cardiac preconditioning in mice through selective activation of HIF-1alpha and AP-1 and iNOS signaling.

    PubMed

    Xi, Lei; Taher, Mohiuddin; Yin, Chang; Salloum, Fadi; Kukreja, Rakesh C

    2004-12-01

    Acute systemic hypoxia induces delayed cardioprotection against ischemia (I)-reperfusion (R) injury via inducible nitric oxide synthase (iNOS)-dependent mechanism. Because CoCl2 is known to elicit hypoxia-like responses, we hypothesized that this chemical would mimic the delayed preconditioning effect in the heart. Adult male mice were pretreated with CoCl2 or saline. The hearts were isolated 24 h later and subjected to 20 min of global I and 30 min of R in Langendorff mode. Myocardial infarct size (% of risk area; mean +/- SE, n=6-8/group) was reduced in mice pretreated with 30 mg/kg CoCl2 (16.1 +/- 3.1% vs. 27.6 +/- 3.3% with saline control; P <0.05) without compromising postischemic cardiac function. Higher doses of CoCl2 failed to induce similar protection. Electrophoretic mobility gel shift assay demonstrated significant enhancement in DNA binding activity of hypoxia-inducible factor 1alpha (HIF-1alpha) and activator protein 1 (AP-1) in nuclear extracts from CoCl2-treated hearts. Activation of HIF-1alpha and AP-1 was evident at 30 min and sustained for the next 4 h after CoCl2 injection. In contrast, CoCl2-induced protection was independent of NF-kappaB activation because no DNA binding or p65 translocation was observed in nuclear extracts. Also, CoCl2-induced cardioprotection was preserved in p50 subunit NF-kappaB-knockout (KO) mice (11.1 +/- 3.0% vs. 25.1 +/- 5.0% in saline-treated p50-KO mice; P <0.05). The infarct-limiting effect of CoCl2 was absent in iNOS-KO mice (20.9 +/- 3.0%). We conclude that in vivo administration of CoCl2 preconditions the heart against I/R injury. The delayed protective effect of CoCl2 is achieved through a distinctive signaling mechanism involving HIF-1alpha, AP-1, and iNOS but independent of NF-kappaB activation.

  3. The Transcription Factor AP-1 Is Required for EGF-induced Activation of Rho-like GTPases, Cytoskeletal Rearrangements, Motility, and In Vitro Invasion of A431 Cells

    PubMed Central

    Malliri, Angeliki; Symons, Marc; Hennigan, Robert F.; Hurlstone, Adam F.L.; Lamb, Richard F.; Wheeler, Tricia; Ozanne, Bradford W.

    1998-01-01

    Human squamous cell carcinomas (SCC) frequently express elevated levels of epidermal growth factor receptor (EGFR). EGFR overexpression in SCC-derived cell lines correlates with their ability to invade in an in vitro invasion assay in response to EGF, whereas benign epidermal cells, which express low levels of EGFR, do not invade. EGF-induced invasion of SCC-derived A431 cells is inhibited by sustained expression of the dominant negative mutant of c-Jun, TAM67, suggesting a role for the transcription factor AP-1 (activator protein-1) in regulating invasion. Significantly, we establish that sustained TAM67 expression inhibits growth factor–induced cell motility and the reorganization of the cytoskeleton and cell-shape changes essential for this process: TAM67 expression inhibits EGF-induced membrane ruffling, lamellipodia formation, cortical actin polymerization and cell rounding. Introduction of a dominant negative mutant of Rac and of the Rho inhibitor C3 transferase into A431 cells indicates that EGF-induced membrane ruffling and lamellipodia formation are regulated by Rac, whereas EGF-induced cortical actin polymerization and cell rounding are controlled by Rho. Constitutively activated mutants of Rac or Rho introduced into A431 or A431 cells expressing TAM67 (TA cells) induce equivalent actin cytoskeletal rearrangements, suggesting that the effector pathways downstream of Rac and Rho required for these responses are unimpaired by sustained TAM67 expression. However, EGF-induced translocation of Rac to the cell membrane, which is associated with its activation, is defective in TA cells. Our data establish a novel link between AP-1 activity and EGFR activation of Rac and Rho, which in turn mediate the actin cytoskeletal rearrangements required for cell motility and invasion. PMID:9817764

  4. Kukoamine A Prevents Radiation-Induced Neuroinflammation and Preserves Hippocampal Neurogenesis in Rats by Inhibiting Activation of NF-κB and AP-1.

    PubMed

    Zhang, Yaqiong; Gao, Lingyue; Cheng, Zhihua; Cai, Jiayi; Niu, Yixuan; Meng, Weihong; Zhao, Qingchun

    2017-02-01

    Impaired hippocampal neurogenesis and neuroinflammation are involved in the pathogenesis of radiation-induced brain injury. Kukoamine A (KuA) was demonstrated to have neuroprotective effects through inhibiting oxidative stress and apoptosis after whole-brain irradiation (WBI) in rats. The aim of this study was to investigate whether administration of KuA would prevent radiation-induced neuroinflammation and the detrimental effect on hippocampal neurogenesis. For this study, male Wistar rats received either sham irradiation or WBI (30 Gy single dose of X-rays) followed by the immediate injection of either KuA or vehicle intravenously. The dose of KuA was 5, 10, and 20 mg/kg, respectively. The levels of pro-inflammatory cytokines were assayed by ELISA kits. The newborn neurons were detected by 5-bromo-2-deoxyuridine (BrdU)/neuronal nuclei (NeuN) double immunofluorescence. Microglial activation was measured by Iba-1 immunofluorescence. The expression of Cox-2 and the activation of nuclear factor κB (NF-κB), activating protein 1(AP-1), and PPARδ were evaluated by western blot. WBI led to a significant increase in the level of TNF-α, IL-1β, and Cox-2, and it was alleviated by KuA administration. KuA attenuated microglial activation in rat hippocampus after WBI. Neurogenesis impairment induced by WBI was ameliorated by KuA. Additionally, KuA alleviated the increased translocation of NF-κB p65 subunit and phosphorylation of c-jun induced by WBI and elevated the expression of PPARδ. These data indicate that KuA could ameliorate the neuroinflammatory response and protect neurogenesis after WBI, partially through regulating the activation of NF-κB, AP-1, and PPARδ.

  5. Cigarette smoke activates human monocytes by an oxidant-AP-1 signaling pathway: implications for steroid resistance.

    PubMed

    Walters, Matthew J; Paul-Clark, Mark J; McMaster, Shaun K; Ito, Kazuhiro; Adcock, Ian M; Mitchell, Jane A

    2005-11-01

    Smoking cigarettes is a major risk factor for the development of cardiovascular and respiratory disease. Moreover, smoking-induced pathophysiology is often resistant to the anti-inflammatory effects of glucocorticoids. The nature of cigarette smoke-induced inflammation is still not defined, although neutrophil recruitment and activation seem to be consistent features. In the current study, we have used a range of approaches to demonstrate that cigarette smoke activates human monocytes and macrophages to release the CXC chemokine CXCL8 [(interleukin-8 (IL-8)]. Furthermore, we show for the first time that cigarette smoke synergizes with proinflammatory cytokines IL-1beta and tumor necrosis factor-alpha, and it is this interaction that confers steroid resistance to smoke-induced CXCL8 release. We go on to show that smoke-induced activation of human cells is an oxidant-mediated phenomenon acting through activator protein-1, but not nuclear factor kappaB, pathway. These observations add significantly to our understanding of smoke as an inflammatory stimulus that has implications for potential the development of treatments of smoking or related disease.

  6. Interaction of glucocorticoid receptor (GR) with estrogen receptor (ER) α and activator protein 1 (AP1) in dexamethasone-mediated interference of ERα activity.

    PubMed

    Karmakar, Sudipan; Jin, Yetao; Nagaich, Akhilesh K

    2013-08-16

    The role of glucocorticoids in the inhibition of estrogen (17-β-estradiol (E2))-regulated estrogen receptor (ER)-positive breast cancer cell proliferation is well established. We and others have seen that synthetic glucocorticoid dexamethasone (Dex) antagonizes E2-stimulated endogenous ERα target gene expression. However, how glucocorticoids negatively regulate the ERα signaling pathway is still poorly understood. ChIP studies using ERα- and glucocorticoid receptor (GR)-positive MCF-7 cells revealed that GR occupies several ERα-binding regions (EBRs) in cells treated with E2 and Dex simultaneously. Interestingly, there was little or no GR loading to these regions when cells were treated with E2 or Dex alone. The E2+Dex-dependent GR recruitment is associated with the displacement of ERα and steroid receptor coactivator-3 from the target EBRs leading to the repression of ERα-mediated transcriptional activation. The recruitment of GR to EBRs requires assistance from ERα and FOXA1 and is facilitated by AP1 binding within the EBRs. The GR binding to EBRs is mediated via direct protein-protein interaction between the GR DNA-binding domain and ERα. Limited mutational analyses indicate that arginine 488 located within the C-terminal zinc finger domain of the GR DNA-binding domain plays a critical role in stabilizing this interaction. Together, the results of this study unravel a novel mechanism involved in glucocorticoid inhibition of ERα transcriptional activity and E2-mediated cell proliferation and thus establish a foundation for future exploitation of the GR signaling pathway in the treatment of ER-positive breast cancer.

  7. Extracellular Signal-Regulated Protein Kinase, c-Jun N-terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1.

    PubMed

    Lesch, Andrea; Rössler, Oliver G; Thiel, Gerald

    2017-01-23

    Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca(2+) /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. This article is protected by copyright. All rights reserved.

  8. Transient receptor potential melastatin-3 (TRPM3)-induced activation of AP-1 requires Ca2+ ions and the transcription factors c-Jun, ATF2, and ternary complex factor.

    PubMed

    Lesch, Andrea; Hui, Xin; Lipp, Peter; Thiel, Gerald

    2015-04-01

    The steroid pregnenolone sulfate activates the transcription factor activator protein-1 (AP-1) via stimulation of transient receptor potential melastatin-3 (TRPM3) channels. Here, we show that the signaling pathway requires an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) levels. The upregulation of AP-1 was attenuated in cells that overexpressed mitogen activated protein kinase phosphatase-1, indicating that Ca(2+) ions prolong the signaling cascade via activation of mitogen activated protein kinases. On the transcriptional level, expression of a dominant-negative mutant of the basic region leucine zipper protein c-Jun, a major constituent of the AP-1 transcription factor complex, or expression of a c-Jun-specific short hairpin RNA attenuated pregnenolone sulfate-induced AP-1 activation. In addition, stimulation of TRPM3 channels increased the transcriptional activation potential of the basic region leucine zipper protein ATF2. Inhibition of ATF2 target gene expression via expression of a dominant-negative mutant of ATF2 or expression of an ATF2-specific short hairpin RNA interfered with TRPM3-mediated stimulation of AP-1. Moreover, we show that a dominant-negative mutant of the ternary complex factor (TCF) Elk-1 attenuated the upregulation of AP-1 following stimulation of TRPM3 channels. Thus, c-Jun, ATF2, and TCFs are required to connect the intracellular signaling cascade elicited by activation of TRPM3 channels with enhanced transcription of AP-1-regulated genes. We conclude that pregnenolone sulfate-induced TRPM3 channel activation changes the gene expression pattern of the cells by activating transcription of c-Jun-, ATF2-, and TCF-controlled genes.

  9. 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.

  10. ATP6AP1 — EDRN Public Portal

    Cancer.gov

    ATP6AP1 is an integral membrane protein composed of at least 10 subunits. It is responsible for acidifying various eukaryotic intracellular organelles. ATP6AP1, also known as vacuolar ATPase or V-ATPase, has a cytosolic V1 domain and a transmembrane V0 domain. The acidification performed by ATP6AP1 is necessary for intracellular processes such as protein sorting, zymogen activation, and receptor-mediated endocytosis.

  11. BRAFV600E induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via AP-1 activation

    PubMed Central

    Tsai, Yo-Ting; Lozanski, Gerard; Lehman, Amy; Sass, Ellen J.; Hertlein, Erin; Salunke, Santosh B.; Chen, Ching-Shih; Grever, Michael R.; Byrd, John C.; Lucas, David M.

    2015-01-01

    A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAFV600E inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. To investigate the biological role of BRAFV600E in B-cell leukemia, we generated a CLL-like B-cell line, OSUCLL, with doxycycline-inducible BRAFV600E expression. Microarray and real-time PCR analysis showed that ABCB1 mRNA is upregulated in these cells, and P-glycoprotein (P-gp) expression as well as function were confirmed by immunoblot and rhodamine exclusion assays. Additionally, pharmacological inhibition of BRAFV600E and MEK alleviated the BRAFV600E-induced ABCB1/P-gp expression. ABCB1 reporter assays and gel shift assays demonstrated that AP-1 activity is crucial in this mechanism. This study therefore uncovers a pathological role for BRAFV600E in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAFV600E and MEK can be used to delay disease progression and occurrence of resistance. PMID:26350141

  12. BRAF(V600E) induces ABCB1/P-glycoprotein expression and drug resistance in B-cells via AP-1 activation.

    PubMed

    Tsai, Yo-Ting; Lozanski, Gerard; Lehman, Amy; Sass, Ellen J; Hertlein, Erin; Salunke, Santosh B; Chen, Ching-Shih; Grever, Michael R; Byrd, John C; Lucas, David M

    2015-09-05

    A subset of patients with chronic lymphocytic leukemia (CLL) and nearly all patients with classic hairy cell leukemia (HCL) harbor somatic BRAF activating mutations. However, the pathological role of activated BRAF in B-cell leukemia development and progression remains unclear. In addition, although HCL patients respond well to the BRAF(V600E) inhibitor vemurafenib, relapses are being observed, suggesting the development of drug resistance in patients with this mutation. To investigate the biological role of BRAF(V600E) in B-cell leukemia, we generated a CLL-like B-cell line, OSUCLL, with doxycycline-inducible BRAF(V600E) expression. Microarray and real-time PCR analysis showed that ABCB1 mRNA is upregulated in these cells, and P-glycoprotein (P-gp) expression as well as function were confirmed by immunoblot and rhodamine exclusion assays. Additionally, pharmacological inhibition of BRAF(V600E) and MEK alleviated the BRAF(V600E)-induced ABCB1/P-gp expression. ABCB1 reporter assays and gel shift assays demonstrated that AP-1 activity is crucial in this mechanism. This study, uncovers a pathological role for BRAF(V600E) in B-cell leukemia, and provides further evidence that combination strategies with inhibitors of BRAF(V600E) and MEK can be used to delay disease progression and occurrence of resistance.

  13. 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

  14. 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

    2016-02-08

    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.

  15. Relative Antioxidant Activities of Quercetin and Its Structurally Related Substances and Their Effects on NF-κB/CRE/AP-1 Signaling in Murine Macrophages

    PubMed Central

    Kim, Byung-Hak; Choi, Jung Sook; Yi, Eun Hee; Lee, Jin-Ku; Won, Cheolhee; Ye, Sang-Kyu; Kim, Myoung-Hwan

    2013-01-01

    Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by the oxidative burst in activated macrophages and neutrophils cause oxidative stress-implicated diseases. Quercetin is flavonoid that occurs naturally in plants and is widely used as a nutritional supplement due to its antioxidant and anti-inflammatory properties. In this study, we investigated antioxidant activities and mechanisms of action in zymosan-induced macrophages of quercetin and quercetin-related flavonoids such as quercitrin, isoquercitrin, quercetin 3-O-β-(2″-galloyl)-rhamnopyranoside (QGR) and quercetin 3-O-β-(2″-galloyl)-glucopyranoside (QGG) as well as gallic acid, a building moiety of QGR and QGG. QGR and QGG exhibited stronger antioxidant activities compared with quercetin, whereas quercitrin, isoquercitrin and gallic acid exhibited weak-to-no antioxidant activities, assessed by 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging, superoxide production, superoxide scavenging, nitric oxide (NO) production, peroxynitrite (ONOO−) scavenging and myeloperoxidase (MPO) activity. Regarding mechanisms, the quercetin-containing flavonoids QGR and QGG differentially targeted compared with quercetin in the NF-κB signaling pathway that inhibited the DNA binding activity of the NF-κB complex without affecting the degradation and phosphorylation of IκBα and NF-κB phosphorylation. In addition, QGR and QGG inhibited CRE and activator protein (AP-1) transcriptional activity and JNK phosphorylation by inhibiting the cAMP/protein kinase A (PKA) and protein kinase C (PKC) signaling in a different manner than quercetin. Our results showed that although QGR and QGG exhibited stronger antioxidant activities than quercetin in macrophages, their mechanisms of action in terms of the NF-κB, PKA and PKC signaling pathways were different. PMID:23649461

  16. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  17. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  18. Telmisartan ameliorates cisplatin-induced nephrotoxicity by inhibiting MAPK mediated inflammation and apoptosis.

    PubMed

    Malik, Salma; Suchal, Kapil; Gamad, Nanda; Dinda, Amit Kumar; Arya, Dharamvir Singh; Bhatia, Jagriti

    2015-02-05

    Nephrotoxicity is a major adverse effect of the widely used anticancer drug cisplatin. Oxidative stress, inflammation and apoptosis are implicated in the pathophysiology of cisplatin-induced acute renal injury. Moreover, cisplatin activates many signal transduction pathways involved in cell injury and death, particularly mitogen activated protein kinase (MAPK) pathway. With this background, we aimed to investigate the protective effect of telmisartan, a widely used antihypertensive drug, in cisplatin-induced nephrotoxicity model in rats. To accomplish this, male albino wistar rats (150-200 g) were divided into 6 groups: Normal, cisplatin-control, telmisartan (2.5, 5 and 10 mg/kg) and telmisartan per se treatment groups. Normal saline or telmisartan was administered orally to rats for 10 days and cisplatin was given on 7th day (8 mg/kg; i.p.) to induce nephrotoxicity. On 10th day, rats were killed and both the kidneys were harvested for biochemical, histopathological and molecular studies. Cisplatin injected rats showed depressed renal function, altered proxidant-antioxidant balance and acute tubular necrosis which was significantly normalized by telmisartan co-treatment. Furthermore, cisplatin administration activated MAPK pathway that caused tubular inflammation and apoptosis in rats. Telmisartan treatment significantly prevented MAPK mediated inflammation and apoptosis. Among the three doses studied telmisartan at 10 mg/kg dose showed maximum nephroprotective effect which could be due to maintenance of cellular redox status and inhibition of MAPK activation.

  19. 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

  20. 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.

  1. 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.

  2. 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 ...

  3. Synergistic transcriptional activation of the mouse urokinase plasminogen activator (uPA) gene and of its enhancer activator protein 1 (AP1) site by cAMP and retinoic acid.

    PubMed Central

    Mira-Y-Lopez, R; Jaramillo, S; Jing, Y

    1998-01-01

    We have investigated the mechanism whereby all-trans retinoic acid (tRA) potentiates the 8-bromo-cAMP (8-BrcAMP)-dependent transcription of the urokinase plasminogen activator (uPA) gene in SC115 mouse mammary carcinoma cells. Photoaffinity labelling experiments showed that tRA did not alter the cellular content of cAMP-dependent protein kinase regulatory subunits I and II. In agreement with this, nuclear run-on analysis in the presence of the translational inhibitor puromycin demonstrated that the effect of 8-BrcAMP and its potentiation by tRA were independent of protein synthesis. A transiently transfected 6.6 kb uPA 5'-flanking region-chloramphenicol acetyltransferase (CAT) fusion gene mimicked the response of the endogenous uPA gene. Thus 1 mM 8-BrcAMP induced a 100-200% increase in CAT content, 100 nM tRA had no effect and 100 nM tRA+1 mM 8-BrcAMP induced a 300-500% increase in cells co-transfected with tRA receptor and/or 9-cis-RA receptor. Analysis of 5'-deleted constructs showed that the tRA effect required at least two cis regions: -2657 to -2186, encompassing the 100 bp uPA enhancer, and -709 to -324, which exhibited silencing activity. Neither region contained a tRA-response element-like motif. Because tRA receptor and 9-cis-RA receptor interact with activator protein 1 (AP1), we tested whether tRA regulated the uPA enhancer AP1 site in the presence of 8-BrcAMP. We found that a dimer of this site fused to a minimal uPA-CAT fusion gene was responsive to 1 mM 8-BrcAMP (100% CAT increase), not responsive to 100 nM tRA, and synergistically responsive to 100 nM tRA+1 mM 8-BrcAMP (240% CAT increase) in cells co-transfected with Fos and Jun. Synergistic activation of the same construct and of the 6.6 kb uPA-CAT fusion gene was also obtained using tRA and 100 nM PMA. We conclude that multiple cis elements, probably including the uPA enhancer AP1 site, mediate the tRA potentiation of uPA transcription. PMID:9560322

  4. Catalytically defective receptor protein tyrosine kinase PTK7 enhances invasive phenotype by inducing MMP-9 through activation of AP-1 and NF-κB in esophageal squamous cell carcinoma cells

    PubMed Central

    Shin, Won-Sik; Hong, Yuri; Lee, Hae Won; Lee, Seung-Taek

    2016-01-01

    Protein tyrosine kinase 7 (PTK7), a member of the catalytically defective receptor protein tyrosine kinase family, is upregulated in various cancers including esophageal squamous cell carcinoma (ESCC). Here, we have explored the molecular mechanism of PTK7-dependent invasiveness in ESCC cells. PTK7 knockdown reduced gelatin degradation and MMP-9 secretion in cultures of ESCC TE-10 cells, and showed reduced levels of MMP9 mRNA using real-time RT-PCR and luciferase reporter assays. PTK7 knockdown decreased not only phosphorylation of NF-κB, IκB, ERK, and JNK, but also nuclear localization of NF-κB and AP-1 consisting of c-Fos and c-Jun. Activation of AP-1 and NF-κB requires PTK7-mediated activation of tyrosine kinases, including Src. In addition, NF-κB activation by PTK7 involves the PI3K/Akt signaling pathway. PTK7-mediated upregulation of MMP9 was also observed in other ESCC cell lines and in three-dimensional cultures of TE-10 cells. Moreover, MMP-9 expression positively correlated with PTK7 expression in ESCC tumor tissue. These findings demonstrate that PTK7 upregulates MMP9 through activation of AP-1 and NF-κB and, thus increases invasive properties of ESCC cells. PMID:27689325

  5. 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.

  6. 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

  7. The role of AP-1 and epigenetics in ALCL.

    PubMed

    Schiefer, Ana-Iris; Vesely, Paul; Hassler, Melanie R; Egger, Gerda; Kenner, Lukas

    2015-06-01

    Anaplastic large cell lymphoma (ALCL) is an aggressive, highly proliferative, T-cell lymphoma with increasing incidence worldwide. Anaplastic Lymphoma Kinase (ALK) fusions occur in about 50% of all cases. Most ALK positive cases of ALCL harbor the t(2;5) translocation that leads to expression of Nucleophosmin-Anaplastic Lymphoma Kinase (NPM-ALK). NPM-ALK induces a variety of oncogenic signaling pathways that lead to malignant transformation of T-cells via Activator Protein-1 (AP-1), STAT3 and other (transcription) factors. In addition to the commonly known AP-1 activators Mitogen-Activated Protein Kinases (MAPKs), there are other signaling pathways, such as PI3K/mTOR/AKT, which are implicated in AP-1 activation/expression in ALCL. The AP-1 factor JUNB was shown to drive ALCL proliferation and the expression of the characteristic ALCL Ki-1 antigen, CD30. cJUN and JUNB target PDGFRB, thereby leading to tumor progression and dissemination. Furthermore, aberrant gene expression in ALCL is frequently accompanied by changes in epigenetic regulatory mechanisms, such as DNA methylation patterns. Here, we discuss the role of AP-1 in the pathogenesis of ALCL and provide an overview of pathological epigenetic changes in ALCL cells.

  8. Excretory-secretory products of Giardia lamblia induce interleukin-8 production in human colonic cells via activation of p38, ERK1/2, NF-κB and AP-1.

    PubMed

    Lee, H-Y; Hyung, S; Lee, N Y; Yong, T-S; Han, S-H; Park, S-J

    2012-04-01

    Giardia lamblia, a pathogen causing diarrhoeal outbreaks, is interesting how it triggers immune response in the human epithelial cells. This study defined the crucial roles of signalling components involved in G. lamblia-induced cytokine production in human epithelial cells. Incubation of the gastrointestinal cell line HT-29 with G. lamblia GS trophozoites triggered production of interleukin (IL)-1β, IL-8 and tumour necrosis factor (TNF)-α. IL-8 production was not significantly decreased by physically separating the HT-29 cells and G. lamblia GS trophozoites. Indeed, treatment of HT-29 with G. lamblia excretory-secretory products (ESP) induced IL-8 production. Electrophoretic mobility gel shift and transfection assays using mutagenized IL-8 promoter reporter plasmids indicated that IL-8 production by G. lamblia ESP occurs through activation of two transcriptional factors, nuclear factor kappaB (NF-κB) and activator protein 1 (AP-1) in HT-29 cells. In addition, activation of two mitogen-activated protein kinases (MAPKs), p38 and ERK1/2, was also detected in the HT-29 cells stimulated with G. lamblia ESP. Selective inhibition of these MAPKs resulted in decreased production of ESP-induced IL-8. These results indicate that activation of p38, ERK1/2 MAPK, NF-κB and AP-1 comprises the signalling pathway responsible for IL-8 production by G. lamblia ESP.

  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. Stem cell factor and interleukin-2/15 combine to enhance MAPK-mediated proliferation of human natural killer cells.

    PubMed

    Benson, Don M; Yu, Jianhua; Becknell, Brian; Wei, Min; Freud, Aharon G; Ferketich, Amy K; Trotta, Rossana; Perrotti, Danilo; Briesewitz, Roger; Caligiuri, Michael A

    2009-03-19

    Stem cell factor (SCF) promotes synergistic cellular proliferation in combination with several growth factors, and appears important for normal natural killer (NK)-cell development. CD34(+) hematopoietic precursor cells (HPCs) require interleukin-15 (IL-15) for differentiation into human NK cells, and this effect can be mimicked by IL-2. Culture of CD34(+) HPCs or some primary human NK cells in IL-2/15 and SCF results in enhanced growth compared with either cytokine alone. The molecular mechanisms responsible for this are unknown and were investigated in the present work. Activation of NK cells by IL-2/15 increases expression of c-kit whose kinase activity is required for synergy with IL-2/15 signaling. Mitogen-activated protein kinase (MAPK) signaling intermediaries that are activated both by SCF and IL-2/15 are enhanced in combination to facilitate earlier cell-cycle entry. The effect results at least in part via enhanced MAPK-mediated modulation of p27 and CDK4. Collectively the data reveal a novel mechanism by which SCF enhances cellular proliferation in combination with IL-2/15 in primary human NK cells.

  11. 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

  12. Florfenicol inhibits allergic airway inflammation in mice by p38 MAPK-mediated phosphorylation of GATA 3.

    PubMed

    Xinxin, Ci; Chi, Chen; Xiao, Chu; Xue, Xu; Yongjun, Yang; Junqing, Cui; Xuming, Deng

    2011-02-01

    Florfenicol has been shown to possess anti-inflammatory activity. However, its possible use for asthma has not yet been studied. First we investigated the anti-inflammatory properties of florfenicol using mice asthma model. BALB/c mice were immunized and challenged by ovalbumin. Treatment with florfenicol caused a marked reduction in inflammatory cells and three Th2 type cytokines in the bronchoalveolar lavage fluids of mice. The levels of ovalbumin-specific IgE and airway hyperresponsiveness were significantly altered after treatment with florfenicol. Histological studies using H&E and AB-PAS staining demonstrate that florfenicol substantially inhibited ovalbumin-induced inflammatory cells infiltration in lung tissue and goblet cell hyperplasia in the airway. These results were similar to those obtained with dexamethasone treatment. We then investigated which signal transduction mechanisms could be implicated in florfenicol activity. Our results suggested that the protective effect of florfenicol was mediated by the inhibition of the p38 MAPK-mediated phosphorylation of GATA 3.

  13. p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis during simulated ischemia.

    PubMed

    Meldrum, K K; Meldrum, D R; Hile, K L; Yerkes, E B; Ayala, A; Cain, M P; Rink, R C; Casale, A J; Kaefer, M A

    2001-08-01

    Ischemia causes renal tubular cell loss through apoptosis; however, the mechanisms of this process remain unclear. Using the renal tubular epithelial cell line LLC-PK(1), we developed a model of simulated ischemia (SI) to investigate the role of p38 MAPK (mitogen-activated protein kinase) in renal cell tumor necrosis factor-alpha (TNF-alpha) mRNA production, protein bioactivity, and apoptosis. Results demonstrate that 60 min of SI induced maximal TNF-alpha mRNA production and bioactivity. Furthermore, 60 min of ischemia induced renal tubular cell apoptosis at all substrate replacement time points examined, with peak apoptotic cell death occurring after either 24 or 48 h. p38 MAPK inhibition abolished TNF-alpha mRNA production and TNF-alpha bioactivity, and both p38 MAPK inhibition and TNF-alpha neutralization (anti-porcine TNF-alpha antibody) prevented apoptosis after 60 min of SI. These results constitute the initial demonstration that 1) renal tubular cells produce TNF-alpha mRNA and biologically active TNF-alpha and undergo apoptosis in response to SI, and 2) p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis after SI.

  14. Decreased expression of aromatase in the Ishikawa and RL95-2 cells by the isoflavone, puerarin, is associated with inhibition of c-jun expression and AP-1 activity.

    PubMed

    Yu, Chaoqin; Li, Yanwei; Chen, Huan; Yang, Shengsheng; Xie, Guangru

    2008-12-01

    Aromatase P450 (P450(arom)) is overexpressed in endometriosis, endometrial cancers and uterine fibroids. With weak estrogen agonists/antagonists and some other enzymatic activities, isoflavones are increasingly advocated as a natural alternative to estrogen replacement therapy (ERT) and are available as dietary supplements. Puerarin is major isoflavonoid compound isolated from Pueraria lobata, a Chinese medicine known as Gegen. Our clinical study shows that puerarin can be used in the treatment of endometriosis, which improves pain and infertility. Assuming that the effect of puerarin on endometriosis may result from the regulation of aromatase expression or activity, we carried out this study to test the effects of puerarin on aromatase in Ishikawa and RL95-2 cell lines. Our data have demonstrated a significant decrease of P450(arom) expression at both mRNA and protein levels by low dose puerarin treatment in both cell lines. Besides, we found that the -410/-401bp and -565/-559bp regions of aromatase promoter II contained the critical cis-acting elements, binding AP-1 and c-jun. We also found that puerarin exerted a time-course effect on the inhibition of c-jun mRNA, which parallelled that of P450(arom). To further confirm if c-jun is responsible for the P450(arom) regulation by puerarin, we knocked down c-jun expression using siRNA and it indicates that c-jun acts as a considerable transcription factor in regulating P450(arom) expression and activity. Accordingly, the suppression of P450(arom) expression and activity by puerarin treatment may associate with the downregulation of transcription factor AP-1 or c-jun.

  15. Tead and AP1 Coordinate Transcription and Motility.

    PubMed

    Liu, Xiangfan; Li, Huapeng; Rajurkar, Mihir; Li, Qi; Cotton, Jennifer L; Ou, Jianhong; Zhu, Lihua J; Goel, Hira L; Mercurio, Arthur M; Park, Joo-Seop; Davis, Roger J; Mao, Junhao

    2016-02-09

    The Tead family transcription factors are the major intracellular mediators of the Hippo-Yap pathway. Despite the importance of Hippo signaling in tumorigenesis, Tead-dependent downstream oncogenic programs and target genes in cancer cells remain poorly understood. Here, we characterize Tead4-mediated transcriptional networks in a diverse range of cancer cells, including neuroblastoma, colorectal, lung, and endometrial carcinomas. By intersecting genome-wide chromatin occupancy analyses of Tead4, JunD, and Fra1/2, we find that Tead4 cooperates with AP1 transcription factors to coordinate target gene transcription. We find that Tead-AP1 interaction is JNK independent but engages the SRC1-3 co-activators to promote downstream transcription. Furthermore, we show that Tead-AP1 cooperation regulates the activity of the Dock-Rac/CDC42 module and drives the expression of a unique core set of target genes, thereby directing cell migration and invasion. Together, our data unveil a critical regulatory mechanism underlying Tead- and AP1-controlled transcriptional and functional outputs in cancer cells.

  16. Helicobacter pylori promotes invasion and metastasis of gastric cancer cells through activation of AP-1 and up-regulation of CACUL1.

    PubMed

    Kong, Ying; Ma, Li-qing; Bai, Pei-song; Da, Rong; Sun, Hong; Qi, Xiao-gai; Ma, Jie-qun; Zhao, Ru-ming; Chen, Nan-zheng; Nan, Ke-jun

    2013-11-01

    Infection with Helicobacter pylori is important in the development and progression of gastric cancer. However, the mechanisms that regulate this activation in gastric tumors remain elusive. CACUL1 has been cloned and identified as a novel gene that is expressed in many types of cancer and is involved in cell cycle regulation and tumor growth. The current study aimed to examine the expression of CACUL1 in gastric cancer samples and analyze its correlation with H. pylori infection. We found that CACUL1 was highly expressed in gastric cancer tissues and negatively correlated with gastric cancer differentiation and TNM stage. In addition, CACUL1 expression was high in H. pylori-infected tissues compared with H. pylori non-infected tissue. We found that H. pylori could up-regulate CACUL1 expression through activating protein 1. The up-regulation of CACUL1 expression could promote matrix metalloproteinase 9 and Slug expression to increase invasion and metastasis of tumor cells. These results suggested that H. pylori-triggered CACUL1 production occurred in an activating protein 1-dependent manner and regulated matrix metalloproteinase 9 and Slug expression to affect the invasion and metastasis of tumor cells. Therefore, CACUL1 is a potential therapeutic target for the treatment of aggressive gastric cancer.

  17. Integrated omic analysis of oropharyngeal carcinomas reveals human papillomavirus (HPV)-dependent regulation of the activator protein 1 (AP-1) pathway.

    PubMed

    Sepiashvili, Lusia; Waggott, Daryl; Hui, Angela; Shi, Wei; Su, Susie; Ignatchenko, Alex; Ignatchenko, Vladimir; Laureano, Marissa; Huang, Shao Hui; Xu, Wei; Weinreb, Ilan; Waldron, John; O'Sullivan, Brian; Irish, Jonathan C; Boutros, Paul C; Liu, Fei-Fei; Kislinger, Thomas

    2014-12-01

    HPV-positive oropharyngeal carcinoma (OPC) patients have superior outcomes relative to HPV-negative patients, but the underlying mechanisms remain poorly understood. We conducted a proteomic investigation of HPV-positive (n = 27) and HPV-negative (n = 26) formalin-fixed paraffin-embedded OPC biopsies to acquire insights into the biological pathways that correlate with clinical behavior. Among the 2,633 proteins identified, 174 were differentially abundant. These were enriched for proteins related to cell cycle, DNA replication, apoptosis, and immune response. The differential abundances of cortactin and methylthioadenosine phosphorylase were validated by immunohistochemistry in an independent cohort of 29 OPC samples (p = 0.023 and p = 0.009, respectively). An additional 1,124 proteins were independently corroborated through comparison to a published proteomic dataset of OPC. Furthermore, utilizing the Cancer Genome Atlas, we conducted an integrated investigation of OPC, attributing mechanisms underlying differential protein abundances to alterations in mutation, copy number, methylation, and mRNA profiles. A key finding of this integration was the identification of elevated cortactin oncoprotein levels in HPV-negative OPCs. These proteins might contribute to reduced survival in these patients via their established role in radiation resistance. Through interrogation of Cancer Genome Atlas data, we demonstrated that activation of the β1-integrin/FAK/cortactin/JNK1 signaling axis and associated differential regulation of activator protein 1 transcription factor target genes are plausible consequences of elevated cortactin protein levels.

  18. 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.

  19. ERK-associated changes of AP-1 proteins during fear extinction.

    PubMed

    Guedea, Anita L; Schrick, Christina; Guzman, Yomayra F; Leaderbrand, Katie; Jovasevic, Vladimir; Corcoran, Kevin A; Tronson, Natalie C; Radulovic, Jelena

    2011-06-01

    Extensive research has unraveled the molecular basis of learning processes underlying contextual fear conditioning, but the mechanisms of fear extinction remain less known. Contextual fear extinction occurs when an aversive stimulus that initially caused fear is no longer present and depends on the activation of the extracellular signal-regulated kinase (ERK), among other molecules. Here we investigated how ERK signaling triggered by extinction affects its downstream targets belonging to the activator protein-1 (AP-1) transcription factor family. We found that extinction, when compared to conditioning of fear, markedly enhanced the interactions of active, phospho-ERK (pERK ) with c-Jun causing alterations of its phosphorylation state. The AP-1 binding of c-Jun was decreased whereas AP-1 binding of JunD, Jun dimerization protein 2 (JDP2) and ERK were significantly enhanced. The increased AP-1 binding of the inhibitory JunD and JDP2 transcription factors was paralleled by decreased levels of the AP-1 regulated proteins c-Fos and GluR2. These changes were specific for extinction and were MEK-dependent. Overall, fear extinction involves ERK/Jun interactions and a decrease of a subset of AP-1-regulated proteins that are typically required for fear conditioning. Facilitating the formation of inhibitory AP-1 complexes may thus facilitate the reduction of fear.

  20. 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

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

    PubMed

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

    2016-07-14

    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, Rab5(GTP)-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.

  2. Inactivation of AP1 proteins by a nuclear serine protease precedes the onset of radiation-induced fibrosing alveolitis.

    PubMed

    Haase, Michael G; Klawitter, Anke; Bierhaus, Angelika; Yokoyama, Kazunari K; Kasper, Michael; Geyer, Peter; Baumann, Michael; Baretton, Gustavo B

    2008-05-01

    Radiation-induced lung damage comprises inflammation (alveolitis) as well as disturbed regulation of cell differentiation and proliferation (fibrosis). The transcriptional regulation of this process is poorly understood. One key transcription factor involved in the regulation of proliferation and differentiation is AP1 (activator protein 1). The present study examined changes in the DNA-binding activity of AP1 after irradiation and defined the underlying molecular mechanisms in an animal model. The right lungs of Fischer rats received a single radiation dose of 20 Gy. Lung tissue was tested for AP1 DNA-binding activity, AP1 mRNA, and levels of AP1 proteins as well as for c-Jun specific proteolytic activity. After an initial increase, the AP1 DNA-binding activity was completely lost starting at 5.5 weeks after irradiation, which is 2.5 weeks before the onset of fibrosing alveolitis. This was not caused by reduction of mRNA levels or size. Instead, a selective nuclear cleavage of c-Jun by a serine protease caused the loss of AP1 activity. Considering the central role of AP1 in cell proliferation and differentiation and the strict timely correlation to the onset of the disease, the complete loss of AP1 function is likely to play a critical role in radiation-induced fibrosing alveolitis.

  3. AP-1 is involved in ICOS gene expression downstream of TCR/CD28 and cytokine receptor signaling.

    PubMed

    Watanabe, Masashi; Nakajima, Shinsuke; Ohnuki, Kazunobu; Ogawa, Shuhei; Yamashita, Masakatsu; Nakayama, Toshinori; Murakami, Yasufumi; Tanabe, Kazunari; Abe, Ryo

    2012-07-01

    It has been proposed that sustained ICOS expression in chronic inflammatory immune conditions, such as autoimmunity and allergy, contributes to symptom exacerbation. Therefore modulation of ICOS gene expression could be a potential therapeutic strategy for such immune diseases. However, the precise molecular mechanisms controlling ICOS gene expression remain poorly understood. In this study, we explored transcription factors involving in ICOS gene expression and examined their roles in a physiological situation. Microarray analysis revealed that one AP-1 molecule, Fos-related antigen-2 (Fra2), was highly correlated with ICOS expression. Ectopic expression of Fra2 and other AP-1 molecules upregulated ICOS expression on T cells. We identified an AP-1-responsive site (AP1-RE) within the ICOS promoter region and demonstrated AP-1 actually binds to AP1-RE upon TCR/CD28 stimulation. Meanwhile, we found several cytokines could upregulate ICOS expression on both naïve and effector T cells in a manner independent of TCR/CD28 stimulation. These cytokine stimuli induced AP-1 binding to AP1-RE. Together, our results indicate AP-1 transcription factors are involved in ICOS gene expression downstream of both TCR/CD28 signaling and cytokine receptor signaling, and suggest AP-1 activation via cytokine receptor signaling may be one of the mechanisms maintaining high level ICOS expression in chronic inflammatory immune responses.

  4. AP1 enhances polyomavirus DNA replication by promoting T-antigen-mediated unwinding of DNA.

    PubMed Central

    Guo, W; Tang, W J; Bu, X; Bermudez, V; Martin, M; Folk, W R

    1996-01-01

    An early step in the initiation of polyomavirus DNA replication is viral large-T-antigen-mediated unwinding of the origin. We report that components of the AP1 transcription factor, Fos and Jun, interact with T antigen in vitro to enhance unwinding of the viral origin. This provides a biochemical basis for the capacity of AP1 to activate viral DNA replication in vivo. PMID:8763994

  5. Adrenergic inducibility of AP-1 binding in the rat pineal gland depends on prior photoperiod.

    PubMed

    Guillaumond, F; Becquet, D; Bosler, O; François-Bellan, A M

    2002-10-01

    The main known function of the pineal gland in mammals is the temporal synchronization of physiological rhythms to seasonal changes of day length (photoperiod). In rat, the transcription factor activating protein-1 (AP-1) displays a circadian rhythm in its DNA binding in the pineal gland, which results from the rhythmic expression of Fra-2. We postulated that, if AP-1 is an important component of pineal gland functioning, then variations in photoperiodic conditions should lead to an adaptation of the AP-1 binding rhythm. Here we show that AP-1 binding patterns adapt to variations in lighting conditions, in the same way as the rhythm of arylalkylamine-N-acetyltransferase (AA-NAT) activity. This adaptation appeared to result from photoperiodic adaptation of the rhythmic fra-2 gene expression and was reflected by an adapted delay between the onset of night and the acrophase of the nocturnal peak. We further showed that photoperiodic adaptation of both the AP-1 binding and AA-NAT activity rhythms resulted from adapted changes in adrenergic inducibility of both variables at night onset. We finally provided evidence that AP-1 shared with the CREM gene encoding the transcriptional repressor protein inducible cAMP early repressor (ICER) the ability to be hypersensitive or subsensitive to adrenergic stimuli, depending on prior photoperiod.

  6. Retinoids interfere with the AP1 signalling pathway in human breast cancer cells.

    PubMed

    Dedieu, Stephane; Lefebvre, Philippe

    2006-06-01

    Retinoic acid and its synthetic analogs exert major effects on many biological processes including cell proliferation and differentiation and are now considered as promising pharmacological agents for prevention and treatment of various cancers. The capacity of retinoids to inhibit AP1-responsive genes seems to be the basis for the chemopreventive and chemotherapeutic effects of these agents against hyperproliferative diseases. However, the molecular basis of retinoid antiproliferative properties remains to this day largely unknown. Here, we showed that retinoids inhibit phorbol ester-induced MMP-1 and MMP-3 expression in human breast cancer cells. Transcriptional interference was observed for both retinoid agonist and antagonist treatments, revealing separated transactivation and transrepression functions of retinoids. In addition, we examined MAP kinases as potential targets of retinoid signalling in human breast cancer cells and demonstrated that retinoids repress AP1-responsive gene expression by inhibiting MKK6/p38 and mainly MEK/ERK signalling pathways. On the contrary, the JNK-dependent pathway was not identified as a molecular relay for AP1 activity and was insensitive to retinoid treatments. Finally, we established that overexpressed c-fos and c-jun partially abolished the ability of retinoids to inhibit AP1 activity, suggesting that c-jun and/or c-fos containing dimers may constitute one target of retinoids for transrepression of AP1. All together, our data help to improve our understanding of how retinoids antagonize AP1 activity and may regulate tumoral cell proliferation.

  7. The RNA-binding protein RBPMS1 represses AP-1 signaling and regulates breast cancer cell proliferation and migration.

    PubMed

    Fu, Jie; Cheng, Long; Wang, Yu; Yuan, Ping; Xu, Xiaojie; Ding, Lihua; Zhang, Hao; Jiang, Kai; Song, Haifeng; Chen, Zhongwu; Ye, Qinong

    2015-01-01

    The activator protein-1 (AP-1) transcription factor complex plays a crucial role in tumor growth and progression. However, how AP-1 transcriptional activity is repressed is not fully understood. Here, we show that RNA-binding protein with multiple splicing 1 (RBPMS1) physically and functionally interacts with AP-1 in vitro and in vivo. The RNA-recognition motif (RRM) and C-terminus of the RBPMS1 isoforms RBPMS1A and RBPMS1C, but not RBPMS1B, interacted with cFos, a member of the AP-1 family that dimerizes with cJun to stimulate AP-1 transcriptional activity. RBPMS1 did not associate with Jun proteins. RBPMS1A and RBPMS1C bound to the basic leucine zipper (bZIP) domain of cFos that mediates dimerization of AP-1 proteins. In addition, RBPMS1A-C interacted with the transcription factor Smad3, which was shown to interact with cJun and increase AP-1 transcriptional activity. RBPMS1 inhibited c-Fos or Smad3-mediated AP-1 transactivation and the expression of AP-1 target genes known to be the key regulators of cancer growth and progression, including vascular endothelial growth factor (VEGF) and cyclin D1. Mechanistically, RBPMS1 blocks the formation of the cFos/cJun or Smad3/cJun complex as well as the recruitment of cFos or Smad3 to the promoters of AP-1 target genes. In cultured cells and a mouse xenograft model, RBPMS1 inhibited the growth and migration of breast cancer cells through c-Fos or Smad3. These data suggest that RBPMS1 is a critical repressor of AP-1 signaling and RBPMS1 activation may be a useful strategy for cancer treatment.

  8. Normal dendrite growth in Drosophila motor neurons requires the AP-1 transcription factor.

    PubMed

    Hartwig, Cortnie L; Worrell, Jason; Levine, Richard B; Ramaswami, Mani; Sanyal, Subhabrata

    2008-09-01

    During learning and memory formation, information flow through networks is regulated significantly through structural alterations in neurons. Dendrites, sites of signal integration, are key targets of activity-mediated modifications. Although local mechanisms of dendritic growth ensure synapse-specific changes, global mechanisms linking neural activity to nuclear gene expression may have profound influences on neural function. Fos, being an immediate-early gene, is ideally suited to be an initial transducer of neural activity, but a precise role for the AP-1 transcription factor in dendrite growth remains to be elucidated. Here we measure changes in the dendritic fields of identified Drosophila motor neurons in vivo and in primary culture to investigate the role of the immediate-early transcription factor AP-1 in regulating endogenous and activity-induced dendrite growth. Our data indicate that (a) increased neural excitability or depolarization stimulates dendrite growth, (b) AP-1 (a Fos, Jun hetero-dimer) is required for normal motor neuron dendritic growth during development and in response to activity induction, and (c) neuronal Fos protein levels are rapidly but transiently induced in motor neurons following neural activity. Taken together, these results show that AP-1 mediated transcription is important for dendrite growth, and that neural activity influences global dendritic growth through a gene-expression dependent mechanism gated by AP-1.

  9. Transcriptional regulation of human novel gene SPATA12 promoter by AP-1 and HSF.

    PubMed

    Li, Dan; Lin, Yiting; Liu, Zhiwen; Zhang, Yunsheng; Rong, Zhuoxian; Liu, Xuanming

    2012-12-10

    Human SPATA12 is a spermatogenesis associated gene and is supposed to function as an inhibitor during male germ cell development. SPATA12 is specifically expressed in spermatocytes, spermatids, and spermatozoa of human testis. In order to understand the regulation mechanism of SPATA12 gene expression, we identified and characterized the SPATA12 gene core promoter region and transcription factor binding sites by using reporter gene assays. AP-1 is founded to be a potential transcriptional activator of SPATA12. The promoter activity of SPATA12 was drastically declined after AP-1 binding site mutation or deletion. We also demonstrated that AP-1 combined with Smad3/4 contributes to the transcriptional regulation of SPATA12 in response to TGF-β1. The expression of SPATA12 could be induced by TGF-β1 in a dose-dependent manner, suggesting that AP-1 as an activator plays a role in the regulation of SPATA12 promoter. We have also shown that heat shock treatment could activate the expression of SPATA12 and transcription factor HSF binding sites in the SPATA12 promoter might be responsible for this heat-induction. These results suggested that AP-1 and HSF may play an important role in regulating SPATA12 promoter activity.

  10. 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.

  11. AP-1 overexpression impairs corticosteroid inhibition of collagen production by fibroblasts isolated from asthmatic subjects.

    PubMed

    Jacques, Eric; Semlali, Abdelhabib; Boulet, Louis Philippe; Chakir, Jamila

    2010-08-01

    Asthma is characterized by airway remodeling associated with an increase in the deposition of ECM proteins such as type I collagen. These components are mainly produced by fibroblasts. Inhaled corticosteroids are considered the cornerstone of asthma therapy. Despite substantial evidence as to the anti-inflammatory action of corticosteroids, their effect on controlling ECM protein deposition in the airways is not completely understood. This study determined the effect of dexamethasone (Dex) on collagen production by bronchial fibroblasts derived from asthmatic and healthy subjects. Expression of procollagen mRNA in fibroblasts from asthmatics and normal controls was determined by quantitative PCR. Regulation of the procollagen-alpha(1)I promoter was evaluated by transient transfections. Transforming growth factor-beta (TGF-beta) protein expression was determined by ELISA. Protein expression of glucocorticoid receptor (GR) and interaction with activator protein-1 (AP-1), a collagen regulatory transcription factor, was assessed by Western blots, coimmunoprecipitations, and EMSA. AP-1 overexpression was performed by transient transfection using c-Fos/c-Jun expression plasmids. Dex significantly downregulated procollagen production and promoter activity in normal fibroblasts but had no effect on asthmatic fibroblasts. AP-1 and GR interaction increased after Dex stimulation in asthmatic fibroblasts. AP-1 overexpression in control fibroblasts abrogated collagen gene response to Dex. These results show that Dex failed to reduce collagen production in fibroblasts from asthmatic subjects. This impaired response may be related to AP-1 overexpression in these cells.

  12. AP-1 transcription factor mediates VEGF-induced endothelial cell migration and proliferation.

    PubMed

    Jia, Jing; Ye, Taiyang; Cui, Pengfei; Hua, Qian; Zeng, Huiyan; Zhao, Dezheng

    2016-05-01

    VEGF, upon binding to its endothelial cell specific receptors VEGF-R1 and VEGF-R2, can induce endothelial cell migration, proliferation and angiogenesis. However, the molecular mechanism of these effects still remains unclear. In this study, we investigated whether VEGF promotes human umbilical vascular endothelial cell (HUVEC) migration and proliferation through activator protein-1 transcription factor (AP-1) family. We first showed that VEGF induces immediate-early genes AP-1 family gene expression differentially with the profound induction of JunB (both mRNA and protein) under various conditions (PBS, DMSO or control adenoviruses). The increase in AP-1 mRNA expression occurs primarily at the transcriptional level. Inhibition of AP-1 DNA binding activity by adenovirus expressing a potent dominant negative form of c-Fos (Afos) significantly attenuated VEGF-induced HUVEC migration and proliferation and cyclin D1 expression. Knockdown of JunB with adenovirus expressing JunB shRNA reduces VEGF-induced JunB expression and attenuated HUVEC migration. However the shJunB-expressing virus has no effect on VEGF-induced cyclin D1 protein expression and proliferation. These results suggest that VEGF-induced endothelial migration is mediated primarily by induction of JunB whereas the promotion of endothelial proliferation by VEGF is mediated by JunB-independent AP-1 family members.

  13. A dual AP-1 and SMAD decoy ODN suppresses tissue fibrosis and scarring in mice.

    PubMed

    Yuan, Hong-Feng; Huang, Hong; Li, Xiang-Yun; Guo, Wei; Xing, Wei; Sun, Zhi-Ya; Liang, Hua-Ping; Yu, Jian; Chen, Dong-Feng; Wang, Zheng-Guo; Hao, Jin; Xu, Xiang

    2013-04-01

    The transforming growth factor-β (TGF-β) signaling pathway promotes tissue fibrosis and scarring through SMAD (small mothers against decapentaplegic)-dependent and SMAD-independent mechanisms. However, inhibition of SMAD-mediated signal transduction alone induces an excessive inflammatory response that impairs the antifibrotic effects of TGF-β inhibitors. In this study, we designed and characterized a dual-functional transcription activator protein 1 (AP-1) and SMAD decoy oligodeoxynucleotide, antifibrosis oligodeoxynucleotide 4 (AFODN4) in vitro and in vivo. AFODN4 binds directly to recombinant AP-1 and SMAD with high affinity. AFODN4 significantly inhibited the DNA-binding and transcriptional activities of both AP-1 and SMAD, as well as the production of fibrotic mediators stimulated by TGF-β1 or TGF-β2 in L929 murine fibroblasts. Local administration of AFODN4 significantly inhibited fibrosis associated with acute dermal wounds in mice. Intriguingly, AFODN4 inhibited AP-1-mediated production of proinflammatory mediators, which can be caused by blockage of SMAD alone in vitro and in vivo. Collectively, these findings suggest that dual inhibition of SMAD and AP-1 signaling by AFODN4 is a useful strategy for the development of new antifibrotic agents.

  14. CXCL12/CXCR4 chemokine signaling in spinal glia induces pain hypersensitivity through MAPKs-mediated neuroinflammation in bone cancer rats.

    PubMed

    Hu, Xue-Ming; Liu, Yan-Nan; Zhang, Hai-Long; Cao, Shou-Bin; Zhang, Ting; Chen, Li-Ping; Shen, Wen

    2015-02-01

    The activation of MAPK pathways in spinal cord and subsequent production of proinflammatory cytokines in glial cells contribute to the development of spinal central sensitization, the basic mechanism underlying bone cancer pain (BCP). Our previous study showed that spinal CXCL12 from astrocytes mediates BCP generation by binding to CXCR4 in both astrocyters and microglia. Here, we verified that CXCL12/CXCR4 signaling contributed to BCP through a MAPK-mediated mechanism. In naïve rats, a single intrathecal administration of CXCL12 considerably induced pain hyperalgesia and phosphorylation expression of spinal MAPK members (including extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase), which could be partially prevented by pre-treatment with CXCR4 inhibitor AMD3100. This CXCL12-induced hyperalgesia was also reduced by MAPK inhibitors. In bone cancer rats, tumor cell inoculation into the tibial cavity caused prominent and persistent pain hyperalgesia, and associated with up-regulation of CXCL12 and CXCR4, activation of glial cells, phosphorylation of MAPKs, and production of proinflammatory cytokines in the spinal cord. These tumor cell inoculation-induced behavioral and neurochemical alterations were all suppressed by blocking CXCL12/CXCR4 signaling or MAPK pathways. Taken together, these results demonstrate that spinal MAPK pathways mediated CXCL12/CXCR4-induced pain hypersensitivity in bone cancer rats, which could be druggable targets for alleviating BCP and glia-derived neuroinflammation. Following tumor cell inoculation, chemokine CXCL12 from astrocytes spreads around the spinal environment, resulting in functional activation of CXCR4-expressing astrocytes and microglia. Once glia are activated, they may initiate MAPK (mitogen-activated protein kinase) pathways, and subsequently produce proinflammatory cytokines and chemokines. Among them, CXCL12 could reinforce the astrocytic and microglial activation in autocrine and paracrine manners

  15. Genome-scale functional profiling of the mammalian AP-1 signaling pathway

    PubMed Central

    Chanda, Sumit K.; White, Suhaila; Orth, Anthony P.; Reisdorph, Richard; Miraglia, Loren; Thomas, Russell S.; DeJesus, Paul; Mason, Daniel E.; Huang, Qihong; Vega, Raquel; Yu, De-Hua; Nelson, Christian G.; Smith, Brendan M.; Terry, Robert; Linford, Alicia S.; Yu, Yang; Chirn, Gung-wei; Song, Chuanzheng; Labow, Mark A.; Cohen, Dalia; King, Frederick J.; Peters, Eric C.; Schultz, Peter G.; Vogt, Peter K.; Hogenesch, John B.; Caldwell, Jeremy S.

    2003-01-01

    Large-scale functional genomics approaches are fundamental to the characterization of mammalian transcriptomes annotated by genome sequencing projects. Although current high-throughput strategies systematically survey either transcriptional or biochemical networks, analogous genome-scale investigations that analyze gene function in mammalian cells have yet to be fully realized. Through transient overexpression analysis, we describe the parallel interrogation of ≈20,000 sequence annotated genes in cancer-related signaling pathways. For experimental validation of these genome data, we apply an integrative strategy to characterize previously unreported effectors of activator protein-1 (AP-1) mediated growth and mitogenic response pathways. These studies identify the ADP-ribosylation factor GTPase-activating protein Centaurin α1 and a Tudor domain-containing hypothetical protein as putative AP-1 regulatory oncogenes. These results provide insight into the composition of the AP-1 signaling machinery and validate this approach as a tractable platform for genome-wide functional analysis. PMID:14514886

  16. AP-1(c-Jun/FosB) mediates xFoxD5b expression in Xenopus early developmental neurogenesis.

    PubMed

    Yoon, Jaeho; Kim, Jung-Ho; Lee, Ok-Joo; Lee, Sung-Young; Lee, Seung-Hwan; Park, Jae-Bong; Lee, Jae-Yong; Kim, Sung-Chan; Kim, Jaebong

    2013-01-01

    AP-1 (activator protein-1) is composed of Jun and Fos proteins and functions in cell proliferation, apoptosis and differentiation. Previous studies have demonstrated that different AP-1 complexes participate in the determination of various cell fates. However, the role of different AP-1 complexes during early vertebrate development is not yet fully understood. In the present study, we demonstrate that AP-1(c-Jun/FosB) regulates neurogenesis via FoxD5b expression. We show that FoxD5 was induced by the inhibition of BMP and that FoxD5b plays roles in neurogenesis. Additionally, we show that the FoxD5b promoter region within -1336 and -1316 contains an AP-1 binding site, which is required for the transcriptional regulation of FoxD5b and is induced by the inhibition of BMP signaling in animal cap explants. Moreover, c-Jun, a component of AP-1, directly binds to the AP-1 binding site of the FoxD5b promoter and induces FoxD5b expression cooperatively with FosB, but not with c-Fos or Fra-1. Altogether, these results suggest that AP-1(c-Jun/FosB) may play a role in neurogenesis via the induction of FoxD5b expression during early vertebrate development.

  17. Contribution of AP-1 interference induced by TAC-101 to tumor growth suppression in a hepatocellular carcinoma model.

    PubMed

    Eshima, Kokoro; Fukaya, Satoshi; Sugimoto, Akiko; Mori, Tomoko; Yokoi, Hiromi; Yamamoto, Yasuji; Sugiura, Shin; Honda, Shizu; Masuko, Norio; Murakami, Koji; Yamasaki, Yasundo; Kagechika, Hiroyuki

    2009-01-01

    TAC-101, 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid, is a synthetic ligand for retinoic acid receptor (RAR)-alpha. Here, we demonstrate the contribution of TAC-101-induced AP-1 interference to stabilization of tumor growth. TAC-101 induced transcriptional activation of RAR, resulting in marked elevation of RARbeta, a representative retinoid response marker, and it also significantly repressed the transcriptional activity of AP-1 in JHH-7 cells. In contrast to JHH-7, JHH-6 is another RARalpha-expressing human hepatocellular carcinoma (HCC) cell line with constitutive activation of AP-1, but it is retinoid insensitive and did not respond to the TAC-101-induced RAR signal. TAC-101 did not inhibit AP-1 activity of the JHH-6 cell line, showing that AP-1 interference by TAC-101 must be in parallel with RAR activation. Interleukin-8 (IL-8), one of the AP-1-regulated factors which correlate with a poor prognosis in HCC patients, was found to be overexpressed in JHH-7 cells. TAC-101 reduced IL-8 production without cytotoxicity and inhibited the progression of HCC in the orthotopic mouse model with decreased tumor IL-8 level. These results suggest that downregulation of the extracellular biomarker for AP-1 interference via the induction of retinoid signals will enhance the pharmacological effect of TAC-101 on HCC and it could be useful as a surrogate biomarker of therapeutic efficacy.

  18. RAS/ERK pathway transcriptional regulation through ETS/AP-1 binding sites.

    PubMed

    Hollenhorst, Peter C

    2012-01-01

    The RAS/RAF/MEK/ERK signaling pathway is activated by mutation in many cancers. Neighboring ETS and AP-1 DNA binding sequences can act as response elements for transcriptional activation by this pathway. ERK phosphorylation of an ETS transcription factor is one mechanism of activating the RAS/ERK gene expression program that can promote cancer cell phenotypes such as proliferation, invasion, and metastasis. Recent genome-wide mapping of ETS proteins over-expressed by chromosomal rearrangement in prostate cancer reveals a second mechanism for activation of this gene expression program. An oncogenic subset of ETS transcription factors can activate RAS/ERK target genes even in the absence of RAS/ERK pathway activation by binding ETS/AP-1 sequences. Thus, regulation of cancer cell invasion and metastasis via ETS/AP-1 sequence elements depends on which ETS protein is bound, and the status of the RAS/ERK pathway. This commentary will focus on what is known about the selectivity of ETS/AP-1 sequences for different ETS transcription factors and the transcriptional consequences of ETS protein selection.

  19. Interaction of amphiphysins with AP-1 clathrin adaptors at the membrane.

    PubMed

    Huser, Sonja; Suri, Gregor; Crottet, Pascal; Spiess, Martin

    2013-02-15

    The assembly of clathrin/AP (adaptor protein)-1-coated vesicles on the trans-Golgi network and endosomes is much less studied than that of clathrin/AP-2 vesicles at the plasma membrane for endocytosis. In vitro, the association of AP-1 with protein-free liposomes had been shown to require phosphoinositides, Arf1 (ADP-ribosylation factor 1)-GTP and additional cytosolic factor(s). We have purified an active fraction from brain cytosol and found it to contain amphiphysin 1 and 2 and endophilin A1, three proteins known to be involved in the formation of AP-2/clathrin coats at the plasma membrane. Assays with bacterially expressed and purified proteins showed that AP-1 stabilization on liposomes depends on amphiphysin 2 or the amphiphysin 1/2 heterodimer. Activity is independent of the SH3 (Src homology 3) domain, but requires interaction of the WDLW motif with γ-adaptin. Endogenous amphiphysin in neurons and transfected protein in cell lines co-localize perinuclearly with AP-1 at the trans-Golgi network. This localization depends on interaction of clathrin and the adaptor sequence in the amphiphysins and is sensitive to brefeldin A, which inhibits Arf1-dependent AP-1 recruitment. Interaction between AP-1 and amphiphysin 1/2 in vivo was demonstrated by co-immunoprecipitation after cross-linking. These results suggest an involvement of amphiphysins not only with AP-2 at the plasma membrane, but also in AP-1/clathrin coat formation at the trans-Golgi network.

  20. Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region.

    PubMed Central

    Moi, P; Chan, K; Asunis, I; Cao, A; Kan, Y W

    1994-01-01

    Hypersensitive site 2 located in the beta-globin locus control region confers high levels of expression to the genes of the beta-globin cluster. A tandem repeat of the consensus sequence for the transcription factors AP1 and NF-E2 (activating protein 1 and nuclear factor erythroid 2, respectively) is present within hypersensitive site 2 and is absolutely required for strong enhancer activity. This sequence binds, in vitro and in vivo, to ubiquitous proteins of the AP1 family and to the recently cloned erythroid-specific transcription factor NF-E2. Using the tandem repeat as a recognition site probe to screen a lambda gt11 cDNA expression library from K562 cells, we isolated several DNA binding proteins. Here, we report the characterization of one of the clones isolated. The gene, which we named Nrf2 (NF-E2-related factor 2), is encoded within a 2.2-kb transcript and predicts a 66-kDa protein with a basic leucine zipper DNA binding domain highly homologous to that of NF-E2. Although Nrf2 is expressed ubiquitously, a role of this protein in mediating enhancer activity of hypersensitive site 2 in erythroid cells cannot be excluded. In this respect, Nrf2 contains a powerful acidic activation domain that may participate in the transcriptional stimulation of beta-globin genes. Images PMID:7937919

  1. 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.

  2. Retinoic acid is a negative regulator of AP-1-responsive genes.

    PubMed Central

    Schüle, R; Rangarajan, P; Yang, N; Kliewer, S; Ransone, L J; Bolado, J; Verma, I M; Evans, R M

    1991-01-01

    We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression. Images PMID:1648728

  3. HIV-1 Nef hijacks clathrin coats by stabilizing AP-1:Arf1 polygons.

    PubMed

    Shen, Qing-Tao; Ren, Xuefeng; Zhang, Rui; Lee, Il-Hyung; Hurley, James H

    2015-10-23

    The lentiviruses HIV and simian immunodeficiency virus (SIV) subvert intracellular membrane traffic as part of their replication cycle. The lentiviral Nef protein helps viruses evade innate and adaptive immune defenses by hijacking the adaptor protein 1 (AP-1) and AP-2 clathrin adaptors. We found that HIV-1 Nef and the guanosine triphosphatase Arf1 induced trimerization and activation of AP-1. Here we report the cryo-electron microscopy structures of the Nef- and Arf1-bound AP-1 trimer in the active and inactive states. A central nucleus of three Arf1 molecules organizes the trimers. We combined the open trimer with a known dimer structure and thus predicted a hexagonal assembly with inner and outer faces that bind the membranes and clathrin, respectively. Hexagons were directly visualized and the model validated by reconstituting clathrin cage assembly. Arf1 and Nef thus play interconnected roles in allosteric activation, cargo recruitment, and coat assembly, revealing an unexpectedly intricate organization of the inner AP-1 layer of the clathrin coat.

  4. A mechanism of AP-1 suppression through interaction of c-Fos with lamin A/C

    PubMed Central

    Ivorra, Carmen; Kubicek, Markus; González, José M.; Sanz-González, Silvia M.; Álvarez-Barrientos, Alberto; O'Connor, José-Enrique; Burke, Brian; Andrés, Vicente

    2006-01-01

    AP-1 (Activating Protein 1) transcription factor activity is tightly regulated at multiple levels, including dimer formation (i.e., Fos/Jun). Here we show that the intermediate filament protein lamin A/C suppresses AP-1 function through direct interaction with c-Fos, and that both proteins can interact and colocalize at the nuclear envelope (NE) in mammalian cells. Perinuclear localization of c-Fos is absent in Lmna-null cells but can be restored by lamin A overexpression. In vitro, preincubation of c-Fos with lamin A prior to the addition of c-Jun inhibits AP-1 DNA-binding activity. In vivo, overexpression of lamin A reduces the formation of c-Fos/c-Jun heterodimers, and suppresses AP-1 DNA-binding and transcriptional activity. Notably, c-Fos colocalizes with lamin A/C at the NE in starvation-synchronized quiescent cells lacking detectable AP-1 DNA binding. In contrast, serum-induced AP-1 DNA-binding activity coincides with abundant nucleoplasmic c-Fos expression without changes in lamin A/C localization. We also found that Lmna-null cells display enhanced proliferation. In contrast, lamin A overexpression causes growth arrest, and ectopic c-Fos partially overcomes lamin A/C-induced cell cycle alterations. We propose lamin A/C-mediated c-Fos sequestration at the NE as a novel mechanism of transcriptional and cell cycle control. PMID:16452503

  5. 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.

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

    PubMed

    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.

  7. Epicatechin induces NF-kappaB, activator protein-1 (AP-1) and nuclear transcription factor erythroid 2p45-related factor-2 (Nrf2) via phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and extracellular regulated kinase (ERK) signalling in HepG2 cells.

    PubMed

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

    2010-01-01

    The dietary flavonoid epicatechin has been reported to exhibit a wide range of biological activities. The objective of the present study was to investigate the time-dependent regulation by epicatechin on the activity of the main transcription factors (NF-kappaB, activator protein-1 (AP-1) and nuclear transcription factor erythroid 2p45-related factor (Nrf2)) related to antioxidant defence and survival and proliferation pathways in HepG2 cells. Treatment of cells with 10 microm-epicatechin induced the NF-kappaB pathway in a time-dependent manner characterised by increased levels of IkappaB kinase (IKK) and phosphorylated inhibitor of kappaB subunit-alpha (p-IkappaBalpha) and proteolytic degradation of IkappaB, which was consistent with an up-regulation of the NF-kappaB-binding activity. Time-dependent activation of the AP-1 pathway, in concert with enhanced c-Jun nuclear levels and induction of Nrf2 translocation and phosphorylation were also demonstrated. Additionally, epicatechin-induced NF-kappaB and Nrf2 were connected to reactive oxygen species intracellular levels and to the activation of cell survival and proliferation pathways, being phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) and extracellular regulated kinase (ERK) associated to Nrf2 modulation and ERK to NF-kappaB induction. These data suggest that the epicatechin-induced survival effect occurs by the induction of redox-sensitive transcription factors through a tight regulation of survival and proliferation pathways.

  8. Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate

    PubMed Central

    Obier, Nadine; Cauchy, Pierre; Assi, Salam A.; Gilmour, Jane; Lie-A-Ling, Michael; Lichtinger, Monika; Hoogenkamp, Maarten; Noailles, Laura; Cockerill, Peter N.; Lacaud, Georges; Kouskoff, Valerie

    2016-01-01

    The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals. PMID:27802171

  9. Cooperative binding of AP-1 and TEAD4 modulates the balance between vascular smooth muscle and hemogenic cell fate.

    PubMed

    Obier, Nadine; Cauchy, Pierre; Assi, Salam A; Gilmour, Jane; Lie-A-Ling, Michael; Lichtinger, Monika; Hoogenkamp, Maarten; Noailles, Laura; Cockerill, Peter N; Lacaud, Georges; Kouskoff, Valerie; Bonifer, Constanze

    2016-12-01

    The transmission of extracellular signals into the nucleus involves inducible transcription factors, but how different signalling pathways act in a cell type-specific fashion is poorly understood. Here, we studied the regulatory role of the AP-1 transcription factor family in blood development using embryonic stem cell differentiation coupled with genome-wide transcription factor binding and gene expression analyses. AP-1 factors respond to MAP kinase signalling and comprise dimers of FOS, ATF and JUN proteins. To examine genes regulated by AP-1 and to examine how it interacts with other inducible transcription factors, we abrogated its global DNA-binding activity using a dominant-negative FOS peptide. We show that FOS and JUN bind to and activate a specific set of vascular genes and that AP-1 inhibition shifts the balance between smooth muscle and hematopoietic differentiation towards blood. Furthermore, AP-1 is required for de novo binding of TEAD4, a transcription factor connected to Hippo signalling. Our bottom-up approach demonstrates that AP-1- and TEAD4-associated cis-regulatory elements form hubs for multiple signalling-responsive transcription factors and define the cistrome that regulates vascular and hematopoietic development by extrinsic signals.

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

    PubMed

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

    2015-09-08

    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.

  11. Alisertib induces G2/M arrest, apoptosis, and autophagy via PI3K/Akt/mTOR- and p38 MAPK-mediated pathways in human glioblastoma cells

    PubMed Central

    Liu, Zheng; Wang, Feng; Zhou, Zhi-Wei; Xia, He-Chun; Wang, Xin-Yu; Yang, Yin-Xue; He, Zhi-Xu; Sun, Tao; Zhou, Shu-Feng

    2017-01-01

    Glioblastoma (GBM) is the most common brain tumor with poor response to current therapeutics. Alisertib (ALS), a second-generation selective Aurora kinase A (AURKA) inhibitor, has shown potent anticancer effects on solid tumors in animal studies. This study aimed to investigate the killing effect of ALS on GBM cell line DAOY and the possible underlying mechanisms using both bioinformatic and cell-based approaches. Our molecular docking showed that ALS preferentially bound AURKA over AURKB via hydrogen bond formation, charge interaction, and π-π stacking. ALS also bound key regulating proteins of cell cycle, apoptosis and autophagy, such as cyclin-dependent kinase 1 (CDK1/CDC2), CDK2, cyclin B1, p27 Kip1, p53, cytochrome C, cleaved caspase 3, Bax, Bcl-2, Bcl-xl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), 5’-adenosine monophosphate-activated protein kinase (AMPK), p38 mitogen-activated protein kinase (MAPK), beclin 1, phosphatase and tensin homolog (PTEN), and microtubule-associated protein light chain 3 (LC3). ALS exhibited potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects on DAOY cells in a concentration-dependent manner. Notably, ALS remarkably induced G2/M arrest mainlyvia regulating the expression of CDK1/CDC2, CDK2, cyclin B1, p27 Kip1, and p53 in DAOY cells. ALS significantly induced the expression of mitochondria-mediated pro-apoptotic proteins such as Baxbut inhibited the expression of anti-apoptotic proteins such as Bcl-2 and Bcl-xl, with a significant increase in the release of cytochrome C and the activation of caspases 3 and 9. ALS also induced PI3K/Akt/mTOR and p38 MAPK signaling pathways while activating the AMPK signaling pathway. Taken together, these findings indicate that ALS exerts a potent inhibitory effect on cell proliferation and induces mitochondria-dependent apoptosis and autophagy with the involvement of PI3K/Akt/mTOR- and p38 MAPK-mediated signaling pathways in

  12. Regulation of Hspb7 by MEF2 and AP-1: implications for Hspb7 in muscle atrophy.

    PubMed

    Tobin, Stephanie Wales; Yang, Dabo; Girgis, John; Farahzad, Ali; Blais, Alexandre; McDermott, John C

    2016-11-01

    Mycocyte enhancer factor 2 (MEF2) and activator protein 1 (AP-1) transcription complexes have been individually implicated in myogenesis, but their genetic interaction has not previously been addressed. Using MEF2A, c-Jun and Fra-1 chromatin immunoprecipitation sequencing (ChIP-seq) data and predicted AP-1 consensus motifs, we identified putative common MEF2 and AP-1 target genes, several of which are implicated in regulating the actin cytoskeleton. Because muscle atrophy results in remodelling or degradation of the actin cytoskeleton, we characterized the expression of putative MEF2 and AP-1 target genes (Dstn, Flnc, Hspb7, Lmod3 and Plekhh2) under atrophic conditions using dexamethasone (Dex) treatment in skeletal myoblasts. Heat shock protein b7 (Hspb7) was induced by Dex treatment and further analyses revealed that loss of MEF2A using siRNA prevented Dex-regulated induction of Hspb7. Conversely, ectopic Fra-2 or c-Jun expression reduced Dex-mediated upregulation of Hspb7 whereas AP-1 depletion enhanced Hspb7 expression. In vivo, expression of Hspb7 and other autophagy-related genes was upregulated in response to atrophic conditions in mice. Manipulation of Hspb7 levels in mice also impacted gross muscle mass. Collectively, these data indicate that MEF2 and AP-1 confer antagonistic regulation of Hspb7 gene expression in skeletal muscle, with implications for autophagy and muscle atrophy.

  13. p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts.

    PubMed

    Furukawa, Fukiko; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yoshida, Katsunori; Sugano, Yasushi; Yamagata, Hideo; Matsushita, Masanori; Seki, Toshihito; Inagaki, Yutaka; Nishizawa, Mikio; Fujisawa, Junichi; Inoue, Kyoichi

    2003-10-01

    Hepatic stellate cells (HSCs) spontaneously transdifferentiate into myofibroblast (MFB)-phenotype on plastic dishes. This response recapitulates the features of activation in vivo. Transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by MFBs. In quiescent HSCs, TGF-beta signaling involves TGF-beta type I receptor (TbetaRI)-mediated phosphorylation of serine residues within the conserved SSXS motif at the C-terminus of Smad2 and Smad3. The middle linker regions of Smad2 and Smad3 also are phosphorylated by mitogen-activated protein kinase (MAPK). This study elucidates the change of Smad3-mediated signals during the transdifferentiation process. By using antibodies highly specific to the phosphorylated C-terminal region and the phosphorylated linker region of Smad3, we found that TGF-beta-dependent Smad3 phosphorylation at the C-terminal region decreased, but that the phosphorylation at the linker region increased in the process of transdifferentiation. TGF-beta activated the p38 MAPK pathway, further leading to Smad3 phosphorylation at the linker region in the cultured MFBs, irrespective of Smad2. The phosphorylation promoted hetero-complex formation and nuclear translocation of Smad3 and Smad4. Once combined with TbetaRI-phosphorylated Smad2, the Smad3 and Smad4 complex bound to plasminogen activator inhibitor-type I promoter could enhance the transcription. In addition, Smad3 phosphorylation mediated by the activated TbetaRI was impaired severely in MFBs during chronic liver injury, whereas Smad3 phosphorylation at the linker region was remarkably induced by p38 MAPK pathway. In conclusion, p38 MAPK-dependent Smad3 phosphorylation promoted extracellular matrix production in MFBs both in vitro and in vivo.

  14. 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.

  15. Brain suppression of AP-1 by inhaled diesel exhaust and reversal by cerium oxide nanoparticles.

    PubMed

    Lung, Shyang; Cassee, Flemming R; Gosens, Ilse; Campbell, Arezoo

    2014-08-01

    One of the uses of cerium oxide nanoparticles (nanoceria, CeO2) is as a diesel fuel additive to improve fuel efficiency. Gene/environment interactions are important determinants in the etiology of age-related disorders. Thus, it is possible that individuals on high-fat diet and genetic predisposition to vascular disease may be more vulnerable to the adverse health effects of particle exposure. The aim of this pilot study was to test the hypothesis that inhalation of diesel exhaust (DE) or diesel exhaust-containing cerium oxide nanoparticles (DCeE) induces stress in the brain of a susceptible animal model. Atherosclerotic prone, apolipoprotein E knockout (ApoE(-/-)) mice fed a high-fat diet, were exposed by inhalation to purified air (control), DE or DCeE. The stress-responsive transcription factor, activator protein-1 (AP-1), was significantly decreased in the cortical and subcortical fraction of the brain after DE exposure. The addition of nanoceria to the diesel fuel reversed this effect. The activation of another stress-related transcription factor (NF-κB) was not inhibited. AP-1 is composed of complexes of the Jun and/or Fos family of proteins. Exposure to DCeE caused c-Jun activation and this may be a mechanism by which addition of nanoceria to the fuel reversed the effect of DE exposure on AP-1 activation. This pilot study demonstrates that exposure to DE does impact the brain and addition of nanoceria may be protective. However, more extensive studies are necessary to determine how DE induced reduction of AP-1 activity and compensation by nanoceria impacts normal function of the brain.

  16. RhoA and p38 MAPK mediate apoptosis induced by cellular cholesterol depletion.

    PubMed

    Calleros, Laura; Lasa, Marina; Rodríguez-Alvarez, Francisco J; Toro, María J; Chiloeches, Antonio

    2006-07-01

    Cholesterol is essential for cell viability, and homeostasis of cellular cholesterol is crucial to various cell functions. Here we examined the effect of cholesterol depletion on apoptosis and the mechanisms underlying this effect in NIH3T3 cells. We show that chronic cholesterol depletion achieved with lipoprotein-deficient serum (LPDS) and 25-hydroxycholesterol (25-HC) treatment resulted in a significant increase in cellular apoptosis and caspase-3 activation. This effect is not due to a deficiency of nonsterol isoprenoids, intermediate metabolites of the cholesterol biosynthetic pathway, but rather to low cholesterol levels, since addition of cholesterol together with LPDS and 25-HC nearly abolished apoptosis, whereas addition of farnesyl pyrophosphate or geranylgeranyl-pyrophosphate did not reverse the cell viability loss induced by LPDS plus 25-HC treatment. These effects were accompanied by an increase in ERK, JNK and p38 MAPK activity. However, only the inhibition of p38 MAPK with the specific inhibitor SB203580 or the overexpression of a kinase defective MKK6 resulted in a significant decrease in apoptosis and caspase-3 cleavage induced by cholesterol depletion. Furthermore, LPDS plus 25-HC increased RhoA activity, and this effect was reversed by addition of exogenous cholesterol. Finally, overexpression of the dominant negative N19RhoA inhibited p38 MAPK phosphorylation and apoptosis induced by low cholesterol levels. Together, our results demonstrate that cholesterol depletion induces apoptosis through a RhoA- and p38 MAPK-dependent mechanism.

  17. Isomahanine induces endoplasmic reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-resistant human oral squamous cell carcinoma cells.

    PubMed

    Utaipan, Tanyarath; Athipornchai, Anan; Suksamrarn, Apichart; Chunsrivirot, Surasak; Chunglok, Warangkana

    2017-02-01

    , the present study demonstrated that isomahanine was able to induce ER stress and trigger p38 MAPK-mediated apoptosis and autophagic cell death in multidrug-resistant OSCC cells. The potential cytotoxic action of isomahanine may provide the development of anticancer candidates for treating multidrug-resistant cancer.

  18. Suppression of endothelial nitric oxide synthase expression and endothelial cell proliferation by an intronic 27-ntmiRNA and it's a novel link to AP-1.

    PubMed

    Li, Yumei; Yan, Limei; Zhang, Wenyu; Hu, Nan; Chen, Wei; Wang, Hui; Kang, Min; Ou, Hesheng

    2015-01-01

    This study aims to investigate the role of activator protein 1 (AP-1) in the effects of 27nt-miRNA on expression of endothelial nitric oxide synthase (eNOS) gene and proliferation of endothelial cells. Cell proliferation was analyzed by cell number counting, colony formation assay and MTT assay. Cell migration and invasion was detected by transwell assay and invasion assay. Expression of eNOS and AP-1 was measured by real-time RT-PCR (mRNA level) and Western blotting (protein level). Luciferase reporter assay was performed to detect the binding of 27nt-miRNA to AP-1. Overexpression of 27nt-miRNA significantly inhibited endothelial cells proliferation, invasion and migration in vitro. And, eNOS and AP-1 expression at mRNA and protein levels were down-regulated by overexpression of 27nt-miRNA. Interestingly, overexpression of AP-1 protein partially restored eNOS expression and endothelial cell proliferation. Furthermore, the luciferase reporter assay demonstrated that AP-1 was a direct target of 27nt-miRNA. These data demonstrate that overexpression of 27nt-miRNA inhibits endothelial cell proliferation, invasion, migration, eNOS expression and AP-1 expression. Moreover, AP-1, a direct target of 27nt-miRNA, reverses the inhibitory effects of 27nt-miRNA. Thus, the effects of 27nt-miRNA might be acted through targeting AP-1.

  19. Regulation of AP-1 and NFAT transcription factors during thymic selection of T cells.

    PubMed Central

    Rincon, M; Flavell, R A

    1996-01-01

    The ability of thymocytes to express cytokine genes changes during the different stages of thymic development. Although CD4- CD8- thymocytes are able to produce a wide spectrum of cytokines in response to a T-cell receptor (TcR)-independent stimulus, as they approach the double-positive (DP) CD4+ CD8+ stage, they lose the ability to produce cytokine. After the DP stage, thymocytes become single-positive CD4+ or CD8+ thymocytes which reacquire the ability to secrete cytokines. In an attempt to understand the molecular basis of this specific regulatin, we use AP-1-luciferase and newly generated NFAT-luciferase transgenic mice to analyze the transcriptional and DNA-binding activities of these two transcription factors that are involved in the regulation of cytokine gene expression. Here, we show that both AP-1 and NFAT transcriptional activities are not inducible in the majority of DP cells but that during the differentiation of DP cells to the mature single-positive stage, thymocytes regain this inducibility. Subpopulation analysis demonstrates that this inducibility is reacquired at the DP stage before the down-modulation of one of the coreceptors. Indeed AP-1 inducibility, just like the ability to express the interleukin-2 gene, is reacquired during the differentiation of DP TcRlow CD69low heat-stable antigen (HSA)high thymocytes to DP TcRhigh CD69high HSAhigh cells, which is considered to be the consequence of the first signal that initiates positive selection. We therefore propose that the inability of DP thymocytes to induce AP-1 and NFAT activities is one of the causes for the lack of cytokine gene expression at this stage and that this inducibility is reacquired at the latest stage of DP differentiation as a consequence of positive selection. This could be a mechanism to prevent the activation of DP thymocytes before selection has taken place. PMID:8622652

  20. Tobacco carcinogen mediated up-regulation of AP-1 dependent pro-angiogenic cytokines in head and neck carcinogenesis.

    PubMed

    Swenson, Wade G; Wuertz, Beverly R K; Ondrey, Frank G

    2011-09-01

    Tobacco is notably genotoxic and associated with head and neck carcinogenesis. Cigarette carcinogens have the capacity to alter early response gene expression in tobacco-related malignancies via genes such as nuclear factor kappa B (NFκB). A number of early response gene activation events are also facilitated by fos/jun activator protein 1 (AP-1) associated pathways. In the present study, we hypothesize that tobacco products may induce microenvironment alterations, promoting angiogenesis and providing a permissive environment for head and neck cancer progression. In an in vitro analysis, we employed immortalized oral keratinocyte (HOK-16B) and laryngeal squamous carcinoma (UM-SCC-11A) cells to investigate interleukin (IL)-8 and vascular endothelial growth factor (VEGF) induction by cigarette smoke condensate (CSC). IL-8 and VEGF expression is based on interactions between NFκB, AP-1, and NF-IL6. We identified at least 1.5-fold dose-dependent induction of AP-1, VEGF, and IL-8 promoter/reporter gene activity after 24 h exposure to CSC. Next, we stably transfected UM-SCC-11A cells with A-Fos, a dominant negative AP-1 protein. Treatment with CSC of the A-Fos cell lines compared to empty vector controls significantly down-regulated AP-1, VEGF, and IL-8 promoter/reporter gene expression. We also performed ELISAs and discovered significant up-regulation of IL-8 and VEGF secretion by UMSCC 11A after treatment with phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, and CSC, which was down-regulated by the A-Fos dominant negative protein. We conclude tobacco carcinogens up-regulate AP-1 activity and AP-1 dependent IL-8 and VEGF gene expression in head and neck cancer. This up-regulation may promote an angiogenic phenotype favoring invasion in both premalignant and squamous cancer cells of the head and neck.

  1. Pro/antioxidant status and AP-1 transcription factor in murine skin following topical exposure to cumene hydroperoxide.

    PubMed

    Murray, A R; Kisin, E R; Kommineni, C; Vallyathan, V; Castranova, V; Shvedova, A A

    2007-07-01

    Organic peroxides, widely used in the chemical and pharmaceutical industries, can act as skin tumor promoters and cause epidermal hyperplasia. They are also known to trigger free radical generation. The present study evaluated the effect of cumene hydroperoxide (Cum-OOH) on the induction of activator protein-1 (AP-1), which is linked to the expression of genes regulating cell proliferation, growth and transformation. Previously, we reported that topical exposure to Cum-OOH caused formation of free radicals and oxidative stress in the skin of vitamin E-deficient mice. The present study used JB6 P+ mouse epidermal cells and AP-1-luciferase reporter transgenic mice to identify whether exposure to Cum-OOH caused activation of AP-1, oxidative stress, depletion of antioxidants and tumor formation during two-stage carcinogenesis. In vitro studies found that exposure to Cum-OOH reduced the level of glutathione (GSH) in mouse epidermal cells (JB6 P+) and caused the induction of AP-1. Mice primed with dimethyl-benz[a]anthracene (DMBA) were topically exposed to Cum-OOH (82.6 micromol) or the positive control, 12-O-tetradecanoylphorbol-13-acetate (TPA, 17 nmol), twice weekly for 29 weeks. Activation of AP-1 in skin was detected as early as 2 weeks following Cum-OOH or TPA exposure. No AP-1 expression was found 19 weeks after initiation. Papilloma formation was observed in both the DMBA-TPA- and DMBA-Cum-OOH-exposed animals, whereas skin carcinomas were found only in the DMBA-Cum-OOH-treated mice. A greater accumulation of peroxidative products (thiobarbituric acid-reactive substances), inflammation and decreased levels of GSH and total antioxidant reserves were also observed in the skin of DMBA-Cum-OOH-exposed mice. These results suggest that Cum-OOH-induced carcinogenesis is accompanied by increased AP-1 activation and changes in antioxidant status.

  2. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    PubMed

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

  3. Pentraxin-2 suppresses c-Jun/AP-1 signaling to inhibit progressive fibrotic disease.

    PubMed

    Nakagawa, Naoki; Barron, Luke; Gomez, Ivan G; Johnson, Bryce G; Roach, Allie M; Kameoka, Sei; Jack, Richard M; Lupher, Mark L; Gharib, Sina A; Duffield, Jeremy S

    2016-12-08

    Pentraxin-2 (PTX-2), also known as serum amyloid P component (SAP/APCS), is a constitutive, antiinflammatory, innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in Col4a3 mutant mice with Alport syndrome, reducing blood markers of kidney failure, enhancing lifespan by 20%, and improving histological signs of disease. Exogenously delivered rhPTX-2 was detected in macrophages but also in tubular epithelial cells, where it counteracted macrophage activation and was cytoprotective for the epithelium. Computational analysis of genes regulated by rhPTX-2 identified the transcriptional regulator c-Jun along with its activator protein-1 (AP-1) binding partners as a central target for the function of rhPTX-2. Accordingly, PTX-2 attenuates c-Jun and AP-1 activity, and reduces expression of AP-1-dependent inflammatory genes in both monocytes and epithelium. Our studies therefore identify rhPTX-2 as a potential therapy for chronic fibrotic disease of the kidney and an important inhibitor of pathological c-Jun signaling in this setting.

  4. Pentraxin-2 suppresses c-Jun/AP-1 signaling to inhibit progressive fibrotic disease

    PubMed Central

    Nakagawa, Naoki; Gomez, Ivan G.; Johnson, Bryce G.; Kameoka, Sei; Jack, Richard M.; Lupher, Mark L.; Gharib, Sina A.; Duffield, Jeremy S.

    2016-01-01

    Pentraxin-2 (PTX-2), also known as serum amyloid P component (SAP/APCS), is a constitutive, antiinflammatory, innate immune plasma protein whose circulating level is decreased in chronic human fibrotic diseases. Here we show that recombinant human PTX-2 (rhPTX-2) retards progression of chronic kidney disease in Col4a3 mutant mice with Alport syndrome, reducing blood markers of kidney failure, enhancing lifespan by 20%, and improving histological signs of disease. Exogenously delivered rhPTX-2 was detected in macrophages but also in tubular epithelial cells, where it counteracted macrophage activation and was cytoprotective for the epithelium. Computational analysis of genes regulated by rhPTX-2 identified the transcriptional regulator c-Jun along with its activator protein–1 (AP-1) binding partners as a central target for the function of rhPTX-2. Accordingly, PTX-2 attenuates c-Jun and AP-1 activity, and reduces expression of AP-1–dependent inflammatory genes in both monocytes and epithelium. Our studies therefore identify rhPTX-2 as a potential therapy for chronic fibrotic disease of the kidney and an important inhibitor of pathological c-Jun signaling in this setting. PMID:27942582

  5. IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes.

    PubMed

    Wang, Xiaoyong; Bi, Zhigang; Chu, Wenming; Wan, Yinsheng

    2005-12-01

    Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.

  6. AP-1 Transcription Factor Serves as a Molecular Switch between Chlamydia pneumoniae Replication and Persistence

    PubMed Central

    Krämer, S.; Crauwels, P.; Bohn, R.; Radzimski, C.; Szaszák, M.; Klinger, M.; Rupp, J.

    2015-01-01

    Chlamydia pneumoniae is a Gram-negative bacterium that causes acute or chronic respiratory infections. As obligate intracellular pathogens, chlamydiae efficiently manipulate host cell processes to ensure their intracellular development. Here we focused on the interaction of chlamydiae with the host cell transcription factor activator protein 1 (AP-1) and its consequence on chlamydial development. During Chlamydia pneumoniae infection, the expression and activity of AP-1 family proteins c-Jun, c-Fos, and ATF-2 were regulated in a time- and dose-dependent manner. We observed that the c-Jun protein and its phosphorylation level significantly increased during C. pneumoniae development. Small interfering RNA knockdown of the c-Jun protein in HEp-2 cells reduced the chlamydial load, resulting in smaller inclusions and significantly lower chlamydial recovery. Furthermore, inhibition of the c-Jun-containing AP-1 complexes using tanshinone IIA changed the replicative infection phenotype into a persistent one. Tanshinone IIA-dependent persistence was characterized by smaller, aberrant inclusions, a strong decrease in the chlamydial load, and significantly reduced chlamydial recovery, as well as by the reversibility of the reduced recovery after the removal of tanshinone IIA. Interestingly, not only was tanshinone IIA treatment accompanied by a significant decrease of ATP levels, but fluorescence live cell imaging analysis by two-photon microscopy revealed that tanshinone IIA treatment also resulted in a decreased fluorescence lifetime of protein-bound NAD(P)H inside the chlamydial inclusion, indicating that chlamydial reticulate bodies have decreased metabolic activity. In all, these data demonstrate that the AP-1 transcription factor is involved in C. pneumoniae development, with tanshinone IIA treatment resulting in persistence. PMID:25895972

  7. Robust dynamic balance of AP-1 transcription factors in a neuronal gene regulatory network

    PubMed Central

    2010-01-01

    Background The octapeptide Angiotensin II is a key hormone that acts via its receptor AT1R in the brainstem to modulate the blood pressure control circuits and thus plays a central role in the cardiac and respiratory homeostasis. This modulation occurs via activation of a complex network of signaling proteins and transcription factors, leading to changes in levels of key genes and proteins. AT1R initiated activity in the nucleus tractus solitarius (NTS), which regulates blood pressure, has been the subject of extensive molecular analysis. But the adaptive network interactions in the NTS response to AT1R, plausibly related to the development of hypertension, are not understood. Results We developed and analyzed a mathematical model of AT1R-activated signaling kinases and a downstream gene regulatory network, with structural basis in our transcriptomic data analysis and literature. To our knowledge, our report presents the first computational model of this key regulatory network. Our simulations and analysis reveal a dynamic balance among distinct dimers of the AP-1 family of transcription factors. We investigated the robustness of this behavior to simultaneous perturbations in the network parameters using a novel multivariate approach that integrates global sensitivity analysis with decision-tree methods. Our analysis implicates a subset of Fos and Jun dependent mechanisms, with dynamic sensitivities shifting from Fos-regulating kinase (FRK)-mediated processes to those downstream of c-Jun N-terminal kinase (JNK). Decision-tree analysis indicated that while there may be a large combinatorial functional space feasible for neuronal states and parameters, the network behavior is constrained to a small set of AP-1 response profiles. Many of the paths through the combinatorial parameter space lead to a dynamic balance of AP-1 dimer forms, yielding a robust AP-1 response counteracting the biological variability. Conclusions Based on the simulation and analysis results, we

  8. 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.

  9. The LIM domain protein nTRIP6 recruits the mediator complex to AP-1-regulated promoters.

    PubMed

    Diefenbacher, Markus E; Reich, Daniela; Dahley, Oliver; Kemler, Denise; Litfin, Margarethe; Herrlich, Peter; Kassel, Olivier

    2014-01-01

    Several LIM domain proteins regulate transcription. They are thought to act through their LIM protein-protein interaction domains as adaptors for the recruitment of transcriptional co-regulators. An intriguing example is nTRIP6, the nuclear isoform of the focal adhesion protein TRIP6. nTRIP6 interacts with AP-1 and enhances its transcriptional activity. nTRIP6 is also essential for the transrepression of AP-1 by the glucocorticoid receptor (GR), by mediating GR tethering to promoter-bound AP-1. Here we report on the molecular mechanism by which nTRIP6 exerts these effects. Both the LIM domains and the pre-LIM region of nTRIP6 are necessary for its co-activator function for AP-1. Discrete domains within the pre-LIM region mediate the dimerization of nTRIP6 at the promoter, which enables the recruitment of the Mediator complex subunits THRAP3 and Med1. This recruitment is blocked by GR, through a competition between GR and THRAP3 for the interaction with the LIM domains of nTRIP6. Thus, nTRIP6 both positively and negatively regulates transcription by orchestrating the recruitment of the Mediator complex to AP-1-regulated promoters.

  10. Apigenin inhibits PMA-induced expression of pro-inflammatory cytokines and AP-1 factors in A549 cells.

    PubMed

    Patil, Rajeshwari H; Babu, R L; Naveen Kumar, M; Kiran Kumar, K M; Hegde, Shubha M; Ramesh, Govindarajan T; Chidananda Sharma, S

    2015-05-01

    Acute and chronic alveolar or bronchial inflammation is thought to be central to the pathogenesis of many respiratory disorders. Cytokines and granulocyte macrophage colony-stimulating factors (GM-CSF) play an important role in chronic inflammation. Activator protein-1 (AP-1) the superfamily of transcription factors is involved in proliferation, differentiation, apoptosis, and transformation including inflammation. Understanding the function and regulation of proinflammatory factors involved in inflammation may provide the novel therapeutic strategies in the treatment of inflammatory diseases. Our aim of the present study is to investigate the pro-inflammatory cytokines and pattern of AP-1 factors expressed during activation of lung adenocarcinoma A549 cells by Phorbol-12-myristate-13-acetate (PMA) and to understand the anti-inflammatory effect of apigenin. A549 cells were treated with and without PMA or apigenin, and the cell viability was assessed by MTT assay. Expressions of inflammatory mediators and different AP-1 factors were analyzed by semi-quantitative RT-PCR. IL-6 protein secreted was analyzed by ELISA, and expressions of IL-1β, c-Jun, and c-Fos proteins were analyzed by Western blotting. Activation of A549 cells by PMA, induced the expression of pro-inflammatory cytokine (IL-1β, IL-2, IL-6, IL-8, and TNF-α) mRNAs and secretion of IL-6 and the expression of specific AP-1 factors (c-Jun, c-Fos, and Fra-1). Treatment of cells with apigenin, significantly inhibited PMA-stimulated mRNA expression of above pro-inflammatory cytokines, AP-1 factors, cyclooxygenase-2, and secretion of IL-6 protein. Results suggested that the AP-1 factors may be involved in inflammation and apigenin has anti-inflammatory effect, which may be useful for therapeutic management of lung inflammatory diseases.

  11. A novel AP-1/miR-101 regulatory feedback loop and its implication in the migration and invasion of hepatoma cells.

    PubMed

    Liu, Jing-Jing; Lin, Xue-Jia; Yang, Xiao-Jing; Zhou, Liangji; He, Shuai; Zhuang, Shi-Mei; Yang, Jine

    2014-10-29

    MicroRNA-101 (miR-101) is frequently downregulated in various cancers. To date, the regulatory networks of miR-101 remain obscure. In this study, we demonstrated that miR-101 was mainly transcribed from human miR-101-2 and mouse miR-101bgene loci. Subsequent analyses revealed that activator protein-1 (AP-1) directly binded to the -17.4 to -16.4 k region upstream of pre-miR-101-2 and activated the expression of miR-101. On the other hand, miR-101 could inhibit the expression of ERK2 and c-Fos, two key factors of the AP-1 pathway, by binding to their 3'-UTRs. Furthermore, reintroduction of miR-101 efficiently suppressed the AP-1 activity and pri-miR-101-2 transcription. These data thus suggest a novel AP-1/miR-101 regulatory circuitry, that is, AP-1 promotes the transcription of miR-101, whereas the expression of miR-101 reduces the level of ERK2 and c-Fos and thereby attenuates the AP-1 signaling. Further investigation disclosed that the AP-1 activator TPA-induced MMP9 activity and the TPA-promoted migration and invasion of hepatoma cells were significantly attenuated by miR-101 but were enhanced by miR-101 inhibitor. Our results suggest that the AP-1/miR-101 feedback loop may prevent the excessive activation of metastatic signals imposed by ERK2/AP-1 and highlight the biological significance of miR-101 downregulation in cancer metastasis.

  12. Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation

    PubMed Central

    Kim, Tae-Hwan; Kwak, Yi-Seong; Kim, Na-Mi; Kim, Seung-Hyung

    2017-01-01

    Torilin, a sesquiterpene isolated from the fruits of Torilis japonica, has shown antimicrobial, anticancer, and anti-inflammatory properties. However, data on the mechanism of torilin action against inflammation is limited. This study aimed at determining the anti-inflammatory property of torilin in LPS-induced inflammation using in vitro model of inflammation. We examined torilin's effect on expression levels of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. The involvement of NF-kB and AP-1, MAP kinases, and adaptor proteins were assessed. Torilin strongly inhibited LPS-induced NO release, iNOS, PGE2, COX-2, NF-α, IL-1β, IL-6, and GM-CSF gene and protein expressions. In addition, MAPKs were also suppressed by torilin pretreatment. Involvement of ERK1/2, P38MAPK, and JNK1/2 was further confirmed by PD98059, SB203580, and SP600125 mediated suppression of iNOS and COX-2 proteins. Furthermore, torilin attenuated NF-kB and AP-1 translocation, DNA binding, and reporter gene transcription. Interestingly, torilin inhibited TAK1 kinase activation with the subsequent suppression of MAPK-mediated JNK, p38, ERK1/2, and AP-1 (ATF-2 and c-jun) activation and IKK-mediated I-κBα degradation, p65/p50 activation, and translocation. Together, the results revealed the suppression of NF-κB and AP-1 regulated inflammatory mediator and cytokine expressions, suggesting the test compound's potential as a candidate anti-inflammatory agent. PMID:28316375

  13. Protein kinase C-theta isoenzyme selective stimulation of the transcription factor complex AP-1 in T lymphocytes.

    PubMed Central

    Baier-Bitterlich, G; Uberall, F; Bauer, B; Fresser, F; Wachter, H; Grunicke, H; Utermann, G; Altman, A; Baier, G

    1996-01-01

    T-lymphocyte stimulation requires activation of several protein kinases, including the major phorbol ester receptor protein kinase C (PKC), ultimately leading to induction of lymphokines, such as interleukin-2 (IL-2). The revelant PKC isoforms which are involved in the activation cascades of nuclear transcription factors involved in IL-2 production have not yet been clearly defined. We have examined the potential role of two representative PKC isoforms in the induction of the IL-2 gene, i.e., PKC-alpha and PKC-theta, the latter being expressed predominantly in hematopoietic cell lines, particularly T cells. Similar to that of PKC-alpha, PKC-theta overexpression in murine EL4 thymoma cells caused a significant increase in phorbol 12-myristate 13-acetate (PMA)-induced transcriptional activation of full-length IL-2-chloramphenicol acetyltransferase (CAT) and NF-AT-CAT but not of NF-IL2A-CAT or NF-kappaB promoter-CAT reporter gene constructs. Importantly, the critical AP-1 enhancer element was differentially modulated by these two distinct PKC isoenzymes, since only PKC-theta but not PKC-alpha overexpression resulted in an approximately 2.8-fold increase in AP-1-collagenase promoter CAT expression in comparison with the vector control. Deletion of the AP-1 enhancer site in the collagenase promoter rendered it unresponsive to PKC-theta. Expression of a constitutively active mutant PKC-theta A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation. Conversely, a catalytically inactive PKC-theta K409R (but not PKC-alpha K368R) mutant abrogated endogenous PMA-mediated activation of AP-1 transcriptional complex. Dominant negative mutant Ha-RasS17N completely inhibited the PKC-O A148E-induced signal, PKC-O. Expression of a constitutively active mutant PKC-O A148E (but not PKC-alpha A25E) was sufficient to induce activation of AP-1 transcription factor complex in the absence of PMA stimulation

  14. 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.

  15. 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.

  16. High glucose-induced, endothelin-dependent fibronectin synthesis is mediated via NF-kappa B and AP-1.

    PubMed

    Chen, Shali; Mukherjee, Suranjana; Chakraborty, Chandan; Chakrabarti, Subrata

    2003-02-01

    Human endothelial cells cultured under high glucose (HG) conditions were shown before to upregulate several basement membrane proteins, including fibronectin (FN), thus mimicking effects of diabetes. Using human macrovascular (HUVEC) and microvascular (HMEC) endothelial cell lines, we evaluated in the present study some of the key molecular signaling events involved in HG-induced FN overexpression. This expression was shown to be dependent on endogenous endothelin (ET) receptor-mediated signaling. We also examined the roles played by protein kinase C (PKC) and the transcription factors nuclear factor kappaB (NF-kappaB) and activating protein (AP)-1 with respect to such changes. HG, PKC activators, and ETs (ET-1 and ET-3) that increased FN expression also caused activation of NF-kappaB and AP-1. Inhibitors of both NF-kappaB and AP-1 prevented HG- and ET-induced FN production. ET receptor blockade also prevented these HG- and ET-mediated changes. The results of this study indicate that glucose-induced increased FN production in diabetes may be mediated via ET-dependent NF-kappaB and AP-1 activation.

  17. NRF-1, and AP-1 regulate the promoter of the human calpain small subunit 1 (CAPNS1) gene.

    PubMed

    Asangani, Irfan A; Rasheed, Suhail A K; Leupold, Jörg H; Post, Stefan; Allgayer, Heike

    2008-02-29

    Ubiquitously expressed micro- and m-calpain are cysteine proteases with broad functions in cell spreading, migration, proliferation, apoptosis, and in tumor invasion. They are heterodimers, with a distinct large 80-kDa catalytic, and a common small 28-kDa regulatory subunit (Capn4/CAPNS1). CAPNS1 is required to maintain stability and activity of both calpains. Despite its biological importance, the transcriptional regulation of this gene has not been studied, and the CAPNS1 promoter has not yet been characterized. In this study, we identified the main transcriptional start site, and cloned and characterized the ~2.0 kb upstream region of the CAPNS1 gene. Deletion analysis identified the core promoter located within region -187/+174. Site-directed mutagenesis, EMSA- and supershift analysis identified Sp1-, NRF-1-, and AP-1-binding elements within the CAPNS1 core promoter. Binding of NRF-1, Sp1 and AP-1 to the natural core promoter was confirmed by chromatin immunoprecipitation (ChIP). Site-directed mutagenesis at the NRF-1 site in HeLa and MCF7 cells substantially reduced core promoter activity by 70%, whereas mutation of the AP-1-binding and Sp1-binding site reduced promoter activity by 50% and 30%, respectively. Double mutation of the NRF-1 and the AP-1 site reduced promoter activity by 90%. In Drosophila SL2 cells, ectopic expression of NRF-1 led to a significant induction of CAPNS1 promoter activity. Furthermore, an siRNA against NRF-1 substantially reduced promoter activity in HeLa cells, which was paralleled by a significant downregulation of CAPNS1 mRNA. These results reveal that especially NRF-1, along with AP-1 and, to a minor extent, an Sp1 site, is essential for human CAPNS1 promoter activity and gene expression.

  18. Duplication of AP1 within the Spinacia oleracea L. AP1/FUL clade is followed by rapid amino acid and regulatory evolution.

    PubMed

    Sather, D Noah; Golenberg, Edward M

    2009-02-01

    The AP1/FUL clade of MADS box genes have undergone multiple duplication events among angiosperm species. While initially identified as having floral meristem identity and floral organ identity function in Arabidopsis, the role of AP1 homologs does not appear to be universally conserved even among eudicots. In comparison, the role of FRUITFULL has not been extensively explored in non-model species. We report on the isolation of three AP1/FUL genes from cultivated spinach, Spinacia oleracea L. Two genes, designated SpAPETALA1-1 (SpAP1-1) and SpAPETALA1-2 (SpAP1-2), cluster as paralogous genes within the Caryophyllales AP1 clade. They are highly differentiated in the 3', carboxyl-end encoding region of the gene following the third amphipathic alpha-helix region, while still retaining some elements of a signature AP1 carboxyl motifs. In situ hybridization studies also demonstrate that the two paralogs have evolved different temporal and spatial expression patterns, and that neither gene is expressed in the developing sepal whorl, suggesting that the AP1 floral organ identity function is not conserved in spinach. The spinach FRUITFULL homolog, SpFRUITFULL (SpFUL), has retained the conserved motif and groups with Caryophyllales FRUITFULL homologs. SpFUL is expressed in leaf as well as in floral tissue, and shows strong expression late in flower development, particularly in the tapetal layer in males, and in the endothecium layer and stigma, in the females. The combined evidence of high rates of non-synonymous substitutions and differential expression patterns supports a scenario in which the AP1 homologs in the spinach AP1/FUL gene family have experienced rapid evolution following duplication.

  19. Involvement of AP-1 in p38MAPK signaling pathway in osteoblast apoptosis induced by high glucose.

    PubMed

    Feng, Z P; Deng, H C; Jiang, R; Du, J; Cheng, D Y

    2015-04-10

    We investigated the effect of p38MAPK/AP-1 (activator protein-1) signaling on the apoptosis of osteoblasts induced by high glucose. A lentivirus vector of small hairpin RNA (shRNA) targeting p38MAPK was constructed in vitro. Osteoblasts MC3T3-E1 cultured in vitro were treated with vehicle, high glucose, p38MAPK-shRNA transfection, p38MAPK inhibitor, and unrelated shRNA transfection. Apoptosis, protein levels of p38MAPK, and activities of AP-1 in MC3T3-E1 osteoblasts were measured using TUNEL and flow cytometry, Western blot analysis, and an electrophoretic mobility shift assay. Compared with the vehicle group, high glucose induced apoptosis of MC3T3-E1 osteoblasts and activated p38MAPK and AP-1. p38MAPK-shRNA transfection blocked the effect of high glucose stimulation, and the p38MAPK inhibitor showed similar effects as those observed in p38MAPK transfection. Unrelated shRNA had no effect on these changes in MC3T3-E1 osteoblasts induced by high glucose. Therefore, our results suggest that p38MAPK-shRNA reduce apoptosis of MC3T3-E1 osteoblasts induced by high glucose by inhibiting the p38MAPK-AP-1 signaling pathway.

  20. 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.

  1. 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

  2. Expression of the Robo4 receptor in endothelial cells is regulated by two AP-1 protein complexes.

    PubMed

    Okada, Yoshiaki; Naruse, Hiroki; Tanaka, Toru; Funahashi, Nobuaki; Regan, Erzsébet Ravasz; Yamakawa, Kazuma; Hino, Nobumasa; Ishimoto, Kenji; Doi, Takefumi; Aird, William C

    2015-11-27

    Roundabout4 (Robo4) is an endothelial cell-specific gene that plays an important role in endothelial cell stability. We previously identified a 3-kb Robo4 promoter and demonstrated the importance of its proximal region in regulating Robo4 gene expression. To investigate the role of the upstream promoter in Robo4 gene regulation, we searched evolutionarily conserved promoter regions by phylogenetic footprinting and identified three conserved promoter regions. The most upstream region included a conserved AP-1 binding motif at position -2875. A mutation in the AP-1 motif significantly decreased Robo4 promoter activity in a transient reporter assay. An electrophoretic mobility shift assay and a chromatin immunoprecipitation assay demonstrated binding of a c-Jun/c-Jun complex and a c-Jun/Fra-1 complex to the AP-1 motif. Knockdown experiments using siRNA revealed that both c-Jun/c-Jun and c-Jun/Fra-1 complexes regulate Robo4 gene expression, and that the c-Jun/c-Jun complex is essential for maximum promoter activation. Collectively, these results indicate that AP-1 complexes regulate Robo4 gene expression in endothelial cells.

  3. An AP1 binding site upstream of the kappa immunoglobulin intron enhancer binds inducible factors and contributes to expression.

    PubMed Central

    Schanke, J T; Marcuzzi, A; Podzorski, R P; Van Ness, B

    1994-01-01

    Expression of the kappa immunoglobulin light chain gene requires developmental- and tissue-specific regulation by trans-acting factors which interact with two distinct enhancer elements. A new protein-DNA interaction has been identified upstream of the intron enhancer, within the matrix-associated region of the J-C intron. The binding activity is greatly inducible in pre-B cells by bacterial lipopolysaccharide and interleukin-1 but specific complexes are found at all stages of B cell development tested. The footprinted binding site is homologous to the consensus AP1 motif. The protein components of this complex are specifically competed by an AP1 consensus motif and were shown by supershift to include c-Jun and c-Fos, suggesting that this binding site is an AP1 motif and that the Jun and Fos families of transcription factors play a role in the regulation of the kappa light chain gene. Mutation of the AP1 motif in the context of the intron enhancer was shown to decrease enhancer-mediated activation of the promoter in both pre-B cells induced with LPS and constitutive expression in mature B cells. Images PMID:7816634

  4. Steroids Do Not Prevent Photoreceptor Degeneration in the Light-Exposed T4R Rhodopsin Mutant Dog Retina Irrespective of AP-1 Inhibition

    PubMed Central

    Gu, Danian; Beltran, William A.; Pearce-Kelling, Sue; Li, Zexiao; Acland, Gregory M.; Aguirre, Gustavo D.

    2009-01-01

    PURPOSE AP-1 has been proposed as a key intermediate linking exposure to light and photoreceptor cell death in rodent light damage models. Inhibition of AP-1 associated with steroid administration also prevents light damage. In this study the role of steroids in inhibiting AP-1 activation and/or in preventing photoreceptor degeneration was examined in the rhodopsin mutant dog model. METHODS The dogs were dark adapted overnight, eyes dilated with mydriatics; the right eye was light occluded and the fundus of the left eye photographed (∼15-17 overlapping frames) with a fundus camera. For biochemical studies, the dogs remained in the dark for 1 to 3 hours after exposure. Twenty-four hours before exposure to light, some dogs were treated with systemic dexamethasone or intravitreal/subconjunctival triamcinolone. AP-1 DNA-binding activity was determined by electrophoresis mobility shift assay (EMSA) and phosphorylation of c-Fos and activation of ERK1/2 were determined by immunoblot analyses. The eyes were collected 1 hour and 2 weeks after exposure to light, for histopathology and immunocytochemistry. RESULTS Inhibition of AP-1 activation, and phosphorylation of ERK1/2 and c-Fos were found after dexamethasone treatment in light-exposed T4R RHO mutant dog retinas. In contrast, increased AP-1 activity and phosphorylation of c-Fos and ERK1/2 were found in triamcinolone-treated mutant retinas. Similar extensive rod degeneration was found after exposure to light with or without treatment, and areas with surviving photoreceptor nuclei consisted primarily of cones. Only with systemic dexamethasone did the RPE cell layer remain. CONCLUSIONS Intraocular or systemic steroids fail to prevent light-induced photoreceptor degeneration in the T4R RHO dog retina. Finding that systemic dexamethasone prevents AP-1 activation, yet does not prevent retinal light damage, further supports the hypothesis that AP-1 is not the critical player in the cell-death signal that occurs in rods. PMID

  5. Mutagenesis of cysteine 81 prevents dimerization of the APS1 subunit of ADP-glucose pyrophosphorylase and alters diurnal starch turnover in Arabidopsis thaliana leaves.

    PubMed

    Hädrich, Nadja; Hendriks, Janneke H M; Kötting, Oliver; Arrivault, Stéphanie; Feil, Regina; Zeeman, Samuel C; Gibon, Yves; Schulze, Waltraud X; Stitt, Mark; Lunn, John E

    2012-04-01

    Many plants, including Arabidopsis thaliana, retain a substantial portion of their photosynthate in leaves in the form of starch, which is remobilized to support metabolism and growth at night. ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the pathway of starch synthesis, the production of ADP-glucose. The enzyme is redox-activated in the light and in response to sucrose accumulation, via reversible breakage of an intermolecular cysteine bridge between the two small (APS1) subunits. The biological function of this regulatory mechanism was investigated by complementing an aps1 null mutant (adg1) with a series of constructs containing a full-length APS1 gene encoding either the wild-type APS1 protein or mutated forms in which one of the five cysteine residues was replaced by serine. Substitution of Cys81 by serine prevented APS1 dimerization, whereas mutation of the other cysteines had no effect. Thus, Cys81 is both necessary and sufficient for dimerization of APS1. Compared to control plants, the adg1/APS1(C81S) lines had higher levels of ADP-glucose and maltose, and either increased rates of starch synthesis or a starch-excess phenotype, depending on the daylength. APS1 protein levels were five- to tenfold lower in adg1/APS1(C81S) lines than in control plants. These results show that redox modulation of AGPase contributes to the diurnal regulation of starch turnover, with inappropriate regulation of the enzyme having an unexpected impact on starch breakdown, and that Cys81 may play an important role in the regulation of AGPase turnover.

  6. Spleen Tyrosine Kinase Regulates AP-1 Dependent Transcriptional Response to Minimally Oxidized LDL

    PubMed Central

    Choi, Soo-Ho; Wiesner, Philipp; Almazan, Felicidad; Kim, Jungsu; Miller, Yury I.

    2012-01-01

    Oxidative modification of low-density lipoprotein (LDL) turns it into an endogenous ligand recognized by pattern-recognition receptors. We have demonstrated that minimally oxidized LDL (mmLDL) binds to CD14 and mediates TLR4/MD-2-dependent responses in macrophages, many of which are MyD88-independent. We have also demonstrated that the mmLDL activation leads to recruitment of spleen tyrosine kinase (Syk) to TLR4 and TLR4 and Syk phosphorylation. In this study, we produced a macrophage-specific Syk knockout mouse and used primary Syk−/− macrophages in our studies. We demonstrated that Syk mediated phosphorylation of ERK1/2 and JNK, which in turn phosphorylated c-Fos and c-Jun, respectively, as assessed by an in vitro kinase assay. c-Jun phosphorylation was also mediated by IKKε. c-Jun and c-Fos bound to consensus DNA sites and thereby completed an AP-1 transcriptional complex and induced expression of CXCL2 and IL-6. These results suggest that Syk plays a key role in TLR4-mediated macrophage responses to host-generated ligands, like mmLDL, with subsequent activation of an AP-1 transcription program. PMID:22384232

  7. miR-155* mediates suppressive effect of PTEN 3'-untranslated region on AP-1/NF-κB pathway in HTR-8/SVneo cells.

    PubMed

    Xue, P; Zheng, M; Diao, Z; Shen, L; Liu, M; Gong, P; Sun, H; Hu, Y

    2013-08-01

    Among miRNAs, miR-155 is a known regulator of immune system. Accumulating studies have revealed the connections between miR-155 and activator protein 1 (AP-1)/nuclear factor (NF)-κB. However, miR-155*, a miR-155 paralog, has so far been less studied. Here we demonstrated that miR-155*, induced by lipopolysaccharide (LPS) in an AP-1/NF-κB dependent manner, played a positive feedback role in AP-1/NF-κB pathway via targeting interleukin-1 receptor-associated kinase M (IRAKM) and NF-κB inhibitor interacting Ras-like 1 (NKIRAS1) in trophoblasts. Our study further proved that miR-155*-targeted PTEN 3'-untranslated region (3'UTR) increased IRAKM and NKIRAS1 expression by competing for miR-155* binding, thereby suppressing AP-1/NF-κB activation induced by LPS.

  8. The AP-1 site is required for basal expression but is not necessary for TPA-response of the human stromelysin gene.

    PubMed Central

    Buttice, G; Quinones, S; Kurkinen, M

    1991-01-01

    We have studied the activity of the AP-1 site, a target for the Fos and Jun family of transcription factors, in the context of the human stromelysin promoter (-1303 to +4). In transiently transfected human HepG2, HeLa and fibroblast cell cultures, point-mutations in any position of the stromelysin AP-1 sequence TGAGTCA (-70 to -64) reduced both the basal level and TPA-induced expression from the stromelysin promoter. TPA-induction fold of the mutant promoters, however, was comparable to that of the wild-type promoter. Similarly, antisense c-Fos mRNA expression reduced basal activity but had no significant effect on the relative TPA-response of the stromelysin promoter. Further, in mouse F9 cells cotransfected with c-Fos and c-Jun expression plasmids, the transfected wild-type stromelysin promoter activity was increased 57-fold whereas no transactivation was detected for an AP-1 mutant stromelysin promoter. In gelshift assays, stromelysin promoter fragments (-101 to -11), containing the mutated AP-1 site, all failed to bind or compete for the in vitro synthesized Fos and Jun proteins. We interpret these data to suggest that the Fos and Jun proteins, or similar activity, and the AP-1 site are required for the basal level expression of the human stromelysin gene. Strikingly, these data also suggest that the stromelysin AP-1 site is not necessary for the TPA-response. Images PMID:1906606

  9. Basolateral sorting of the Mg²⁺ transporter CNNM4 requires interaction with AP-1A and AP-1B.

    PubMed

    Hirata, Yusuke; Funato, Yosuke; Miki, Hiroaki

    2014-12-12

    Ancient conserved domain protein/cyclin M (CNNM) 4 is an evolutionarily conserved Mg(2+) transporter that localizes at the basolateral membrane of the intestinal epithelia. Here, we show the complementary importance of clathrin adaptor protein (AP) complexes AP-1A and AP-1B in basolateral sorting of CNNM4. We first confirmed the basolateral localization of both endogenous and ectopically expressed CNNM4 in Madin-Darby Canine Kidney cells, which form highly polarized epithelia in culture. Single knockdown of μ1B, a cargo-recognition subunit of AP-1B, did not affect basolateral localization, but simultaneous knockdown of the μ1A subunit of AP-1A abrogated localization. Mutational analyses showed the importance of three conserved dileucine motifs in CNNM4 for both basolateral sorting and interaction with μ1A and μ1B. These results imply that CNNM4 is sorted to the basolateral membrane by the complementary function of AP-1A and AP-1B.

  10. 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.

  11. NF-κB-to-AP-1 Switch: A Mechanism Regulating Transition From Endothelial Barrier Injury to Repair in Endotoxemic Mice

    PubMed Central

    Liu, Gang; Ye, Xiaobing; Miller, Edmund J.; Liu, Shu Fang

    2014-01-01

    Endothelial barrier disruption is a hallmark of multiple organ injury (MOI). However, mechanisms governing the restoration of endothelial barrier function are poorly understood. Here, we uncovered an NF-κB-to-AP-1 switch that regulates the transition from barrier injury to repair following endotoxemic MOI. Endothelial NF-κB mediates barrier repair by inhibiting endothelial cell (EC) apoptosis. Blockade of endothelial NF-κB pathway activated the activator protein (AP)-1 pathway (NF-κB-to-AP-1 switch), which compensated for the anti-apoptotic and barrier-repair functions of NF-κB. The NF-κB-to-AP-1 switch occurred at 24 hours (injury to repair transition phase), but not at 48 hours (repair phase) post-LPS, and required an inflammatory signal within the endothelium. In the absence of an inflammatory signal, the NF-κB-to-AP-1 switch failed, resulting in enhanced EC apoptosis, augmented endothelial permeability, and impeded transition from barrier injury to recovery. The NF-κB-to-AP-1 switch is a protective mechanism to ensure timely transition from endothelial barrier injury to repair, accelerating barrier restoration following MOI. PMID:24986487

  12. Bacterial Fucose-Rich Polysaccharide Stabilizes MAPK-Mediated Nrf2/Keap1 Signaling by Directly Scavenging Reactive Oxygen Species during Hydrogen Peroxide-Induced Apoptosis of Human Lung Fibroblast Cells

    PubMed Central

    Roy Chowdhury, Sougata; Sinha, Tridib Kumar; Sen, Ramkrishna; Basak, Ratan Kumar; Adhikari, Basudam; Bhattacharyya, Arindam

    2014-01-01

    Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and 1H/13C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities. PMID:25412177

  13. Bacterial fucose-rich polysaccharide stabilizes MAPK-mediated Nrf2/Keap1 signaling by directly scavenging reactive oxygen species during hydrogen peroxide-induced apoptosis of human lung fibroblast cells.

    PubMed

    Roy Chowdhury, Sougata; Sengupta, Suman; Biswas, Subir; Sinha, Tridib Kumar; Sen, Ramkrishna; Basak, Ratan Kumar; Adhikari, Basudam; Bhattacharyya, Arindam

    2014-01-01

    Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼ 42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and (1)H/(13)C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities.

  14. Peptidoglycan induces interleukin-6 expression through the TLR2 receptor, JNK, c-Jun, and AP-1 pathways in microglia.

    PubMed

    Lin, Hsiao-Yun; Tang, Chih-Hsin; Chen, Jia-Hong; Chuang, Jing-Yuan; Huang, Ssu-Ming; Tan, Tzu-Wei; Lai, Chih-Ho; Lu, Dah-Yuu

    2011-06-01

    We recently reported that peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium, induces NF-κB activation and microglia activation. However, PGN-regulated AP-1 activation and cytokine expression in microglia remains unclear. This study investigated how PGN influences the signaling pathway involved in IL-6 production in microglia. IL-6 mRNA and protein level up-regulation were increased by PGN in a concentration- and time-dependent manner. In addition, PGN increased toll-like receptor-2 (TLR2) expression, but not TLR4 receptor up-regulation. Administration of TLR2 siRNA or TLR2 neutralized antibody effectively inhibited PGN-induced IL-6 expression. In contrast, PGN-induced IL-6 mRNA and protein up-regulation were attenuated by the SAPK/JNK (c-Jun N-terminal kinases) inhibitor SP600125. Treatment of microglia with PGN increased levels of JNK phosphorylation and c-Jun phosphorylation, and up-regulated of JNK kinase activity. Treatment of microglia with AP-1 inhibitors (Tanshinone IIA and curcumin) effectively reduced PGN-induced IL-6 expression. PGN also significantly increased c-Fos and phospho-c-Jun translocation to nucleus. In line with this, PGN also increased AP-1-DNA complexes formation, as determined by the electrophoretic mobility shift assay. Furthermore, PGN also increased IL-6 transcription activity determined by transfection with IL-6 promoter construct plasmid. Co-transfection with dominant negative mutant of JNK (DN-JNK), or treatment with SP600125, curcumin, or Tanshinone IIA effectively antagonized PGN-increased IL-6 transcription activity. Our data demonstrate that PGN-induced IL-6 expression is mediated by AP-1 activation through the TLR2 and JNK/c-Jun pathways in microglia.

  15. Overt expression of AP-1 reduces alpha myosin heavy chain expression and contributes to heart failure from chronic volume overload.

    PubMed

    Freire, Grace; Ocampo, Catherina; Ilbawi, Nadim; Griffin, Andrew J; Gupta, Madhu

    2007-10-01

    Reduced expression of alpha-MHC plays a significant role in cardiac contractile dysfunction from hemodynamic overload. Previously, Pur proteins and YY1 have been shown to play a role in alpha-MHC repression during heart failure induced by pressure overload and by spontaneous hypertension, respectively. This was not observed in volume-overload-induced heart failure, suggesting additional regulatory mechanisms for alpha-MHC repression. The present study was performed to identify volume overload responsive transcription factors involved in alpha-MHC gene regulation. DNA binding activity of several transcription factors was evaluated in a functionally characterized rat model of heart failure induced by aorto-caval shunt. After 10 weeks of shunt, severe LV dilatation and reduced LV function were accompanied by increased expression of ANF and beta-MHC, and decreased expression of alpha-MHC. This was associated with dramatic (10-fold) activation of AP-1 together with increased expression of c-fos and c-jun. AP-1 activation was not observed following 4 weeks of shunt when cardiac function was preserved. In cultured cardiomyocytes, induction of AP-1 by PMA attenuated alpha-MHC mRNA by 60%. Transient transfection assays mapped PMA responsive sequence to -582 to -588 bp of alpha-MHC promoter. Deletion or mutation of these nucleotides had minimal effect on basal promoter activity but played a dominant role in PMA-mediated repression of alpha-MHC promoter activity. Over-expression of c-fos and c-jun in cardiomyocytes inhibited alpha-MHC promoter activity in a concentration dependent manner. Data suggest a repressive role of AP-1 in alpha-MHC expression and its possible involvement in the transition from compensatory hypertrophy to heart failure in chronic volume overload.

  16. AP-1-Targeted Inhibition of Macrophage Function and Lipopolysaccharide/D-Galactosamine-Induced Hepatitis by Phyllanthus acidus Methanolic Extract.

    PubMed

    Hossen, Muhammad Jahangir; Kim, Mi-Yeon; Kim, Jong-Hoon; Cho, Jae Youl

    2015-01-01

    Traditionally, Phyllanthus acidus (Phyllanthaceae) has been used for the treatment of rheumatism, bronchitis, asthma, respiratory disorders, and hepatitis. Recently, we showed that a methanol extract of Phyllanthaceae (Pa-ME) has a potent anti-inflammatory activity in RAW264.7 cells and strongly ameliorates HCl / EtOH -induced gastric ulcers in mice by targeting the Src/Syk of NF-κB. In the present study, we explored the molecular mechanism of Pa-ME on the AP-1 activation pathway and evaluated its potential hepatoprotective effects. To do this, we employed lipopolysaccharide (LPS)-stimulated RAW264.7 cells and U937 cells and an LPS/D-galactosamine (D- GaIN )-induced acute hepatitis mouse model. We utilized a multitude of assays, including immunoblotting analysis, reporter gene assays, and mRNA expression analysis, to determine the effect of Pa-ME on the AP-1 pathway. Pa-ME strikingly suppressed the production of LPS-induced pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). Furthermore, Pa-ME also strongly inhibited activator protein-1 (AP-1) activation and mitogen-activated protein kinase (MAPK) phosphorylation in LPS-stimulated RAW264.7 macrophages cells and the U937 monocyte like human cell line. Moreover, pre-treatment with Pa-ME exhibited strong hepatoprotective and curative effects in an LPS/D-Gal-induced mouse hepatitis model as evidenced by a decrease in elevated serum AST and ALT levels and the amelioration of histological damage. Taken together, our data suggest that Pa-ME might play a crucial ethnopharmacological role as a hepatoprotective herbal remedy by suppressing MAPK signaling and the activity of the downstream transcription factor AP-1.

  17. 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

  18. Loss-of-Function Mutations in YY1AP1 Lead to Grange Syndrome and a Fibromuscular Dysplasia-Like Vascular Disease.

    PubMed

    Guo, Dong-Chuan; Duan, Xue-Yan; Regalado, Ellen S; Mellor-Crummey, Lauren; Kwartler, Callie S; Kim, Dong; Lieberman, Kenneth; de Vries, Bert B A; Pfundt, Rolph; Schinzel, Albert; Kotzot, Dieter; Shen, Xuetong; Yang, Min-Lee; Bamshad, Michael J; Nickerson, Deborah A; Gornik, Heather L; Ganesh, Santhi K; Braverman, Alan C; Grange, Dorothy K; Milewicz, Dianna M

    2017-01-05

    Fibromuscular dysplasia (FMD) is a heterogeneous group of non-atherosclerotic and non-inflammatory arterial diseases that primarily involves the renal and cerebrovascular arteries. Grange syndrome is an autosomal-recessive condition characterized by severe and early-onset vascular disease similar to FMD and variable penetrance of brachydactyly, syndactyly, bone fragility, and learning disabilities. Exome-sequencing analysis of DNA from three affected siblings with Grange syndrome identified compound heterozygous nonsense variants in YY1AP1, and homozygous nonsense or frameshift YY1AP1 variants were subsequently identified in additional unrelated probands with Grange syndrome. YY1AP1 encodes yin yang 1 (YY1)-associated protein 1 and is an activator of the YY1 transcription factor. We determined that YY1AP1 localizes to the nucleus and is a component of the INO80 chromatin remodeling complex, which is responsible for transcriptional regulation, DNA repair, and replication. Molecular studies revealed that loss of YY1AP1 in vascular smooth muscle cells leads to cell cycle arrest with decreased proliferation and increased levels of the cell cycle regulator p21/WAF/CDKN1A and disrupts TGF-β-driven differentiation of smooth muscle cells. Identification of YY1AP1 mutations as a cause of FMD indicates that this condition can result from underlying genetic variants that significantly alter the phenotype of vascular smooth muscle cells.

  19. Induction of the mammalian stress response gene GADD153 by oxidative stress: role of AP-1 element.

    PubMed Central

    Guyton, K Z; Xu, Q; Holbrook, N J

    1996-01-01

    GADD153 is a CCAAT/enhancer-binding-protein-related gene that may function to control cellular growth in response to stress signals. In this study, a variety of oxidant treatments were shown to stimulate endogenous GADD153 mRNA expression and to transcriptionally activate a GADD153 promoter-reporter gene construct in transfected HeLa cells. Both commonalities and distinctions in the induction of GADD153 by H2O2 and the thiol-reactive compound arsenite were demonstrated. GADD153 mRNA induction by both H2O2 and arsenite was potentiated by GSH depletion, and completely inhibited by N-acetyl-cysteine. o-Phenanthroline and mannitol blocked GADD153 induction by H2O2, indicating that iron-generated hydroxyl radical mediates this induction. Concordantly, GSH peroxidase overexpression in WI38 cells attenuated GADD153 mRNA induction by H2O2. However, GADD153 induction by arsenite was only modestly reduced in the same cells, suggesting a lesser contribution of peroxides to gene activation by arsenite. We also demonstrated that oxidative stress participates in the induction of GADD153 by UVC (254 nm) irradiation. Finally, both promoter-deletion analysis and point mutation of the AP-1 site in an otherwise intact promoter support a significant role for AP-1 in transcriptional activation of GADD153 by UVC or oxidant treatment. Indeed, exposure of cells to oxidants or UVC stimulated binding of Fos and Jun to the GADD153 AP-1 element. Together, these results demonstrate that both free-radical generation and thiol modification can transcriptionally activate GADD153, and that AP-1 is critical to oxidative regulation of this gene. This study further supports a role for the GADD153 gene product in the cellular response to oxidant injury. PMID:8670069

  20. Erg and AP-1 as determinants of glucocorticoid response in acute lymphoblastic leukemia.

    PubMed

    Chen, D W-C; Saha, V; Liu, J-Z; Schwartz, J-M; Krstic-Demonacos, M

    2013-06-20

    Glucocorticoids (GCs) are among the most widely prescribed medications in clinical practice. The beneficial effects of GCs in acute lymphoblastic leukemia (ALL) are based on their ability to induce apoptosis, but the underlying transcriptional mechanisms remain poorly defined. Computational modeling has enormous potential in the understanding of biological processes such as apoptosis and the discovery of novel regulatory mechanisms. We here present an integrated analysis of gene expression kinetic profiles using microarrays from GC sensitive and resistant ALL cell lines and patients, including newly generated and previously published data sets available from the Gene Expression Omnibus. By applying time-series clustering analysis in the sensitive ALL CEM-C7-14 cells, we identified 358 differentially regulated genes that we classified into 15 kinetic profiles. We identified GC response element (GRE) sequences in 33 of the upregulated known or potential GC receptor (GR) targets. Comparative study of sensitive and resistant ALL showed distinct gene expression patterns and indicated unexpected similarities between sensitivity-restored and resistant ALL. We found that activator protein 1 (AP-1), Ets related gene (Erg) and GR pathways were differentially regulated in sensitive and resistant ALL. Erg protein levels were substantially higher in CEM-C1-15-resistant cells, c-Jun was significantly induced in sensitive cells, whereas c-Fos was expressed at low levels in both. c-Jun was recruited on the AP-1 site on the Bim promoter, whereas a transient Erg occupancy on the GR promoter was detected. Inhibition of Erg and activation of GR lead to increased apoptosis in both sensitive and resistant ALL. These novel findings significantly advance our understanding of GC sensitivity and can be used to improve therapy of leukemia.

  1. Baicalein reduces angiogenesis in the inflammatory microenvironment via inhibiting the expression of AP-1

    PubMed Central

    Huang, Yujie; Miao, Zhaorui; Hu, Yang; Yuan, Yang; Zhou, Yuxin; Wei, Libin; Zhao, Kai; Guo, Qinglong; Lu, Na

    2017-01-01

    Increasing clinical and experimental studies have demonstrated that refractory chronic inflammation will result in malignant tumor and anti-angiogenic therapy may be an effective way to thwart the progression. Baicalein, one of the major active flavanoids found in Scutellaria baicalensis Georgi, has been exhibited potent anti-inflammation and anti-tumor effects by reducing angiogenesis. However, the exact mechanism of baicalein on endothelial cells in inflammatory microenvironment was not clear yet. Here, we investigated the anti-angiogenic effect of baicalein by incubating human umbilical vein endothelial cells (HUVECs) with THP-1 conditioned medium in vitro. The tube formation of HUVECs and microvessel outgrowth of rat aorta were attenuated, as well as the number of newly formed blood vessels in chicken chorioallantoic membrane (CAM) was reduced by baicalein. This anti-angiogenic effect was mainly on account of the inhibited motility, migration and invasion of HUVECs. In addition, mechanistic studies showed that baicalein could bind to AP-1 directly and the expression of c-Jun and c-Fos in HUVECs was reduced, accompanied by their increased proteasomal degradation. Besides, baicalein suppressed the nuclear translation, heterodimer formation and DNA binding affinity of c-Jun and c-Fos. What's more, the anti-angiogenic effect of baicalein was further confirmed by matrigel plug assay in vivo. Taken together, our study demonstrated that baicalein could exert its anti-angiogenic effect in the inflammation microenvironment via inhibiting the transcriptional activity of AP-1, which suggested that baicalein might be an alternative treatment against refractory chronic inflammation. PMID:27903990

  2. The V-ATPase accessory protein Atp6ap1b mediates dorsal forerunner cell proliferation and left-right asymmetry in zebrafish.

    PubMed

    Gokey, Jason J; Dasgupta, Agnik; Amack, Jeffrey D

    2015-11-01

    Asymmetric fluid flows generated by motile cilia in a transient 'organ of asymmetry' are involved in establishing the left-right (LR) body axis during embryonic development. The vacuolar-type H(+)-ATPase (V-ATPase) proton pump has been identified as an early factor in the LR pathway that functions prior to cilia, but the role(s) for V-ATPase activity are not fully understood. In the zebrafish embryo, the V-ATPase accessory protein Atp6ap1b is maternally supplied and expressed in dorsal forerunner cells (DFCs) that give rise to the ciliated organ of asymmetry called Kupffer's vesicle (KV). V-ATPase accessory proteins modulate V-ATPase activity, but little is known about their functions in development. We investigated Atp6ap1b and V-ATPase in KV development using morpholinos, mutants and pharmacological inhibitors. Depletion of both maternal and zygotic atp6ap1b expression reduced KV organ size, altered cilia length and disrupted LR patterning of the embryo. Defects in other ciliated structures-neuromasts and olfactory placodes-suggested a broad role for Atp6ap1b during development of ciliated organs. V-ATPase inhibitor treatments reduced KV size and identified a window of development in which V-ATPase activity is required for proper LR asymmetry. Interfering with Atp6ap1b or V-ATPase function reduced the rate of DFC proliferation, which resulted in fewer ciliated cells incorporating into the KV organ. Analyses of pH and subcellular V-ATPase localizations suggested Atp6ap1b functions to localize the V-ATPase to the plasma membrane where it regulates proton flux and cytoplasmic pH. These results uncover a new role for the V-ATPase accessory protein Atp6ap1b in early development to maintain the proliferation rate of precursor cells needed to construct a ciliated KV organ capable of generating LR asymmetry.

  3. AP1 transcription factors are required to maintain the peripheral taste system

    PubMed Central

    Shandilya, Jayasha; Gao, Yankun; Nayak, Tapan K; Roberts, Stefan G E; Medler, Kathryn F

    2016-01-01

    The sense of taste is used by organisms to achieve the optimal nutritional requirement and avoid potentially toxic compounds. In the oral cavity, taste receptor cells are grouped together in taste buds that are present in specialized taste papillae in the tongue. Taste receptor cells are the cells that detect chemicals in potential food items and transmit that information to gustatory nerves that convey the taste information to the brain. As taste cells are in contact with the external environment, they can be damaged and are routinely replaced throughout an organism's lifetime to maintain functionality. However, this taste cell turnover loses efficiency over time resulting in a reduction in taste ability. Currently, very little is known about the mechanisms that regulate the renewal and maintenance of taste cells. We therefore performed RNA-sequencing analysis on isolated taste cells from 2 and 6-month-old mice to determine how alterations in the taste cell-transcriptome regulate taste cell maintenance and function in adults. We found that the activator protein-1 (AP1) transcription factors (c-Fos, Fosb and c-Jun) and genes associated with this pathway were significantly downregulated in taste cells by 6 months and further declined at 12 months. We generated conditional c-Fos-knockout mice to target K14-expressing cells, including differentiating taste cells. c-Fos deletion caused a severe perturbation in taste bud structure and resulted in a significant reduction in the taste bud size. c-Fos deletion also affected taste cell turnover as evident by a decrease in proliferative marker, and upregulation of the apoptotic marker cleaved-PARP. Thus, AP1 factors are important regulators of adult taste cell renewal and their downregulation negatively impacts taste maintenance. PMID:27787515

  4. Transport of the cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1.

    PubMed

    Matsudaira, Tatsuyuki; Niki, Takahiro; Taguchi, Tomohiko; Arai, Hiroyuki

    2015-08-15

    The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

  5. AP-1A controls secretory granule biogenesis and trafficking of membrane secretory granule proteins.

    PubMed

    Bonnemaison, Mathilde; Bäck, Nils; Lin, Yimo; Bonifacino, Juan S; Mains, Richard; Eipper, Betty

    2014-10-01

    The adaptor protein 1A complex (AP-1A) transports cargo between the trans-Golgi network (TGN) and endosomes. In professional secretory cells, AP-1A also retrieves material from immature secretory granules (SGs). The role of AP-1A in SG biogenesis was explored using AtT-20 corticotrope tumor cells expressing reduced levels of the AP-1A μ1A subunit. A twofold reduction in μ1A resulted in a decrease in TGN cisternae and immature SGs and the appearance of regulated secretory pathway components in non-condensing SGs. Although basal secretion of endogenous SG proteins was unaffected, secretagogue-stimulated release was halved. The reduced μ1A levels interfered with the normal trafficking of carboxypeptidase D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), integral membrane enzymes that enter immature SGs. The non-condensing SGs contained POMC products and PAM-1, but not CPD. Based on metabolic labeling and secretion experiments, the cleavage of newly synthesized PAM-1 into PHM was unaltered, but PHM basal secretion was increased in sh-μ1A PAM-1 cells. Despite lacking a canonical AP-1A binding motif, yeast two-hybrid studies demonstrated an interaction between the PAM-1 cytosolic domain and AP-1A. Coimmunoprecipitation experiments with PAM-1 mutants revealed an influence of the luminal domains of PAM-1 on this interaction. Thus, AP-1A is crucial for normal SG biogenesis, function and composition.

  6. Assessment of costimulation and coinhibition in a triple parameter T cell reporter line: Simultaneous measurement of NF-κB, NFAT and AP-1.

    PubMed

    Jutz, Sabrina; Leitner, Judith; Schmetterer, Klaus; Doel-Perez, Iago; Majdic, Otto; Grabmeier-Pfistershammer, Katharina; Paster, Wolfgang; Huppa, Johannes B; Steinberger, Peter

    2016-03-01

    Engagement of the T cell receptor complex reprograms T cells for proliferation, cytokine production and differentiation towards effector cells. This process depends on activating costimulatory signals and is counteracted by coinhibitory molecules. Three transcription factors, namely NF-κB, NFAT and AP-1, have a major role in inducing the transcriptional program that is required for T cell activation and differentiation. Here we describe the generation of a triple parameter reporter based on the human Jurkat T cell line, where response elements for NF-κB, NFAT and AP-1 drive the expression of the fluorescent proteins CFP, eGFP and mCherry, respectively. The emission spectra of these proteins allow simultaneous assessment of NF-κB, NFAT and AP-1 activity in response to stimulation. Ligation of the TCR complex induced moderate reporter activity, which was strongly enhanced upon coengagement of the costimulatory receptors CD2 or CD28. Moreover, we have generated and tested triple parameter reporter cells that harbor costimulatory and inhibitory receptors not endogenously expressed in the Jurkat cells. In these experiments we could show that engagement of the costimulatory molecule 4-1BB enhances NF-κB and AP-1 activity, whereas coinhibition via PD-1 or BTLA strongly reduced the activation of NF-κB and NFAT. Engagement of BTLA significantly inhibited AP-1, whereas PD-1 had little effect on the activation of this transcription factor. Our triple parameter reporter T cell line is an excellent tool to assess the effect of costimulatory and coinhibitory receptors on NF-κB, NFAT and AP-1 activity and has a wide range of applications beyond the evaluation of costimulatory pathways.

  7. LL202 protects against dextran sulfate sodium-induced experimental colitis in mice by inhibiting MAPK/AP-1 signaling

    PubMed Central

    Zhao, Yue; Hu, Yang; Li, Zhiyu; Guo, Qinglong; Zhao, Kai; Lu, Na

    2016-01-01

    LL202, a newly-synthesized flavonoid derivative, has been reported to inhibit inflammatory-induced angiogenesis. However, the exact role of LL202 in inflammation along with its mechanism has not been explored. In this study, we investigated the anti-inflammatory effect of LL202 on intestinal inflammation by establishing dextran sulfate sodium (DSS)-induced experimental colitis. LL202 attenuated DSS-induced body weight loss, colon length shortening and colonic pathological damage. The inflammatory cells infiltration, myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities were decreased by LL202 in a dose-dependent manner. LL202 reduced the production of pro-inflammatory cytokines in serum and colon of DSS-induced mice as well. Mechanically, LL202 could decrease the expression and nuclear translation of AP-1 to protect against DSS-induced colitis. In lipopolysaccharide (LPS)-induced THP-1 cells, LL202 markedly decreased the secretion, mRNA level and protein expression of IL-1β, IL-6 and TNF-α via inhibiting ERK/JNK/p38 MAPK pathways and the nuclear translocation of AP-1. Furthermore, these findings were confirmed in LPS-induced bone marrow derived macrophages (BMDM). In conclusion, our study demonstrated that LL202 could exert its anti-inflammatory effect via inhibiting MAPK/AP-1 signaling, which suggested that LL202 might be a potential effective drug for the treatment of inflammatory bowel diseases. PMID:27590510

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection.

    PubMed

    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.

  13. The inflammation-related gene S100A12 is positively regulated by C/EBPβ and AP-1 in pigs.

    PubMed

    Li, Xinyun; Tang, Juan; Xu, Jing; Zhu, Mengjin; Cao, Jianhua; Liu, Ying; Yu, Mei; Zhao, Shuhong

    2014-08-08

    S100A12 is involved in the inflammatory response and is considered an important marker for many inflammatory diseases in humans. Our previous studies indicated that the S100A12 gene was abundant in the immune tissues of pigs and was significantly upregulated during infection with Haemophilus parasuis (HPS) or porcine circovirus type 2 (PCV2). In this study, the mechanism of transcriptional regulation of S100A12 was investigated in pigs. Our results showed that S100A12, CCAAT/enhancer-binding protein beta (C/EBPβ) and activator protein-1 (AP-1) genes were up-regulated in PK-15 (ATCC, CCL-33) cells when treated with LPS or Poly I: C. Additionally, the promoter activity and expression level of the S100A12 gene were significantly upregulated when C/EBPβ or AP-1 were overexpressed. We utilized electromobility shift assays (EMSA) to confirm that C/EBPβ and AP-1 could directly bind the S100A12 gene promoter. We also found that the transcriptional activity and expression levels of C/EBPβ and AP-1 could positively regulate each other. Furthermore, the promoter activity of the S100A12 gene was higher when C/EBPβ and AP-1 were cotransfected than when they were transfected individually. We concluded that the S100A12 gene was cooperatively and positively regulated by C/EBPβ and AP-1 in pigs. Our study offers new insight into the transcriptional regulation of the S100A12 gene.

  14. Inhibition of MAPK-mediated ACE expression by compound C66 prevents STZ-induced diabetic nephropathy.

    PubMed

    Pan, Yong; Huang, Yi; Wang, Zhe; Fang, Qilu; Sun, Yusheng; Tong, Chao; Peng, Kesong; Wang, Yangwei; Miao, Lining; Cai, Lu; Zhao, Yunjie; Liang, Guang

    2014-02-01

    A range of in vitro, experimental and clinical intervention studies have implicated an important role for hyperglycaemia-induced activation of the renin-angiotensin system (RAS) in the development and progression of diabetic nephropathy (DN). Blockade of RAS by angiotensin converting enzyme (ACE) inhibitors is an effective strategy in treating diabetic kidney diseases. However, few studies demonstrate the mechanism by which hyperglycaemia up-regulates the expression of ACE gene. Our previous studies have identified a novel curcumin analogue, (2E,6E)-2,6-bis(2-(trifluoromethyl)benzylidene)cyclohexanone (C66), which could inhibit the high glucose (HG)-induced phosphorylation of mitogen-activated protein kinases in mouse macrophages. In this study, we found that the renal protection of C66 in diabetic mice was associated with mitogen-activated protein kinase (MAPK) inactivation and ACE/angiotensin II (Ang II) down-regulation. Generally, MAPKs have been considered as a downstream signalling of Ang II and a mediator for Ang II-induced pathophysiological actions. However, using C66 and specific inhibitors as small molecule probes, in vitro experiments demonstrate that the MAPK signalling pathway regulates ACE expression under HG stimulation, which contributes to renal Ang II activation and the development of DN. This study indicates that C66 is a potential candidate of DN therapeutic agents, and more importantly, that reduction in ACE expression by MAPKs inhibition seems to be an alternative strategy for the treatment of DN.

  15. Dioscin suppresses human laryngeal cancer cells growth via induction of cell-cycle arrest and MAPK-mediated mitochondrial-derived apoptosis and inhibition of tumor invasion.

    PubMed

    Si, Lingling; Zheng, Lingli; Xu, Lina; Yin, Lianhong; Han, Xu; Qi, Yan; Xu, Youwei; Wang, Changyuan; Peng, Jinyong

    2016-03-05

    The anti-cancer effects of dioscin have been widely reported. However, its effect on laryngeal cancer remains unknown. In the present paper, our results showed that dioscin markedly caused cell apoptosis and DNA damage, increased reactive oxygen species (ROS) level, induced S-phase arrest, and inhibited invasion of human laryngeal cancer HEp-2 and TU212 cells. Mechanism investigation showed that dioscin markedly up-regulated p53 level, and down-regulated cyclin-dependent kinase 2 (CDK2) and Cyclin A levels. In addition, dioscin significantly down-regulated the levels of p-ERK, Bcl-2, up-regulated the levels of p-JNK, p-p38, Bax, cleaved caspase-3/-9, and caused Cytochrome c release. Furthermore, U0126, an ERK1/2 inhibitor, markedly down-regulated Bcl-2 level, up-regulated the levels of Bax, cleaved caspase-3/9, and enhanced Cytochrome c release inducted by dioscin. While, SP600125 (one JNK inhibitor) and SB203580 (one p38 inhibitor) markedly up-regulated Bcl-2 level, down-regulated the levels of Bax, cleaved caspase-3/9, and obviously boosted Cytochrome c release induced by dioscin. Interestingly, dioscin also markedly down-regulated the levels of MMP2 and MMP9 associated with tumor invasion. Taken together, our study indicated that dioscin suppressed laryngeal cancer cells growth via inducting cell-cycle arrest, MAPK-mediated mitochondrial- derived apoptosis and inhibiting tumor invasion, which could be used as one potential candidate for the treatment of laryngeal cancer in the future.

  16. Epigallocatechin-3-gallate attenuates lipopolysaccharide-induced mastitis in rats via suppressing MAPK mediated inflammatory responses and oxidative stress.

    PubMed

    Chen, Jinglou; Xu, Jun; Li, Jingjing; Du, Lifen; Chen, Tao; Liu, Ping; Peng, Sisi; Wang, Mingwei; Song, Hongping

    2015-05-01

    Green tea (Camellia sinensis) is an extremely popular beverage worldwide. Epigallocatechin-3-gallate (EGCG) is one of the major catechins isolated from green tea and contributes to its beneficial therapeutic functions including antioxidant, anti-inflammatory and anti-cancer effects. However, the effect of EGCG on mastitis is not yet known. This study was to investigate the protective potential of EGCG against mastitis in rats. The rat mastitis model was induced by injecting lipopolysaccharide (LPS) into the duct of mammary gland. The mammary gland was collected after the experimental period. The levels of mammary oxidative stress and inflammatory responses were assessed by measuring the local activities of antioxidant enzymes and the levels of inflammatory cytokines. The mammary expressions of mitogen-activated protein kinases (MAPKs), nuclear factor κB-p65 (NFκB-p65) and hypoxia-inducible factor-1α (HIF-1α) were evaluated by western blot analysis. It was found that EGCG obviously normalized LPS-induced low activities of antioxidant enzymes as well as decreased the high levels of inflammatory cytokines. Additionally, EGCG inhibited the mammary over-expression of MAPKs, NFκB-p65 and HIF-1α. These results indicated that EGCG was able to attenuate LPS-induced mastitis in rats by suppressing MAPK related oxidative stress and inflammatory responses.

  17. AP-1 regulates sphingosine kinase 1 expression in a positive feedback manner in glomerular mesangial cells exposed to high glucose.

    PubMed

    Huang, Kaipeng; Huang, Juan; Chen, Cheng; Hao, Jie; Wang, Shaogui; Huang, Junying; Liu, Peiqing; Huang, Heqing

    2014-03-01

    Our previous studies have confirmed that the sphingosine kinase 1 (SphK1)-sphingosine 1-phosphate (S1P) signaling pathway in the kidney under diabetic conditions is closely correlated with the pathogenesis of diabetic nephropathy (DN). The activation of SphK1-S1P pathway by high glucose (HG) can increase the expression of fibronectin (FN), an important fibrotic component, in glomerular mesangial cells (GMCs) by promoting the DNA-binding activity of transcription factor AP-1. However, the mechanism responsible for the sustained activation of SphK1-S1P pathway remains unclear. Given the binding motifs for AP-1 within the first intron of the SphK1 gene, we speculated that the activated AP-1 in the kidney under HG condition possibly regulates SphK1 expression in a positive feedback manner, thereby promoting the sustained activation of SphK1-S1P pathway and mediating the pathological progression of DN. Here, we observed the effect of AP-1 on SphK1 expression in GMCs and explored the molecular mechanism involved in the sustained activation of SphK1-S1P pathway. We found two consensus binding motifs for AP-1 in the promoter sequences and non-coding region downstream of the transcriptional initiation of the rat SphK1 gene by chromatin immunoprecipitation assay. The treatment of GMCs with both HG and S1P significantly increased the protein expression of c-Jun and c-Fos, and obviously enhanced the phosphorylation of c-Jun at Ser63 and Ser73, and c-Fos at Ser32. Knockdown of c-Jun and c-Fos with siRNAs substantially inhibited the expression of SphK1 and FN, whereas overexpression of c-Jun and c-Fos significantly increased the expression of SphK1 and FN. Curcumin treatment greatly decreased the levels of c-Jun, c-Fos, SphK1, and FN in the kidney tissues of diabetic rats. SiRNAs targeting SphK1 and S1P2 receptor respectively inhibited the phosphorylation of c-Jun (ser63 and ser73) and c-Fos (ser32), as well as FN expression under both normal and HG conditions. Our data

  18. Aclacinomycin A sensitizes K562 chronic myeloid leukemia cells to imatinib through p38MAPK-mediated erythroid differentiation.

    PubMed

    Lee, Yueh-Lun; Chen, Chih-Wei; Liu, Fu-Hwa; Huang, Yu-Wen; Huang, Huei-Mei

    2013-01-01

    Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and

  19. The transcription factor Ets21C drives tumor growth by cooperating with AP-1

    PubMed Central

    Toggweiler, Janine; Willecke, Maria; Basler, Konrad

    2016-01-01

    Tumorigenesis is driven by genetic alterations that perturb the signaling networks regulating proliferation or cell death. In order to block tumor growth, one has to precisely know how these signaling pathways function and interplay. Here, we identified the transcription factor Ets21C as a pivotal regulator of tumor growth and propose a new model of how Ets21C could affect this process. We demonstrate that a depletion of Ets21C strongly suppressed tumor growth while ectopic expression of Ets21C further increased tumor size. We confirm that Ets21C expression is regulated by the JNK pathway and show that Ets21C acts via a positive feed-forward mechanism to induce a specific set of target genes that is critical for tumor growth. These genes are known downstream targets of the JNK pathway and we demonstrate that their expression not only depends on the transcription factor AP-1, but also on Ets21C suggesting a cooperative transcriptional activation mechanism. Taken together we show that Ets21C is a crucial player in regulating the transcriptional program of the JNK pathway and enhances our understanding of the mechanisms that govern neoplastic growth. PMID:27713480

  20. 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.

  1. The mTOR/AP-1/VEGF signaling pathway regulates vascular endothelial cell growth.

    PubMed

    Wang, Shuo; Lu, Jiawei; You, Qingsheng; Huang, Hua; Chen, Yingying; Liu, Kun

    2016-08-16

    Vascular restenosis is a common adverse event following percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG). The atypical Ser/Thr protein kinase mammalian target of rapamycin (mTOR) plays an important role in cell differentiation and apoptosis. Vascular restenosis caused by excessive endothelial cell proliferation can be inhibited by local application of the mTOR inhibitor rapamycin (RAPA); however, RAPA can also suppress normal vascular endothelial cell growth by blocking mTOR/VEGF signaling, although the underlying mechanism is still unclear. Here, endogenous mTOR, AP-1, and VEGF were inhibited or overexpressed to investigate the mechanism underlying the effects of RAPA. Inhibition of AP-1 or mTOR with AP-1-siRNA or RAPA treatment respectively, decreased vascular endothelial cell proliferation, upregulation of AP-1 or mTOR increased cell proliferation, and VEGF overexpression increased, while RAPA-induced mTOR inhibition decreased vascular endothelial cell proliferation, the results indicate that combining mTOR downregulation and VEGF upregulation might both inhibit restenosis and maintain normal vascular endothelial cell growth after PCI or CABG, suggest the mTOR/AP-1/VEGF pathway might play a crucial role in regulating vascular endothelial cell growth.

  2. ARH cooperates with AP-1B in the exocytosis of LDLR in polarized epithelial cells.

    PubMed

    Kang, Richard S; Fölsch, Heike

    2011-04-04

    The autosomal recessive hypercholesterolemia protein (ARH) is well known for its role in clathrin-mediated endocytosis of low-density lipoprotein receptors (LDLRs). During uptake, ARH directly binds to the FxNPxY signal in the cytoplasmic tail of LDLR. Interestingly, the same FxNPxY motif is used in basolateral exocytosis of LDLR from recycling endosomes (REs), which is facilitated by the epithelial-specific clathrin adaptor AP-1B. However, AP-1B directly interacts with neither the FxNPxY motif nor the second more distally located YxxØ sorting motif of LDLR. Here, we show that ARH colocalizes and cooperates with AP-1B in REs. Knockdown of ARH in polarized epithelial cells leads to specific apical missorting of truncated LDLR, which encodes only the FxNPxY motif (LDLR-CT27). Moreover, a mutation in ARH designed to disrupt the interaction of ARH with AP-1B specifically abrogates exocytosis of LDLR-CT27. We conclude that in addition to its role in endocytosis, ARH cooperates with AP-1B in basolateral exocytosis of LDLR from REs.

  3. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis.

    PubMed

    Delevoye, Cédric; Hurbain, Ilse; Tenza, Danièle; Sibarita, Jean-Baptiste; Uzan-Gafsou, Stéphanie; Ohno, Hiroshi; Geerts, Willie J C; Verkleij, Arie J; Salamero, Jean; Marks, Michael S; Raposo, Graça

    2009-10-19

    Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1- and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type-specific positioning of endosomes that facilitate endosome-LRO contacts and are required for organelle maturation.

  4. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis

    PubMed Central

    Delevoye, Cédric; Hurbain, Ilse; Tenza, Danièle; Sibarita, Jean-Baptiste; Uzan-Gafsou, Stéphanie; Ohno, Hiroshi; Geerts, Willie J.C.; Verkleij, Arie J.; Salamero, Jean; Marks, Michael S.

    2009-01-01

    Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type–specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1– and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type–specific positioning of endosomes that facilitate endosome–LRO contacts and are required for organelle maturation. PMID:19841138

  5. Interactions between SIRT1 and AP-1 reveal a mechanistic insight into the growth promoting properties of alumina (Al2O3) nanoparticles in mouse skin epithelial cells.

    PubMed

    Dey, Swatee; Bakthavatchalu, Vasudevan; Tseng, Michael T; Wu, Peng; Florence, Rebecca L; Grulke, Eric A; Yokel, Robert A; Dhar, Sanjit Kumar; Yang, Hsin-Sheng; Chen, Yumin; St Clair, Daret K

    2008-10-01

    The physicochemical properties of nanomaterials differ from those of the bulk material of the same composition. However, little is known about the underlying effects of these particles in carcinogenesis. The purpose of this study was to determine the mechanisms involved in the carcinogenic properties of nanoparticles using aluminum oxide (Al(2)O(3)/alumina) nanoparticles as the prototype. Well-established mouse epithelial JB6 cells, sensitive to neoplastic transformation, were used as the experimental model. We demonstrate that alumina was internalized and maintained its physicochemical composition inside the cells. Alumina increased cell proliferation (53%), proliferating cell nuclear antigen (PCNA) levels, cell viability and growth in soft agar. The level of manganese superoxide dismutase, a key mitochondrial antioxidant enzyme, was elevated, suggesting a redox signaling event. In addition, the levels of reactive oxygen species and the activities of the redox sensitive transcription factor activator protein-1 (AP-1) and a longevity-related protein, sirtuin 1 (SIRT1), were increased. SIRT1 knockdown reduces DNA synthesis, cell viability, PCNA levels, AP-1 transcriptional activity and protein levels of its targets, JunD, c-Jun and BcL-xl, more than controls do. Immunoprecipitation studies revealed that SIRT1 interacts with the AP-1 components c-Jun and JunD but not with c-Fos. The results identify SIRT1 as an AP-1 modulator and suggest a novel mechanism by which alumina nanoparticles may function as a potential carcinogen.

  6. 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.

  7. Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Delta 12,14-prostaglandin J2.

    PubMed

    Pérez-Sala, Dolores; Cernuda-Morollón, Eva; Cañada, F Javier

    2003-12-19

    Cyclopentenone prostaglandins may interfere with cellular functions by multiple mechanisms. The cyclopentenone 15-deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2) has been reported to inhibit the activity of the transcription factor AP-1 in several experimental settings. We have explored the possibility of a direct interaction of 15d-PGJ2 with AP-1 proteins. Here we show that 15d-PGJ2 covalently modifies c-Jun and directly inhibits the DNA binding activity of AP-1. The modification of c-Jun occurs both in vitro and in intact cells as detected by labeling with biotinylated 15d-PGJ2 and mass spectrometry analysis. Attachment of the cyclopentenone prostaglandin occurs at cysteine 269, which is located in the c-Jun DNA binding domain. In addition, 15d-PGJ2 can promote the oligomerization of a fraction of c-Jun through the formation of intermolecular disulfide bonds or 15d-PGJ2-bonded dimers. Our results identify a novel site of interaction of 15d-PGJ2 with the AP-1 activation pathway that may contribute to the complex effects of cyclopentenone prostaglandins on the cellular response to pro-inflammatory agents. They also show the first evidence for the induction of protein cross-linking by 15d-PGJ2.

  8. NFκB- and AP-1-mediated DNA looping regulates matrix metalloproteinase-9 transcription in TNF-α-treated human leukemia U937 cells.

    PubMed

    Chen, Ying-Jung; Chang, Long-Sen

    2015-10-01

    The aim of this study is to explore the spatial association of critical genomic elements in the effect of TNF-α on matrix metalloproteinase-9 (MMP-9) expression in human leukemia U937 cells. TNF-α up-regulated MMP-9 protein expression and mRNA level in U937 cells, and Akt-mediated-NFκB/p65 activation and JNK-mediated c-Jun activation were proven to be involved in TNF-α-induced MMP-9 up-regulation. Promoter luciferase activity assay revealed that NFκB (nt-600) and AP-1 (nt-79) binding sites were crucial for TNF-α-induced transcription of MMP-9 gene. The results of a chromatin immunoprecipitation assay indicated that TNF-α reduced histone deacetylase-1 (HDAC-1) recruitment but increased p300 (a histone acetyltransferase) recruitment to MMP-9 promoter regions surrounding NFκB and AP-1 binding sites. Consistently, TNF-α increased enrichment of the acetylated histone H3 mark on MMP-9 promoter regions. DNA affinity purification assay revealed that p300 and HDAC1 could bind oligonucleotides containing AP-1/c-Jun and NFκB/p65 binding sites. Chromosome conformation capture assay showed that TNF-α stimulated chromosomal loops in the MMP-9 promoter via NFκB/p65 and AP-1/c-Jun. The p300-associated acetyltransferase activity was crucial for p65/c-Jun-mediated DNA looping, and inhibition of HDAC activity increased the level of DNA looping. Reduction in the level of DNA looping eliminated all TNF-α-stimulated MMP-9 up-regulation. Taken together, our data suggest that p65/c-Jun-mediated DNA looping is involved in TNF-α-induced MMP-9 up-regulation and that the recruitment of p300 or HDAC1 to NFκB and AP-1 binding sites modifies the level of DNA looping.

  9. Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation.

    PubMed

    González-Ramos, Marta; Mora, Inés; de Frutos, Sergio; Garesse, Rafael; Rodríguez-Puyol, Manuel; Olmos, Gemma; Rodríguez-Puyol, Diego

    2012-06-01

    The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.

  10. 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.

  11. Possible roles of the AP-1 site in the cytosolic T3 binding protein promoter and insights into its physiological significance.

    PubMed

    Suzuki, S; Nishio, S-I; Ishii, H; Sekido, T; Takeshige, K; Ohkubo, Y; Hiwatashi, D; Takeda, T; Komatsu, M

    2013-07-01

    Cytosolic 3,5,3'-triiodo-l-thyronine-binding protein plays pivotal roles in the regulation of intracellular 3,5,3'-triiodo-l-thyronine concentration in vivo. The expression of the protein, which is identical to μ-crystallin, is regulated by various factors. To elucidate the mechanisms of its expression, we evaluated the promoter transactivity and insulin signaling via the AP-1 site in the promoter. The isolated 600 bp human and 1976 bp mouse 5'-flanking regions were cloned in a luciferase reporter plasmid. The luciferase activity was estimated in GH3, dRLh-84, HEK293, and insulin receptor-overexpressing CHO-IR cells. The effects of 12-O-tetradecanoylphorbol 13-acetate and insulin on μ-crystallin mRNA expression were evaluated in various cells. The region between -200 and the transcriptional start site was crucial for constitutive expression in μ-crystallin-expressing dRLh-84 cells. This region contained an AP-1 site. 12-O-Tetradecanoylphorbol 13-acetate increased the level of μ-crystallin mRNA expression in HEK 293 cells. The compound also increased luciferase activity through the promoter. Mutation in the AP1 site diminished the response to the compound. The promoter was also activated by insulin treatment in CHO-IR cells. Insulin treatment increased μ-crystallin mRNA expression in Raw264.7 cells, but decreased in HEK293, P19, and dRLH-84 cells. The expression of μ-crystallin was regulated through the AP-1 site in the promoter. The signals related to AP-1 activation, such as insulin signaling may have diverse effects on μ-crystallin mRNA expression.

  12. The AP-1 μ adaptin is required for KNOLLE localization at the cell plate to mediate cytokinesis in Arabidopsis.

    PubMed

    Teh, Ooi-Kock; Shimono, Yuki; Shirakawa, Makoto; Fukao, Yoichiro; Tamura, Kentaro; Shimada, Tomoo; Hara-Nishimura, Ikuko

    2013-06-01

    Formation of clathrin-coated vesicles (CCVs) requires the scaffolding adaptor protein (AP) complexes, which are conserved across all eukaryotes. The Arabidopsis genome encodes five AP complexes (AP-1 to AP-5), and each complex consists of four subunits. In this study, we characterized the poorly defined AP-1 complex by using genetics, proteomics and live cell imaging. We showed that the AP-1 µ adaptin subunit (AP1M2) was localized to the trans-Golgi network (TGN) and interacted physically with the AP-1 subunits in Arabidopsis. During treatment with brefeldin A (BFA), the functional fluorophore-tagged AP1M2 relocated to the BFA compartment. The AP1M2 loss-of-function mutant ap1m2 displayed deleterious growth defects, which were particularly evident in the compromised cytokinesis that was revealed by the presence of cell wall stubs in multinucleate cells. Immunolocalization of the cytokinesis-specific syntaxin KNOLLE (KN) in ap1m2 showed that KN was mislocalized and aggregated around the division plane, while a secretory marker targeting to the cell plate remained unaffected. Taken together, we propose that the AP-1 complex is required for cell plate-targeted trafficking of KN in dividing plant cells, and that it has a common role in mediating plant and yeast/animal cytokinesis systems which are fundamentally different.

  13. Functions of adaptor protein (AP)-3 and AP-1 in tyrosinase sorting from endosomes to melanosomes.

    PubMed

    Theos, Alexander C; Tenza, Danièle; Martina, José A; Hurbain, Ilse; Peden, Andrew A; Sviderskaya, Elena V; Stewart, Abigail; Robinson, Margaret S; Bennett, Dorothy C; Cutler, Daniel F; Bonifacino, Juan S; Marks, Michael S; Raposo, Graça

    2005-11-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies.

  14. Highly efficient gene silencing using perfect complementary artificial miRNA targeting AP1 or heteromeric artificial miRNA targeting AP1 and CAL genes

    PubMed Central

    Park, Wonkeun; Zhai, Jixian; Lee, Jung-Youn

    2009-01-01

    Gene silencing is a useful technique for elucidating biological function of genes by knocking down their expression. A recently developed artificial microRNAs (amiRNAs) exploits an endogenous gene silencing mechanism that processes natural miRNA precursors to small silencing RNAs that target transcripts for degradation. Based on natural miRNA structures, amiRNAs are commonly designed such that they have a few mismatching nucleotides with respect to their target sites as well as within mature amiRNA duplexes. In this study, we performed an analysis in which the conventional and modified form of an amiRNA was compared side by side. We showed that the amiRNA containing 5′ mismatch with its amiRNA* and perfect complementarity to its target gene acted as a highly potent gene silencing agent against AP1, achieving a desired null mutation effect. In addition, a simultaneous silencing of two independent genes, AP1 and CAL1 wastested by employing a multimeric form of amiRNAs. Advantages and potential disadvantages of using amiRNAs with perfect complementarity to the target gene are discussed. The results presented here should be helpful in designing more specific and effective gene silencing agents. PMID:19066901

  15. AP1S3 Mutations Are Associated with Pustular Psoriasis and Impaired Toll-like Receptor 3 Trafficking

    PubMed Central

    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

    2014-01-01

    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

  16. JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells.

    PubMed

    Maślikowski, Bart M; Wang, Lizhen; Wu, Ying; Fielding, Ben; Bédard, Pierre-André

    2017-01-01

    The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant negative mutant TAM67 induced senescence, adipogenesis, or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEFs) whereas inhibition of JunD by short hairpin RNA (shRNA) specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEFs expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe sets upregulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPβ-regulated mediator of apoptosis in gamma interferon (IFN-γ)-induced cell death. Here, we show that inhibition of DAPK1 abrogates cell death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPβ was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPβ abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by chromatin immunoprecipitation (ChIP) assays, JunD was not detected on the DAPK1 promoter under any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells.

  17. AP-1/σ1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory

    PubMed Central

    Glyvuk, Nataliya; Tsytsyura, Yaroslav; Geumann, Constanze; D'Hooge, Rudi; Hüve, Jana; Kratzke, Manuel; Baltes, Jennifer; Böning, Daniel; Klingauf, Jürgen; Schu, Peter

    2010-01-01

    Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1–σ1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1–σ1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three σ1 subunit isoforms: A, B and C. The expressions of σ1A and σ1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from σ1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The σ1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation. PMID:20203623

  18. 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

  19. Cigarette smoke extract-induced proliferation of normal human urothelial cells via the MAPK/AP-1 pathway

    PubMed Central

    Geng, Hao; Zhao, Li; Liang, Zhaofeng; Zhang, Zhiqiang; Xie, Dongdong; Bi, Liangkuan; Wang, Yi; Zhang, Tao; Cheng, Lei; Yu, Dexin; Zhong, Caiyun

    2017-01-01

    Bladder cancer (BC) is universally acknowledged as a significant public health issue, worldwide. Numerous studies have demonstrated that cigarette smoke is the primary risk factor for BC. However, the mechanism of cigarette smoke-induced BC has not been fully elucidated. Sustained epithelial cell hyperplasia has been identified as a preneoplastic lesion during the formation of BC. The aim of the present study was to investigate whether exposure to cigarette smoke extract (CSE) induced proliferation in normal human urothelial SV-HUC-1 cells. Furthermore, the role of the mitogen-activated protein kinase (MAPK)/activator protein-1 (AP-1) pathway in the CSE-induced proliferation of SV-HUC-1 cells was also investigated. The present study revealed that the expression of phosphorylated-extracellular signal regulated protein kinase (ERK)1/2, Jun N-terminal kinase (JNK) and p38 was significantly increased following exposure to CSE in SV-HUC-1 cells. Furthermore, CSE increased the expression of the proliferation markers, cyclin D1 and proliferating cell nuclear antigen. By contrast, CSE attenuated the expression of p21. In addition, the inhibitors of ERK1/2 and JNK reversed the aforementioned effects of CSE. However, p38 inhibition did not reverse CSE-induced proliferation. In conclusion, the results of the present study demonstrated that exposure to CSE induced proliferation in normal human urothelial cells. Furthermore, the results also indicated that the ERK1/2 and JNK pathways are important for the regulation of proliferation via the AP-1 proteins. PMID:28123584

  20. Structure and regulation of the versican promoter: the versican promoter is regulated by AP-1 and TCF transcription factors in invasive human melanoma cells.

    PubMed

    Domenzain-Reyna, Clelia; Hernández, Daniel; Miquel-Serra, Laia; Docampo, María José; Badenas, Celia; Fabra, Angels; Bassols, Anna

    2009-05-01

    Versican is a large chondroitin sulfate proteoglycan of the extracellular matrix that is involved in a variety of cellular processes. We showed previously that versican, which is overexpressed in cutaneous melanomas as well as in premalignant lesions, contributes to melanoma progression, favoring the detachment of cells and the metastatic dissemination. Here, we investigated the transcriptional regulation of the versican promoter in melanoma cell lines with different levels of biological aggressiveness and stages of differentiation. We show that versican promoter up-regulation accounts for the differential expression levels of mRNA and protein detected in the invasive SK-mel-131 human melanoma cells. The activity of the versican promoter increased 5-fold in these cells in comparison with that measured in non-invasive MeWo melanoma cells. Several transcriptional regulatory elements were identified in the proximal promoter, including AP-1, Sp1, AP-2, and two TCF-4 sites. We show that promoter activation is mediated by the ERK/MAPK and JNK signaling pathways acting on the AP-1 site, suggesting that BRAF mutation present in SK-mel-131 cells impinge upon the up-regulation of the versican gene through signaling elicited by the ERK/MAPK pathway. This is the first time the AP-1 transcription factor family has been shown to be related to the regulation of versican expression. Furthermore, deletion of the TCF-4 binding sites caused a 60% decrease in the promoter activity in SK-mel-131 cells. These results showing that AP-1 and TCF-4 binding sites are the main regulatory regions directing versican production provide new insights into versican promoter regulation during melanoma progression.

  1. 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.

  2. DIXDC1 increases the invasion and migration ability of non-small-cell lung cancer cells via the PI3K-AKT/AP-1 pathway.

    PubMed

    Xu, Zhonghai; Liu, Di; Fan, Chuifeng; Luan, Lan; Zhang, Xiupeng; Wang, Enhua

    2014-11-01

    DIX domain containing 1 (DIXDC1), is a human homolog of Ccd1, a recently identified DIX domain containing protein in zebrafish. DIXDC1 protein was detected in human colorectal adenocarcinoma tissues and was found to be correlated with a high cell proliferation index. We demonstrated DIXDC1 overexpression in 55% (92/167) of non-small cell lung cancer (NSCLC) cases, compared to adjacent noncancerous lung tissues (P < 0.01). Overexpression of DIXDC1 was associated with lymph node metastasis and more advanced TNM stage (P < 0.001 and P = 0.001, respectively). Kaplan-Meier survival curves and log-rank testing indicated that overexpression of DIXDC1 correlated with worse overall survival in NSCLC (P = 0.031). DIXDC1 was more abundant in seven NSCLC lines than the bronchial cell line HBE, and modulation of its expression regulated AP-1 activity; MMP2, MMP7, and MMP9 protein and mRNA; and invasion ability. Metalloproteinase induction was reversed by PI3K/AKT and AP-1 inhibition. These results suggest DIXDC1 is associated with stage and prognosis in NSCLC, and may promote invasion and migration through PI3K-AKT/AP-1-dependent activation of metalloproteinases.

  3. Chrysin Inhibits Tumor Promoter-Induced MMP-9 Expression by Blocking AP-1 via Suppression of ERK and JNK Pathways in Gastric Cancer Cells

    PubMed Central

    Xia, Yong; Lian, Sen; Khoi, Pham Ngoc; Yoon, Hyun Joong; Joo, Young Eun; Chay, Kee Oh; Kim, Kyung Keun; Do Jung, Young

    2015-01-01

    Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients’ survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells. PMID:25875631

  4. Chrysin inhibits tumor promoter-induced MMP-9 expression by blocking AP-1 via suppression of ERK and JNK pathways in gastric cancer cells.

    PubMed

    Xia, Yong; Lian, Sen; Khoi, Pham Ngoc; Yoon, Hyun Joong; Joo, Young Eun; Chay, Kee Oh; Kim, Kyung Keun; Do Jung, Young

    2015-01-01

    Cell invasion is a crucial mechanism of cancer metastasis and malignancy. Matrix metalloproteinase-9 (MMP-9) is an important proteolytic enzyme involved in the cancer cell invasion process. High expression levels of MMP-9 in gastric cancer positively correlate with tumor aggressiveness and have a significant negative correlation with patients' survival times. Recently, mechanisms suppressing MMP-9 by phytochemicals have become increasingly investigated. Chrysin, a naturally occurring chemical in plants, has been reported to suppress tumor metastasis. However, the effects of chrysin on MMP-9 expression in gastric cancer have not been well studied. In the present study, we tested the effects of chrysin on MMP-9 expression in gastric cancer cells, and determined its underlying mechanism. We examined the effects of chrysin on MMP-9 expression and activity via RT-PCR, zymography, promoter study, and western blotting in human gastric cancer AGS cells. Chrysin inhibited phorbol-12-myristate 13-acetate (PMA)-induced MMP-9 expression in a dose-dependent manner. Using AP-1 decoy oligodeoxynucleotides, we confirmed that AP-1 was the crucial transcriptional factor for MMP-9 expression. Chrysin blocked AP-1 via suppression of the phosphorylation of c-Jun and c-Fos through blocking the JNK1/2 and ERK1/2 pathways. Furthermore, AGS cells pretreated with PMA showed markedly enhanced invasiveness, which was partially abrogated by chrysin and MMP-9 antibody. Our results suggest that chrysin may exert at least part of its anticancer effect by controlling MMP-9 expression through suppression of AP-1 activity via a block of the JNK1/2 and ERK1/2 signaling pathways in gastric cancer AGS cells.

  5. Disruption of AP1S1, Causing a Novel Neurocutaneous Syndrome, Perturbs Development of the Skin and Spinal Cord

    PubMed Central

    Drouin, Christian A.; Lapointe, Line; Boudreau, Michèle; Meloche, Caroline; Drouin, Régen; Hudson, Thomas J.; Drapeau, Pierre; Cossette, Patrick

    2008-01-01

    Adaptor protein (AP) complexes regulate clathrin-coated vesicle assembly, protein cargo sorting, and vesicular trafficking between organelles in eukaryotic cells. Because disruption of the various subunits of the AP complexes is embryonic lethal in the majority of cases, characterization of their function in vivo is still lacking. Here, we describe the first mutation in the human AP1S1 gene, encoding the small subunit σ1A of the AP-1 complex. This founder splice mutation, which leads to a premature stop codon, was found in four families with a unique syndrome characterized by mental retardation, enteropathy, deafness, peripheral neuropathy, ichthyosis, and keratodermia (MEDNIK). To validate the pathogenic effect of the mutation, we knocked down Ap1s1 expression in zebrafish using selective antisens morpholino oligonucleotides (AMO). The knockdown phenotype consisted of perturbation in skin formation, reduced pigmentation, and severe motility deficits due to impaired neural network development. Both neural and skin defects were rescued by co-injection of AMO with wild-type (WT) human AP1S1 mRNA, but not by co-injecting the truncated form of AP1S1, consistent with a loss-of-function effect of this mutation. Together, these results confirm AP1S1 as the gene responsible for MEDNIK syndrome and demonstrate a critical role of AP1S1 in development of the skin and spinal cord. PMID:19057675

  6. AP-1 clathrin adaptor and CG8538/Aftiphilin are involved in Notch signaling during eye development in Drosophila melanogaster.

    PubMed

    Kametaka, Satoshi; Kametaka, Ai; Yonekura, Shinichi; Haruta, Mineyuki; Takenoshita, Seiichi; Goto, Satoshi; Waguri, Satoshi

    2012-02-01

    Clathrin adaptor protein complex-1 (AP-1) and its accessory proteins play a role in the sorting of integral membrane proteins at the trans-Golgi network and endosomes. Their physiological functions in complex organisms, however, are not fully understood. In this study, we found that CG8538p, an uncharacterized Drosophila protein, shares significant structural and functional characteristics with Aftiphilin, a mammalian AP-1 accessory protein. The Drosophila Aftiphilin was shown to interact directly with the ear domain of γ-adaptin of Drosophila AP-1, but not with the GAE domain of Drosophila GGA. In S2 cells, Drosophila Aftiphilin and AP-1 formed a complex and colocalized at the Golgi compartment. Moreover, tissue-specific depletion of AP-1 or Aftiphilin in the developing eyes resulted in a disordered alignment of photoreceptor neurons in larval stage and roughened eyes with aberrant ommatidia in adult flies. Furthermore, AP-1-depleted photoreceptor neurons showed an intracellular accumulation of a Notch regulator, Scabrous, and downregulation of Notch by promoting its degradation in the lysosomes. These results suggest that AP-1 and Aftiphilin are cooperatively involved in the intracellular trafficking of Notch during eye development in Drosophila.

  7. Antroquinonol from Antrodia Camphorata suppresses breast tumor migration/invasion through inhibiting ERK-AP-1- and AKT-NF-κB-dependent MMP-9 and epithelial-mesenchymal transition expressions.

    PubMed

    Lee, Wai-Theng; Lee, Tzong-Huei; Cheng, Chia-Hsiung; Chen, Ku-Chung; Chen, Yen-Chou; Lin, Cheng-Wei

    2015-04-01

    Antroquinonol (ANQ) is an ubiquinon derivative isolated from the mycelium of Antrodia camphorata. However, the effect of ANQ on breast cancer treatment is unknown. We found that ANQ significantly suppressed the migration and invasion of breast cancer MDA-MB-231 cells, and inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced invasiveness by MCF7 cells. ANQ inhibiting MMP-9 gene expression and enzymatic activity occurred at transcriptional regulation. Mechanistically, activation of ERK and AKT is crucial for MMP-9 gene expression, and the addition of ANQ suppressed phosphorylation of ERK and AKT. The induction of the AP-1 and NF-κB pathway participated in MMP-9 gene expression. Suppression of ERK inhibited AP-1, whereas blocking AKT diminished NF-κB activity, and treatment with ANQ suppressed both AP-1 and NF-κB signaling. Moreover, ANQ suppressed EMT protein expression, and inhibited TPA-induced EMT through downregulating the ERK-AP-1 and AKT-NF-κB signaling cascades. Together, our data showed for the first time that ANQ inhibited breast cancer invasiveness by suppressing ERK-AP-1- and AKT-NF-κB-dependent MMP-9 and EMT expressions.

  8. Overexpression of BpAP1 induces early flowering and produces dwarfism in Betula platyphylla × Betula pendula.

    PubMed

    Huang, Haijiao; Wang, Shan; Jiang, Jing; Liu, Guifeng; Li, Huiyu; Chen, Su; Xu, Huanwen

    2014-08-01

    The involvement of APETALA1 (AP1) in the flowering transition has been the focus of much research. Here, we produced Betula platyphylla × Betula pendula (birch) lines that overexpressed BpAP1 using Agrobacterium-mediated transformation; we obtained five independent 35S::BpAP1 transgenic lines. Polymerase chain reaction (PCR), Southern, northern and western analyses were used to identify the transformants. As determined by quantitative real-time PCR (qRT-PCR), BpAP1 expression in roots, shoots, leaves and terminal buds of 35S::BpAP1 transgenic lines was significantly higher than that in the wild type (WT, P < 0.01). The average height of 2-year-old 35S::BpAP1 plants was significantly lower (41.17%) than that of non-transgenic plants. In the 35S::BpAP1 lines, inflorescences emerged successively beginning 2 months after transplanting. In addition, the length-diameter ratio of fully developed male and female inflorescences were both significantly less than those of the WT (P < 0.05), i.e. the morphological characteristic was stubby. The male inflorescences emerged early, with empty, draped anthers, and pollen was rarely produced, whereas the female floret structure was not different from WT. The pistils developed normally and could accept pollen, leading to the production of hybrid progeny (F1 ). F1 plants completed flowering within only 1 year after sowing. We demonstrate that BpAP1 can be inherited through sexual reproduction. Overexpression of BpAP1 caused early flowering and dwarfism; these lines had an obviously shortened juvenile phase. These results greatly increase our understanding of the mechanisms underlying the flowering transition and enhance genetic studies of birch traits, and they open up new possibilities for the breeding of birch and other woody plants.

  9. Pseudomonas aeruginosa quorum-sensing signaling molecule N-3-oxododecanoyl homoserine lactone induces matrix metalloproteinase 9 expression via the AP1 pathway in rat fibroblasts.

    PubMed

    Nakagami, Gojiro; Minematsu, Takeo; Morohoshi, Tomohiro; Yamane, Takumi; Kanazawa, Toshiki; Huang, Lijuan; Asada, Mayumi; Nagase, Takashi; Ikeda, Shin-ichi; Ikeda, Tsukasa; Sanada, Hiromi

    2015-01-01

    Quorum sensing is a cell-to-cell communication mechanism, which is responsible for regulating a number of bacterial virulence factors and biofilm maturation and therefore plays an important role for establishing wound infection. Quorum-sensing signals may induce inflammation and predispose wounds to infection by Pseudomonas aeruginosa; however, the interaction has not been well investigated. We examined the effects of the P. aeruginosa las quorum-sensing signal, N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), on matrix metalloproteinase (MMP) 9 expression in Rat-1 fibroblasts. 3OC12-HSL upregulated the expression of the MMP9 gene bearing an activator protein-1 (AP-1) binding site in the promoter region. We further investigated the mechanism underlying this effect. c-Fos gene expression increased rapidly after exposure to 3OC12-HSL, and nuclear translocation of c-Fos protein was observed; both effects were reduced by pretreatment with an AP-1 inhibitor. These results suggest that 3OC12-HSL can alter MMP9 gene expression in fibroblasts via the AP-1 signaling pathway.

  10. SANE's Measurement of the Proton's Virtual Photon Spin Asymmetry, Ap1, at Large Bjorken x

    NASA Astrophysics Data System (ADS)

    Mulholland, Jonathan Robert Lee

    The experiment SANE (Spin Asymmetries of the Nucleon Experiment) measured inclusive double polarization electron asymmetries on a proton target at the Continuous Electron Beam Accelerator Facility at the Thomas Jefferson National Laboratory in Newport News Virgina. Polarized electrons were scattered from a solid 14NH3 polarized target provided by the University of Virginia target group. Measurements were taken with the target polarization oriented at 80° and 180° relative to the beam direction, and beam energies of 4.7 and 5.9 GeV were used. Scattered electrons were detected by a multi-component novel non-magnetic detector package constructed for this experiment. Asymmetries measured at the two target orientations allow for the extraction of the virtual Compton asymmetries Ap1 and Ap2 as well as the spin structure functions gp1 and gp2 . This work addresses the extraction of the virtual Compton asymmetry Ap1 in the deep inelastic regime. The analysis uses data in the kinematic range from Bjorken x of 0.30 to 0.55, separated into four Q2 bins from 1.9 to 4.7 GeV2.

  11. 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

    2015-01-23

    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.

  12. 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

  13. Promotion of RAD51-mediated Homologous DNA Pairing by the RAD51AP1-UAF1 Complex

    PubMed Central

    Liang, Fengshan; Longerich, Simonne; Miller, Adam S.; Tang, Caroline; Buzovetsky, Olga; Xiong, Yong; Maranon, David G.; Wiese, Claudia; Kupfer, Gary M.; Sung, Patrick

    2017-01-01

    Summary 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 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

  14. 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.

  15. Xylosyltransferase-1 expression is refractory to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1β in nucleus pulposus cells: novel regulation by AP-1, Sp1, and Sp3.

    PubMed

    Ye, Wei; Zhou, Jie; Markova, Dessislava Z; Tian, Ye; Li, Jun; Anderson, D Greg; Shapiro, Irving M; Risbud, Makarand V

    2015-02-01

    We investigated whether expression of xylosyltransferase-1 (XT-1), a key enzyme in glycosaminoglycan biosynthesis, is responsive to disk degeneration and to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1β in nucleus pulposus (NP) cells. Analysis of human NP tissues showed that XT-1 expression is unaffected by degeneration severity; XT-1 and Jun, Fos, and Sp1 mRNA were positively correlated. Cytokines failed to inhibit XT-1 promoter activity and expression. However, cytokines decreased activity of XT-1 promoters containing deletion and mutation of the -730/-723 bp AP-1 motif, prompting us to investigate the role of AP-1 and Sp1/Sp3 in the regulation of XT-1 in healthy NP cells. Overexpression and suppression of AP-1 modulated XT-1 promoter activity. Likewise, treatment with the Sp1 inhibitors WP631 and mithramycin A or cotransfection with the plasmid DN-Sp1 decreased XT-1 promoter activity. Inhibitors of AP-1 and Sp1 and stable knockdown of Sp1 and Sp3 resulted in decreased XT-1 expression in NP cells. Genomic chromatin immunoprecipitation analysis showed AP-1 binding to motifs located at -730/-723 bp and -684/-677 bp and Sp1 binding to -227/-217 bp and -124/-114 bp in XT-1 promoter. These results suggest that XT-1 expression is refractory to the disease process and to inhibition by inflammatory cytokines and that signaling through AP-1, Sp1, and Sp3 is important in the maintenance of XT-1 levels in NP cells.

  16. 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.

  17. 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.

  18. Purinergic P2Y2 Receptor Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: NEW AP-1 TRANSCRIPTION FACTOR SITE AND NEGATIVE REGULATOR.

    PubMed

    Liu, Yiwei; Zhang, Lingxin; Wang, Chuan; Roy, Shama; Shen, Jianzhong

    2016-01-22

    We recently reported that the P2Y2 receptor (P2Y2R) is the predominant nucleotide receptor expressed in human coronary artery endothelial cells (HCAEC) and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here, we report the role of a newly identified AP-1 consensus sequence in the TF gene promoter and its original binding components in P2Y2R regulation of TF transcription. Using bioinformatics tools, we found that a novel AP-1 site at -1363 bp of the human TF promoter region is highly conserved across multiple species. Activation of P2Y2R increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this distal AP-1 site all significantly suppressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2, and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2 but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation through Src, leading to Fra-1 activation, whereas Rho/JNK mediated P2Y2R-induced activation of c-Jun and ATF-2. These findings reveal the molecular basis for P2Y G protein-coupled receptor control of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy for controlling vascular inflammation and thrombogenicity associated with endothelial dysfunction.

  19. OXIDATIVE STRESS ACTIVATES ANION EXCHANGE PROTEIN 2 AND AP-1 IN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Anion exchange protein 2 (AE2) is a membrane-bound protein that mediates chloride-bicarbonate exchange. In addition to regulating intracellular pH and cell volume, AE2 exports superoxide (O.) to the extracellular matrix in an HCO-dependent process. Given this ability to export O....

  20. Curcumin attenuates carcinogenesis by down regulating proinflammatory cytokine interleukin-1 (IL-1α and IL-1β) via modulation of AP-1 and NF-IL6 in lymphoma bearing mice.

    PubMed

    Das, Laxmidhar; Vinayak, Manjula

    2014-05-01

    Interleukin-1 (IL-1α and IL-1β) is a prototypic, potent, multifunctional proinflammatory cytokine affecting almost all cell types. Expression of IL-1 is up regulated in different tumor phenotypes and is implicated as an important factor in tumor progression via expression of metastatic, angiogenic genes and growth factors. Therefore, down regulation of expression of IL-1 may be able to inhibit cancer progression. Mechanism of transcriptional regulation of mouse IL-1α is not yet reported. AP-1 binding site at -12 to -6 on human IL-1α promotor is highly conserved in rat IL-1α gene and regulates its expression. Based on in silico analysis, regions -12 to -6bp is found to be conserved in human and mouse IL-1α gene promotor and therefore selected to study activation of IL-1α. Further, the regions -12 to -6bp in mouse IL-1α gene promotor corresponding to AP-1 binding element show 3'→5' orientation, necessary for AP-1 binding. The present work is focused on long term effect of curcumin on expression of IL-1α and IL-1β in liver of lymphoma bearing mice. Transcriptional regulation of IL-1α and IL-1β was analyzed by AP-1 and NF-IL-6 respectively. Elevated expression and protein level of IL-1α and IL-1β were found in lymphoma bearing mice compared to normal, which were significantly down regulated by curcumin treatment. Similarly, curcumin treatment down regulated activation of IL-1α and IL-1β via AP-1 and NF-IL-6 respectively. The findings conclude that curcumin attenuates carcinogenesis by down regulating proinflammatory cytokine interleukin-1 (IL-1α and IL-1β) via modulation of AP-1 and NF-IL6 respectively in lymphoma bearing mice.

  1. Determinants of half-site spacing preferences that distinguish AP-1 and ATF/CREB bZIP domains.

    PubMed Central

    Kim, J; Struhl, K

    1995-01-01

    The AP-1 and ATF/CREB families of eukaryotic transcription factors are dimeric DNA-binding proteins that contain the bZIP structural motif. The AP-1 and ATF/CREB proteins are structurally related and recognize identical half-sites (TGAC), but they differ in their requirements for half-site spacing. AP-1 proteins such as yeast GCN4 preferentially bind to sequences with overlapping half-sites, whereas ATF/CREB proteins bind exclusively to sequences with adjacent half-sites. Here we investigate the distinctions between AP-1 and ATF/CREB proteins by determining the DNA-binding properties of mutant and hybrid proteins. First, analysis of GCN4-ATF1 hybrid proteins indicates that a short surface spanning the basic and fork regions of the bZIP domain is the major determinant of half-site spacing. Replacement of two GCN4 residues on this surface (Ala244 and Leu247) by their ATF1 counterparts largely converts GCN4 into a protein with ATF/CREB specificity. Secondly, analysis of a Fos derivative containing the GCN4 leucine zipper indicates that Fos represents a novel intermediate between AP-1 and ATF/CREB proteins. Thirdly, we examine the effects of mutations in the invariant arginine residue of GCN4 (Arg243) that contacts the central base pair(s) of the target sites. While most mutations abolish DNA binding, substitution of a histidine residue results in a GCN4 derivative with ATF/CREB binding specificity. These results suggest that the AP-1 and ATF/CREB proteins differ in positioning a short surface that includes the invariant arginine and that AP-1 proteins may represent a subclass (and perhaps evolutionary offshoot) of ATF/CREB proteins that can tolerate overlapping half-sites. Images PMID:7630732

  2. Redox regulation of an AP-1-like transcription factor, YapA, in the fungal symbiont Epichloe festucae.

    PubMed

    Cartwright, Gemma M; Scott, Barry

    2013-10-01

    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.

  3. 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.

  4. Gallic Acid-g-Chitosan Modulates Inflammatory Responses in LPS-Stimulated RAW264.7 Cells Via NF-κB, AP-1, and MAPK Pathways.

    PubMed

    Ahn, Chang-Bum; Jung, Won-Kyo; Park, Sun-Joo; Kim, Yong-Tae; Kim, Won-Suk; Je, Jae-Young

    2016-02-01

    Chitosan is a naturally occurring polysaccharide, which has exhibited antioxidant, antimicrobial, and anti-cancer activities among others. Modification of chitosan by grafting phenolic compounds is a good strategy for improvement of bioactivities of chitosan. We investigated the anti-inflammatory action of gallic acid-grafted-chitosan (GAC) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. GAC inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) by inhibiting inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 macrophages. GAC also suppressed the production and mRNA expression of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). GAC inactivated nuclear factor-κB (NF-κB) via inhibiting the phosphorylation and degradation of the NF-κB inhibitor, IκB. In addition, GAC suppresses the activation of activator protein-1 (AP-1) through the phosphorylation of mitogen-activated protein kinase (MAPK) such as extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). These results suggest that GAC has the potential anti-inflammatory action by downregulating transcriptional factors (NF-κB and AP-1) through MAPK signaling pathways.

  5. A single Beta adaptin contributes to AP1 and AP2 complexes and clathrin function in Dictyostelium

    PubMed Central

    Sosa, R. Thomas; Weber, Michelle M.; Wen, Yujia; O’Halloran, Theresa J.

    2011-01-01

    The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric Assembly Proteins, also called Adaptor Proteins (AP’s), each of which contains a beta subunit. We identified a single beta subunit, named β1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding β1/2 resulted in severe defects in growth, cytokinesis, and development. Additionally, cells lacking β1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of β1/2 were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of β1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the beta subunit in the stability of the medium subunits. Dictyostelium β1/2 could resemble a common ancestor of the more specialized β1 and β2 subunits of the vertebrate AP complexes. Our results support the essential contribution a single beta subunit to the stability and function AP1 and AP2 in a simple eukaryote. PMID:22050483

  6. SAG/ROC2 E3 ligase regulates skin carcinogenesis by stage-dependent targeting of c-Jun/AP1 and IkappaB-alpha/NF-kappaB.

    PubMed

    Gu, Qingyang; Bowden, G Tim; Normolle, Daniel; Sun, Yi

    2007-09-10

    Sensitive to apoptosis gene (SAG)/regulator of cullins-2-Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase regulates cellular functions through ubiquitination and degradation of protein substrates. We report that, when expressed in mouse epidermis driven by the K14 promoter, SAG inhibited TPA-induced c-Jun levels and activator protein-1 (AP-1) activity in both in vitro primary culture, in vivo transgenic mice, and an AP-1- luciferase reporter mouse model. After AP-1 inactivation, epidermal proliferation induced by 7,12-dimethylbenz(a)-anthracene/12-O-tetradecanoylphorbol-13-acetate at the early stage of carcinogenesis was substantially inhibited. Later stage tumor formation was also substantially inhibited with prolonged latency and reduced frequency of tumor formation. Interestingly, SAG expression increased tumor size, not because of accelerated proliferation, but caused by reduced apoptosis resulting, at least in part, from nuclear factor kappaB (NF-kappaB) activation. Thus, SAG, in a manner depending on the availability of F-box proteins, demonstrated early-stage suppression of tumor formation by promoting c-Jun degradation, thereby inhibiting AP-1, and later-stage enhancement of tumor growth, by promoting inhibitor of kappaBalpha degradation to activate NF-kappaB and inhibit apoptosis.

  7. [6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

    PubMed

    Radhakrishnan, E K; Bava, Smitha V; Narayanan, Sai Shyam; Nath, Lekshmi R; Thulasidasan, Arun Kumar T; Soniya, Eppurathu Vasudevan; Anto, Ruby John

    2014-01-01

    We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

  8. Celastrol ameliorates HIV-1 Tat-induced inflammatory responses via NF-kappaB and AP-1 inhibition and heme oxygenase-1 induction in astrocytes.

    PubMed

    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.

  9. 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.

  10. ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation

    PubMed Central

    Jansen, Eric J. R.; Timal, Sharita; Ryan, Margret; Ashikov, Angel; van Scherpenzeel, Monique; Graham, Laurie A.; Mandel, Hanna; Hoischen, Alexander; Iancu, Theodore C.; Raymond, Kimiyo; Steenbergen, Gerry; Gilissen, Christian; Huijben, Karin; van Bakel, Nick H. M.; Maeda, Yusuke; Rodenburg, Richard J.; Adamowicz, Maciej; Crushell, Ellen; Koenen, Hans; Adams, Darius; Vodopiutz, Julia; Greber-Platzer, Susanne; Müller, Thomas; Dueckers, Gregor; Morava, Eva; Sykut-Cegielska, Jolanta; Martens, Gerard J. M.; Wevers, Ron A.; Niehues, Tim; Huynen, Martijn A.; Veltman, Joris A.; Stevens, Tom H.; Lefeber, Dirk J.

    2016-01-01

    The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function. PMID:27231034

  11. ATP6AP1 deficiency causes an immunodeficiency with hepatopathy, cognitive impairment and abnormal protein glycosylation.

    PubMed

    Jansen, Eric J R; Timal, Sharita; Ryan, Margret; Ashikov, Angel; van Scherpenzeel, Monique; Graham, Laurie A; Mandel, Hanna; Hoischen, Alexander; Iancu, Theodore C; Raymond, Kimiyo; Steenbergen, Gerry; Gilissen, Christian; Huijben, Karin; van Bakel, Nick H M; Maeda, Yusuke; Rodenburg, Richard J; Adamowicz, Maciej; Crushell, Ellen; Koenen, Hans; Adams, Darius; Vodopiutz, Julia; Greber-Platzer, Susanne; Müller, Thomas; Dueckers, Gregor; Morava, Eva; Sykut-Cegielska, Jolanta; Martens, Gerard J M; Wevers, Ron A; Niehues, Tim; Huynen, Martijn A; Veltman, Joris A; Stevens, Tom H; Lefeber, Dirk J

    2016-05-27

    The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

  12. Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes

    PubMed Central

    2012-01-01

    Background Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. Methods In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. Results Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. Conclusion From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases. PMID:22251375

  13. 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.

  14. Ramalin inhibits VCAM-1 expression and adhesion of monocyte to vascular smooth muscle cells through MAPK and PADI4-dependent NF-kB and AP-1 pathways.

    PubMed

    Park, Bongkyun; Yim, Joung-Han; Lee, Hong-Kum; Kim, Byung-Oh; Pyo, Suhkneung

    2015-01-01

    Cell adhesion molecules play a critical role in inflammatory processes and atherosclerosis. In this study, we investigated the effect of ramalin, a chemical compound from the Antarctic lichen Ramalina terebrata, on vascular cell adhesion molecule-1 (VCAM-1) expression induced by TNF-α in vascular smooth muscular cells (VSMCs). Pretreatment of VSMCs with ramalin (0.1-10 μg/mL) concentration-dependently inhibited TNF-α-induced VCAM-1 expression. Additionally, ramalin inhibited THP-1 (human acute monocytic leukemia cell line) cell adhesion to TNF-α-stimulated VSMCs. Ramalin suppressed TNF-α-induced production of reactive oxygen species (ROS), PADI4 expression, and phosphorylation of p38, ERK, and JNK. Moreover, ramalin inhibited TNF-α-induced translocation of NF-κB and AP-1. Inhibition of PADI4 expression by small interfering RNA or the PADI4-specific inhibitor markedly attenuated TNF-α-induced activation of NF-κB and AP-1 and VCAM-1 expression in VSMCs. Our study provides insight into the mechanisms underlying ramalin activity and suggests that ramalin may be a potential therapeutic agent to modulate inflammation within atherosclerosis.

  15. The Effects of NF-κB and c-Jun/AP-1 on the Expression of Prothrombotic and Proinflammatory Molecules Induced by Anti-β2GPI in Mouse.

    PubMed

    Xia, Longfei; Xie, Hongxiang; Yu, Yinjing; Zhou, Hong; Wang, Ting; Yan, Jinchuan

    2016-01-01

    Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β2GPI/β2GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β2GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β2GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β2GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β2GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β2GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.

  16. Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals

    PubMed Central

    Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

    2016-01-01

    The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38. PMID:27058530

  17. Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals.

    PubMed

    Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

    2016-04-06

    The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38.

  18. Functions of Adaptor Protein (AP)-3 and AP-1 in Tyrosinase Sorting from Endosomes to MelanosomesD⃞

    PubMed Central

    Theos, Alexander C.; Tenza, Danièle; Martina, José A.; Hurbain, Ilse; Peden, Andrew A.; Sviderskaya, Elena V.; Stewart, Abigail; Robinson, Margaret S.; Bennett, Dorothy C.; Cutler, Daniel F.; Bonifacino, Juan S.; Marks, Michael S.; Raposo, Graça

    2005-01-01

    Specialized cells exploit adaptor protein complexes for unique post-Golgi sorting events, providing a unique model system to specify adaptor function. Here, we show that AP-3 and AP-1 function independently in sorting of the melanocyte-specific protein tyrosinase from endosomes to the melanosome, a specialized lysosome-related organelle distinguishable from lysosomes. AP-3 and AP-1 localize in melanocytes primarily to clathrin-coated buds on tubular early endosomes near melanosomes. Both adaptors recognize the tyrosinase dileucine-based melanosome sorting signal, and tyrosinase largely colocalizes with each adaptor on endosomes. In AP-3-deficient melanocytes, tyrosinase accumulates inappropriately in vacuolar and multivesicular endosomes. Nevertheless, a substantial fraction still accumulates on melanosomes, concomitant with increased association with endosomal AP-1. Our data indicate that AP-3 and AP-1 function in partially redundant pathways to transfer tyrosinase from distinct endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. PMID:16162817

  19. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    SciTech Connect

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.

  20. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    DOE PAGES

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; ...

    2015-08-31

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintainingmore » wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Finally, our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression.« less

  1. NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability

    PubMed Central

    Parplys, Ann C.; Zhao, Weixing; Sharma, Neelam; Groesser, Torsten; Liang, Fengshan; Maranon, David G.; Leung, Stanley G.; Grundt, Kirsten; Dray, Eloïse; Idate, Rupa; Østvold, Anne Carine; Schild, David; Sung, Patrick; Wiese, Claudia

    2015-01-01

    NUCKS1 (nuclear casein kinase and cyclin-dependent kinase substrate 1) is a 27 kD chromosomal, vertebrate-specific protein, for which limited functional data exist. Here, we demonstrate that NUCKS1 shares extensive sequence homology with RAD51AP1 (RAD51 associated protein 1), suggesting that these two proteins are paralogs. Similar to the phenotypic effects of RAD51AP1 knockdown, we find that depletion of NUCKS1 in human cells impairs DNA repair by homologous recombination (HR) and chromosome stability. Depletion of NUCKS1 also results in greatly increased cellular sensitivity to mitomycin C (MMC), and in increased levels of spontaneous and MMC-induced chromatid breaks. NUCKS1 is critical to maintaining wild type HR capacity, and, as observed for a number of proteins involved in the HR pathway, functional loss of NUCKS1 leads to a slow down in DNA replication fork progression with a concomitant increase in the utilization of new replication origins. Interestingly, recombinant NUCKS1 shares the same DNA binding preference as RAD51AP1, but binds to DNA with reduced affinity when compared to RAD51AP1. Our results show that NUCKS1 is a chromatin-associated protein with a role in the DNA damage response and in HR, a DNA repair pathway critical for tumor suppression. PMID:26323318

  2. MAPK-mediated enhanced expression of vacuolar H(+)-ATPase confers the improved adaption to NaCl stress in a halotolerate peppermint (Mentha piperita L.).

    PubMed

    Li, Zhe; Zhen, Zhen; Guo, Kai; Harvey, Paul; Li, Jishun; Yang, Hetong

    2016-03-01

    Vacuolar H(+)-ATPase (V-H(+)-ATPase) has been proved to be of importance in maintenance of ion homeostasis inside plant cells under NaCl stress. In this study, the expression levels and salt-tolerate function of V-H(+)-ATPase genes were investigated in the roots and leaves of a halotolerate peppermint (Mentha × piperita L.) Keyuan-1 treated with different concentrations of NaCl. Results showed that the expressions of V-H(+)-ATPase in the transcriptional, protein and activity levels were significantly enhanced in the halotolerate peppermint Keyuan-1 compared to the wild-type (WT) peppermint under 50, 100, and 150 mM NaCl treatment. Moreover, inhibition experiments exhibited that V-H(+)-ATPase activity played vital roles in the salt tolerance of peppermint Keyuan-1 to 150 mM NaCl stress through increasing the vacuolar H(+) pumping activity and Na(+) compartmentalization capacity. Furthermore, results of Western blots showed that the activity of a mitogen-activated protein kinase (MAPK) was significantly increased under different concentrations of NaCl in the halotolerate peppermint Keyuan-1, which was much higher than that of WT peppermint. Further experiments with inhibitors suggested that this MAPK protein was responsible for the enhanced expression of V-H(+)-ATPase in the halotolerate peppermint Keyuan-1. In response to NaCl stress, increase of cytoplasmic calcium concentration ([Ca(2+)]cyt) occurred upstream of MAPK activation in the halotolerate peppermint Keyuan-1. In all, these findings demonstrated that increased V-H(+)-ATPase activity was positively correlated with the enhanced salt tolerance in the halotolerate peppermint Keyuan-1, providing the theoretic basis for the further development and utilization of peppermint in saline areas.

  3. Role of AP-1 family proteins in regulation of inducible nitric oxide synthase (iNOS) in human neutrophils.

    PubMed

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr; Iwaniuk, Agnieszka

    2013-01-01

    The aim of the study was to assess the activity of AP-1 family proteins, e.g. Fra-1, Fra-2, JunB, JunD, and FosB, engaged in the regulation of inducible nitric oxide synthase (iNOS) expression and the production of NO by neutrophils (PMN) exposed to N-nitrosodimethylamine (NDMA) xenobiotic. Isolated human PMN were incubated in the presence of NDMA. iNOS mRNA expression was then analyzed using Northern blot and the expression of other proteins in the cytoplasmic and nuclear fractions were assessed using Western blot. The obtained results indicate that NDMA increased iNOS mRNA and protein expression in human PMN. Furthermore, it increased the expression of Fra-1, Fra-2, JunB, and JunD in the cytoplasmic fraction, and FosB expression in the fractions of analyzed cells. As a consequence of inhibiting p38 pathway and JNK, reduced iNOS expression and NO production was noted in PMN exposed to NDMA. Inhibition of the p38 pathway resulted in reduced expression of all analyzed proteins in the cytoplasmic fraction of PMN exposed to NDMA. Furthermore, increased Fra-2 expression and reduced FosB expression were found in the nuclear fraction of those cells. Inhibiting ERK5 pathway resulted in increased JunB expression in both fractions of the analyzed cells. Therefore, no changes in the expression of analyzed proteins in the presence of NDMA were observed in PMN pre-incubated with JNK pathway inhibitor. In conclusion, the results here indicate a role of Fra-1, Fra-2, JunB, JunD, and FosB transcription factors in the regulation of iNOS expression and NO production by human neutrophils exposed to NDMA.

  4. Amelioration of severe TNBS induced colitis by novel AP-1 and NF- κ B inhibitors in rats.

    PubMed

    El-Salhy, Magdy; Umezawa, Kazuo; Gilja, Odd Helge; Hatlebakk, Jan G; Gundersen, Doris; Hausken, Trygve

    2014-01-01

    AP-1 and NF-κ B inhibitors, namely, DTCM-G and DHMEQ, were investigated in male Wistar rats with severe colitis, induced by TNBS. The animals were randomized into 3 groups. The control group received 0.5 mL of 0.5% of the vehicle i.p., the DTCM-G group received 22.5 mg/kg body weight DTCM-G in 0.5% i.p., and the DHMEQ group received 15 mg/kg body weight DHMEQ i.p., all twice daily for 5 days. The body weight losses and mortality rates were significantly higher in the control group than those in DTCM-G-treated and DHMEQ-treated groups. The endoscopic inflammation scores in the control, DTCM-G-treated, and DHMEQ-treated groups were 6.3 ± 0.7, 1.0 ± 0.3, and 0.7 ± 0.3, respectively (P = 0.004 and 0.02, resp.). The inflammation scores as assessed by the macroscopic appearance were 4.3 ± 0.8, 0.7 ± 0.3, and 1.2 ± 0.4 in the control, DTCM-G-treated, and DHMEQ-treated groups, respectively (P = 0.01 and 0.009, resp.). The histopathological inflammation scores were 6.4 ± 0.7, 2.0 ± 1.0, and 2.2 ± 0.6 in the control, DTCM-G-treated, and DHMEQ-treated groups, respectively (P = 0.03 and 0.01, resp.). It was concluded that DTCM-G and DHMEQ exhibit strong anti-inflammatory and anticancer activities with no apparent toxicity, which make them excellent drug candidates for clinical use in inflammatory bowel diseases.

  5. MAPK-mediated regulation of growth and essential oil composition in a salt-tolerant peppermint (Mentha piperita L.) under NaCl stress.

    PubMed

    Li, Zhe; Wang, Wenwen; Li, Guilong; Guo, Kai; Harvey, Paul; Chen, Quan; Zhao, Zhongjuan; Wei, Yanli; Li, Jishun; Yang, Hetong

    2016-11-01

    Peppermint (Mentha × piperita L.) is an important and commonly used flavoring agent worldwide, and salinity is a major stress that limits plant growth and reduces crop productivity. This work demonstrated the metabolic responses of essential oil production including the yield and component composition, gene expression, enzyme activity, and protein activation in a salt-tolerant peppermint Keyuan-1 with respect to NaCl stress. Our results showed that Keyuan-1 maintained normal growth and kept higher yield and content of essential oils under NaCl stress than wild-type (WT) peppermint.Gas chromatography-mass spectrometry (GC-MS) and qPCR results showed that compared to WT seedlings, a 150-mM NaCl stress exerted no obvious changes in essential oil composition, transcriptional level of enzymes related to essential oil metabolism, and activity of pulegone reductase (Pr) in Keyuan-1 peppermint which preserved the higher amount of menthol and menthone as well as the lower content of menthofuran upon the 150-mM NaCl stress. Furthermore, it was noticed that a mitogen-activated protein kinase (MAPK) protein exhibited a time-dependent activation in the Keyuan-1 peppermint and primarily involved in the modulation of the essential oil metabolism in the transcript and enzyme levels during the 12-day treatment of 150 mM NaCl. In all, our data elucidated the effect of NaCl on metabolic responses of essential oil production, and demonstrated the MAPK-dependent regulation mechanism of essential oil biosynthesis in the salt-tolerant peppermint, providing scientific basis for the economic and ecological utilization of peppermint in saline land.

  6. Tissue-specific up-regulation of arginase I and II induced by p38 MAPK mediates endothelial dysfunction in type 1 diabetes mellitus

    PubMed Central

    Pernow, J; Kiss, A; Tratsiakovich, Y; Climent, B

    2015-01-01

    Background and Purpose Emerging evidence suggests a selective up-regulation of arginase I in diabetes causing coronary artery disease; however, the mechanisms behind this up-regulation are still unknown. Activated p38 MAPK has been reported to increase arginase II in various cardiovascular diseases. We therefore tested the role of p38 MAPK in the regulation of arginase I and II expression and its effect on endothelial dysfunction in diabetes mellitus. Experimental Approach Endothelial function was determined in septal coronary (SCA), left anterior descending coronary (LAD) and mesenteric (MA) arteries from healthy and streptozotocin-induced diabetic Wistar rats by wire myographs. Arginase activity and protein levels of arginase I, II, phospho-p38 MAPK and phospho-endothelial NOS (eNOS) (Ser1177) were determined in these arteries from diabetic and healthy rats treated with a p38 MAPK inhibitor in vivo. Key Results Diabetic SCA and MA displayed impaired endothelium-dependent relaxation, which was prevented by arginase and p38 MAPK inhibition while LAD relaxation was not affected. Arginase I, phospho-p38 MAPK and eNOS protein expression was increased in diabetic coronary arteries. In diabetic MA, however, increased expression of arginase II and phospho-p38 MAPK, increased arginase activity and decreased expression of eNOS were observed. All these effects were reversed by p38 MAPK inhibition. Conclusions and Implications Diabetes-induced activation of p38 MAPK causes endothelial dysfunction via selective up-regulation of arginase I expression in coronary arteries and arginase II expression in MA. Therefore, regional differences appear to exist in the arginase isoforms contributing to endothelial dysfunction in type 1 diabetes mellitus. PMID:26140333

  7. Resveratrol modulates phorbol ester-induced pro-inflammatory signal transduction pathways in mouse skin in vivo: NF-kappaB and AP-1 as prime targets.

    PubMed

    Kundu, Joydeb Kumar; Shin, Young Kee; Surh, Young-Joon

    2006-11-30

    Functional abnormalities of intracellular signaling network cause the disruption in homeostasis maintained by critical cellular components, thereby accelerating premalignant and malignant transformation. Multiple lines of evidence suggest that an elevated expression of cyclooxygenase-2 (COX-2) is causally linked to tumorigenesis. The exposure to oxidative/pro-inflammatory stimuli turns on signaling arrays mediated by diverse classes of kinases and transcription factors, which may lead to aberrant expression of COX-2. We have attempted to unravel the signal transduction pathways involved in elevated COX-2 expression in mouse skin stimulated with a prototype tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its modulation by resveratrol, a phytoalexin known to exert potential chemopreventive effects. Our study revealed that topical application of TPA induced COX-2 expression in mouse skin via activation of nuclear factor-kappaB (NF-kappaB), which is regulated by upstream IkappaB kinase (IKK) or differentially by mitogen-activated protein (MAP) kinases. Besides NF-kappaB, the p38 MAP kinase-mediated activation of activator protein-1 (AP-1) has also been attributed to TPA-induced COX-2 expression in mouse skin. Among the MAP kinases, extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase have been shown to regulate TPA-induced NF-kappaB activation, while p38 MAP kinase and c-Jun-N-terminal kinase are preferentially involved in TPA-induced activation of AP-1 in mouse skin in vivo. This commentary focuses on resveratrol modulation of intracellular signaling pathways involved in aberrant COX-2 expression in TPA-stimulated mouse skin to delineate molecular mechanisms underlying antitumor promoting effects of resveratrol.

  8. Kaempferol suppresses cell metastasis via inhibition of the ERK-p38-JNK and AP-1 signaling pathways in U-2 OS human osteosarcoma cells.

    PubMed

    Chen, Hui-Jye; Lin, Chung-Ming; Lee, Chao-Ying; Shih, Nai-Chen; Peng, Shu-Fen; Tsuzuki, Minoru; Amagaya, Sakae; Huang, Wen-Wen; Yang, Jai-Sing

    2013-08-01

    Kaempferol is a natural flavonoid that possesses anti-proliferative and apoptosis-inducing activities in several cancer cell lines. In the present study, we investigated the anti-metastatic activity of kaempferol and its molecular mechanism(s) of action in human osteosarcoma cells. Kaempferol displayed inhibitory effects on the invasion and adhesion of U-2 osteosarcoma (OS) cells in a concentration-dependent manner by Matrigel Transwell assay and cell adhesion assay. Kaempferol also inhibited the migration of U-2 OS cells in a concentration-dependent manner at different treatment time points by wound-healing assay. Additional experiments showed that kaempferol treatment reduced the enzymatic activities and protein levels of matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator (uPA) by gelatin and casein-plasminogen zymography assays and western blot analyses. Kaempferol also downregulated the mRNA levels of MMP-2 and MMP-9 by quantitative PCR analyses. Furthermore, kaempferol was able to reduce the protein phosphorylation of ERK, p38 and JNK by western blotting. By electrophoretic mobility-shift assay (EMSA), we demonstrated that kaempferol decreased the DNA binding activity of AP-1, an action likely to result in the reduced expression of MMP-2, MMP-9 and uPA. Collectively, our data showed that kaempferol attenuated the MAPK signaling pathways including ERK, JNK and p38 and resulted in the decreased DNA binding ability of AP-1, and hence, the downregulation in the expression and enzymatic activities of MMP-2, MMP-9 and uPA, contributing to the inhibition of metastasis of U-2 OS cells. Our results suggest a potential role of kaempferol in the therapy of tumor metastasis of OS.

  9. Potentiation of Schaffer-Collateral CA1 Synaptic Transmission by eEF2K and p38 MAPK Mediated Mechanisms

    PubMed Central

    Weng, Weiguang; Chen, Ying; Wang, Man; Zhuang, Yinghan; Behnisch, Thomas

    2016-01-01

    The elongation factor 2 kinase (eEF2K), likewise known as CaMKIII, has been demonstrated to be involved in antidepressant responses of NMDA receptor antagonists. Even so, it remains open whether direct inhibition of eEF2K without altering up-stream or other signaling pathways affects hippocampal synaptic transmission and neuronal network synchrony. Inhibition of eEF2K by the selective and potent eEF2K inhibitor A-484954 induced a fast pre-synaptically mediated enhancement of synaptic transmission and synchronization of neural network activity. The eEF2K-inhibition mediated potentiation of synaptic transmission of hippocampal CA1 neurons is most notably independent of protein synthesis and does not rely on protein kinase C, protein kinase A or mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase 1/2. Moreover, the strengthening of synaptic transmission in the response to the inhibition of eEF2K was strongly attenuated by the inhibition of p38 MAPK. In addition, we show the involvement of barium-sensitive and more specific the TWIK-related potassium-1 (TREK-1) channels in the eEF2K-inhibition mediated potentiation of synaptic transmission. These findings reveal a novel pathway of eEF2K mediated regulation of hippocampal synaptic transmission. Further research is required to study whether such compounds could be beneficial for the development of mood disorder treatments with a fast-acting antidepressant response. PMID:27826228

  10. 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 Central

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

    2009-01-01

    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

  11. 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.

  12. Luteolin and luteolin-7-O-glucoside inhibit lipopolysaccharide-induced inflammatory responses through modulation of NF-κB/AP-1/PI3K-Akt signaling cascades in RAW 264.7 cells.

    PubMed

    Park, Chung Mu; Song, Young-Sun

    2013-12-01

    Luteolin is a flavonoid found in abundance in celery, green pepper, and dandelions. Previous studies have shown that luteolin is an anti-inflammatory and anti-oxidative agent. In this study, the anti-inflammatory capacity of luteolin and one of its glycosidic forms, luteolin-7-O-glucoside, were compared and their molecular mechanisms of action were analyzed. In lipopolysaccharide (LPS)-activated RAW 264.7 cells, luteolin more potently inhibited the production of nitric oxide (NO) and prostaglandin E2 as well as the expression of their corresponding enzymes (inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) than luteolin-7-O-glucoside. The molecular mechanisms underlying these effects were investigated to determine whether the inflammatory response was related to the transcription factors, nuclear factor (NF)-κB and activator protein (AP)-1, or their upstream signaling molecules, mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K). Luteolin attenuated the activation of both transcription factors, NF-κB and AP-1, while luteolin-7-O-glucoside only impeded NF-κB activation. However, both flavonoids inhibited Akt phosphorylation in a dose-dependent manner. Consequently, luteolin more potently ameliorated LPS-induced inflammation than luteolin-7-O-glucoside, which might be attributed to the differentially activated NF-κB/AP-1/PI3K-Akt pathway in RAW 264.7 cells.

  13. Luteolin and luteolin-7-O-glucoside inhibit lipopolysaccharide-induced inflammatory responses through modulation of NF-κB/AP-1/PI3K-Akt signaling cascades in RAW 264.7 cells

    PubMed Central

    Park, Chung Mu

    2013-01-01

    Luteolin is a flavonoid found in abundance in celery, green pepper, and dandelions. Previous studies have shown that luteolin is an anti-inflammatory and anti-oxidative agent. In this study, the anti-inflammatory capacity of luteolin and one of its glycosidic forms, luteolin-7-O-glucoside, were compared and their molecular mechanisms of action were analyzed. In lipopolysaccharide (LPS)-activated RAW 264.7 cells, luteolin more potently inhibited the production of nitric oxide (NO) and prostaglandin E2 as well as the expression of their corresponding enzymes (inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) than luteolin-7-O-glucoside. The molecular mechanisms underlying these effects were investigated to determine whether the inflammatory response was related to the transcription factors, nuclear factor (NF)-κB and activator protein (AP)-1, or their upstream signaling molecules, mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K). Luteolin attenuated the activation of both transcription factors, NF-κB and AP-1, while luteolin-7-O-glucoside only impeded NF-κB activation. However, both flavonoids inhibited Akt phosphorylation in a dose-dependent manner. Consequently, luteolin more potently ameliorated LPS-induced inflammation than luteolin-7-O-glucoside, which might be attributed to the differentially activated NF-κB/AP-1/PI3K-Akt pathway in RAW 264.7 cells. PMID:24353826

  14. 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

  15. SKF-82958 is a subtype-selective estrogen receptor-alpha (ERalpha ) agonist that induces functional interactions between ERalpha and AP-1.

    PubMed

    Walters, Marian R; Dutertre, Martin; Smith, Carolyn L

    2002-01-18

    The transcriptional activity of estrogen receptors (ERs) can be regulated by ligands as well as agents such as dopamine, which stimulate intracellular signaling pathways able to communicate with these receptors. We examined the ability of SKF-82958 (SKF), a previously characterized full dopamine D1 receptor agonist, to stimulate the transcriptional activity of ERalpha and ERbeta. Treatment of HeLa cells with SKF-82958 stimulated robust ERalpha-dependent transcription from an estrogen-response element-E1b-CAT reporter in the absence of estrogen, and this was accompanied by increased receptor phosphorylation. However, induction of ERbeta-directed gene expression under the same conditions was negligible. In our cell model, SKF treatment did not elevate cAMP levels nor enhance transcription from a cAMP-response element-linked reporter. Control studies revealed that SKF-82958, but not dopamine, competes with 17beta-estradiol for binding to ERalpha or ERbeta with comparable relative binding affinities. Therefore, SKF-82958 is an ERalpha-selective agonist. Transcriptional activation of ERalpha by SKF was more potent than expected from its relative binding activity, and further examination revealed that this synthetic compound induced expression of an AP-1 target gene in a tetradecanoylphorbol-13-acetate-response element (TRE)-dependent manner. A putative TRE site upstream of the estrogen-response element and the amino-terminal domain of the receptor contributed to, but were not required for, SKF-induced expression of an ERalpha-dependent reporter gene. Overexpression of the AP-1 protein c-Jun, but not c-Fos, strongly enhanced SKF-induced ERalpha target gene expression but only when the TRE was present. These studies provide information on the ability of a ligand that weakly stimulates ERalpha to yield strong stimulation of ERalpha-dependent gene expression through cross-talk with other intracellular signaling pathways producing a robust combinatorial response within the

  16. B:2a:p1.5 meningococcal strains likely arisen from capsular switching event still spreading in Spain.

    PubMed

    Castilla, Jesús; Vázquez, Julio A; Salcedo, Celia; García Cenoz, Manuel; García Irure, José Javier; Torroba, Luis; Beristain, Xabier; Abad, Raquel; Barricarte, Aurelio

    2009-02-01

    Eighteen clustered cases of meningococcal disease associated with B:2a:P1.5 strains doubled the annual incidence up to 4.3 x 10(5) in Navarra, Spain, in 2007. Eleven percent of cases were fatalities, and 74% of cases were individuals 10 to 24 years old. This is the third cluster associated with this strain in northern Spain since 2001.

  17. Basolateral sorting of chloride channel 2 is mediated by interactions between a dileucine motif and the clathrin adaptor AP-1

    PubMed Central

    de la Fuente-Ortega, Erwin; Gravotta, Diego; Bay, Andres Perez; Benedicto, Ignacio; Carvajal-Gonzalez, Jose Maria; Lehmann, Guillermo L.; Lagos, Carlos F.; Rodríguez-Boulan, Enrique

    2015-01-01

    In spite of the many key cellular functions of chloride channels, the mechanisms that mediate their subcellular localization are largely unknown. ClC-2 is a ubiquitous chloride channel usually localized to the basolateral domain of epithelia that regulates cell volume, ion transport, and acid–base balance; mice knocked out for ClC-2 are blind and sterile. Previous work suggested that CLC-2 is sorted basolaterally by TIFS812LL, a dileucine motif in CLC-2's C-terminal domain. However, our in silico modeling of ClC-2 suggested that this motif was buried within the channel's dimerization interface and identified two cytoplasmically exposed dileucine motifs, ESMI623LL and QVVA635LL, as candidate sorting signals. Alanine mutagenesis and trafficking assays support a scenario in which ESMI623LL acts as the authentic basolateral signal of ClC-2. Silencing experiments and yeast three-hybrid assays demonstrated that both ubiquitous (AP-1A) and epithelium-specific (AP-1B) forms of the tetrameric clathrin adaptor AP-1 are capable of carrying out basolateral sorting of ClC-2 through interactions of ESMI623LL with a highly conserved pocket in their γ1-σ1A hemicomplex. PMID:25739457

  18. Regulation of Inflammatory Cytokine Expression in Pulmonary Epithelial Cells by Pre-B-cell Colony-enhancing Factor via a Nonenzymatic and AP-1-dependent Mechanism*

    PubMed Central

    Liu, Peng; Li, Hailong; Cepeda, Javier; Xia, Yue; Kempf, Jessica A.; Ye, Hong; Zhang, Li Qin; Ye, Shui Qing

    2009-01-01

    Although our previous studies found Pre-B-cell colony-enhancing factor (PBEF) as a highly up-regulated gene in acute lung injury that could stimulate expressions of other inflammatory cytokines, the underlying molecular mechanisms remain to be fully elucidated. Growing evidence indicates that PBEF is a nicotinamide phosphoribosyltransferase involved in the mammalian salvage pathway of NAD synthesis. This study was designed to determine whether the effect of PBEF to stimulate expressions of inflammatory cytokines depends on its enzymatic activity. We prepared two human PBEF mutant (H247E and H247A) recombinant proteins and overexpressing constructs for their overexpressions in A549 cells and confirmed that enzymatic activities of both mutants were nearly or completely abolished. Two mutants stimulated interleukin-8 (IL-8) expression at both the mRNA level and protein level just as equally effective as the wild-type PBEF did. These effects were due to the increased transcription, not the mRNA stability, of the IL-8 gene. Reporter gene assays and gel shift experiments indicated that AP-1 transcription factor is required to mediate these effects. SB203580, a p38 MAPK pathway inhibitor, and JNK inhibitor 1 can attenuate these effects. Both PBEF mutants similarly stimulated the expression of two other inflammatory cytokines: IL-16 and CCR3. These results indicate that PBEF stimulated expression of IL-8, IL-16, and CCR3 via its non-enzymatic activity. This effect is AP-1-dependent, in part via the p38 MAPK pathway and the JNK pathway. This finding reveals a new insight, which may manifest a novel role of PBEF in the pathogenesis of acute lung injury and other inflammatory disorders. PMID:19654329

  19. 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

  20. Possible role of lipid peroxidation in the induction of NF-kappa B and AP-1 in RFL-6 cells by crocidolite asbestos: evidence following protection by vitamin E.

    PubMed

    Faux, S P; Howden, P J

    1997-09-01

    Asbestos fibers cause persistent induction of the oxidative stress sensitive transcription factors nuclear factor kappa-B (NF-kappa B) and activator protein-1 (AP-1) in mammalian cells. These transcription factors play an important role in the regulation of cellular activity. Lipid peroxidation, mediated by reactive oxygen species, is thought to be a possible mechanism in the pathogenicity of asbestos fibers. These studies were designed to determine if crocidolite asbestos-induced lipid peroxidation plays a role in the mechanism of formation of NF-kappa B and AP-1. Treatment of a rat lung fibroblast cell line (RFL-6) with crocidolite asbestos in the presence and absence of the membrane antioxidant vitamin E decreased the levels of crocidolite-induced AP-1 and NF-kappa B to background levels. Preincubation of RFL-6 cells with 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, prior to exposure to crocidolite, abrogated crocidolite-induced NF-kappa B DNA-binding activity to background levels. Coincubation with indomethacin, a cyclooxygenase inhibitor, had no effect on NF-kappa B DNA-binding activity induced by crocidolite. However, nordihydroguaiaretic acid, a lipoxygenase inhibitor, decreased levels of NF-kappa B to background levels. This would suggest that lipoxygenase metabolites of arachidonic acid, produced following lipid peroxidation, are involved in the cellular signalling events to NF-kappa B transcription factor induction by asbestos.

  1. Tumorigenesis by Meis1 overexpression is accompanied by a change of DNA target-sequence specificity which allows binding to the AP-1 element

    PubMed Central

    Dardaei, Leila; Penkov, Dmitry; Mathiasen, Lisa; Bora, Pranami; Morelli, Marco J.; Blasi, Francesco

    2015-01-01

    Meis1 overexpression induces tumorigenicity but its activity is inhibited by Prep1 tumor suppressor. Why does overexpression of Meis1 cause cancer and how does Prep1 inhibit? Tumor profiling and ChIP-sequencing data in a genetically-defined set of cell lines show that: 1) The number of Meis1 and Prep1 DNA binding sites increases linearly with their concentration resulting in a strong increase of “extra” target genes. 2) At high concentration, Meis1 DNA target specificity changes such that the most enriched consensus becomes that of the AP-1 regulatory element, whereas the specific OCTA consensus is not enriched because diluted within the many extra binding sites. 3) Prep1 inhibits Meis1 tumorigenesis preventing the binding to many of the “extra” genes containing AP-1 sites. 4) The overexpression of Prep1, but not of Meis1, changes the functional genomic distribution of the binding sites, increasing seven fold the number of its “enhancer” and decreasing its “promoter” targets. 5) A specific Meis1 “oncogenic” and Prep1 “tumor suppressing” signature has been identified selecting from the pool of genes bound by each protein those whose expression was modified uniquely by the “tumor-inducing” Meis1 or tumor-inhibiting Prep1 overexpression. In both signatures, the enriched gene categories are the same and are involved in signal transduction. However, Meis1 targets stimulatory genes while Prep1 targets genes that inhibit the tumorigenic signaling pathways. PMID:26259236

  2. NF-κB/AP-1-Targeted Inhibition of Macrophage-Mediated Inflammatory Responses by Depigmenting Compound AP736 Derived from Natural 1,3-Diphenylpropane Skeleton

    PubMed Central

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

    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

  3. Dehydroascorbic acid taken up by glucose transporters stimulates estradiol production through inhibition of JNK/c-Jun/AP1 signaling in JAR cells.

    PubMed

    Wang, Yongjie; Tang, Chao; Wu, Minglan; Pan, Yibin; Ruan, Hongfeng; Chen, Linling; Yao, Hongyi; Zhu, Haibin; Wu, Ximei

    2014-08-01

    We have previously demonstrated that the reduced form of vitamin C (l-ascorbic acid, AA) is able to induce the production of both steroid and peptide hormones in human choriocarcinoma cells. Here, we attempted to investigate the role and underlying mechanism of the oxidized form of vitamin C, dehydroascorbic acid (DHA), in steroidogenesis in primary human cytotrophoblasts and human choriocarcinoma cells. Messenger RNA and protein levels of steroidogenic enzymes including P450 cholesterol side-chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1), 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) and aromatase were examined by quantitative RT-PCR and western blots, respectively. Progesterone (P4) and estradiol (E2) levels were determined by enzyme immunoassays. Knockdown of c-Jun was achieved by lentivirus-mediated shRNA, and signaling pathways implicated in DHA-induced steroidogenesis were examined by western blots and dual-luciferase assays. DHA dose-dependently induced the expression of steroidogenic enzymes including 3β-HSD1, 17β-HSD1 and aromatase at both mRNA and protein levels, and subsequently increased the production of E2 but not P4. These effects were synergized by diethylmaleate, a glutathione-depleting compound, and α-tocopherol, a reducing agent, but robustly attenuated by inhibition of DHA transportation by phloretin or 2-deoxy-d-glucose. DHA time-dependently inhibited JNK and c-Jun phosphorylation, and dose-dependently reduced AP1 reporter activity. JNK signaling pathway-specific inhibitor SP600125 and c-Jun shRNA both significantly increased the expression of steroidogenic enzymes and E2 production regardless of the presence or absence of DHA. These findings suggest that DHA is able to induce steroidogenesis through inhibition of JNK/c-Jun/AP1 signaling, and may therefore play indispensable roles in pregnancy maintenance.

  4. The euAP1 protein MPF3 represses MPF2 to specify floral calyx identity and displays crucial roles in Chinese lantern development in Physalis.

    PubMed

    Zhao, Jing; Tian, Ying; Zhang, Ji-Si; Zhao, Man; Gong, Pichang; Riss, Simone; Saedler, Rainer; He, Chaoying

    2013-06-01

    The Chinese lantern phenotype or inflated calyx syndrome (ICS) is a postfloral morphological novelty in Physalis. Its origin is associated with the heterotopic expression of the MADS box gene 2 from Physalis floridana (MPF2) in floral organs, yet the process underlying its identity remains elusive. Here, we show that MPF3, which is expressed specifically in floral tissues, encodes a core eudicot APETALA1-like (euAP1) MADS-domain protein. MPF3 was primarily localized to the nucleus, and it interacted with MPF2 and some floral MADS-domain proteins to selectively bind the CC-A-rich-GG (CArG) boxes in the MPF2 promoter. Downregulating MPF3 resulted in a dramatic elevation in MPF2 in the calyces and androecium, leading to enlarged and leaf-like floral calyces; however, the postfloral lantern was smaller and deformed. Starch accumulation in pollen was blocked. MPF3 MPF2 double knockdowns showed normal floral calyces and more mature pollen than those found in plants in which either MPF3 or MPF2 was downregulated. Therefore, MPF3 specifies calyx identity and regulates ICS formation and male fertility through interactions with MPF2/MPF2. Furthermore, both genes were found to activate Physalis floridana invertase gene 4 homolog, which encodes an invertase cleaving Suc, a putative key gene in sugar partitioning. The novel role of the MPF3-MPF2 regulatory circuit in male fertility is integral to the origin of ICS. Our results shed light on the evolution and development of ICS in Physalis and on the functional evolution of euAP1s in angiosperms.

  5. Lead induces the expression of endoplasmic reticulum chaperones GRP78 and GRP94 in vascular endothelial cells via the JNK-AP-1 pathway.

    PubMed

    Shinkai, Yasuhiro; Yamamoto, Chika; Kaji, Toshiyuki

    2010-04-01

    Lead, a ubiquitous heavy metal, is an important industrial and environmental pollutant that can target the vascular endothelium. To clarify the effects of lead on the unfolded protein response (UPR) and their significance in cytotoxicity, we examined the expression and function of endoplasmic reticulum (ER) chaperones glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) in vascular endothelial cells. We used bovine aortic endothelial cells as an in vitro model of the vascular endothelium. Exposure of vascular endothelial cells to lead nitrate resulted in a marked induction of GRP78 and GRP94 messenger RNA levels. In response to lead, the expression of GRP78 and GRP94 proteins also significantly increased in a dose- and time-dependent manner. In addition, small interfering RNA (siRNA)-mediated knockdown of GRP78 significantly enhanced lead-induced cytotoxicity. Compared with other metal(loid)s, including cadmium chloride, zinc sulfate, copper sulfate, and sodium arsenite, lead nitrate was found to be the most potent metal to induce these chaperones in endothelial cells. In the examined UPR pathways, lead increased the phosphorylation of inositol-requiring enzyme 1 (IRE1) and c-jun N-terminal kinase (JNK). Interestingly, the lead-induced upregulation of GRP78 and GRP94 was almost completely blocked by the JNK inhibitor SP600125 or activator protein-1 (AP-1) inhibitor curcumin. Taken together, these results suggest that lead induces ER stress, but the induction of GRP78 and GRP94 expression via the JNK-AP-1 pathway functions as a defense mechanism against lead-induced cytotoxicity in vascular endothelial cells.

  6. EPO gene expression promotes proliferation, migration and invasion via the p38MAPK/AP-1/MMP-9 pathway by p21WAF1 expression in vascular smooth muscle cells.

    PubMed

    Park, Sung Lyea; Won, Se Yeon; Song, Jun-Hui; Kambe, Taiho; Nagao, Masaya; Kim, Wun-Jae; Moon, Sung-Kwon

    2015-03-01

    The use of recombinant human erythropoietin (rHuEpo) can lead to hypertrophy and hyperplasia, and has induced the proliferation of vascular smooth muscle cells (VSMCs). The effect of the EPO gene in the migration and invasion of VSMCs remains unclear. In this study, overexpression of the EPO gene increased the DNA synthesis and phosphorylation of ERK1/2 and p38MAPK in VSMCs. In addition, EPO gene expression induced the migration and invasion of VSMCs via the expression of MMP-9 by the activation of NF-κB and AP-1 binding. A blockade of p38MAPK by specific p38MAPK inhibitor SB203580 led to a suppression of the increased DNA synthesis, migration, and invasion of VSMCs that was induced by the EPO gene. SB203580 treatment blocked the increased expression of MMP-9 through the binding activity of AP-1. Transfection of the EPO gene with VSMCs was associated with the up-regulation of cyclin D1/CDK4, cyclin E/CDK2, and p21WAF1, and with the down-regulation of p27KIP1. The specific suppression of p21WAF1 expression by siRNA rescued the enhancement of DNA synthesis via the phosphorylation of p38MAPK and the increase in migration and invasion through AP-1-mediated MMP-9 expression in EPO gene transfectants. These novel findings demonstrate that p21WAF1 regulates the proliferation, migration and invasion of VSMC induced by EPO gene.

  7. Berberine attenuates high glucose-induced proliferation and extracellular matrix accumulation in mesangial cells: involvement of suppression of cell cycle progression and NF-κB/AP-1 pathways.

    PubMed

    Lan, Tian; Wu, Teng; Chen, Cheng; Chen, Xiaolan; Hao, Jie; Huang, Junying; Wang, Lijing; Huang, Heqing

    2014-03-25

    Berberine has been shown to have renoprotective effects on diabetes through attenuating TGF-β1 and fibronectin (FN) expression. However, how berberine regulates TGF-β1 and FN is not fully clear. Here we investigated whether berberine inhibited TGF-β1 and FN expression in high glucose-cultured mesangial cells. Berberine significantly inhibited mesangial cell proliferation and hypertrophy by increasing the cell population in G1-phase and reducing that in S-phase. In addition, berberine reversed high glucose-induced down-regulation of cyclin-dependent kinase inhibitor p21(Waf1)/(Cip1) and p27(Kip1). Berberine inhibited p65 translocation to the nucleus and c-jun phosphorylation induced by high glucose. Furthermore, berberine attenuated high glucose-induced expression of TGF-β1 and FN. Using a luciferase reporter assay, we found that high glucose-induced transcription activity of NF-κB and AP-1 was blocked by berberine. Electrophoretic mobility shift assay showed that high glucose increased that NF-κB and AP-1 DNA binding activity. These data indicate that berberine inhibited mesangial cell proliferation and hypertrophy by modulating cell cycle progress. In addition, berberine suppressed high glucose-induced TGF-β1 and FN expression by blocking NF-κB/AP-1 pathways.

  8. Signaling through P2X7 receptor in human T cells involves p56lck, MAP kinases, and transcription factors AP-1 and NF-kappa B.

    PubMed

    Budagian, Vadim; Bulanova, Elena; Brovko, Luba; Orinska, Zane; Fayad, Raja; Paus, Ralf; Bulfone-Paus, Silvia

    2003-01-17

    ATP-gated ion channel P2X receptors are expressed on the surface of most immune cells and can trigger multiple cellular responses, such as membrane permeabilization, cytokine production, and cell proliferation or apoptosis. Despite broad distribution and pleiotropic activities, signaling pathways downstream of these ionotropic receptors are still poorly understood. Here, we describe intracellular signaling events in Jurkat cells treated with millimolar concentrations of extracellular ATP. Within minutes, ATP treatment resulted in the phosphorylation and activation of p56(lck) kinase, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase but not p38 kinase. These effects were wholly dependent upon the presence of extracellular Ca(2+) ions in the culture medium. Nevertheless, calmodulin antagonist calmidazolium and CaM kinase inhibitor KN-93 both had no effect on the activation of p56(lck) and ERK, whereas a pretreatment of Jurkat cells with MAP kinase kinase inhibitor P098059 was able to abrogate phosphorylation of ERK. Further, expression of c-Jun and c-Fos proteins and activator protein (AP-1) DNA binding activity were enhanced in a time-dependent manner. In contrast, DNA binding activity of NF-kappa B was reduced. ATP failed to stimulate the phosphorylation of ERK and c-Jun N-terminal kinase and activation of AP-1 in the p56(lck)-deficient isogenic T cell line JCaM1, suggesting a critical role for p56(lck) kinase in downstream signaling. Regarding the biological significance of the ATP-induced signaling events we show that although extracellular ATP was able to stimulate proliferation of both Jurkat and JCaM1 cells, an increase in interleukin-2 transcription was observed only in Jurkat cells. The nucleotide selectivity and pharmacological profile data supported the evidence that the ATP-induced effects in Jurkat cells were mediated through the P2X7 receptor. Taken together, these results demonstrate the ability of extracellular ATP to activate

  9. CXCL12 induces connective tissue growth factor expression in human lung fibroblasts through the Rac1/ERK, JNK, and AP-1 pathways.

    PubMed

    Lin, Chien-Huang; Shih, Chung-Huang; Tseng, Chih-Chieh; Yu, Chung-Chi; Tsai, Yuan-Jhih; Bien, Mauo-Ying; Chen, Bing-Chang

    2014-01-01

    CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression.

  10. CXCL12 Induces Connective Tissue Growth Factor Expression in Human Lung Fibroblasts through the Rac1/ERK, JNK, and AP-1 Pathways

    PubMed Central

    Tseng, Chih-Chieh; Yu, Chung-Chi; Tsai, Yuan-Jhih; Bien, Mauo-Ying; Chen, Bing-Chang

    2014-01-01

    CXCL12 (stromal cell-derived factor-1, SDF-1) is a potent chemokine for homing of CXCR4+ fibrocytes to injury sites of lung tissue, which contributes to pulmonary fibrosis. Overexpression of connective tissue growth factor (CTGF) plays a critical role in pulmonary fibrosis. In this study, we investigated the roles of Rac1, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein-1 (AP-1) in CXCL12-induced CTGF expression in human lung fibroblasts. CXCL12 caused concentration- and time-dependent increases in CTGF expression and CTGF-luciferase activity. CXCL12-induced CTGF expression was inhibited by a CXCR4 antagonist (AMD3100), small interfering RNA of CXCR4 (CXCR4 siRNA), a dominant negative mutant of Rac1 (RacN17), a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor (PD98059), a JNK inhibitor (SP600125), a p21-activated kinase inhibitor (PAK18), c-Jun siRNA, and an AP-1 inhibitor (curcumin). Treatment of cells with CXCL12 caused activations of Rac1, Rho, ERK, and c-Jun. The CXCL12-induced increase in ERK phosphorylation was inhibited by RacN17. Treatment of cells with PD98059 and SP600125 both inhibited CXCL12-induced c-Jun phosphorylation. CXCL12 caused the recruitment of c-Jun and c-Fos binding to the CTGF promoter. Furthermore, CXCL12 induced an increase in α-smooth muscle actin (α-SMA) expression, a myofibroblastic phenotype, and actin stress fiber formation. CXCL12-induced actin stress fiber formation and α-SMA expression were respectively inhibited by AMD3100 and CTGF siRNA. Taken together, our results suggest that CXCL12, acting through CXCR4, activates the Rac/ERK and JNK signaling pathways, which in turn initiates c-Jun phosphorylation, and recruits c-Jun and c-Fos to the CTGF promoter and ultimately induces CTGF expression in human lung fibroblasts. Moreover, overexpression of CTGF mediates CXCL12-induced α-SMA expression. PMID:25121739

  11. Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors*

    PubMed Central

    Stahlschmidt, Wiebke; Robertson, Mark J.; Robinson, Phillip J.; McCluskey, Adam; Haucke, Volker

    2014-01-01

    Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane. PMID:24407285

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

    PubMed Central

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

    2016-01-01

    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

  13. Clathrin terminal domain-ligand interactions regulate sorting of mannose 6-phosphate receptors mediated by AP-1 and GGA adaptors.

    PubMed

    Stahlschmidt, Wiebke; Robertson, Mark J; Robinson, Phillip J; McCluskey, Adam; Haucke, Volker

    2014-02-21

    Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.

  14. o,p'-DDT induces cyclooxygenase-2 gene expression in murine macrophages: Role of AP-1 and CRE promoter elements and PI3-kinase/Akt/MAPK signaling pathways

    SciTech Connect

    Han, Eun Hee; Kim, Ji Young; Kim, Hyung-Kyun; Hwang, Yong Pil; Jeong, Hye Gwang

    2008-12-01

    Dichlorodiphenyltrichloroethane (DDT) has been used as an insecticide to prevent the devastation of malaria in tropical zones. However, many reports suggest that DDT may act as an endocrine disruptor and may have possible carcinogenic effects. Cyclooxygenase-2 (COX-2) acts as a link between inflammation and carcinogenesis through its involvement in tumor promotion. In the present study, we examined the effect of o,p'-DDT on COX-2 gene expression and analyzed the molecular mechanism of its activity in murine RAW 264.7 macrophages. Exposure to o,p'-DDT markedly enhanced the production of prostaglandin E{sub 2} (PGE{sub 2}), a major COX-2 metabolite, in murine macrophages. Furthermore, o,p'-DDT dose-dependently increased the levels of COX-2 protein and mRNA. Transfection with human COX-2 promoter construct, electrophoretic mobility shift assays and DNA-affinity protein-binding assay experiments revealed that o,p'-DDT activated the activator protein 1 (AP-1) and cyclic AMP response element (CRE) sites, but not the NF-{kappa}B site. Phosphatidylinositol 3 (PI3)-kinase, its downstream signaling molecule, Akt, and mitogen-activated protein kinases (MAPK) were also significantly activated by the o,p'-DDT-induced AP-1 and CRE activation. These results demonstrate that o,p'-DDT induced COX-2 expression via AP-1 and CRE activation through the PI3-K/Akt/ERK, JNK, and p38 MAP kinase pathways. These findings provide further insight into the signal transduction pathways involved in the carcinogenic effects of o,p'-DDT.

  15. Control of E-cadherin apical localisation and morphogenesis by a SOAP-1/AP-1/clathrin pathway in C. elegans epidermal cells.

    PubMed

    Gillard, Ghislain; Shafaq-Zadah, Massiullah; Nicolle, Ophélie; Damaj, Raghida; Pécréaux, Jacques; Michaux, Grégoire

    2015-05-01

    E-cadherin (E-cad) is the main component of epithelial junctions in multicellular organisms, where it is essential for cell-cell adhesion. The localisation of E-cad is often strongly polarised in the apico-basal axis. However, the mechanisms required for its polarised distribution are still largely unknown. We performed a systematic RNAi screen in vivo to identify genes required for the strict E-cad apical localisation in C. elegans epithelial epidermal cells. We found that the loss of clathrin, its adaptor AP-1 and the AP-1 interactor SOAP-1 induced a basolateral localisation of E-cad without affecting the apico-basal diffusion barrier. We further found that SOAP-1 controls AP-1 localisation, and that AP-1 is required for clathrin recruitment. Finally, we also show that AP-1 controls E-cad apical delivery and actin organisation during embryonic elongation, the final morphogenetic step of embryogenesis. We therefore propose that a molecular pathway, containing SOAP-1, AP-1 and clathrin, controls the apical delivery of E-cad and morphogenesis.

  16. Overexpression of AtAP1M3 regulates flowering time and floral development in Arabidopsis and effects key flowering-related genes in poplar.

    PubMed

    Chen, Zhong; Ye, Meixia; Su, Xiaoxing; Liao, Weihua; Ma, Huandi; Gao, Kai; Lei, Bingqi; An, Xinmin

    2015-08-01

    APETALA1 plays a crucial role in the transition from vegetative to reproductive phase and in floral development. In this study, to determine the effect of AP1 expression on flowering time and floral organ development, transgenic Arabidopsis and poplar overexpressing of AtAP1M3 (Arabidopsis AP1 mutant by dominant negative mutation) were generated. Transgenic Arabidopsis with e35Spro::AtAP1M3 displayed phenotypes with delayed-flowering compared to wild-type and flowers with abnormal sepals, petals and stamens. In addition, transgenic Arabidopsis plants exhibited reduced growth vigor compared to the wild-type plants. Ectopic expression of AtAP1M3 in poplar resulted in up- or down-regulation of some endogenous key flowering-related genes, including floral meristems identity gene LFY, B-class floral organ identity genes AP3 and PI, flowering pathway integrator FT1 and flower repressors TFL1 and SVP. These results suggest that AtAP1M3 regulates flowering time and floral development in plants.

  17. Thrombin mediates migration of rat brain astrocytes via PLC, Ca²⁺, CaMKII, PKCα, and AP-1-dependent matrix metalloproteinase-9 expression.

    PubMed

    Lin, Chih-Chung; Lee, I-Ta; Wu, Wen-Bin; Liu, Chiung-Ju; Hsieh, Hsi-Lung; Hsiao, Li-Der; Yang, Chien-Chung; Yang, Chuen-Mao

    2013-12-01

    Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca(2+)-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca(2+) concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca(2+)/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.

  18. Two Mechanisms Regulate Keratin K15 Expression In Keratinocytes: Role of PKC/AP-1 and FOXM1 Mediated Signalling

    PubMed Central

    Bose, Amrita; Teh, Muy-Teck; Hutchison, Iain L.; Wan, Hong; Leigh, Irene M.; Waseem, Ahmad

    2012-01-01

    Background Keratin 15 (K15) is a type I keratin that is used as a marker of stem cells. Its expression is restricted to the basal layer of stratified epithelia, and the bulge in hair follicles. However, in certain clinical situations including oral lichen planus, K15 is induced in suprabasal layers, which is inconsistent with the role of a stem cell marker. This study provides insights into the mechanisms of K15 expression in the basal and differentiating keratinocytes. Methodology/Principal Findings Human keratinocytes were differentiated by three different methods; suspension in methylcellulose, high cell density and treatment with phorbol ester. The expression of mRNA was determined by quantitative PCR and protein by western blotting and immunostaining. Keratinocytes in suspension suppressed β1-integrin expression, induced differentiation-specific markers and K15, whereas FOXM1 (a cell cycle regulated protein) and K14 were downregulated. Rescuing β1-integrin by either fibronectin or the arginine-glycine-aspartate peptide suppressed K15 but induced K14 and FOXM1 expression. Specific inhibition of PKCδ, by siRNA, and AP-1 transcription factor, by TAM67 (dominant negative c-Jun), suppressed K15 expression, suggesting that PKC/AP-1 pathway plays a role in the differentiation-specific expression of K15. The basal cell-specific K15 expression may involve FOXM1 because ectopic expression of the latter is known to induce K15. Using chromatin immunoprecipitation, we have identified a single FOXM1 binding motif in the K15 promoter. Conclusions/Significance The data suggests that K15 is induced during terminal differentiation mediated by the down regulation of β1-integrin. However, this cannot be the mechanism of basal/stem cell-specific K15 expression in stratified epithelia, because basal keratinocytes do not undergo terminal differentiation. We propose that there are two mechanisms regulating K15 expression in stratified epithelia; differentiation-specific involving

  19. Norepinephrine Controls Effector T Cell Differentiation through β2-Adrenergic Receptor-Mediated Inhibition of NF-κB and AP-1 in Dendritic Cells.

    PubMed

    Takenaka, Maisa Carla; Araujo, Leandro Pires; Maricato, Juliana Terzi; Nascimento, Vanessa M; Guereschi, Marcia Grando; Rezende, Rafael Machado; Quintana, Francisco J; Basso, Alexandre S

    2016-01-15

    Despite accumulating evidence indicating that neurotransmitters released by the sympathetic nervous system can modulate the activity of innate immune cells, we still know very little about how norepinephrine impacts signaling pathways in dendritic cells (DC) and the consequence of that in DC-driven T cell differentiation. In this article, we demonstrate that β2-adrenergic receptor (β2AR) activation in LPS-stimulated DC does not impair their ability to promote T cell proliferation; however, it diminishes IL-12p70 secretion, leading to a shift in the IL-12p70/IL-23 ratio. Although β2AR stimulation in DC induces protein kinase A-dependent cAMP-responsive element-binding protein phosphorylation, the effect of changing the profile of cytokines produced upon LPS challenge occurs in a protein kinase A-independent manner and, rather, is associated with inhibition of the NF-κB and AP-1 signaling pathways. Moreover, as a consequence of the inverted IL-12p70/IL-23 ratio following β2AR stimulation, LPS-stimulated DC promoted the generation of CD4(+) T cells that, upon TCR engagement, produced lower amounts of IFN-γ and higher levels of IL-17. These findings provide new insights into molecular and cellular mechanisms by which β2AR stimulation in murine DC can influence the generation of adaptive immune responses and may explain some aspects of how sympathetic nervous system activity can modulate immune function.

  20. 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

  1. JNK/ERK-AP-1/Runx2 induction "paves the way" to cartilage load-ignited chondroblastic differentiation.

    PubMed

    Papachristou, Dionysios J; Pirttiniemi, Pertti; Kantomaa, Tuomo; Papavassiliou, Athanasios G; Basdra, Efthimia K

    2005-09-01

    Chondro-osteogenesis and subsequently skeletal morphology are greatly influenced by mechanical loads. The exact mechanism(s) by which mechanical stimuli are transduced in chondrocytes remains obscure and appears to be equally complex with similar signal transducing systems. Here we investigated whether and to what extent the MAPK (JNK/ERK)-AP-1/Runx2 signaling pathways are engaged in this phenomenon, and assessed their involvement in the functional biology of articular cartilage. For this purpose, 14-day-old female Wistar rats were divided into 2 groups: the first group was fed hard diet (simulating physiologic temporomandibular joint (TMJ) loading), while the second group was fed soft diet (reduced TMJ loading). On day 21 (experiment initiation day - weaning day), biopsies from condyles of both groups were obtained after 6, 12 and 48 h of functional TMJ loading. Immunohistochemical methodology was employed to evaluate the expression levels of pc-Jun, c-Fos, JNK2, p-JNK, p-ERK and Runx2 due to alteration in functional load. Our data demsonstrate that the protein levels of all the aforementioned molecules were markedly increased in animals fed with the hard diet, throughout the experimental procedure. These results indicate that functional cartilage loading induces the AP-1 and Runx2 transcription factors through the JNK and ERK MAPK cascades. In as much as the above signaling mediators/effectors are considered to be crucial in the differentiation/maturation process of cartilage tissue, we pose that functional mechanical loading of condylar cartilage serves to "fine tune" chondroblastic differentiation/maturation.

  2. Suppression of acetylpolyamine oxidase by selected AP-1 members regulates DNp73 abundance: mechanistic insights for overcoming DNp73-mediated resistance to chemotherapeutic drugs

    PubMed Central

    Bunjobpol, W; Dulloo, I; Igarashi, K; Concin, N; Matsuo, K; Sabapathy, K

    2014-01-01

    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

  3. Transcriptional Network Analysis in Muscle Reveals AP-1 as a Partner of PGC-1α in the Regulation of the Hypoxic Gene Program

    PubMed Central

    Baresic, Mario; Salatino, Silvia; Kupr, Barbara

    2014-01-01

    Skeletal muscle tissue shows an extraordinary cellular plasticity, but the underlying molecular mechanisms are still poorly understood. Here, we use a combination of experimental and computational approaches to unravel the complex transcriptional network of muscle cell plasticity centered on the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a regulatory nexus in endurance training adaptation. By integrating data on genome-wide binding of PGC-1α and gene expression upon PGC-1α overexpression with comprehensive computational prediction of transcription factor binding sites (TFBSs), we uncover a hitherto-underestimated number of transcription factor partners involved in mediating PGC-1α action. In particular, principal component analysis of TFBSs at PGC-1α binding regions predicts that, besides the well-known role of the estrogen-related receptor α (ERRα), the activator protein 1 complex (AP-1) plays a major role in regulating the PGC-1α-controlled gene program of the hypoxia response. Our findings thus reveal the complex transcriptional network of muscle cell plasticity controlled by PGC-1α. PMID:24912679

  4. Transcriptional network analysis in muscle reveals AP-1 as a partner of PGC-1α in the regulation of the hypoxic gene program.

    PubMed

    Baresic, Mario; Salatino, Silvia; Kupr, Barbara; van Nimwegen, Erik; Handschin, Christoph

    2014-08-01

    Skeletal muscle tissue shows an extraordinary cellular plasticity, but the underlying molecular mechanisms are still poorly understood. Here, we use a combination of experimental and computational approaches to unravel the complex transcriptional network of muscle cell plasticity centered on the peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a regulatory nexus in endurance training adaptation. By integrating data on genome-wide binding of PGC-1α and gene expression upon PGC-1α overexpression with comprehensive computational prediction of transcription factor binding sites (TFBSs), we uncover a hitherto-underestimated number of transcription factor partners involved in mediating PGC-1α action. In particular, principal component analysis of TFBSs at PGC-1α binding regions predicts that, besides the well-known role of the estrogen-related receptor α (ERRα), the activator protein 1 complex (AP-1) plays a major role in regulating the PGC-1α-controlled gene program of the hypoxia response. Our findings thus reveal the complex transcriptional network of muscle cell plasticity controlled by PGC-1α.

  5. The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter

    PubMed Central

    1992-01-01

    Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at - 150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c- jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter. PMID:1740667

  6. Stimulatory heterotrimeric G protein augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 human lung cancer cells.

    PubMed

    Choi, Yoon Jung; Kim, So Young; Oh, Jung Min; Juhnn, Yong Sung

    2009-08-31

    Stimulatory heterotrimeric GTP-binding proteins (Gs protein) stimulate cAMP generation in response to various signals, and modulate various cellular phenomena such as proliferation and apoptosis. This study aimed to investigate the effect of Gs proteins on gamma ray-induced apoptosis of lung cancer cells and its molecular mechanism, as an attempt to develop a new strategy to improve the therapeutic efficacy of gamma radiation. Expression of constitutively active mutant of the alpha subunit of Gs (GalphasQL) augmented gamma ray-induced apoptosis via mitochondrial dependent pathway when assessed by clonogenic assay, FACS analysis of PI stained cells, and western blot analysis of the cytoplasmic translocation of cytochrome C and the cleavage of caspase-3 and ploy(ADP-ribose) polymerase (PARP) in H1299 human lung cancer cells. GalphasQL up-regulated the Bak expression at the levels of protein and mRNA. Treatment with inhibitors of PKA (H89), SP600125 (JNK inhibitor), and a CRE-decoy blocked GalphasQL-stimulated Bak reporter luciferase activity. Expression of GalphasQL increased basal and gamma ray-induced luciferase activity of cAMP response element binding protein (CREB) and AP-1, and the binding of CREB and AP-1 to Bak promoter. Furthermore, prostaglandin E2, a Galphas activating signal, was found to augment gamma ray-induced apoptosis, which was abolished by treatment with a prostanoid receptor antagonist. These results indicate that Galphas augments gamma ray-induced apoptosis by up-regulation of Bak expression via CREB and AP-1 in H1299 lung cancer cells, suggesting that the efficacy of radiotherapy of lung cancer may be improved by modulating Gs signaling pathway.

  7. 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.

  8. Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity.

    PubMed Central

    Riechmann, J L; Meyerowitz, E M

    1997-01-01

    The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) combinatorially specify the identity of Arabidopsis floral organs. AP1/AP1, AG/AG, and AP3/PI dimers bind to similar CArG box sequences; thus, differences in DNA-binding specificity among these proteins do not seem to be the origin of their distinct organ identity properties. To assess the overall contribution that specific DNA binding could make to their biological specificity, we have generated chimeric genes in which the amino-terminal half of the MADS domain of AP1, AP3, PI, and AG was substituted by the corresponding sequences of human SRF and MEF2A proteins. In vitro DNA-binding assays reveal that the chimeric proteins acquired the respective, and distinct, DNA-binding specificity of SRF or MEF2A. However, ectopic expression of the chimeric genes reproduces the dominant gain-of-function phenotypes exhibited by plants ectopically expressing the corresponding Arabidopsis wild-type genes. In addition, both the SRF and MEF2 chimeric genes can complement the pertinent ap1-1, ap3-3, pi-1, or ag-3 mutations to a degree similar to that of AP1, AP3, PI, and AG when expressed under the control of the same promoter. These results indicate that determination of floral organ identity by the MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. In addition, the DNA-binding experiments show that either one of the two MADS domains of a dimer can be sufficient to confer a particular DNA-binding specificity to the complex and that sequences outside the amino-terminal basic region of the MADS domain can, in some cases, contribute to the DNA-binding specificity of the proteins. Images PMID:9243505

  9. Effects of AP-1 and NF-κB inhibitors on colonic endocrine cells in rats with TNBS-induced colitis

    PubMed Central

    El-Salhy, Magdy; Umezawa, Kazuo

    2016-01-01

    Interactions between intestinal neuroendocrine peptides/amines and the immune system appear to have an important role in the pathophysiology of inflammatory bowel disease (IBD). The present study investigated the effects of activator protein (AP)-1 and nuclear factor (NF)-κB inhibitors on inflammation-induced alterations in enteroendocrine cells. A total of 48 male Wistar rats were divided into the following four groups (n=12 rats/group): Control, trinitrobenzene sulfonic acid (TNBS)-induced colitis only (TNBS group), TNBS-induced colitis with 3-[(dodecylthiocarbonyl)-methyl]-glutarimide (DTCM-G) treatment (DTCM-G group), and TNBS-induced colitis with dehydroxymethylepoxyquinomicin (DHMEQ) treatment (DHMEQ group). A total of 3 days following administration of TNBS, the rats were treated as follows: The control and TNBS groups received 0.5 ml vehicle (0.5% carboxymethyl cellulose; CMC), respectively; the DTCM-G group received DTCM-G (20 mg/kg body weight) in 0.5% CMC; and the DHMEQ group received DHMEQ (15 mg/kg body weight) in 0.5% CMC. All injections were performed intraperitoneally twice daily for 5 days. The rats were sacrificed, and tissue samples obtained from the colon were examined histopathologically and immunohistochemically. Inflammation was evaluated using a scoring system. In addition, the sections were immunostained for chromogranin A (CgA), serotonin, peptide YY (PYY), oxyntomodulin, pancreatic polypeptide (PP) and somatostatin, and immunostaining was quantified using image-analysis software. The density of cells expressing CgA, PYY and PP was significantly lower in the TNBS group compared with in the control group, whereas the density of cells expressing serotonin, oxyntomodulin and somatostatin was significantly higher in the TNBS group compared with in the control group. None of the endocrine cell types differed significantly between the control group and either the DTCM-G or DHMEQ groups. All of the colonic endocrine cell types were affected in

  10. Induction of Connective Tissue Growth Factor Expression by Hypoxia in Human Lung Fibroblasts via the MEKK1/MEK1/ERK1/GLI-1/GLI-2 and AP-1 Pathways

    PubMed Central

    Cheng, Yi; Lin, Chien-huang; Chen, Jing-Yun; Li, Chien-Hua; Liu, Yu-Tin; Chen, Bing-Chang

    2016-01-01

    Several reports have indicated that hypoxia, GLI, and connective tissue growth factor (CTGF) contribute to pulmonary fibrosis in idiopathic pulmonary fibrosis. We investigated the participation of mitogen-activated protein kinase kinase (MEK) kinase 1 (MEKK1)/MEK1/ERK1/GLI-1/2 and activator protein-1 (AP-1) signaling in hypoxia-induced CTGF expression in human lung fibroblasts. Hypoxia time-dependently increased CTGF expression, which was attenuated by the small interfering RNA (siRNA) of GLI-1 (GLI-1 siRNA) and GLI-2 (GLI-2 siRNA) in both human lung fibroblast cell line (WI-38) and primary human lung fibroblasts (NHLFs). Moreover, GLI-1 siRNA and GLI-2 siRNA attenuated hypoxia-induced CTGF-luciferase activity, and the treatment of cells with hypoxia induced GLI-1 and GLI-2 translocation. Furthermore, hypoxia-induced CTGF expression was reduced by an MEK inhibitor (PD98059), MEK1 siRNA, ERK inhibitor (U0126), ERK1 siRNA, and MEKK1 siRNA. Both PD98059 and U0126 significantly attenuated hypoxia-induced CTGF-luciferase activity. Hypoxia time-dependently increased MEKK1, ERK, and p38 MAPK phosphorylation. Moreover, SB203580 (a p38 MAPK inhibitor) also apparently inhibited hypoxia-induced CTGF expression. The treatment of cells with hypoxia induced ERK, GLI-1, or GLI-2 complex formation. Hypoxia-induced GLI-1 and GLI-2 translocation into the nucleus was significantly attenuated by U0126. In addition, hypoxia-induced ERK Tyr204 phosphorylation was impeded by MEKK1 siRNA. Moreover, hypoxia-induced CTGF-luciferase activity was attenuated by cells transfected with AP-1 site mutation in a CTGF construct. Exposure to hypoxia caused a time-dependent phosphorylation of c-Jun, but not of c-Fos. Chromatin immunoprecipitation (ChIP) revealed that hypoxia induced the recruitment of c-Jun, GLI-1, and GLI-2 to the AP-1 promoter region of CTGF. Hypoxia-treated cells exhibited an increase in α-smooth muscle actin (α-SMA) and collagen production, which was blocked by GLI-1 siRNA and

  11. Human kidney anion exchanger 1 interacts with adaptor-related protein complex 1 {mu}1A (AP-1 mu1A)

    SciTech Connect

    Sawasdee, Nunghathai; Junking, Mutita; Ngaojanlar, Piengpaga; Sukomon, Nattakan; Ungsupravate, Duangporn; Limjindaporn, Thawornchai; Akkarapatumwong, Varaporn; Noisakran, Sansanee; Yenchitsomanus, Pa-thai

    2010-10-08

    Research highlights: {yields} Trafficking defect of kAE1 is a cause of dRTA but trafficking pathway of kAE1 has not been clearly described. {yields} Adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) was firstly reported to interact with kAE1. {yields} The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. {yields} AP-1 mu1A knockdown showed a marked reduction of kAE1 on the cell membrane and its accumulation in endoplasmic reticulum. {yields} AP-1 mu1A has a critical role in kAE1 trafficking to the plasma membrane. -- Abstract: Kidney anion exchanger 1 (kAE1) mediates chloride (Cl{sup -}) and bicarbonate (HCO{sub 3}{sup -}) exchange at the basolateral membrane of kidney {alpha}-intercalated cells. Impaired trafficking of kAE1 leads to defect of the Cl{sup -}/HCO{sub 3}{sup -} exchange at the basolateral membrane and failure of proton (H{sup +}) secretion at the apical membrane, causing a kidney disease - distal renal tubular acidosis (dRTA). To gain a better insight into kAE1 trafficking, we searched for proteins physically interacting with the C-terminal region of kAE1 (Ct-kAE1), which contains motifs crucial for intracellular trafficking, by a yeast two-hybrid (Y2H) system. An adaptor-related protein complex 1 {mu}1A (AP-1 mu1A) subunit was found to interact with Ct-kAE1. The interaction between either Ct-kAE1 or full-length kAE1 and AP-1 mu1A were confirmed in human embryonic kidney (HEK) 293T by co-immunoprecipitation, affinity co-purification, co-localization, yellow fluorescent protein (YFP)-based protein fragment complementation assay (PCA) and GST pull-down assay. The interacting site for AP-1 mu1A on Ct-kAE1 was found to be Y904DEV907, a subset of YXXO motif. Interestingly, suppression of endogenous AP-1 mu1A in HEK 293T by small interfering RNA (siRNA) decreased membrane localization of kAE1 and increased its intracellular accumulation, suggesting for the first time that AP-1 mu1A is involved in the kAE1

  12. The designed protein M(II)-Gly-Lys-His-Fos(138-211) specifically cleaves the AP-1 binding site containing DNA.

    PubMed

    Harford, C; Narindrasorasak, S; Sarkar, B

    1996-04-09

    A new specific DNA cleavage protein, Gly-Lys-His-Fos(138-211), was designed, expressed, and characterized. The DNA-binding component of the design uses the basic and leucine zipper regions of the leucine zipper Fos, which are represented by Fos(138-211). The DNA cleavage moiety was provided by the design of the amino-terminal Cu(II)-, Ni(II)-binding site GKH at the amino terminus of Fos(138-211). Binding of Cu(II) or Ni(II) by the protein activates its cleavage ability. The GKH motif was predicted to form a specific amino-terminal Cu(II)-, Ni(II)-binding motif as previously defined [Predki, P. F., Harford, C., Brar, P., & Sarkar, B. (1992) Biochem. J. 287, 211 -215]. This prediction was verified as the tripeptide, GKH, and the expressed protein, GKH-Fos(138-211), were both shown to be capable of binding Cu(II) and Ni(II). The designed protein upon heterodimerization with Jun(248-334) was shown to bind to and cleave several forms of DNA which contained an AP-1 binding site. The cleavage was shown to be specific. This design demonstrates the versatility of the amino-terminal Cu(II)-, Ni(II)-binding motif and the variety of motifs which can be generated. The site of cleavage by GKH-Fos(138-211) on DNA provides further information regarding the bending of DNA upon binding to Fos-Jun heterodimers.

  13. 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.

  14. Curcumin nanoparticles ameliorate ICAM-1 expression in TNF-α-treated lung epithelial cells through p47 (phox) and MAPKs/AP-1 pathways.

    PubMed

    Yen, Feng-Lin; Tsai, Ming-Horng; 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.

  15. ZN2+-INDUCED IL-8 EXPRESSION INVOLVES AP-1, JNK, AND ERK ACTIVITIES IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    Exposure to zinc-laden particulate matter (PM) in ambient and occupational settings has been associated with proinflammatory responses in the lung. IL-8 is an important proinflammatory cytokine in the human lung and is induced in human airway epithelial cells exposed to zin...

  16. A novel bipartite UNC-101/AP-1 μ1 binding signal mediates KVS-4/Kv2.1 somatodendritic distribution in Caenorhabditis elegans.

    PubMed

    Zhou, Xin; Zeng, Jia; Ouyang, Chenxi; Luo, Qianyun; Yu, Miao; Yang, Zhenrong; Wang, Hui; Shen, Kang; Shi, Anbing

    2016-01-01

    Potassium channels such as Kv2.1 are targeted to specific subcellular compartments to fulfill various functions. However, the mechanisms for their localization are poorly understood. Here, we show that KVS-4/Kv2.1 somatodendritic localization in Caenorhabditis elegansDA9 neuron requires UNC-101(AP-1 μ subunit). We define a bipartite sorting signal within KVS-4 consisting of a C-terminal EQMIL and N-terminal WNIIE motifs. The bipartite signal is sufficient to target nonpolarized transmembrane protein MIG-13 into DA9 somatodendritic compartments. Furthermore, we found that AP-1 interacts with the bipartite signal through UNC-101/AP-1 μ N-terminal predicted Longin-like domain. Our results provide new insight into the mechanisms of Kv2.1 post-Golgi sorting and targeting.

  17. 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.

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

    PubMed Central

    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. 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. Chikusetsusaponin IVa Methyl Ester Isolated from the Roots of Achyranthes japonica Suppresses LPS-Induced iNOS, TNF-α, IL-6, and IL-1β Expression by NF-κB and AP-1 Inactivation.

    PubMed

    Lee, Hae-Jun; Shin, Ji-Sun; Lee, Woo-Seok; Shim, Heon-Yong; Park, Ji-Min; Jang, Dae-Sik; Lee, Kyung-Tae

    2016-01-01

    We investigated the effect of chikusetsusaponin IVa (CS) and chikusetsusaponin IVa methyl ester (CS-ME) from the roots of Achyranthes japonica NAKAI on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in RAW264.7 macrophages. CS-ME more potently inhibited LPS-induced NO and PGE2 production than CS. CS-ME concentration-dependently inhibited LPS-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 and IL-1β production in RAW264.7 macrophages and mouse peritoneal macrophages. Consistent with these findings, CS-ME suppressed LPS-induced expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 at protein level as well as iNOS, COX-2, TNF-α, IL-6, and IL-1β at mRNA level. In addition, CS-ME suppressed LPS-induced transcriptional activity of nuclear factor (NF)-κB and activator protein (AP)-1. The anti-inflammatory properties of CS-ME might result from suppression of iNOS, COX-2, TNF-α, IL-6, and IL-1β expression through downregulation of NF-κB and AP-1 in macrophages.

  1. Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways

    PubMed Central

    Yang, Lichao; Guo, Han; Li, Ying; Meng, Xianglan; Yan, Lu; Dan Zhang; Wu, Sangang; Zhou, Hao; Peng, Lu; Xie, Qiang; Jin, Xin

    2016-01-01

    The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 μg/ml) in the presence or absence of OEA (10, 20 and 40 μM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases. PMID:27721381

  2. Differential expression of AP-1 transcription factor genes c-fos and c-jun in the helminth parasites Taenia crassiceps and Taenia solium.

    PubMed

    Morales-Montor, J; Escobedo, G; Rodriguez-Dorantes, M; Téllez-Ascencio, N; Cerbón, M A; Larralde, C

    2004-08-01

    Homologues of c-fos and c-jun from total DNA of Taenia crassiceps and Taenia solium were cloned and sequenced. The amino acid alignment analysis revealed that c-fos DNAs from T. crassiceps and T. solium were highly homologous (96%), and both have high homology compared to several mammalian c-fos proteins (93% to mouse, 96% to rat and 86% to human). The c-jun protein alignment showed higher homology (T. crassiceps and T. solium have 98%), when compared with mouse, rat and human, being 92%, 98% and 93% respectively. RT-PCR amplification of the parasite's total RNA, showed that T. crassiceps expressed both AP-1 complex genes, while T. solium only expressed c-fos. Southern blot hybridization analysis confirmed the true origin of each amplified gene. AP-1 transcription gene expression is regulated by oestradiol in the same fashion as their mammalian counterparts only in T. crassiceps. To study if AP-1 genes are involved in a physiological function of the cyst, reproduction was studied in vitro. Oestradiol treatment stimulated reproduction in T. crassiceps but not in T. solium cysticerci. This is the first report of the detection and functionality of AP-1 transcription factor genes in any species of helminth parasite.

  3. Polarized sorting of the copper transporter ATP7B in neurons mediated by recognition of a dileucine signal by AP-1.

    PubMed

    Jain, Shweta; Farías, Ginny G; Bonifacino, Juan S

    2015-01-15

    Neurons are highly polarized cells having distinct somatodendritic and axonal domains. Here we report that polarized sorting of the Cu(2+) transporter ATP7B and the vesicle-SNARE VAMP4 to the somatodendritic domain of rat hippocampal neurons is mediated by recognition of dileucine-based signals in the cytosolic domains of the proteins by the σ1 subunit of the clathrin adaptor AP-1. Under basal Cu(2+) conditions, ATP7B was localized to the trans-Golgi network (TGN) and the plasma membrane of the soma and dendrites but not the axon. Mutation of a dileucine-based signal in ATP7B or overexpression of a dominant-negative σ1 mutant resulted in nonpolarized distribution of ATP7B between the somatodendritic and axonal domains. Furthermore, addition of high Cu(2+) concentrations, previously shown to reduce ATP7B incorporation into AP-1-containing clathrin-coated vesicles, caused loss of TGN localization and somatodendritic polarity of ATP7B. These findings support the notion of AP-1 as an effector of polarized sorting in neurons and suggest that altered polarity of ATP7B in polarized cell types might contribute to abnormal copper metabolism in the MEDNIK syndrome, a neurocutaneous disorder caused by mutations in the σ1A subunit isoform of AP-1.

  4. 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.

  5. 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

  6. Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog*

    PubMed Central

    Park, Richard; Wang'ondu, Ruth; Heston, Lee; Shedd, Duane; Miller, George

    2011-01-01

    Nuclear aggresomes induced by proteins containing an expanded polyglutamine (polyQ) tract are pathologic hallmarks of certain neurodegenerative diseases. Some GFP fusion proteins lacking a polyQ tract may also induce nuclear aggresomes in cultured cells. Here we identify single missense mutations within the basic DNA recognition region of Bam HI Z E B virus replication activator (ZEBRA), an Epstein-Barr virus (EBV)-encoded basic zipper protein without a polyQ tract, that efficiently induced the formation of nuclear aggresomes. Wild-type (WT) ZEBRA was diffusely distributed within the nucleus. Four non-DNA-binding mutants, Z(R179E), Z(R183E), Z(R190E), and Z(K178D) localized to the periphery of large intranuclear spheres, to discrete nuclear aggregates, and to the cytoplasm. Other non-DNA-binding mutants, Z(N182K), Z(N182E), and Z(S186E), did not exhibit this phenotype. The interior of the spheres contained promyelocytic leukemia and HSP70 proteins. ZEBRA mutants directly induced the nuclear aggresome pathway in cells with and without EBV. Specific cellular proteins (SC35 and HDAC6) and viral proteins (WT ZEBRA, Rta, and BMLF1) but not other cellular or viral proteins were recruited to nuclear aggresomes. Co-transfection of WT ZEBRA with aggresome-inducing mutants Z(R183E) and Z(R179E) inhibited late lytic viral protein expression and lytic viral DNA amplification. This is the first reported instance in which nuclear aggresomes are induced by single missense mutations in a viral or cellular protein. We discuss conformational changes in the mutant viral AP-1 proteins that may lead to formation of nuclear aggresomes. PMID:21233201

  7. AP-1 transcription factors, mucin-type molecules and MMPs regulate the IL-11 mediated invasiveness of JEG-3 and HTR-8/SVneo trophoblastic cells.

    PubMed

    Suman, Pankaj; Godbole, Geeta; Thakur, Ravi; Morales-Prieto, Diana M; Modi, Deepak N; Markert, Udo R; Gupta, Satish K

    2012-01-01

    This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo

  8. AP-1 Transcription Factors, Mucin-Type Molecules and MMPs Regulate the IL-11 Mediated Invasiveness of JEG-3 and HTR-8/SVneo Trophoblastic Cells

    PubMed Central

    Suman, Pankaj; Godbole, Geeta; Thakur, Ravi; Morales-Prieto, Diana M.; Modi, Deepak N.; Markert, Udo R.; Gupta, Satish K.

    2012-01-01

    This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo

  9. Recuperating Lung Decoction Attenuates the Oxidative Stress State of Chronic Obstructive Pulmonary Disease by Inhibiting the MAPK/AP-1 Signal Pathway and Regulating γ-GCS

    PubMed Central

    Shi, Qi; Yan, Yue; Kong, Yanhua; Meng, YanYan; Wang, Tiezhu; Zhang, Xing; Bao, Haipeng

    2017-01-01

    Purpose/Objective. To evaluate the effects of Recuperating Lung Decoction (RLD) on the indices of oxidative stress in a rat model of COPD and detect the indices of the MAPK/AP-1/γ-GCS signal pathway for a further survey of the possible targeting site of RLD. Methods/Materials. The rats of COPD were treated with RLD. The protein levels of glutathione (GSH), oxidized glutathione (GSSG), 8-hydroxy-2-deoxyguanosine (8-OHdG), and 4-hydroxynonenal (4-HNE) were measured. In addition, the levels of key signaling molecules (extracellular signal-regulated kinases [ERK], the c-jun N-terminal kinase [JNKs signal pathway], and p38 MAP kinase [p38MAPK], AP-1 proteins [C-fos, C-jun], and γ-glutamyl-cysteine synthetase [γ-GCS-h]) of the MAPK/AP-1/γ-GCS-h signal pathway were assessed. Results. After treatment, the protein level of GSH and the ratio of GSH/GSSG were increased and the amounts of 8-OHdG and 4-HNE were decreased significantly in lung tissues when compared with the nontreated COPD group. Further results showed that the RLD could effectively inhibit the MAPK pathway by inactivation of p38MAPK and ERK and could also downregulate the AP-1 and the γ-GCS-h genes expressions in both protein and mRNA levels. Conclusion. RLD might improve the state of oxidative stress by downregulation of the expression of γ-GCS-h gene by inhibition of the MAPK/AP-1 pathway, thereafter enhancing the ability of antioxidation in COPD.

  10. SAG/ROC2/RBX2 E3 ligase promotes UVB-induced skin hyperplasia, but not skin tumors, by simultaneously targeting c-Jun/AP-1 and p27.

    PubMed

    He, Hongbin; Gu, Qingyang; Zheng, Min; Normolle, Daniel; Sun, Yi

    2008-04-01

    Sensitive to apoptosis gene (SAG)/regulator of cullins-2/RING box protein 2 is a stress-responsive RING component of Skp-1/Cullins/F-box protein E3 ubiquitin ligase. When overexpressed, SAG inhibits apoptosis induced by reactive oxygen species or hypoxia. Here, we report that SAG overexpression inhibits ultraviolet (UV) B-induced apoptosis in mouse JB6 epidermal cells. Using a transgenic mouse model, in which SAG expression was targeted primarily to epidermis by a K14 promoter, we showed that, at the early stage of UVB skin carcinogenesis (10 weeks post-UVB exposure), c-Jun, p27, p53, c-Fos and cyclin D1 were strongly induced. While having no effect on UVB-induced p53, c-Fos and cyclin D1, SAG-transgenic expression reduced the levels of c-Jun and p27 and inhibited AP-1 activity. The net outcome of SAG-mediated inhibition of c-Jun/AP-1 (pro-tumor promotion) and of p27 (antiproliferation) increased skin hyperplasia, with no apparent effect on apoptosis, as evidenced by increased skin thickness, and increased rate of DNA synthesis, but hardly any apoptosis. Although skin hyperplasia was promoted, SAG-transgenic expression had no significant effect on tumor formation in the later stage of UVB carcinogenesis. Thus, by simultaneously targeting c-Jun and p27, SAG accelerates UVB-induced skin hyperplasia, but not carcinogenesis.

  11. TNF-α modulates genome-wide redistribution of ΔNp63α/TAp73 and NF-κB c-REL interactive binding on TP53 and AP-1 motifs to promote an oncogenic gene program in squamous cancer

    PubMed Central

    Si, Han; Lu, Hai; Yang, Xinping; Mattox, Austin; Jang, Minyoung; Bian, Yansong; Sano, Eleanor; Viadiu, Hector; Yan, Bin; Yau, Christina; Ng, Sam; Lee, Steven K.; Romano, Rose-Anne; Davis, Sean; Walker, Robert L.; Xiao, Wenming; Sun, Hongwei; Wei, Lai; Sinha, Satrajit; Benz, Christopher C; Stuart, Joshua M.; Meltzer, Paul S.; Van Waes, Carter; Chen, Zhong

    2016-01-01

    The Cancer Genome Atlas (TCGA) network study of 12 cancer types (PanCancer 12) revealed frequent mutation of TP53, and amplification and expression of related TP63 isoform ΔNp63 in squamous cancers. Further, aberrant expression of inflammatory genes and TP53/p63/p73 targets were detected in the PanCancer 12 project, reminiscent of gene programs co-modulated by cREL/ΔNp63/TAp73 transcription factors we uncovered in head and neck squamous cell carcinomas (HNSCC). However, how inflammatory gene signatures and cREL/p63/p73 targets are co-modulated genome-wide is unclear. Here, we examined how inflammatory factor TNF-α broadly modulates redistribution of cREL with ΔNp63α/TAp73 complexes and signatures genome-wide in the HNSCC model UM-SCC46 using chromatin immunoprecipitation sequencing (ChIP-seq). TNF-α enhanced genome-wide co-occupancy of cREL with ΔNp63α on TP53/p63 sites, while unexpectedly promoting redistribution of TAp73 from TP53 to Activator Protein-1 (AP-1) sites. cREL, ΔNp63α, and TAp73 binding and oligomerization on NF-κB, TP53 or AP-1 specific sequences were independently validated by ChIP-qPCR, oligonucleotide-binding assays, and analytical ultracentrifugation. Function of the binding activity was confirmed using TP53, AP-1, and NF-κB specific response elements, or p21, SERPINE1, and IL-6 promoter luciferase reporter activities. Concurrently, TNF-α regulated a broad gene network with co-binding activities for cREL, ΔNp63α, and TAp73 observed upon array profiling and RT-PCR. Overlapping target gene signatures were observed in squamous cancer subsets and in inflamed skin of transgenic mice overexpressing ΔNp63α. Furthermore, multiple target genes identified in this study were linked to TP63 and TP73 activity and increased gene expression in large squamous cancer samples from PanCancer 12 TCGA by CircleMap. PARADIGM inferred pathway analysis revealed the network connection of TP63 and NF-κB complexes through an AP-1 hub, further supporting

  12. Ascofuranone inhibits lipopolysaccharide–induced inflammatory response via NF-kappaB and AP-1, p-ERK, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages

    PubMed Central

    Park, Jun-Young; Chung, Tae-Wook; Jeong, Yun-Jeong; Kwak, Choong-Hwan; Ha, Sun-Hyung; Kwon, Kyung-Min; Abekura, Fukushi; Cho, Seung-Hak; Lee, Young-Choon; Ha, Ki-Tae; Magae, Junji; Chang, Young-Chae; Kim, Cheorl-Ho

    2017-01-01

    The natural fungal compound ascofuranone (5-chloro-3-[(2E,6E)-7-[(2S)-5,5-dimethyl-4-oxo-tetrahydrofuran-2-yl]-3-methyl-octa-2,6-dienyl]-2,4-dihydroxy-6-methyl-benzaldehyde, MW 420.93) (AF) isolated from Ascochyta viciae has been known to promote cell cycle arrest and inhibit invasion of tumor cells. We have previously studied a structurally similar compound ascochlorin (ASC; MW 404.93) with regard to its anti-inflammatory activity in LPS- stimulated RAW 264.7 macrophages. In order to examine the relationship between the anti-inflammatory activities and the molecular differences between AF and ASC, the activity of AF is herein studied, because ASC has a unique trimethyl oxocyclohexyl structure, while AF has a unique dimethyl-oxo-tetrahydrofuran structure. AF dose-dependently inhibited the production of NO and iNOS and the COX-2 mRNA and protein levels in RAW 264.7 cells. In addition, AF suppressed mRNA expression levels of inflammatory cytokines such as TNF-α, IL-6, and IL-1β, as assessed by RT-PCR. AF (30–50 μg/ml) treatment clearly inhibited the nuclear translocation of NF-κB, AP-1 (p-c-Jun) from the cytosolic space. Phosphorylation of IκB, which functions to maintain the activity of NF-κB, was decreased by AF treatment. Moreover, AF suppressed the binding of NF-κB (p65). Inhibition of IkBa phosphorylation and degradation inhibits nuclear translocation of p65. Immunofluorescence confocal microscopy analysis also revealed that translocation of NF-κB and AP-1 (p-c-Jun) was decreased upon AF treatment. AF specifically decreased the expression level of p-ERK, but not the expression level of p-p38 or p-JNK. Given these results, we suggest that AF suppresses the inflammatory response by targeting p-ERK. This indicates that AF is a negative regulator of LPS-stimulated nuclear translocation of NF-κB and AP-1 (p-c-Jun) in RAW 264.7 macrophages, and specifically it targets p-ERK. Therefore, AF and ASC exert their effects in different ways, most probably because

  13. Ascofuranone inhibits lipopolysaccharide-induced inflammatory response via NF-kappaB and AP-1, p-ERK, TNF-α, IL-6 and IL-1β in RAW 264.7 macrophages.

    PubMed

    Park, Jun-Young; Chung, Tae-Wook; Jeong, Yun-Jeong; Kwak, Choong-Hwan; Ha, Sun-Hyung; Kwon, Kyung-Min; Abekura, Fukushi; Cho, Seung-Hak; Lee, Young-Choon; Ha, Ki-Tae; Magae, Junji; Chang, Young-Chae; Kim, Cheorl-Ho

    2017-01-01

    The natural fungal compound ascofuranone (5-chloro-3-[(2E,6E)-7-[(2S)-5,5-dimethyl-4-oxo-tetrahydrofuran-2-yl]-3-methyl-octa-2,6-dienyl]-2,4-dihydroxy-6-methyl-benzaldehyde, MW 420.93) (AF) isolated from Ascochyta viciae has been known to promote cell cycle arrest and inhibit invasion of tumor cells. We have previously studied a structurally similar compound ascochlorin (ASC; MW 404.93) with regard to its anti-inflammatory activity in LPS- stimulated RAW 264.7 macrophages. In order to examine the relationship between the anti-inflammatory activities and the molecular differences between AF and ASC, the activity of AF is herein studied, because ASC has a unique trimethyl oxocyclohexyl structure, while AF has a unique dimethyl-oxo-tetrahydrofuran structure. AF dose-dependently inhibited the production of NO and iNOS and the COX-2 mRNA and protein levels in RAW 264.7 cells. In addition, AF suppressed mRNA expression levels of inflammatory cytokines such as TNF-α, IL-6, and IL-1β, as assessed by RT-PCR. AF (30-50 μg/ml) treatment clearly inhibited the nuclear translocation of NF-κB, AP-1 (p-c-Jun) from the cytosolic space. Phosphorylation of IκB, which functions to maintain the activity of NF-κB, was decreased by AF treatment. Moreover, AF suppressed the binding of NF-κB (p65). Inhibition of IkBa phosphorylation and degradation inhibits nuclear translocation of p65. Immunofluorescence confocal microscopy analysis also revealed that translocation of NF-κB and AP-1 (p-c-Jun) was decreased upon AF treatment. AF specifically decreased the expression level of p-ERK, but not the expression level of p-p38 or p-JNK. Given these results, we suggest that AF suppresses the inflammatory response by targeting p-ERK. This indicates that AF is a negative regulator of LPS-stimulated nuclear translocation of NF-κB and AP-1 (p-c-Jun) in RAW 264.7 macrophages, and specifically it targets p-ERK. Therefore, AF and ASC exert their effects in different ways, most probably because

  14. The γ/σ1 and α/σ2 Hemicomplexes of Clathrin Adaptors AP-1 and AP-2 Harbor the Dileucine Recognition Site

    PubMed Central

    Doray, Balraj; Lee, Intaek; Knisely, Jane; Bu, Guojun

    2007-01-01

    The clathrin adaptors AP-1 and AP-2 bind cargo proteins via two types of motifs: tyrosine-based Yxxφ and dileucine-based [DE]XXXL[LI]. Although it is well established that Yxxφ motifs bind to the μ subunits of AP-1 or AP-2, dileucine motifs have been reported to bind to either the μ or β subunits of these adaptors as well as the γ/σ1 hemicomplex of AP-1. To clarify this controversy, the various subunits of AP-1 and AP-2 were expressed individually and in hemicomplex form in insect cells, and they were used in glutathione S-transferase pull-down assays to determine their binding properties. We report that the γ/σ1 or α/σ2 hemicomplexes bound the dileucine-based motifs of several proteins quite strongly, whereas binding by the β1/μ1 and β2/μ2 hemicomplexes, and the individual β or μ subunits, was extremely weak or undetectable. The γ/σ1 and α/σ2 hemicomplexes displayed substantial differences in their preference for particular dileucine-based motifs. Most strikingly, an aspartate at position −4 compromised binding to the γ/σ1 hemicomplex, whereas minimally affecting binding to α/σ2. There was an excellent correlation between binding to the α/σ2 hemicomplex and in vivo internalization mediated by the dileucine-based sorting signals. These findings provide new insights into the trafficking mechanisms of D/EXXXL[LI]-mediated sorting signals. PMID:17360967

  15. MetAP1 and MetAP2 drive cell selectivity for a potent anti-cancer agent in synergy, by controlling glutathione redox state

    PubMed Central

    Frottin, Frédéric; Bienvenut, Willy V.; Bignon, Jérôme; Jacquet, Eric; Jacome, Alvaro Sebastian Vaca; Van Dorsselaer, Alain; Cianferani, Sarah; Carapito, Christine; Meinnel, Thierry; Giglione, Carmela

    2016-01-01

    Fumagillin and its derivatives are therapeutically useful because they can decrease cancer progression. The specific molecular target of fumagillin is methionine aminopeptidase 2 (MetAP2), one of the two MetAPs present in the cytosol. MetAPs catalyze N-terminal methionine excision (NME), an essential pathway of cotranslational protein maturation. To date, it remains unclear the respective contribution of MetAP1 and MetAP2 to the NME process in vivo and why MetAP2 inhibition causes cell cycle arrest only in a subset of cells. Here, we performed a global characterization of the N-terminal methionine excision pathway and the inhibition of MetAP2 by fumagillin in a number of lines, including cancer cell lines. Large-scale N-terminus profiling in cells responsive and unresponsive to fumagillin treatment revealed that both MetAPs were required in vivo for M[VT]X-targets and, possibly, for lower-level M[G]X-targets. Interestingly, we found that the responsiveness of the cell lines to fumagillin was correlated with the ability of the cells to modulate their glutathione homeostasis. Indeed, alterations to glutathione status were observed in fumagillin-sensitive cells but not in cells unresponsive to this agent. Proteo-transcriptomic analyses revealed that both MetAP1 and MetAP2 accumulated in a cell-specific manner and that cell sensitivity to fumagillin was related to the levels of these MetAPs, particularly MetAP1. We suggest that MetAP1 levels could be routinely checked in several types of tumor and used as a prognostic marker for predicting the response to treatments inhibiting MetAP2. PMID:27542228

  16. Polarized Traffic of LRP1 Involves AP1B and SNX17 Operating on Y-dependent Sorting Motifs in Different Pathways

    PubMed Central

    Donoso, Maribel; Cancino, Jorge; Lee, Jiyeon; van Kerkhof, Peter; Retamal, Claudio; Bu, Guojun; Gonzalez, Alfonso; Cáceres, Alfredo

    2009-01-01

    Low-density lipoprotein receptor–related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17). In the biosynthetic pathway, Y29 but not N26 from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N26A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptor's exit from BSE. An endocytic Y63ATL66 motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL86,87), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes. PMID:19005208

  17. Discs large 1 (Dlg1) scaffolding protein participates with clathrin and adaptator protein complex 1 (AP-1) in forming Weibel-Palade bodies of endothelial cells.

    PubMed

    Philippe, Monique; Léger, Thibaut; Desvaux, Raphaëlle; Walch, Laurence

    2013-05-03

    Weibel-Palade bodies (WPBs) are specific cigar-shaped granules that store von Willebrand factor (VWF) for its regulated secretion by endothelial cells. The first steps of the formation of these granules at the trans-Golgi network specifically require VWF aggregation and an external scaffolding complex that contains the adaptator protein complex 1 (AP-1) and clathrin. Discs large 1 (Dlg1) is generally considered to be a modular scaffolding protein implicated in the control of cell polarity in a large variety of cells by specific recruiting of receptors, channels, or signaling proteins to specialized zones of the plasma membrane. We propose here that in endothelial cells, Dlg1, in a complex with AP-1 and clathrin, participates in the biogenesis of WPBs. Supporting data show that Dlg1 colocalizes with microtubules, intermediate filaments, and Golgi markers. Tandem mass spectrometry experiments led to the identification of clathrin as an Dlg1-interacting partner. Interaction was confirmed by in situ proximity ligation assays. Furthermore, AP-1 and VWF immunoprecipitate and colocalize with Dlg1 in the juxtanuclear zone. Finally, Dlg1 depletion by siRNA duplexes disrupts trans-Golgi network morphology and WPB formation. Our results provide the first evidence for an unexpected role of Dlg1 in controlling the formation of specific secretory granules involved in VWF exocytosis in endothelial cells.

  18. Treatment with novel AP-1 and NF-κB inhibitors restores the colonic endocrine cells to normal levels in rats with DSS-induced colitis

    PubMed Central

    EL-SALHY, MAGDY; UMEZAWA, KAZUO

    2016-01-01

    The aim of this study was to determine the effects of two anti-inflammatory agents on the abnormalities in colonic endocrine cells in dextran sodium sulfate (DSS)-induced colitis. Colitis was induced in male Wistar rats (n=45) using DSS; a further 15 rats without colitis were included in a healthy control group. The animals with DSS-induced colitis were randomly divided into 3 treatment groups as follows: i) DSS group, rats were treated with 0.5 ml of 0.5% carboxymethyl cellulose (CMC); ii) DSS-G group, rats were treated with 3-[(dodecyl thiocarbonyl)-methyl]-glutarimide (DTCM-G), a novel activator protein 1 (AP-1) inhibitor, 20 mg/kg in CMC; and iii) DSS-Q group, rats were treated with dehydroxymethylepoxyquinomicin, a nuclear factor κB (NF-κB) inhibitor, 15 mg/kg in CMC. The treatments were administered intraperitoneally, twice daily for 5 days, after which the animals were sacrificed and tissue samples from the colon were immunostained for chromogranin A (CgA), serotonin, peptide YY (PYY), enteroglucagon, pancreatic polypeptide (PP), somatostatin, leukocytes, B/T lymphocytes, B lymphocytes, T lymphocytes, macrophages/monocytes and mast cells. The densities of these endocrine and immune cells were quantified by computer-aided image analysis. The densities of CgA-, serotonin-, PYY- and enteroglucagon-producing cells were significantly higher, and those of PP- and somatostatin-producing cells were significantly lower in the DSS-G, DSS-Q and control groups than in the DSS group. The densities of all the immune cells were lower in the DSS-G, DSS-Q and control groups than in the DSS group. The densities of all endocrine cell types and immune cells in both the DSS groups treated with anti-inflammatory agents were restored to control levels. In conclusion, our data demonstrate that there is an interaction between endocrine and immune cells during inflammation. This interaction with subsequent changes in endocrine cells is responsible for the clinical manifestation of

  19. Treatment with novel AP-1 and NF-κB inhibitors restores the colonic endocrine cells to normal levels in rats with DSS-induced colitis.

    PubMed

    El-Salhy, Magdy; Umezawa, Kazuo

    2016-03-01

    The aim of this study was to determine the effects of two anti-inflammatory agents on the abnormalities in colonic endocrine cells in dextran sodium sulfate (DSS)-induced colitis. Colitis was induced in male Wistar rats (n=45) using DSS; a further 15 rats without colitis were included in a healthy control group. The animals with DSS-induced colitis were randomly divided into 3 treatment groups as follows: i) DSS group, rats were treated with 0.5 ml of 0.5% carboxymethyl cellulose (CMC); ii) DSS‑G group, rats were treated with 3-[(dodecylthiocarbonyl)‑methyl]‑glutarimide (DTCM‑G), a novel activator protein 1 (AP-1) inhibitor, 20 mg/kg in CMC; and iii) DSS‑Q group, rats were treated with dehydroxymethylepoxyquinomicin, a nuclear factor κB (NF-κB) inhibitor, 15 mg/kg in CMC. The treatments were administered intraperitoneally, twice daily for 5 days, after which the animals were sacrificed and tissue samples from the colon were immunostained for chromogranin A (CgA), serotonin, peptide YY (PYY), enteroglucagon, pancreatic polypeptide (PP), somatostatin, leukocytes, B/T lymphocytes, B lymphocytes, T lymphocytes, macrophages/monocytes and mast cells. The densities of these endocrine and immune cells were quantified by computer‑aided image analysis. The densities of CgA-, serotonin-, PYY- and enteroglucagon-producing cells were significantly higher, and those of PP- and somatostatin-producing cells were significantly lower in the DSS‑G, DSS‑Q and control groups than in the DSS group. The densities of all the immune cells were lower in the DSS‑G, DSS‑Q and control groups than in the DSS group. The densities of all endocrine cell types and immune cells in both the DSS groups treated with anti‑inflammatory agents were restored to control levels. In conclusion, our data demonstrate that there is an interaction between endocrine and immune cells during inflammation. This interaction with subsequent changes in endocrine cells is responsible for the

  20. Indoxyl sulfate enhances IL-1β-induced E-selectin expression in endothelial cells in acute kidney injury by the ROS/MAPKs/NFκB/AP-1 pathway.

    PubMed

    Shen, Wen-Ching; Liang, Chan-Jung; Huang, Tao-Ming; Liu, Chen-Wei; Wang, Shu-Huei; Young, Guang-Huar; Tsai, Jaw-Shiun; Tseng, Ying-Chin; Peng, Yu-Sen; Wu, Vin-Cent; Chen, Yuh-Lien

    2016-11-01

    Uremic toxins are considered a risk factor for cardiovascular disorders in kidney diseases, but it is not known whether, under inflammatory conditions, they affect adhesion molecule expression on endothelial cells, which may play a critical role in acute kidney injury (AKI). In the present study, in cardiovascular surgery-related AKI patients, who are known to have high plasma levels of the uremic toxin indoxyl sulfate (IS), plasma levels of IL-1β were found to be positively correlated with plasma levels of the adhesion molecule E-selectin. In addition, high E-selectin and IL-1β expression were seen in the kidney of ischemia/reperfusion mice in vivo. We also examined the effects of IS on E-selectin expression by IL-1β-treated human umbilical vein endothelial cells (HUVECs) and the underlying mechanism. IS pretreatment of HUVECs significantly increased IL-1β-induced E-selectin expression, monocyte adhesion, and the phosphorylation of mitogen-activated protein kinases (ERK, p38, and JNK) and transcription factors (NF-κB and AP-1), and phosphorylation was decreased by pretreatment with inhibitors of ERK1/2 (PD98059), p38 MAPK (SB202190), and JNK (SP600125). Furthermore, IS increased IL-1β-induced reactive oxygen species (ROS) production and this effect was inhibited by pretreatment with N-acetylcysteine (a ROS scavenger) or apocynin (a NADPH oxidase inhibitor). Gel shift assays and ChIP-PCR demonstrated that IS enhanced E-selectin expression in IL-1-treated HUVECs by increasing NF-κB and AP-1 DNA-binding activities. Moreover, IS-enhanced E-selectin expression in IL-1β-treated HUVECs was inhibited by Bay11-7082, a NF-κB inhibitor. Thus, IS may play an important role in the development of cardiovascular disorders in kidney diseases during inflammation by increasing endothelial expression of E-selectin.

  1. Functional and evolutionary analysis of the AP1/SEP/AGL6 superclade of MADS-box genes in the basal eudicot Epimedium sagittatum

    PubMed Central

    Sun, Wei; Huang, Wenjun; Li, Zhineng; Song, Chi; Liu, Di; Liu, Yongliang; Hayward, Alice; Liu, Yifei; Huang, Hongwen; Wang, Ying

    2014-01-01

    Background and Aims MADS-box transcriptional regulators play important roles during plant development. Based on phylogenetic reconstruction, the AP1/SEP/AGL6 superclade of floral MADS-box genes underwent one or two duplication events in the common ancestor of the core eudicots. However, the functional evolution of the AP1/SEP/AGL6 superclade in basal eudicots remains uncharacterized. Epimedium sagittatum is a basal eudicot species valued for its medicinal properties and showing unique floral morphology. In this study, structural and functional variation of FUL-like (AP1 subfamily), SEP-like and AGL6-like genes in this species was investigated to further our understanding of flower evolution in angiosperms. Detailed investigations into the microsynteny and evolutionary history of the floral A and E class MADS-box genes in eudicots were undertaken and used to trace their genomic rearrangements. Methods One AP1-like gene, two SEP-like genes and one AGL6-like gene were cloned from E. sagittatum. Their expression patterns were examined using quantitative RT-PCR in different vegetative and reproductive organs at two developmental stages. Yeast two-hybrid assays were carried out among AP1/SEP/AGL6 superclade, AP3/PI and AGAMOUS subfamily members for elucidation of dimerization patterns. In addition, possible formation of a ternary complex involving B class proteins with the A class protein EsFUL-like, the E class SEP-like protein EsAGL2-1 or the AGL6-class protein EsAGL6 were detected using yeast three-hybrid assays. Transgenic Arabidopsis or tobacco plants expressing EsFUL-like, EsAGL2-1 and EsAGL6-like under the cauliflower mosaic virus (CaMV) 35S promoter were generated and analysed. Genomic studies of AP1 syntenic regions in arabidopsis, columbine, strawberry, papaya, peach, grapevine and tomato were conducted for microsyntenic analyses. Key Results Sequence and phylogenetic analyses showed that EsFUL-like is a member of the AP1 (A class) subfamily, EsAGL2-1 and Es

  2. Dietary turmeric modulates DMBA-induced p21{sup ras}, MAP kinases and AP-1/NF-{kappa}B pathway to alter cellular responses during hamster buccal pouch carcinogenesis

    SciTech Connect

    Garg, Rachana; Ingle, Arvind; Maru, Girish

    2008-11-01

    The chemopreventive efficacy of turmeric has been established in experimental systems. However, its mechanism(s) of action are not fully elucidated in vivo. The present study investigates the mechanism of turmeric-mediated chemoprevention in 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis at 2, 4, 6, 10 and 12 weeks. Dietary turmeric (1%) led to decrease in DMBA-induced tumor burden and multiplicity, and enhanced the latency period in parallel, to its modulatory effects on oncogene products and various cellular responses during HBP tumorigenesis. DMBA-induced expression of ras oncogene product, p21 and downstream target, the mitogen-activated protein kinases were significantly decreased by turmeric during HBP carcinogenesis. Turmeric also diminished the DMBA-induced mRNA expression of proto-oncogenes (c-jun, c-fos) and NF-{kappa}B, leading to decreased protein levels and in further attenuation of DMBA-induced AP-1/NF-{kappa}B DNA-binding in the buccal pouch nuclear extracts. Besides, buccal pouch of hamsters receiving turmeric diet showed significant alterations in DMBA-induced effects: (a) decrease in cell proliferation (diminished PCNA and Bcl2 expression), (b) enhanced apoptosis (increased expression of Bax, caspase-3 and apoptotic index), (c) decrease in inflammation (levels of Cox-2, the downstream target of AP-1/NF-{kappa}B, and PGE2) and (d) aberrant expression of differentiation markers, the cytokeratins (1, 5, 8, and 18). Together, the protective effects of dietary turmeric converge on augmenting apoptosis of the initiated cells and decreasing cell proliferation in DMBA-treated animals, which in turn, is reflected in decreased tumor burden, multiplicity and enhanced latency period. Some of these biomarkers are likely to be helpful in monitoring clinical trials and evaluating drug effect measurements.

  3. AP-1/σ1B-Dependent SV Protein Recycling Is Regulated in Early Endosomes and Is Coupled to AP-2 Endocytosis.

    PubMed

    Kratzke, Manuel; Candiello, Ermes; Schmidt, Bernhard; Jahn, Olaf; Schu, Peter

    2015-08-01

    Adaptor protein (AP)-1/σ1B(-/-) mice have reduced synaptic-vesicle (SV) recycling and increased endosomes. Mutant mice have impaired spatial memory, and σ1B-deficient humans have a severe mental retardation. In order to define these σ1B(-/-) 'bulk' endosomes and to determine their functions in SV recycling, we developed a protocol to separate them from the majority of the neuronal endosomes. The σ1B(-/-) 'bulk' endosomes proved to be classic early endosomes with an increase in the phospholipid phosphatidylinositol 3-phosphate (PI-3-P), which recruits proteins mediating protein sorting out of early endosomes into different routes. σ1B deficiency induced alterations in the endosomal proteome reveals two major functions: SV protein storage and sorting into endolysosomes. Alternative endosomal recycling pathways are not up-regulated, but certain SV proteins are misrouted. Tetraspanins are enriched in σ1B(-/-) synaptosomes, but not in their endosomes or in their clathrin-coated-vesicles (CCVs), indicating AP-1/σ1B-dependent sorting. Synapses contain also more AP-2 CCV, although it is expected that they contain less due to reduced SV recycling. Coat composition of these AP-2 CCVs is altered, and thus, they represent a subpopulation of AP-2 CCVs. Association of calmodulin-dependent protein kinase (CaMK)-IIα, -δ and casein kinase (CK)-IIα with the endosome/SV pool is altered, as well as 14-3-3η, indicating changes in specific signalling pathways regulating synaptic plasticity. The accumulation of early endosomes and endocytotic AP-2 CCV indicates the regulation of SV recycling via early endosomes by the interdependent regulation of AP-2-mediated endocytosis and AP-1/σ1B-mediated SV reformation.

  4. Apigenin-7-O-β-D-glucuronide inhibits LPS-induced inflammation through the inactivation of AP-1 and MAPK signaling pathways in RAW 264.7 macrophages and protects mice against endotoxin shock.

    PubMed

    Hu, Weicheng; Wang, Xinfeng; Wu, Lei; Shen, Ting; Ji, Lilian; Zhao, Xihong; Si, Chuan-Ling; Jiang, Yunyao; Wang, Gongcheng

    2016-02-01

    Apigenin-7-O-β-D-glucuronide (AG), an active flavonoid derivative isolated from the agricultural residue of Juglans sigillata fruit husks, possesses multiple pharmacological activities, including anti-oxidant, anti-complement, and aldose reductase inhibitory activities. To date, no report has identified the anti-inflammatory mechanisms of AG. This study was therefore designed to characterize the molecular mechanisms of AG on lipopolysaccharide (LPS)-induced inflammatory cytokines in RAW 264.7 cells and on endotoxin-induced shock in mice. AG suppressed the release of nitric oxide (NO), prostaglandin E2 (PGE2), and tumour necrosis factor-α (TNF-α) in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner without affecting cell viability. Additionally, AG suppressed LPS-induced mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α. AG treatment decreased the translocation of c-Jun into the nucleus, and decreased activator protein-1 (AP-1)-mediated luciferase activity through the inhibition of both p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) phosphorylation. Consistent with the in vitro observations, AG protected mice from LPS-induced endotoxin shock by inhibiting proinflammatory cytokine production. Taken together, these results suggest that AG may be used as a source of anti-inflammatory agents as well as a dietary complement for health promotion.

  5. The Novel Organic Arsenical Darinaparsin Induces MAPK-Mediated and SHP1-Dependent Cell Death in T-cell Lymphoma and Hodgkin’s Lymphoma Cells and Human Xenograft Models

    PubMed Central

    Ravi, Dashnamoorthy; Bhalla, Savita; Gartenhaus, Ronald B; Crombie, Jennifer; Kandela, Irawati; Sharma, Jaya; Mazar, Andrew; Evens, Andrew M.

    2014-01-01

    Purpose Darinaparsin (Zio-101) is a novel organic arsenical compound with encouraging clinical activity in relapsed/refractory T-cell lymphoma (TCL) and Hodgkin lymphoma (HL), however little is known regarding its mechanism of action. Experimental Design TCL cell lines (Jurkat, Hut78, and HH) and HL cell lines (L428, L540, and L1236) were examined for in vitro cell death by MTT assay and Annexin-V based flow cytometry. Jurkat and L540-derived xenografts in SCID mice were examined for in vivo tumor inhibition and survival. Biological effects of darinaparsin on MAPK pathway were investigated using pharmacological inhibitors, RNA interference (RNAi) and transient transfection for overexpression for SHP1 and MEK. Results Darinaparsin treatment resulted in dose-dependent cytotoxicity and apoptosis in all TCL and HL cell lines. Additionally, darinaparsin had more rapid, higher, and sustained intracellular arsenic levels compared with arsenic trioxide via mass spectrometry. In vivo experiments with Jurkat (TCL) and L540 (HL)-derived lymphoma xenografts showed significant inhibition of tumor growth and improved survival in darinaparsin-treated SCID mice. Biologically, darinaparsin caused phosphorylation of ERK (and relevant downstream substrates) primarily by decreasing the inhibitory SHP1 phosphatase and co-immunoprecipitation showed significant ERK/SHP1 interaction. Furthermore, ERK shRNA knockdown or constitutive overexpression of SHP1 resulted in increased apoptosis, while co-treatment with pharmacologic MEK inhibitors resulted in synergistic cell death. Conversely, SHP1 blockade (via pharmacologic inhibition or RNAi) as well as MEK constitutive activation decreased darinaparsin-related cell death. Conclusions Altogether, these data show that darinaparsin is highly active in HL and TCL and its activity is dependent primarily on MAPK mechanisms. PMID:25316819

  6. Low fucose containing bacterial polysaccharide facilitate mitochondria-dependent ROS-induced apoptosis of human lung epithelial carcinoma via controlled regulation of MAPKs-mediated Nrf2/Keap1 homeostasis signaling.

    PubMed

    Chowdhury, Sougata Roy; Sengupta, Suman; Biswas, Subir; Sen, Ramkrishna; Sinha, Tridib Kumar; Basak, Ratan Kumar; Adhikari, Basudam; Bhattacharyya, Arindam

    2015-12-01

    Reactive oxygen species (ROS), the key mediators of cellular oxidative stress and redox dysregulation involved in cancer initiation and progression, have recently emerged as promising targets for anticancer drug discovery. Continuous free radical assault upsets homeostasis in cellular redox system and regulates the associated signaling pathways to mediate stress-induced cell death. This study investigates the dose-specific pro-oxidative behavior of a bacterial fucose polysaccharide, which attenuated proliferation of different cancer cells. In the fermentation process, Bacillus megaterium RB-05 [GenBank Accession Number HM371417] was found to biosynthesize a polysaccharide with low-fucose content (4.9%), which conferred the maximum anti-proliferative activity (750 µg/mL) against human lung cancer epithelial cells (A549) during preliminary screening. Structural elucidation and morphological characterization of the duly purified polysaccharide was done using HPLC, GC-MS, (1)H/(13)C NMR, and microscopy. The polysaccharide exhibited concentration- and time-dependent anti-proliferative effects against A549 cells by inducing intracellular ROS level and regulating the mitochondrial membrane-permeability following the apoptotic pathway. This process encompasses activation of caspase-8/9/3/7, increase in the ratio of Bax/Bcl2 ratio, translocation of Bcl2-associated X protein (Bax) and cytochrome c, decrease in expression of anti-apoptotic members of Bcl2 family, and phosphorylation of mitogen activated protein kinases (MAPKs). Apoptosis was attenuated upon pretreatment with specific caspase-inhibitors. Simultaneously, during apoptosis, the ROS-mediated stress as well as activated MAPKs triggered nuclear translocation of transcription factors like nuclear factor (erythroid-derived)-like 2 (Nrf2) and promoted further transcription of downstream cytoprotective genes, which somehow perturbed the chemotherapeutic efficacy of the polysaccharide, although using CuPP, a chemical

  7. IL-10 Inhibits the NF-κB and ERK/MAPK-Mediated Production of Pro-Inflammatory Mediators by Up-Regulation of SOCS-3 in Trypanosoma cruzi-Infected Cardiomyocytes

    PubMed Central

    Siffo, Sofía; Mirkin, Gerardo A.; Goren, Nora B.

    2013-01-01

    Trypanosoma cruzi (T. cruzi) infection produces an intense inflammatory response which is critical for the control of the evolution of Chagas’ disease. Interleukin (IL)-10 is one of the most important anti-inflammatory cytokines identified as modulator of the inflammatory reaction. This work shows that exogenous addition of IL-10 inhibited ERK1/2 and NF-κB activation and reduced inducible nitric oxide synthase (NOS2), metalloprotease (MMP) -9 and MMP-2 expression and activities, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-6 expression, in T. cruzi-infected cardiomyocytes. We found that T. cruzi and IL-10 promote STAT3 phosphorylation and up-regulate the expression of suppressor of cytokine signalling (SOCS)-3 thereby preventing NF-κB nuclear translocation and ERK1/2 phosphorylation. Specific knockdown of SOCS-3 by small interfering RNA (siRNA) impeded the IL-10-mediated inhibition of NF-κB and ERK1/2 activation. As a result, the levels of studied pro-inflammatory mediators were restored in infected cardiomyocytes. Our study reports the first evidence that T. cruzi up- regulates SOCS-3 expression and highlights the relevance of IL-10 in the modulation of pro-inflammatory response of cardiomyocytes in Chagas’ disease. PMID:24260222

  8. Notch-1 Confers Chemoresistance in Lung Adenocarcinoma to Taxanes through AP-1/microRNA-451 Mediated Regulation of MDR-1.

    PubMed

    Huang, Jiayuan; Chen, Yitian; Li, Junyang; Zhang, Kai; Chen, Jing; Chen, Dongqin; Feng, Bing; Song, Haizhu; Feng, Jifeng; Wang, Rui; Chen, Longbang

    2016-01-01

    We previously demonstrated that expression of Notch-1 is associated with poor prognosis in lung adenocarcinoma (LAD) patients. The aim of this study is to reveal whether Notch-1 was associated with Taxanes-resistant LAD and, the underlying mechanisms. We collected 39 patients of advanced LAD treated with Taxanes and found that positive Notch-1 expression is closely related to LAD lymph node metastasis, recurrence and poorer prognosis, and Notch-1 acts as an independent poor prognostic factor in LAD by multivariate analysis with Cox regression model. Then, by using the Docetaxel (DTX)-resistant LAD cell lines that we established previously, we found that Notch-1 contributes to resistance of LAD cells to DTX in vitro, and inhibition of Notch-1 sensitizes LAD to DTX in vivo. We further demonstrated that Notch-1 mediates chemoresistance response and strengthens proliferation capacity in LAD cells partially through negative regulation of miR-451 by transcription factor AP-1. Moreover, we found that MDR-1 is a direct target of miR-451 and influences chemoresistance of LAD cells. Taken together, our data revealed a novel Notch-1/AP-1/miR-451/MDR-1 signaling axis, and suggested a new therapeutic strategy of combining DTX with Notch inhibitors to treat DTX-resistant LAD.

  9. Human cytomegalovirus contains a tegument protein that enhances transcription from promoters with upstream ATF and AP-1 cis-acting elements.

    PubMed Central

    Liu, B; Stinski, M F

    1992-01-01

    The tegument proteins of human cytomegalovirus are introduced into cells as components of infectious virus. The tegument proteins may affect viral and cellular transcription prior to the synthesis of the immediate-early viral regulatory proteins. The phosphorylated tegument protein of 71 kDa (pp71) is reported to be encoded by the UL82 gene. The UL82 gene products transactivated promoters containing upstream ATF or AP-1 binding sites. In contrast, the phosphorylated tegument protein of 65 kDa (pp65), encoded by the UL83 gene, had no detectable effect on these promoters. Enhancement by UL82 of downstream transcription was directly proportional to the number of upstream ATF sites. Response to UL82 transactivation was abolished by mutation of the ATF site. Mutation in the carboxy-terminal region of UL82 also eliminated transactivation. Even though the major immediate-early promoter of human cytomegalovirus is a strong enhancer-containing promoter, UL82 further enhanced its transcription as much as 20-fold. The mechanism of UL82 enhancement of transcription from viral or cellular promoters is not known, but the enhancement may be mediated by triggering one of the protein kinase signaling pathways, increasing the affinity of ATF or AP-1 for the target sequence, or stabilizing the complex between the eucaryotic transcription factor and the target sequence. Images PMID:1318413

  10. Oxidant exposure induces cysteine-rich protein 61 (CCN1) via c-Jun/AP-1 to reduce collagen expression in human dermal fibroblasts.

    PubMed

    Qin, Zhaoping; Robichaud, Patrick; He, Tianyuan; Fisher, Gary J; Voorhees, John J; Quan, Taihao

    2014-01-01

    Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin.

  11. Actions of Mycobacterium sp. strain AP1 on the saturated- and aromatic-hydrocarbon fractions of fuel oil in a marine medium.

    PubMed

    Vila, Joaquim; Grifoll, Magdalena

    2009-10-01

    The pyrene-degrading Mycobacterium sp. strain AP1 grew in nutrient-supplemented artificial seawater with a heavy fuel oil as the sole carbon source, causing the complete removal of all linear (C(12) to C(40)) and branched alkanes from the aliphatic fraction, as well as an extensive degradation of the three- and four-ring polycyclic aromatic hydrocarbons (PAHs) phenanthrene (95%), anthracene (80%), fluoranthene (80%), pyrene (75%), and benzo(a)anthracene (30%). Alkylated PAHs, which are more abundant in crude oils than the nonsubstituted compounds, were selectively attacked at extents that varied from more than 90% for dimethylnaphthalenes, methylphenanthrenes, methylfluorenes, and methyldibenzothiophenes to about 30% for monomethylated fluoranthenes/pyrenes and trimethylated phenanthrenes and dibenzothiophenes. Identification of key metabolites indicated the utilization of phenanthrene, pyrene, and fluoranthene by known assimilatory metabolic routes, while other components were cooxidized. Detection of mono- and dimethylated phthalic acids demonstrated ring cleavage and further oxidation of alkyl PAHs. The extensive degradation of the alkanes, the two-, three-, and four-ring PAHs, and their 1-, 2-, and 3-methyl derivatives from a complex mixture of hydrocarbons by Mycobacterium sp. strain AP1 illustrates the great substrate versatility of alkane- and PAH-degrading mycobacteria.

  12. Oxidant Exposure Induces Cysteine-Rich Protein 61 (CCN1) via c-Jun/AP-1 to Reduce Collagen Expression in Human Dermal Fibroblasts

    PubMed Central

    Qin, Zhaoping; Robichaud, Patrick; He, Tianyuan; Fisher, Gary J.; Voorhees, John J.; Quan, Taihao

    2014-01-01

    Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin. PMID:25536346

  13. The ABCs of flower development: mutational analysis of AP1/FUL-like genes in rice provides evidence for a homeotic (A)-function in grasses.

    PubMed

    Wu, Feng; Shi, Xiaowei; Lin, Xuelei; Liu, Yuan; Chong, Kang; Theißen, Günter; Meng, Zheng

    2017-01-01

    The well-known ABC model describes the combinatorial interaction of homeotic genes in specifying floral organ identities. While the B- and C-functions are highly conserved throughout flowering plants and even in gymnosperms, the A-function, which specifies the identity of perianth organs (sepals and petals in eudicots), remains controversial. One reason for this is that in most plants that have been investigated thus far, with Arabidopsis being a remarkable exception, one does not find recessive mutants in which the identity of both types of perianth organs is affected. Here we report a comprehensive mutational analysis of all four members of the AP1/FUL-like subfamily of MADS-box genes in rice (Oryza sativa). We demonstrate that OsMADS14 and OsMADS15, in addition to their function of specifying meristem identity, are also required to specify palea and lodicule identities. Because these two grass-specific organs are very likely homologous to sepals and petals of eudicots, respectively, we conclude that there is a floral homeotic (A)-function in rice as defined previously. Together with other recent findings, our data suggest that AP1/FUL-like genes were independently recruited to fulfil the (A)-function in grasses and some eudicots, even though other scenarios cannot be excluded and are discussed.

  14. Suppression of Inflammatory Responses by Black Rice Extract in RAW 264.7 Macrophage Cells via Downregulation of NF-kB and AP-1 Signaling Pathways.

    PubMed

    Limtrakul, Pornngarm; Yodkeeree, Supachai; Pitchakarn, Pornsiri; Punfa, Wanisa

    2015-01-01

    Anthocyanin, a phenolic compound, has been reported to have an anti-inflammatory effect against lipopolysaccharide (LPS) induced changes in immune cells. However, little is known about the molecular mechanisms underlying its anti-inflammatory effects. Few research studies have concerned the anti-inflammation properties of colored rice extract as a functional material. Therefore, the purpose of this study was to examine anti-inflammatory effects of the polar fraction of black rice whole grain extracts (BR-WG-P) that features a high anthocyanin content. Our results showed that BR-WG-P significantly inhibited LPS-induced pro- inflammatory mediators, including production of NO and expression of iNOS and COX-2. In addition, secretion of pro-inflammatory cytokines including TNF-α and IL-6 was also significantly inhibited. Moreover, BR-WG-P and anthocyanin inhibited NF-kB and AP-1 translocation into the nucleus. BR-WG-P also decreased the phosphorylation of ERK, p38 and JNK in a dose dependent manner. These results suggested that BR-WG-P might suppress LPS-induced inflammation via the inhibition of the MAPK signaling pathway leading to decrease of NF-kB and AP-1 translocation. All of these results indicate that BR-WG-P exhibits therapeutic potential associated with the anthocyanin content in the extract for treating inflammatory diseases associated with cancer.

  15. Progesterone and the Repression of Myometrial Inflammation: The Roles of MKP-1 and the AP-1 System

    PubMed Central

    Lei, K.; Georgiou, E. X.; Chen, L.; Yulia, A.; Sooranna, S. R.; Brosens, J. J.; Bennett, P. R.

    2015-01-01

    Progesterone (P4) maintains uterine quiescence during pregnancy and its functional withdrawal is associated with increased prostaglandin synthesis and the onset of labor. In primary human myometrial cells, the glucocorticoid receptor (GR) rather than the P4 receptor mediates P4 antagonism of IL-1β-induced cyclooxygenase-2 (COX-2) expression, the rate-limiting enzyme in prostaglandin synthesis. We now report that P4 also acts via GR to induce MAPK phosphatase (MKP)-1 and knockdown of MKP-1 impairs the ability of P4 to repress IL-1β-dependent COX-2 induction. Microarray analysis revealed that P4 repressed preferentially activator protein-1-responsive genes in response to IL-1β. Consistent with these observations, we found that the ability of P4 to reduce c-Jun activation was lost upon GR as well as MKP-1 knockdown. Interestingly, c-Jun levels in human myometrial cells declined upon GR and MKP-1 knockdown, which suggests the presence of an activator protein-1 feedback loop. This is supported by our observation that c-Jun levels declined after an initial rise in primary myometrial cells treated with phorbol 12-myrisatate 13-acetate, a potent activator of c-Jun N-terminal kinase. Finally, we show that MKP-1 is an intermediate in P4-mediated repression of some but not all IL-1β-responsive genes. For example, P4 repression of IL11 and IRAK3 was maintained upon MKP-1 knockdown. Taken together, the data show that P4 acts via GR to drive MKP-1 expression, which in turn inhibits IL-1β-dependent c-Jun activation and COX-2 expression. PMID:26280733

  16. 2,3,5,6-Tetramethylpyrazine (TMP) down-regulated arsenic-induced heme oxygenase-1 and ARS2 expression by inhibiting Nrf2, NF-κB, AP-1 and MAPK pathways in human proximal tubular cells.

    PubMed

    Gong, Xuezhong; Ivanov, Vladimir N; Hei, Tom K

    2016-09-01

    Our recent study demonstrated that sodium arsenite at a clinically relevant dose induced nephrotoxicity in human renal proximal tubular epithelial cell line HK-2, which could be inhibited by natural product 2,3,5,6-tetramethylpyrazine (TMP) with antioxidant activity. The present study demonstrated that arsenic exposure resulted in protein and enzymatic induction of heme oxygenase-1 (HO-1) in dose- and time-dependent manners in HK-2 cells. Blocking HO-1 enzymatic activity by zinc protoporphyrin (ZnPP) augmented arsenic-induced apoptosis, ROS production and mitochondrial dysfunction, suggesting a critical role for HO-1 as a renal protectant in this procession. On the other hand, TMP, upstream of HO-1, inhibited arsenic-induced ROS production and ROS-dependent HO-1 expression. TMP also prevented mitochondria dysfunction and suppressed activation of the intrinsic apoptotic pathway in HK-2 cells. Our results revealed that the regulation of arsenic-induced HO-1 expression was performed through multiple ROS-dependent signal pathways and the corresponding transcription factors, including p38 MAPK and JNK (but not ERK), AP-1, Nrf2 and NF-κB. TMP inhibited arsenic-induced activations of JNK, p38 MAPK, ERK, AP-1 and Nrf2 and block HO-1 protein expression. The present study, furthermore, demonstrated arsenic-induced expression of arsenic response protein 2 (ARS2) that was regulated by p38 MAPK, ERK and NF-κB. To our knowledge, this is the first report showing that ARS2 involved in arsenic-induced nephrotoxicity, while TMP pretreatment prevented such an up-regulation of ARS2 in HK-2 cells. Given ARS2 and HO-1 sharing the similar regulation mechanism, we speculated that ARS2 might also mediate cell survival in this procession. In summary, our study highlighted a role of HO-1 in the protection against arsenic-induced cytotoxicity downstream from the primary targets of TMP and further indicated that TMP may be used as a potential therapeutic agent in the treatment of arsenic

  17. Transforming growth factor-β3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

    PubMed

    Zhang, Xu; Lui, Wing-Yee

    2015-06-01

    Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-β) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-β3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-β3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-β3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-β3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-β3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-β3-induced JAM-B protein degradation. In addition, TGF-β3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-β3 promotes the degradation of JAM-B transcript and TGF-β3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-β3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES.

  18. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes

    PubMed Central

    Sitaram, Anand; Dennis, Megan K.; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S.; Sviderskaya, Elena V.; Bennett, Dorothy C.; Raposo, Graça; Bonifacino, Juan S.; Marks, Michael S.

    2012-01-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine–based sorting signal in the pigment cell–specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1– and AP-3–favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs. PMID:22718909

  19. Differential recognition of a dileucine-based sorting signal by AP-1 and AP-3 reveals a requirement for both BLOC-1 and AP-3 in delivery of OCA2 to melanosomes.

    PubMed

    Sitaram, Anand; Dennis, Megan K; Chaudhuri, Rittik; De Jesus-Rojas, Wilfredo; Tenza, Danièle; Setty, Subba Rao Gangi; Wood, Christopher S; Sviderskaya, Elena V; Bennett, Dorothy C; Raposo, Graça; Bonifacino, Juan S; Marks, Michael S

    2012-08-01

    Cell types that generate unique lysosome-related organelles (LROs), such as melanosomes in melanocytes, populate nascent LROs with cargoes that are diverted from endosomes. Cargo sorting toward melanosomes correlates with binding via cytoplasmically exposed sorting signals to either heterotetrameric adaptor AP-1 or AP-3. Some cargoes bind both adaptors, but the relative contribution of each adaptor to cargo recognition and their functional interactions with other effectors during transport to melanosomes are not clear. Here we exploit targeted mutagenesis of the acidic dileucine-based sorting signal in the pigment cell-specific protein OCA2 to dissect the relative roles of AP-1 and AP-3 in transport to melanosomes. We show that binding to AP-1 or AP-3 depends on the primary sequence of the signal and not its position within the cytoplasmic domain. Mutants that preferentially bound either AP-1 or AP-3 each trafficked toward melanosomes and functionally complemented OCA2 deficiency, but AP-3 binding was necessary for steady-state melanosome localization. Unlike tyrosinase, which also engages AP-3 for optimal melanosomal delivery, both AP-1- and AP-3-favoring OCA2 variants required BLOC-1 for melanosomal transport. These data provide evidence for distinct roles of AP-1 and AP-3 in OCA2 transport to melanosomes and indicate that BLOC-1 can cooperate with either adaptor during cargo sorting to LROs.

  20. EV71 induces COX-2 expression via c-Src/PDGFR/PI3K/Akt/p42/p44 MAPK/AP-1 and NF-kappaB in rat brain astrocytes.

    PubMed

    Tung, Wei-Hsuan; Lee, I-Ta; Hsieh, Hsi-Lung; Yang, Chuen-Mao

    2010-08-01

    Enterovirus 71 (EV71) induces the expression of cyclooxgenase (COX)-2 served as a major neurotoxic factor in CNS injury. However, the mechanisms underlying EV71-initiated intracellular signaling pathways leading to COX-2 expression remain unknown. Therefore, we investigated the mechanisms underlying EV71-induced COX-2 expression and prostaglandin E(2) (PGE(2)) production in rat brain astrocytes (RBA)-1, determined by Western blotting, RT-PCR, and promoter assay. Here, we reported that EV71-induced COX-2 expression and PGE(2) production were attenuated by pretreatment with the inhibitors of c-Src (PP1), PDGFR (AG1296), PI3K (Wortmannin), MEK1/2 (PD98059), NF-kappaB (helenalin), and AP-1 (Tanshinone) and transfection with shRNA or siRNA of c-Src, PDGFR, p85, c-Jun, c-Fos, ERK1, or ERK2. We further observed that EV71-induced activation of Akt and p42/p44 MAPK were mediated via c-Src and PDGFR. Pretreatment with PP1 attenuated EV71-stimulated phosphorylation of Src, PDGFR, Akt, and p42/p44 MAPK. Inhibition of PI3K by Wortmannin attenuated EV71-induced Akt and p42/p44 MAPK phosphorylation, but had no effect on PDGFR phosphorylation, suggesting that PDGFR is an upstream and p42/p44 MAPK is a downstream component of PI3K/Akt in these responses. EV71-stimulated NF-kappaB translocation from the cytoplasm to the nucleus, IkappaBalpha degradation and NF-kappaB promoter activity were attenuated by pretreatment with helenalin, but not AG1296, Wortmannin, and PD98059. EV71-induced c-Jun mRNA expression was attenuated by pretreatment with PD98059, AG1296, or Wortmannin. These results demonstrate that in RBA-1 cells, EV71-induced COX-2 expression associated with PGE(2) production is mediated through activation of c-Src/PDGFR/PI3K/Akt/p42/p44 MAPK to initiate the expression of AP-1.

  1. Establishment and characterization of cetuximab resistant head and neck squamous cell carcinoma cell lines: focus on the contribution of the AP-1 transcription factor

    PubMed Central

    Boeckx, Carolien; Blockx, Lina; de Beeck, Ken Op; Limame, Ridha; Camp, Guy Van; Peeters, Marc; Vermorken, Jan B; Specenier, Pol; Wouters, An; Baay, Marc; Lardon, Filip

    2015-01-01

    Background: After an initial response to EGFR targeted therapy, secondary resistance almost invariably ensues, thereby limiting the clinical benefit of the drug. Hence, it has been recognized that the successful implementation of targeted therapy in the treatment of HNSCC cancer is very much dependent on predictive biomarkers for patient selection. Methods: We generated an in vitro model of acquired cetuximab resistance by chronically exposing three HNSCC cell lines to increasing cetuximab doses. Gene expression profiles of sensitive parental cells and resistant daughter cells were compared using microarray analysis. Growth inhibitory experiments were performed with an HB-EGF antibody and the MMP inhibitor, both in combination with cetuximab. Characteristics of EMT were analyzed using migration and invasion assays, immunofluorescent vimentin staining and qRT-PCR for several genes involved in this process. The function of the transcription factor AP-1 was investigated using qRT-PCR for several genes upregulated or downregulated in cetuximab resistant cells. Furthermore, anchorage-independent growth was investigated using the soft agar assay. Results: Gene expression profiling shows that cetuximab resistant cells upregulate several genes, including interleukin 8, the EGFR ligand HB-EGF and the metalloproteinase ADAM19. Cytotoxicity experiments with neutralizing HB-EGF antibody could not induce any growth inhibition, whereas an MMP inhibitor inhibited cell growth in cetuximab resistant cells. However, no synergetic effects combined with cetuximab could be observed. Cetuximab resistant cells showed traits of EMT, as witnessed by increased migratory potential, increased invasive potential, increased vimentine expression and increased expression of several genes involved in EMT. Furthermore, expression of upregulated genes could be repressed by the treatment with apigenin. The cetuximab resistant LICR-HN2 R10.3 cells tend to behave differently in cell culture, forming

  2. The role of Candida albicans AP-1 protein against host derived ROS in in vivo models of infection.

    PubMed

    Jain, Charu; Pastor, Kelly; Gonzalez, Arely Y; Lorenz, Michael C; Rao, Reeta P

    2013-01-01

    Candida albicans is a major fungal pathogen of humans, causing mucosal infections that are difficult to eliminate and systemic infections that are often lethal primarily due to defects in the host's innate status. Here we demonstrate the utility of Caenorhabditis elegans, a model host to study innate immunity, by exploring the role of reactive oxygen species (ROS) as a critical innate response against C. albicans infections. Much like a human host, the nematode's innate immune response is activated to produce ROS in response to fungal infection. We use the C. albicans cap1 mutant, which is susceptible to ROS, as a tool to dissect this physiological innate immune response and show that cap1 mutants fail to cause disease and death, except in bli-3 mutant worms that are unable to produce ROS because of a defective NADPH oxidase. We further validate the ROS-mediated host defense mechanism in mammalian phagocytes by demonstrating that chemical inhibition of the NADPH oxidase in cultured macrophages enables the otherwise susceptible cap1 mutant to resists ROS-mediated phagolysis. Loss of CAP1 confers minimal attenuation of virulence in a disseminated mouse model, suggesting that CAP1-independent mechanisms contribute to pathogen survival in vivo. Our findings underscore a central theme in the process of infection-the intricate balance between the virulence strategies employed by C. albicans and the host's innate immune system and validates C. elegans as a simple model host to dissect this balance at the molecular level.

  3. Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells

    PubMed Central

    Vuong, Linh M.; Chellappa, Karthikeyani; Dhahbi, Joseph M.; Deans, Jonathan R.; Fang, Bin; Bolotin, Eugene; Titova, Nina V.; Hoverter, Nate P.; Spindler, Stephen R.; Waterman, Marian L.

    2015-01-01

    The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/β-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/β-catenin/TCF4 and AP-1 pathways. PMID:26240283

  4. 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.

  5. Ceramide synthases CerS4 and CerS5 are upregulated by 17β-estradiol and GPER1 via AP-1 in human breast cancer cells.

    PubMed

    Wegner, Marthe-Susanna; Wanger, Ruth Anna; Oertel, Stephanie; Brachtendorf, Sebastian; Hartmann, Daniela; Schiffmann, Susanne; Marschalek, Rolf; Schreiber, Yannick; Ferreirós, Nerea; Geisslinger, Gerd; Grösch, Sabine

    2014-12-15

    Ceramide synthases (CerS) are important enzymes of the sphingolipid pathway, responsible for the production of ceramides with distinct chain lengths. In human breast cancer tissue, we detected a significant increase in CerS4 and CerS6 mRNA in estrogen receptor positive (ER+) cancer tissue. To clarify the molecular mechanism of this upregulation, we cloned CerS2, -4, -5 and CerS6 promoter and 3'-UTR fragments into luciferase reporter gene plasmids and determined luciferase activity in MCF-7 (ERα/β) and MDA-MB-231 (ERβ) cells after 17β-estradiol treatment. Only the activities of CerS4 and CerS5 promoter Luc constructs, as well as CerS2- and CerS5-3'-UTR Luc constructs increased after estradiol treatment in MCF-7 cells, and this could be inhibited by the anti-estrogen fulvestrant. Co-transfection with the G protein-coupled estrogen receptor 1 (GPER1) also enhanced CerS2, CerS4 and CerS6 promoter activity whereas CerS5 promoter activity was inhibited in both cell lines. Promoter deletion and mutation constructs from CerS4 and CerS5 promoters revealed that estradiol and GPER1 mediate their effects on both promoters by activating AP-1, most likely through dimerization of c-Jun and c-Fos. At least we could show, that cell proliferation induced by estradiol could be blocked by co-treatment with Fumonisin B1, indicating that upregulation of CerS in breast cancer cells by estrogen is important for cell proliferation and possibly tumor development. In conclusion, our data highlight transcriptional and posttranscriptional mechanisms regulating CerS expression in human cells which provide the basis for further studies investigating the regulation of CerS expression and ceramide synthesis after diverse stimuli in physiological and pathophysiological processess.

  6. The Expression of Fos, Jun and AP-1 DNA Binding Activity in Rat Supraoptic Nucleus Neurons Following Acute Versus Repeated Osmotic Stimulation

    DTIC Science & Technology

    1995-06-22

    stimulation. This pattern has been observed previously in the hippocampus after treatment with the seizure-inducing drug , metrazole (Sonnenberg et al... fosB , and fra-1 and -2. fra refers to ~OS­ ~elated ~ntigen. Western blot experiments and employment of less stringent nucleic acid hybridization...fos, fra-l and fosB , only form heterodimeric complexes with Jun-related proteins (Nakabeppu et al., 1988; Rauscher et al., 1988b) The AP-l site of many

  7. TE-domestication and horizontal transfer in a putative Nef-AP1mu mimic of HLA-A cytoplasmic domain re-trafficking.

    PubMed

    Murray, Joseph S; Murray, Elaina H

    2016-01-01

    Genes of the major histocompatibility complex (MHC; also called HLA in human) are polymorphic elements in the genomes of sharks to humans. Class-I and class-II MHC loci appear responsible for much of the genetic linkage to myriad disease states via the capacity to bind short (~8-15 a.a.) peptides of a given pathogen's proteome, or in some cases, the altered proteomes of cancerous cells, and even (in autoimmunity) certain nominal 'self' peptides (Janeway, 2004).(1) Unfortunately, little is known about how the canonical structure of the MHC-I/-II peptide-presenting gene evolved, particularly since beyond ~500 Mya (sharks) no paralogs exist.(2,3) We previously reported that HLA-A isotype alleles with the α1-helix, R65 motif, are wide-spread in phylogeny, but that the α 2-helix, H151R motif, has apparently segregated out of most species. Surprisingly, an uncharacterized orf in T. syrichta (Loc-103275158) encoded R151, but within a truncated A-23 like gene containing 5'- and 3'- footprints of the transposon (TE), tigger-1; the extant tarsier A-23 allele is totally missing exon-3 and part-of exon-4; together, suggesting TE-mediated inactivation of an intact/ancestral A-23 allele (Murray, 2015a).(4) The unique Loc-103275158 orf encodes a putative 15-exon transcript with no apparent paralogs throughout phylogeny. However, an HLA-A11 like gene in M. leucophaeus with a shortened C-terminal domain, and an HLA-A like orf in C. atys with two linked α1/α2/α3 domains, both contain a second transmembrane segment, which is conserved in Loc-103275158. Thus, we could model the putative protein with its Nef-like tail domain docked to its MHC-I like α3 domain (i.e., on the same side of a membrane). This modeled tertiary structure is strikingly similar to the solved structure of the Nef:MHC-I CD:AP1mu transporter (Jia, 2012).(5) Nef:AP1mu binds the CD of MHC-I in trafficking MHC-I away from the trans-golgi and into the endocytic pathway in HIV-1 infected cells. The CD loop of the

  8. Iron, oxidative stress, and virulence: roles of iron-sensitive transcription factor Sre1 and the redox sensor ChAp1 in the maize pathogen Cochliobolus heterostrophus.

    PubMed

    Zhang, Ning; MohdZainudin, Nur A I; Scher, Keren; Condon, Bradford J; Horwitz, Benjamin A; Turgeon, B Gillian

    2013-12-01

    The gene SRE1, encoding the GATA transcription factor siderophore biosynthesis repressor (Sre1), was identified in the genome of the maize pathogen Cochliobolus heterostrophus and deleted. Mutants were altered in sensitivity to iron, oxidative stress, and virulence to the host. To gain insight into mechanisms of this combined regulation, genetic interactions among SRE1 (the nonribosomal peptide synthetase encoding gene NPS6, which is responsible for extracellular siderophore biosynthesis) and ChAP1 (encoding a transcription factor regulating redox homeostasis) were studied. To identify members of the Sre1 regulon, expression of candidate iron and oxidative stress-related genes was assessed in wild-type (WT) and sre1 mutants using quantitative reverse-transcription polymerase chain reaction. In sre1 mutants, NPS6 and NPS2 genes, responsible for siderophore biosynthesis, were derepressed under iron replete conditions, whereas the high-affinity reductive iron uptake pathway associated gene, FTR1, was not, in contrast to outcomes with other well-studied fungal models. C. heterostrophus L-ornithine-N(5)- monooxygenase (SIDA2), ATP-binding cassette (ABC6), catalase (CAT1), and superoxide dismutase (SOD1) genes were also derepressed under iron-replete conditions in sre1 mutants. Chap1nps6 double mutants were more sensitive to oxidative stress than either Chap1 or nps6 single mutants, while Chap1sre1 double mutants showed a modest increase in resistance compared with single Chap1 mutants but were much more sensitive than sre1 mutants. These findings suggest that the NPS6 siderophore indirectly contributes to redox homeostasis via iron sequestration, while Sre1 misregulation may render cells more sensitive to oxidative stress. The double-mutant phenotypes are consistent with a model in which iron sequestration by NPS6 defends the pathogen against oxidative stress. C. heterostrophus sre1, nps6, Chap1, Chap1nps6, and Chap1sre1 mutants are all reduced in virulence toward the

  9. Cell-specific expression of the macrophage scavenger receptor gene is dependent on PU.1 and a composite AP-1/ets motif.

    PubMed Central

    Moulton, K S; Semple, K; Wu, H; Glass, C K

    1994-01-01

    The type I and II scavenger receptors (SRs) are highly restricted to cells of monocyte origin and become maximally expressed during the process of monocyte-to-macrophage differentiation. In this report, we present evidence that SR genomic sequences from -245 to +46 bp relative to the major transcriptional start site were sufficient to confer preferential expression of a reporter gene to cells of monocyte and macrophage origin. This profile of expression resulted from the combinatorial actions of multiple positive and negative regulatory elements. Positive transcriptional control was primarily determined by two elements, located 181 and 46 bp upstream of the major transcriptional start site. Transcriptional control via the -181 element was mediated by PU.1/Spi-1, a macrophage and B-cell-specific transcription factor that is a member of the ets domain gene family. Intriguingly, the -181 element represented a relatively low-affinity binding site for Spi-B, a closely related member of the ets domain family that has been shown to bind with relatively high affinity to other PU.1/Spi-1 binding sites. These observations support the idea that PU.1/Spi-1 and Spi-B regulate overlapping but nonidentical sets of genes. The -46 element represented a composite binding site for a distinct set of ets domain proteins that were preferentially expressed in monocyte and macrophage cell lines and that formed ternary complexes with members of the AP-1 gene family. In concert, these observations suggest a model for how interactions between cell-specific and more generally expressed transcription factors function to dictate the appropriate temporal and cell-specific patterns of SR expression during the process of macrophage differentiation. Images PMID:8007948

  10. Functional Conservation and Divergence of Four Ginger AP1/AGL9 MADS–Box Genes Revealed by Analysis of Their Expression and Protein–Protein Interaction, and Ectopic Expression of AhFUL Gene in Arabidopsis

    PubMed Central

    Song, Juanjuan; Sun, Wei; Xia, Kuaifei; Liao, Jingping; Zhang, Mingyong

    2014-01-01

    Alpinia genus are known generally as ginger–lilies for showy flowers in the ginger family, Zingiberaceae, and their floral morphology diverges from typical monocotyledon flowers. However, little is known about the functions of ginger MADS–box genes in floral identity. In this study, four AP1/AGL9 MADS–box genes were cloned from Alpinia hainanensis, and protein–protein interactions (PPIs) and roles of the four genes in floral homeotic conversion and in floral evolution are surveyed for the first time. AhFUL is clustered to the AP1lineage, AhSEP4 and AhSEP3b to the SEP lineage, and AhAGL6–like to the AGL6 lineage. The four genes showed conserved and divergent expression patterns, and their encoded proteins were localized in the nucleus. Seven combinations of PPI (AhFUL–AhSEP4, AhFUL–AhAGL6–like, AhFUL–AhSEP3b, AhSEP4–AhAGL6–like, AhSEP4–AhSEP3b, AhAGL6–like–AhSEP3b, and AhSEP3b–AhSEP3b) were detected, and the PPI patterns in the AP1/AGL9 lineage revealed that five of the 10 possible combinations are conserved and three are variable, while conclusions cannot yet be made regarding the other two. Ectopic expression of AhFUL in Arabidopsis thaliana led to early flowering and floral organ homeotic conversion to sepal–like or leaf–like. Therefore, we conclude that the four A. hainanensis AP1/AGL9 genes show functional conservation and divergence in the floral identity from other MADS–box genes. PMID:25461565

  11. Identification of a Novel Metabolite in the Degradation of Pyrene by Mycobacterium sp. Strain AP1: Actions of the Isolate on Two- and Three-Ring Polycyclic Aromatic Hydrocarbons

    PubMed Central

    Vila, Joaquim; López, Zaira; Sabaté, Jordi; Minguillón, Cristina; Solanas, Anna M.; Grifoll, Magdalena

    2001-01-01

    Mycobacterium sp. strain AP1 grew with pyrene as a sole source of carbon and energy. The identification of metabolites accumulating during growth suggests that this strain initiates its attack on pyrene by either monooxygenation or dioxygenation at its C-4, C-5 positions to give trans- or cis-4,5-dihydroxy-4,5-dihydropyrene, respectively. Dehydrogenation of the latter, ortho cleavage of the resulting diol to form phenanthrene 4,5-dicarboxylic acid, and subsequent decarboxylation to phenanthrene 4-carboxylic acid lead to degradation of the phenanthrene 4-carboxylic acid via phthalate. A novel metabolite identified as 6,6′-dihydroxy-2,2′-biphenyl dicarboxylic acid demonstrates a new branch in the pathway that involves the cleavage of both central rings of pyrene. In addition to pyrene, strain AP1 utilized hexadecane, phenanthrene, and fluoranthene for growth. Pyrene-grown cells oxidized the methylenic groups of fluorene and acenaphthene and catalyzed the dihydroxylation and ortho cleavage of one of the rings of naphthalene and phenanthrene to give 2-carboxycinnamic and diphenic acids, respectively. The catabolic versatility of strain AP1 and its use of ortho cleavage mechanisms during the degradation of polycyclic aromatic hydrocarbons (PAHs) give new insight into the role that pyrene-degrading bacterial strains may play in the environmental fate of PAH mixtures. PMID:11722898

  12. HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s) alternative to the pPGI-pPGM-AGP pathway.

    PubMed

    Bahaji, Abdellatif; Baroja-Fernández, Edurne; Sánchez-López, Angela María; Muñoz, Francisco José; Li, Jun; Almagro, Goizeder; Montero, Manuel; Pujol, Pablo; Galarza, Regina; Kaneko, Kentaro; Oikawa, Kazusato; Wada, Kaede; Mitsui, Toshiaki; Pozueta-Romero, Javier

    2014-01-01

    In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.

  13. Brain-derived neurotrophic factor and epidermal growth factor activate neuronal m-calpain via mitogen-activated protein kinase-dependent phosphorylation.

    PubMed

    Zadran, Sohila; Jourdi, Hussam; Rostamiani, Karoline; Qin, Qingyu; Bi, Xiaoning; Baudry, Michel

    2010-01-20

    Calpain is a calcium-dependent protease that plays a significant role in synaptic plasticity, cell motility, and neurodegeneration. Two major calpain isoforms are present in brain, with mu-calpain (calpain1) requiring micromolar calcium concentrations for activation and m-calpain (calpain2) needing millimolar concentrations. Recent studies in fibroblasts indicate that epidermal growth factor (EGF) can activate m-calpain independently of calcium via mitogen-activated protein kinase (MAPK)-mediated phosphorylation. In neurons, MAPK is activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate m-calpain by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells, both of which express BDNF and EGF receptors. Calpain activation was monitored by quantitative analysis of spectrin degradation and by a fluorescence resonance energy transfer (FRET)-based assay, which assessed the truncation of a calpain-specific peptide flanked by the FRET fluorophore pair DABCYL and EDANS. In both cell types, BDNF and EGF rapidly elicited calpain activation, which was completely blocked by MAPK and calpain inhibitors. BDNF stimulated m-calpain but not mu-calpain serine phosphorylation, an effect also blocked by MAPK inhibitors. Remarkably, BDNF- and EGF-induced calpain activation was preferentially localized in dendrites and dendritic spines of hippocampal neurons and was associated with actin polymerization, which was prevented by calpain inhibition. Our results indicate that, in cultured neurons, both BDNF and EGF activate m-calpain by MAPK-mediated phosphorylation. These results strongly support a role for calpain in synaptic plasticity and may explain why m-calpain, although widely expressed in CNS, requires nonphysiological calcium levels for activation.

  14. 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.

  15. 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.

  16. Drosophila Jun kinase regulates expression of decapentaplegic via the ETS-domain protein Aop and the AP-1 transcription factor DJun during dorsal closure.

    PubMed

    Riesgo-Escovar, J R; Hafen, E

    1997-07-01

    During Drosophila embryogenesis, ectodermal cells of the lateral epithelium stretch in a coordinated fashion to internalize the amnioserosa cells and close the embryo dorsally. This process, dorsal closure, requires two signaling pathways: the Drosophila Jun-amino-terminal kinase (DJNK) pathway and the Dpp pathway. We have identified mutations in DJun and show that DJNK controls dorsal closure by activating DJun and inactivating the ETS repressor Aop/Yan by phosphorylation. DJun and Aop regulate dpp expression in the most dorsal row of cells. Secreted Dpp then instructs more ventrally located cells to stretch. Our results provide a causal link between the DJNK and Dpp pathways during dorsal closure. Interestingly, in vertebrates, transforming growth factor-beta and c-Jun regulate collagenase gene expression during wound healing, a process that also involves the closing of an epithelial sheath.

  17. Downregulation of feline sarcoma-related protein inhibits cell migration, invasion and epithelial-mesenchymal transition via the ERK/AP-1 pathway in bladder urothelial cell carcinoma

    PubMed Central

    Hu, Xudong; Zhang, Zhiqiang; Liang, Zhaofeng; Xie, Dongdong; Zhang, Tao; Yu, Dexin; Zhong, Caiyun

    2017-01-01

    Feline sarcoma-related protein (Fer) is a nuclear and cytoplasmic non-receptor protein tyrosine kinase and Fer overexpression is associated with various biological processes. However, the clinicopathological characteristics and molecular mechanisms of Fer expression in bladder urothelial cell carcinoma (UCC) have yet to be elucidated. The present study demonstrated that Fer was significantly upregulated in bladder UCC tissues and cell lines. A clinicopathological analysis suggested that Fer expression was significantly associated with tumor stage, histological grade and lymph node status, and Fer expression was a prognostic factor for overall survival in a multivariate analysis. Furthermore, small interfering RNA (siRNA) was used to silence the expression of the Fer gene in human bladder UCC T24 cells, and was shown to significantly reduce the migration and invasion of the cells. It was also observed that Fer-siRNA caused the T24 cells to acquire an epithelial cobblestone phenotype, and was able to reverse the epithelial-mesenchymal transition of the cells. Subsequently, Fer-knockdown was shown to deactivate the extracellular signal-regulated kinase/activator protein-1 signaling pathway in T24 cells. These results indicated, for the first time, that Fer has a critical role in bladder UCC progression and may be a potential therapeutic target for bladder UCC metastasis. PMID:28356947

  18. QSAR study of ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-4-(trifluoromethyl) pyrimidine-5-carboxylate: an inhibitor of AP-1 and NF-kappa B mediated gene expression based on support vector machines.

    PubMed

    Liu, H X; Zhang, R S; Yao, X J; Liu, M C; Hu, Z D; Fan, B T

    2003-01-01

    The support vector machine, as a novel type of learning machine, for the first time, was used to develop a QSAR model of 57 analogues of ethyl 2-[(3-methyl-2,5-dioxo(3-pyrrolinyl))amino]-4-(trifluoromethyl)pyrimidine-5-carboxylate (EPC), an inhibitor of AP-1 and NF-kappa B mediated gene expression, based on calculated quantum chemical parameters. The quantum chemical parameters involved in the model are Kier and Hall index (order3) (KHI3), Information content (order 0) (IC0), YZ Shadow (YZS) and Max partial charge for an N atom (MaxPCN), Min partial charge for an N atom (MinPCN). The mean relative error of the training set, the validation set, and the testing set is 1.35%, 1.52%, and 2.23%, respectively, and the maximum relative error is less than 5.00%.

  19. Anti-inflammatory effects of novel AP-1 and NF-κB inhibitors in dextran-sulfate-sodium-induced colitis in rats.

    PubMed

    El-Salhy, Magdy; Umezawa, Kazuo

    2016-06-01

    The aim of the present study was to elucidate the anti-inflammatory effects of the two novel anti-inflammatory substances, 3-[(dodecylthiocarbonyl)‑methyl]-glutarimide (DTCM-G) and dehydroxymethylepoxyquinomicin (DHMEQ), on DSS-induced colitis in rats. For this purpose, rats with dextran sulfate sodium (DSS)-induced colitis were randomly divided into 3 groups with 10 animals in each group as follows: i) the control group, which received 0.5 ml of 0.5% carboxymethyl cellulose (CMC; vehicle), ii) rats that received DTCM-G (20 mg/kg body weight in 0.5% CMC; the DTCM-G group), and iii) rats that received DHMEQ (15 mg/kg body weight in 0.5% CMC; the DHMEQ group). The animals were sacrificed after the 5-day treatment period, and tissue samples were taken from their colons and sectioned for histological evaluation. The tissue sections were stained with hematoxylin and eosin, and immunostained for leukocytes, lymphocytes, macrophages/monocytes and mast cells. The disease activity index (DAI), histological grading of colitis, and densities of several types of submucosal immune cells were compared between the controls, and the DTCM-G and DHMEQ groups. The DAI values were significantly lower in both the DTCM-G and DHMEQ groups than in the control group. The total scores for the histological grading of colitis were also significant