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Sample records for activated c-jun n-terminal

  1. Ozone exposure triggers insulin resistance through muscle c-Jun N-terminal kinase activation.

    PubMed

    Vella, Roxane E; Pillon, Nicolas J; Zarrouki, Bader; Croze, Marine L; Koppe, Laetitia; Guichardant, Michel; Pesenti, Sandra; Chauvin, Marie-Agnès; Rieusset, Jennifer; Géloën, Alain; Soulage, Christophe O

    2015-03-01

    A growing body of evidence suggests that exposure to traffic-related air pollution is a risk factor for type 2 diabetes. Ozone, a major photochemical pollutant in urban areas, is negatively associated with fasting glucose and insulin levels, but most aspects of this association remain to be elucidated. Using an environmentally realistic concentration (0.8 parts per million), we demonstrated that exposure of rats to ozone induced whole-body insulin resistance and oxidative stress, with associated endoplasmic reticulum (ER) stress, c-Jun N-terminal kinase (JNK) activation, and disruption of insulin signaling in skeletal muscle. Bronchoalveolar lavage fluids from ozone-treated rats reproduced this effect in C2C12 myotubes, suggesting that toxic lung mediators were responsible for the phenotype. Pretreatment with the chemical chaperone 4-phenylbutyric acid, the JNK inhibitor SP600125, or the antioxidant N-acetylcysteine alleviated insulin resistance, demonstrating that ozone sequentially triggered oxidative stress, ER stress, and JNK activation to impair insulin signaling in muscle. This study is the first to report that ozone plays a causative role in the development of insulin resistance, suggesting that it could boost the development of diabetes. We therefore provide a potential mechanism linking pollutant exposure and the increased incidence of metabolic diseases. PMID:25277399

  2. Activation of c-Jun transcription factor by substitution of a charged residue in its N-terminal domain.

    PubMed Central

    Hoeffler, W K; Levinson, A D; Bauer, E A

    1994-01-01

    C-Jun is a cellular transcription factor that can control gene expression in response to treatment of cells with phorbol esters, growth factors, and expression of some oncogenes. The ability of c-Jun to catalyze the transcription of certain genes is controlled, in part, by changes in the phosphorylation state of specific amino acids in c-Jun. One of the major sites that is phosphorylated during signal response is Ser73. Here we show that substitution of a negatively charged aspartic acid residue at 73 constitutively increased transcriptional activity of c-Jun. The Asp73 substitution also enhanced its availability to bind to DNA in a whole cell extract without altering its intrinsic DNA binding activity since the intrinsic activity was unaltered for the c-Jun mutant proteins expressed in a bacterial system. The negatively charged Asp substitution may mimic the negative charge of a phosphorylated serine at 73. The substitution of an uncharged alanine at 73 resulted in lowered activities. The N-terminal end of c-Jun containing these substitutions was fused to the DNA-binding region of the bovine papilloma virus E2 protein, and was able to confer the same activation properties to the fusion protein at the heterologous E2 DNA-binding site. Ser73 lies in a region of c-Jun previously proposed to bind an uncharacterized inhibitor, perhaps related to a protein of approximately 17.5 kD that coprecipitates along with our c-Jun or the JunE2 fusion products. Images PMID:8165146

  3. Costunolide-induced apoptosis in human leukemia cells: involvement of c-jun N-terminal kinase activation.

    PubMed

    Choi, Jung-Hye; Lee, Kyung-Tae

    2009-10-01

    The authors previously reported that costunolide, an active compound isolated from the stem bark of Magnolia sieboldii, induced apoptosis via reactive oxygen species (ROS) and Bcl-2-dependent mitochondrial permeability transition in human leukemia cells. In the present study, the authors investigated whether mitogen-activated protein kinases (MAPKs) are involved in the costunolide-induced apoptosis in human promonocytic leukemia U937 cells. Treatment with costunolide resulted in the significant activation of c-Jun N-terminal kinase (JNK), but not of extracellular-signal-related kinase (ERK1/2) or p38. In vitro kinase assays showed that JNK activity was low in untreated cells but increased dramatically after 30 min of costunolide treatment. U937 cells co-treated with costunolide and sorbitol, a JNK activator, exhibited higher levels of cell death. In addition, inhibition of the JNK pathway using a dominant-negative mutation of c-jun and JNK inhibitor SP600125, significantly prevented costunolide-induced apoptosis. Furthermore, pretreatment with the antioxidant NAC (N-acetyl-L-cysteine) blocked the costunolide-stimulated activation of JNK while the overexpression of Bcl-2 failed to reverse JNK activation. Pretreatment with SP600125 recovered the costunolide-suppressed Bcl-2 expression. These results indicate that costunolide-induced JNK activation acts downstream of ROS but upstream of Bcl-2, and suggest that ROS-mediated JNK activation plays a key role in costunolide-induced apoptosis. Moreover, the administration of costunolide (intraperitoneally once a day for 7 d) significantly suppressed tumor growth and increased survival in 3LL Lewis lung carcinoma-bearing model. PMID:19801848

  4. Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress.

    PubMed Central

    Dougherty, Christopher J; Kubasiak, Lori A; Prentice, Howard; Andreka, Peter; Bishopric, Nanette H; Webster, Keith A

    2002-01-01

    Reperfusion injury occurs when ischaemic tissue is reperfused. It involves the generation and release of reactive oxygen that activates numerous signalling pathways and initiates cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approx. 30% of the myocytes. Although JNK activation has been described in a number of models of ischaemia/reperfusion, the contribution of JNK activation to cell fate has not been established. Here we report that the activation of JNK by reoxygenation correlates with myocyte survival. Transfection of myocytes with JNK pathway interfering plasmid vectors or infection with adenoviral vectors support the hypothesis that JNK is protective. Transfection or infection with JNK inhibitory mutants increased the rates of apoptosis by almost 2-fold compared with control cultures grown aerobically or subjected to hypoxia and reoxygenation. Caspase 9 activity, measured by LEHD cleavage, increased >3-fold during reoxygenation and this activity was enhanced significantly at all times in cultures infected with dominant negative JNK adenovirus. Hypoxia-reoxygenation mediated a biphasic (2.6- and 2.9-fold) activation of p38 mitogen-activated protein kinase, as well as a small increase of tumour necrosis factor alpha (TNFalpha) secretion, but treatments with the p38 MAPK-specific inhibitor SB203580 or saturating levels of a TNFalpha-1 blocking antibody provided only partial protection against apoptosis. The results suggest that JNK activation is protective and that the pathway is largely independent of p38 MAPK or secreted TNFalpha. PMID:11879182

  5. Design, Synthesis, and Structure-Activity Relationship of Substrate Competitive, Selective, and in Vivo Active Triazole and Thiadiazole inhibitors of the c-Jun N-Terminal Kinase

    PubMed Central

    De, Surya K.; Stebbins, John L.; Chen, Li-Hsing; Riel-Mehan, Megan; Machleidt, Thomas; Dahl, Russell; Yuan, Hongbin; Emdadi, Aras; Barile, Elisa; Chen, Vida; Murphy, Ria; Pellecchia, Maurizio

    2009-01-01

    We report comprehensive structure activity relationship studies on a novel series of c-Jun N-terminal kinase (JNK) inhibitors. The compounds are substrate competitive inhibitors that bind to the docking site of the kinase. The reported medicinal chemistry and structure-based optimizations studies resulted in the discovery of selective and potent thiadiazole JNK inhibitors that displays promising in vivo activity in mouse models of insulin insensitivity. PMID:19271755

  6. cJun N-terminal kinase (JNK) phosphorylation of serine 36 is critical for p66Shc activation

    PubMed Central

    Khalid, Sana; Drasche, Astrid; Thurner, Marco; Hermann, Martin; Ashraf, Muhammad Imtiaz; Fresser, Friedrich; Baier, Gottfried; Kremser, Leopold; Lindner, Herbert; Troppmair, Jakob

    2016-01-01

    p66Shc-dependent ROS production contributes to many pathologies including ischemia/reperfusion injury (IRI) during solid organ transplantation. Inhibiting p66Shc activation may provide a novel therapeutic approach to prevent damage, which is poorly managed by antioxidants in vivo. Previous work suggested that pro-oxidant and a pro-apoptotic function of p66Shc required mitochondrial import, which depended on serine 36 phosphorylation. PKCß has been proposed as S36 kinase but cJun N-terminal kinases (JNKs) may also phosphorylate this residue. To simulate the early stages of ischemia/reperfusion (IR) we either used H2O2 treatment or hypoxia/reoxygenation (HR). As during reperfusion in vivo, we observed increased JNK and p38 activity in mouse embryonic fibroblasts (MEFs) and HL-1 cardiomyocytes along with significantly increased p66ShcS36 phosphorylation, ROS production and cell damage. Application of specific inhibitors caused a pronounced decrease in p66ShcS36 phosphorylation only in the case of JNK1/2. Moreover, S36 phosphorylation of recombinant p66Shc by JNK1 but not PKCß was demonstrated. We further confirmed JNK1/2-dependent regulation of p66ShcS36 phosphorylation, ROS production and cell death using JNK1/2 deficient MEFs. Finally, the low ROS phenotype of JNK1/2 knockout MEFs was reversed by the phosphomimetic p66ShcS36E mutant. Inhibiting JNK1/2-regulated p66Shc activation may thus provide a therapeutic approach for the prevention of oxidative damage. PMID:26868434

  7. Enzyme kinetics and interaction studies for human JNK1β1 and substrates activating transcription factor 2 (ATF2) and c-Jun N-terminal kinase (c-Jun).

    PubMed

    Figuera-Losada, Mariana; LoGrasso, Philip V

    2012-04-13

    c-Jun N-terminal kinase (JNK) is a stress signal transducer linked to cell death, and survival. JNK1 has been implicated in obesity, glucose intolerance, and insulin resistance. In this study we report the kinetic mechanism for JNK1β1 with transcription factors ATF2 and c-Jun along with interaction kinetics for these substrates. JNK1β1 followed a random sequential mechanism forming a ternary complex between JNK-substrate-ATP. K(m) for ATF2 and c-Jun was 1.1 and 2.8 μM, respectively. Inhibition studies using adenosine 5'-(β,γ-methylenetriphosphate) and a peptide derived from JNK interacting protein 1 (JIP1) supported the proposed kinetic mechanism. Biolayer interferometry studies showed that unphosphorylated JNK1β1 bound to ATF2 with similar affinity as it did to c-Jun (K(D) = 2.60 ± 0.34 versus 1.00 ± 0.35 μM, respectively). The presence of ATP increased the affinity of unphosphorylated JNK1β1 for ATF2 and c-Jun, to 0.80 ± 0.04 versus 0.65 ± 0.07 μM, respectively. Phosphorylation of JNK1β1 decreased the affinity of the kinase for ATF2 to 11.0 ± 1.1 μM and for c-Jun to 17.0 ± 7.5 μM in the absence of ATP. The presence of ATP caused a shift in the K(D) of the active kinase for ATF2 to 1.70 ± 0.25 μM and for c-Jun of 3.50 ± 0.95 μM. These results are the first kinetic and biochemical characterization of JNK1β1 and uncover some of the differences in the enzymatic activity of JNK1β1 compared with other variants and suggest that ATP binding or JNK phosphorylation could induce changes in the interactions with substrates, activators, and regulatory proteins. PMID:22351776

  8. Activation of c-Jun N-terminal kinase and apoptosis in endothelial cells mediated by endogenous generation of hydrogen peroxide

    NASA Technical Reports Server (NTRS)

    Ramachandran, Anup; Moellering, Douglas; Go, Young-Mi; Shiva, Sruti; Levonen, Anna-Liisa; Jo, Hanjoong; Patel, Rakesh P.; Parthasarathy, Sampath; Darley-Usmar, Victor M.

    2002-01-01

    Reactive oxygen species have been implicated in the activation of signal transduction pathways. However, extracellular addition of oxidants such as hydrogen peroxide (H2O2) often requires concentrations that cannot be readily achieved under physiological conditions to activate biological responses such as apoptosis. Explanations for this discrepancy have included increased metabolism of H2O2 in the extracellular environment and compartmentalization within the cell. We have addressed this issue experimentally by examining the induction of apoptosis of endothelial cells induced by exogenous addition of H2O2 and by a redox cycling agent, 2,3-dimethoxy-1,4-naphthoquinone, that generates H2O2 in cells. Here we show that low nanomolar steady-state concentrations (0.1-0.5 nmol x min(-1) x 10(6) cells) of H2O2 generated intracellularly activate c-Jun N terminal kinase and initiate apoptosis in endothelial cells. A comparison with bolus hydrogen peroxide suggests that the low rate of intracellular formation of this reactive oxygen species results in a similar profile of activation for both c-Jun N terminal kinase and the initiation of apoptosis. However, a detailed analysis reveals important differences in both the duration and profile for activation of these signaling pathways.

  9. Monosodium Urate in the Presence of RANKL Promotes Osteoclast Formation through Activation of c-Jun N-Terminal Kinase

    PubMed Central

    Choe, Jung-Yoon; Park, Ki-Yeun; Kim, Seong-Kyu

    2015-01-01

    The aim of this study was to clarify the role of monosodium urate (MSU) crystals in receptor activator of nuclear factor kB ligand- (RANKL-) RANK-induced osteoclast formation. RAW 264.7 murine macrophage cells were incubated with MSU crystals or RANKL and differentiated into osteoclast-like cells as confirmed by staining for tartrate-resistant acid phosphatase (TRAP) and actin ring, pit formation assay, and TRAP activity assay. MSU crystals in the presence of RANKL augmented osteoclast differentiation, with enhanced mRNA expression of NFATc1, cathepsin K, carbonic anhydrase II, and matrix metalloproteinase-9 (MMP-9), in comparison to RAW 264.7 macrophages incubated in the presence of RANKL alone. Treatment with both MSU crystals and RANKL induced osteoclast differentiation by activating downstream molecules in the RANKL-RANK pathway including tumor necrosis factor receptor-associated factor 6 (TRAF-6), JNK, c-Jun, and NFATc1. IL-1b produced in response to treatment with both MSU and RANKL is involved in osteoclast differentiation in part through the induction of TRAF-6 downstream of the IL-1b pathway. This study revealed that MSU crystals contribute to enhanced osteoclast formation through activation of RANKL-mediated pathways and recruitment of IL-1b. These findings suggest that MSU crystals might be a pathologic causative agent of bone destruction in gout. PMID:26347587

  10. Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway.

    PubMed Central

    Logan, S K; Falasca, M; Hu, P; Schlessinger, J

    1997-01-01

    The signaling events which mediate activation of c-Jun N-terminal kinase (JNK) are not yet well characterized. To broaden our understanding of upstream mediators which link extracellular signals to the JNK pathway, we investigated the role of phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-mediated JNK activation. In this report we demonstrate that a dominant negative form of PI 3-kinase as well as the inhibitor wortmannin blocks EGF-induced JNK activation dramatically. However, wortmannin does not have an effect on JNK activation induced by UV irradiation or osmotic shock. In addition, a membrane-targeted, constitutively active PI 3-kinase (p110beta) was shown to produce in vivo products and to activate JNK, while a kinase-mutated form of this protein showed no activation. On the basis of these experiments, we propose that PI 3-kinase activity plays a role in EGF-induced JNK activation in these cells. PMID:9315636

  11. Inhibition of Apoptosis in Prostate Cancer Cells by Androgens Is Mediated through Downregulation of c-Jun N-terminal Kinase Activation1

    PubMed Central

    Lorenzo, Petra Isabel; Saatcioglu, Fahri

    2008-01-01

    Androgen deprivation induces the regression of prostate tumors mainly due to an increase in the apoptosis rate; however, the molecular mechanisms underlying the antiapoptotic actions of androgens are not completely understood. We have studied the antiapoptotic effects of androgens in prostate cancer cells exposed to different proapoptotic stimuli. Terminal deoxynucleotidyl transferase-mediated nick-end labeling and nuclear fragmentation analyses demonstrated that androgens protect LNCaP prostate cancer cells from apoptosis induced by thapsigargin, the phorbol ester 12-O-tetradecanoyl-13-phorbol-acetate, or UV irradiation. These three stimuli require the activation of the c-Jun N-terminal kinase (JNK) pathway to induce apoptosis and in all three cases, androgen treatment blocks JNK activation. Interestingly, okadaic acid, a phosphatase inhibitor that causes apoptosis in LNCaP cells, induces JNK activation that is also inhibited by androgens. Actinomycin D, the antiandrogen bicalutamide or specific androgen receptor (AR) knockdown by small interfering RNA all blocked the inhibition of JNK activation mediated by androgens indicating that this activity requires AR-dependent transcriptional activation. These data suggest that the crosstalk between AR and JNK pathways may have important implications in prostate cancer progression and may provide targets for the development of new therapies. PMID:18472959

  12. The loss of c-Jun N-terminal protein kinase activity prevents the amyloidogenic cleavage of amyloid precursor protein and the formation of amyloid plaques in vivo.

    PubMed

    Mazzitelli, Sonia; Xu, Ping; Ferrer, Isidre; Davis, Roger J; Tournier, Cathy

    2011-11-23

    Phosphorylation plays a central role in the dynamic regulation of the processing of the amyloid precursor protein (APP) and the production of amyloid-β (Aβ), one of the clinically most important factors that determine the onset of Alzheimer's disease (AD). This has led to the hypothesis that aberrant Aβ production associated with AD results from regulatory defects in signal transduction. However, conflicting findings have raised a debate over the identity of the signaling pathway that controls APP metabolism. Here, we demonstrate that activation of the c-Jun N-terminal protein kinase (JNK) is essential for mediating the apoptotic response of neurons to Aβ. Furthermore, we discovered that the functional loss of JNK signaling in neurons significantly decreased the number of amyloid plaques present in the brain of mice carrying familial AD-linked mutant genes. This correlated with a reduction in Aβ production. Biochemical analyses indicate that the phosphorylation of APP at threonine 668 by JNK is required for γ-mediated cleavage of the C-terminal fragment of APP produced by β-secretase. Overall, this study provides genetic evidence that JNK signaling is required for the formation of amyloid plaques in vivo. Therefore, inhibition of increased JNK activity associated with aging or with a pathological condition constitutes a potential strategy for the treatment of AD. PMID:22114267

  13. TNF-α increases the expression and activity of vitamin D receptor in keratinocytes: role of c-Jun N-terminal kinase

    PubMed Central

    Ziv, Ester; Koren, Ruth; Zahalka, Muayad A.; Ravid, Amiram

    2016-01-01

    ABSTRACT Several inflammatory mediators increase calcitriol production by epidermal keratinocytes. In turn calcitriol attenuates the keratinocyte inflammatory response. Since the effect of the in-situ generated calcitriol depends also on the sensitivity to the hormone we studied the effect of inflammatory cytokines on the response of HaCaT human keratinocytes to calcitriol by examining the expression and transcriptional activity of VDR. Treatment with TNF, but not with IL-1β or interferon γ, increased VDR protein level, while decreasing the level of its heterodimerization partner RXRα. This was associated with increased VDR mRNA levels. c-Jun N-terminal kinase, but not P38 MAPK or NFκB, was found to participate in the upregulation of VDR by TNF. The functional significance of the modulation of VDR and RXRα levels by TNF is manifested by increased induction of VDR target gene CYP24A1 by calcitriol. Calcitriol, in turn, inhibited the enhanced expression of VDR by TNF. In conclusion, the inflammatory cytokine TNF increases the response of keratinocytes to calcitriol through upregulation of its receptor VDR, which in turn is subject to negative feedback by the hormone accelerating the return of the keratinocyte vitamin D system to its basal activity. We surmise that the increased generation and sensitivity to calcitriol in keratinocytes play a role in the resolution of epidermal inflammation. PMID:27195054

  14. TNF-α increases the expression and activity of vitamin D receptor in keratinocytes: role of c-Jun N-terminal kinase.

    PubMed

    Ziv, Ester; Koren, Ruth; Zahalka, Muayad A; Ravid, Amiram

    2016-01-01

    Several inflammatory mediators increase calcitriol production by epidermal keratinocytes. In turn calcitriol attenuates the keratinocyte inflammatory response. Since the effect of the in-situ generated calcitriol depends also on the sensitivity to the hormone we studied the effect of inflammatory cytokines on the response of HaCaT human keratinocytes to calcitriol by examining the expression and transcriptional activity of VDR. Treatment with TNF, but not with IL-1β or interferon γ, increased VDR protein level, while decreasing the level of its heterodimerization partner RXRα. This was associated with increased VDR mRNA levels. c-Jun N-terminal kinase, but not P38 MAPK or NFκB, was found to participate in the upregulation of VDR by TNF. The functional significance of the modulation of VDR and RXRα levels by TNF is manifested by increased induction of VDR target gene CYP24A1 by calcitriol. Calcitriol, in turn, inhibited the enhanced expression of VDR by TNF. In conclusion, the inflammatory cytokine TNF increases the response of keratinocytes to calcitriol through upregulation of its receptor VDR, which in turn is subject to negative feedback by the hormone accelerating the return of the keratinocyte vitamin D system to its basal activity. We surmise that the increased generation and sensitivity to calcitriol in keratinocytes play a role in the resolution of epidermal inflammation. PMID:27195054

  15. Fluorescence polarization-based assays for detecting compounds binding to inactive c-Jun N-terminal kinase 3 and p38α mitogen-activated protein kinase.

    PubMed

    Ansideri, Francesco; Lange, Andreas; El-Gokha, Ahmed; Boeckler, Frank M; Koch, Pierre

    2016-06-15

    Two fluorescein-labeled pyridinylimidazoles were synthesized and evaluated as probes for the binding affinity determination of potential kinase inhibitors to the c-Jun N-terminal kinase 3 (JNK3) and p38α mitogen-activated protein kinase (MAPK). Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using 1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-5-yl)-3-(4-((4-(4-(4-fluorophenyl)-2-(methylthio)-1H-imidazol-5-yl)pyridin-2-yl)amino)phenyl)thiourea (5) as an FP probe (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM). The validation of the assays with known inhibitors of JNK3 and p38α MAPK revealed that both FP assays correlate very well with inhibition data received by the activity assays. This, in addition to the viability of both FP-based binding assays for the high-throughput screening procedure, makes the assays suitable as inexpensive prescreening protocols for JNK3 and p38α MAPK inhibitors. PMID:26954235

  16. TRPM7 Activates m-Calpain by Stress-Dependent Stimulation of p38 MAPK and c-Jun N-Terminal Kinase

    PubMed Central

    Su, Li-Ting; Chen, Hsiang-Chin; González-Pagán, Omayra; Overton, Jeffrey D.; Xie, Jia; Yue, Lixia; Runnels, Loren W.

    2010-01-01

    Summary TRPM7 is a Ca2+ and Mg2+ permeant ion channel in possession of its own kinase domain. In a previous study we showed that overexpression of the channel-kinase in HEK-293 cells produced cell rounding and loss of adhesion which was dependent upon the Ca2+-dependent protease m-calpain. The TRPM7-elicited change in cell morphology was channel-dependent and occurred without any significant increase in cytosolic Ca2+. Here we demonstrate that overexpression of TRPM7 increased levels of cellular reactive oxygen species (ROS) and nitric oxide (NO), causing the activation of p38 MAP kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK). Application of inhibitors of p38 MAPK and JNK blocked TRPM7-induced cell rounding and activation of m-calpain, without affecting the phosphorylation state of the protease. Overexpression of TRPM7 increased intracellular Mg2+; however, when the concentrations of either external Ca2+ or Mg2+ was increased to favor permeation of one divalent cation over the other, a similar increase in cell rounding and calpain activity was detected, indicating that TRPM7-mediated activation of m-calpain is not dependent on the nature of the divalent conducted by the channel. Application of inhibitors of nitric oxide synthase and mitochondrial-derived ROS reduced TRPM7-induced increases in nitric oxide and ROS production, blocked the change in cell morphology, and reduced cellular calpain activity. Collectively, our data reveal that excessive TRPM7 channel activity causes oxidative and nitrosative stress, producing cell rounding mediated by p38 MAPK/JNK dependent activation of m-calpain. PMID:20070945

  17. THE GAP JUNCTION INHIBITOR 2-AMINOETHOXY-DIPHENYL-BORATE PROTECTS AGAINST ACETAMINOPHEN HEPATOTOXICITY BY INHIBITING CYTOCHROME P450 ENZYMES AND C-JUN N-TERMINAL KINASE ACTIVATION

    PubMed Central

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao; Farhood, Anwar; Vinken, Mathieu; Jaeschke, Hartmut

    2013-01-01

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5h after APAP. However, the protection was completely lost when 2-APB was given 4–6h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. PMID:24070586

  18. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation.

    PubMed

    Du, Kuo; Williams, C David; McGill, Mitchell R; Xie, Yuchao; Farhood, Anwar; Vinken, Mathieu; Jaeschke, Hartmut

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4-6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. PMID:24070586

  19. Auraptene increases the production of amyloid-β via c-Jun N-terminal kinase-dependent activation of γ-secretase.

    PubMed

    Jung, Cha-Gyun; Uhm, Kyung-Ok; Horike, Hirofumi; Kim, Mi-Jeong; Misumi, Sachiyo; Ishida, Akimasa; Ueda, Yoshimoto; Choi, Eun-Kyoung; Kim, Yong-Sun; Michikawa, Makoto; Hida, Hideki

    2015-01-01

    Amyloid-β (Aβ) peptide plays a major role in the pathogenesis of Alzheimer's disease (AD), and is generated by β- and γ-secretase-mediated proteolytic processing of amyloid-β protein precursor (AβPP). In the present study, we investigated the effect of 118 natural compounds on Aβ production in the medium of HEK293 cells stably expressing human AβPP695 (HEK293-AβPP) using Aβ42 sandwich ELISA to find natural compounds that can modulate Aβ production. We found that a coumarin derivative of citrus fruits, auraptene, increased Aβ production. Treatment of HEK293-AβPP cells and rat primary cortical neurons with auraptene significantly increased the secretion of Aβ40, Aβ42, and the Aβ42/40 ratio. However, auraptene did not change the protein levels of the AβPP processing enzymes, a disintegrin and metalloproteinases 10 (ADAM10, α-secretase), β-site AβPP cleaving enzyme-1 (BACE-1, β-secretase), and presenilin 1 (PS1, γ-secretase component). Auraptene increased the activity of γ-secretase but not that of α- and β-secretase. Furthermore, auraptene enhanced γ-secretase-mediated production of Aβ from AβPP or AβPP-C99, but not through α- and β-secretase. Auraptene also phosphorylated c-Jun N-terminal kinase (JNK), and pretreatment with the JNK inhibitor, SP600125, reduced auraptene-induced γ-secretase activity. Overall, our results suggest that auraptene-mediated activation of JNK may contribute to the production of Aβ by promoting γ-secretase activity. PMID:25147119

  20. Downregulation of Cellular c-Jun N-Terminal Protein Kinase and NF-κB Activation by Berberine May Result in Inhibition of Herpes Simplex Virus Replication

    PubMed Central

    Song, Siwei; Qiu, Min; Chu, Ying; Chen, Deyan; Wang, Xiaohui; Su, Airong

    2014-01-01

    Berberine is a quaternary ammonium salt from the protoberberine group of isoquinoline alkaloids. Some reports show that berberine exhibits anti-inflammatory, antitumor, and antiviral properties by modulating multiple cellular signaling pathways, including p53, nuclear factor κB (NF-κB), and mitogen-activated protein kinase. In the present study, we investigated the antiviral effect of berberine against herpes simplex virus (HSV) infection. Current antiherpes medicines such as acyclovir can lessen the recurring activation when used early at infection but are unable to prevent or cure infections where treatment has selected for resistant mutants. In searching for new antiviral agents against herpesvirus infection, we found that berberine reduced viral RNA transcription, protein synthesis, and virus titers in a dose-dependent manner. To elucidate the mechanism of its antiviral activity, the effect of berberine on the individual steps of viral replication cycle of HSV was investigated via time-of-drug addition assay. We found that berberine acted at the early stage of HSV replication cycle, between viral attachment/entry and genomic DNA replication, probably at the immediate-early gene expression stage. We further demonstrated that berberine significantly reduced HSV-induced NF-κB activation, as well as IκB-α degradation and p65 nuclear translocation. Moreover, we found that berberine also depressed HSV-induced c-Jun N-terminal kinase (JNK) phosphorylation but had little effect on p38 phosphorylation. Our results suggest that the berberine inhibition of HSV infection may be mediated through modulating cellular JNK and NF-κB pathways. PMID:24913175

  1. Activation of the cJun N-terminal kinase (JNK) pathway by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1).

    PubMed

    Eliopoulos, A G; Young, L S

    1998-04-01

    Expression of the oncogenic Epstein-Barr virus (EBV)-encoded Latent Membrane Protein 1 (LMP1) activates signalling on the NF-kappaB axis through two distinct domains in the cytoplasmic C-terminus of the protein, namely CTAR1 (aa 187-231) and CTAR2 (aa 351-386). Whilst this effect is responsible for some of the functional consequences of LMP1 expression, additional LMP1-mediated signalling pathways may exist which contribute to the pleiotropic activities of this protein. In this study we provide evidence of a kinase cascade being activated by LMP1. Thus, we demonstrate that stable or transient expression of the LMP1 prototype from B95.8 in cells of epithelial or B cell origin activates the c-Jun N-terminal kinase (JNK, also known as the stress-activated protein kinase, SAPK) pathway, an effect which was found to be mediated through CTAR2 but not CTAR1. LMP1 from the Cao viral strain or LMP1 homologues from the simian EBV naturally infecting baboons and rhesus monkeys were also able to activate JNK. This phenomenon translates to induction of AP-1, a transcription factor which is readily activated by growth factors and mitogens. Interestingly, an LMP1/ CD40 chimaera comprising of the N-terminus and transmembrane domain of LMP1 and the cytoplasmic tail of CD40 which shares a common TRAF binding motif with CTAR1, effectively induced JNK. As NF-kappaB and JNK are co-activated in LMP1-expressing cells, we investigated whether the two pathways are overlapping or independent. We have found that inhibition of NF-kappaB by metabolic inhibitors or a constitutively active mutated IkappaBalpha does not impair the ability of LMP1 to signal on the JNK axis. Conversely, whilst a dominant negative mutated SEK (JNKK) inhibited LMP1-induced JNK activation, it did not affect NF-kappa-B suggesting that these two LMP1-mediated pathways are divergent. PMID:9582021

  2. c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea.

    PubMed

    Anttonen, Tommi; Herranen, Anni; Virkkala, Jussi; Kirjavainen, Anna; Elomaa, Pinja; Laos, Maarja; Liang, Xingqun; Ylikoski, Jukka; Behrens, Axel; Pirvola, Ulla

    2016-01-01

    Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death. PMID:27257624

  3. c-Jun N-Terminal Phosphorylation: Biomarker for Cellular Stress Rather than Cell Death in the Injured Cochlea123

    PubMed Central

    Anttonen, Tommi; Herranen, Anni; Virkkala, Jussi; Kirjavainen, Anna; Elomaa, Pinja; Laos, Maarja; Liang, Xingqun; Ylikoski, Jukka; Behrens, Axel

    2016-01-01

    Prevention of auditory hair cell death offers therapeutic potential to rescue hearing. Pharmacological blockade of JNK/c-Jun signaling attenuates injury-induced hair cell loss, but with unsolved mechanisms. We have characterized the c-Jun stress response in the mouse cochlea challenged with acoustic overstimulation and ototoxins, by studying the dynamics of c-Jun N-terminal phosphorylation. It occurred acutely in glial-like supporting cells, inner hair cells, and the cells of the cochlear ion trafficking route, and was rapidly downregulated after exposures. Notably, death-prone outer hair cells lacked c-Jun phosphorylation. As phosphorylation was triggered also by nontraumatic noise levels and none of the cells showing this activation were lost, c-Jun phosphorylation is a biomarker for cochlear stress rather than an indicator of a death-prone fate of hair cells. Preconditioning with a mild noise exposure before a stronger traumatizing noise exposure attenuated the cochlear c-Jun stress response, suggesting that the known protective effect of sound preconditioning on hearing is linked to suppression of c-Jun activation. Finally, mice with mutations in the c-Jun N-terminal phosphoacceptor sites showed partial, but significant, hair cell protection. These data identify the c-Jun stress response as a paracrine mechanism that mediates outer hair cell death. PMID:27257624

  4. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    SciTech Connect

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao; Farhood, Anwar; Vinken, Mathieu; Jaeschke, Hartmut

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  5. The c-Jun N-terminal kinase (JNK) pathway is activated in human interstitial cystitis (IC) and rat protamine sulfate induced cystitis

    PubMed Central

    Zhao, Jiang; Wang, Liang; Dong, Xingyou; Hu, Xiaoyan; Zhou, Long; Liu, Qina; Song, Bo; Wu, Qingjian; Li, Longkun

    2016-01-01

    The pathogenesis of bladder pain syndrome/interstitial cystitis (BPS/IC) is currently unclear. However, inflammation has been suggested to play an important role in BPS/IC. JNK downstream signaling plays an important role in numerous chronic inflammatory diseases. However, studies of the JNK pathway in BPS/IC are limited. In this study, we investigated the role of the JNK pathway in human BPS/IC and rat protamine sulfate (PS)-induced cystitis and examined the effect of the selective JNK inhibitor SP600125 on rat bladder cystitis. In our study, we demonstrated that the JNK signaling pathway was activated (the expression of JNK, c-Jun, p-JNK, p-c-Jun, IL-6 and TNF-α were significantly increasing in BPS/IC compared to the non-BPS/IC patients) and resulted in inflammation in human BPS/IC. Further animal models showed that the JNK pathway played an important role in the pathogenesis of cystitis. JNK inhibitors, SP600125, effectively inhibited the expression of p-JNK, p-c-Jun, IL-6 and TNF-α. The inhibition of these pathways had a protective effect on PS-induced rat cystitis by significantly decreasing histological score and mast cell count and improving bladder micturition function (micturition frequency significantly decreasing and bladder capacity significantly increasing). Therefore, JNK inhibition could be used as a potential treatment for BPS/IC. PMID:26883396

  6. Ketamine inhibits tumor necrosis factor-{alpha} and interleukin-6 gene expressions in lipopolysaccharide-stimulated macrophages through suppression of toll-like receptor 4-mediated c-Jun N-terminal kinase phosphorylation and activator protein-1 activation

    SciTech Connect

    Wu, G.-J.; Chen, T.-L.; Ueng, Y.-F.; Chen, R.-M.

    2008-04-01

    Our previous study showed that ketamine, an intravenous anesthetic agent, has anti-inflammatory effects. In this study, we further evaluated the effects of ketamine on the regulation of tumor necrosis factor-{alpha} (TNF-{alpha}) and interlukin-6 (IL-6) gene expressions and its possible signal-transducing mechanisms in lipopolysaccharide (LPS)-activated macrophages. Exposure of macrophages to 1, 10, and 100 {mu}M ketamine, 100 ng/ml LPS, or a combination of ketamine and LPS for 1, 6, and 24 h was not cytotoxic to macrophages. A concentration of 1000 {mu}M of ketamine alone or in combined treatment with LPS caused significant cell death. Administration of LPS increased cellular TNF-{alpha} and IL-6 protein levels in concentration- and time-dependent manners. Meanwhile, treatment with ketamine concentration- and time-dependently alleviated the enhanced effects. LPS induced TNF-{alpha} and IL-6 mRNA syntheses. Administration of ketamine at a therapeutic concentration (100 {mu}M) significantly inhibited LPS-induced TNF-{alpha} and IL-6 mRNA expressions. Application of toll-like receptor 4 (TLR4) small interfering (si)RNA into macrophages decreased cellular TLR4 levels. Co-treatment of macrophages with ketamine and TLR4 siRNA decreased the LPS-induced TNF-{alpha} and IL-6 productions more than alone administration of TLR4 siRNA. LPS stimulated phosphorylation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos from the cytoplasm to nuclei. However, administration of ketamine significantly decreased LPS-induced activation of c-Jun N-terminal kinase and translocation of c-Jun and c-Fos. LPS increased the binding of nuclear extracts to activator protein-1 consensus DNA oligonucleotides. Administration of ketamine significantly ameliorated LPS-induced DNA binding activity of activator protein-1. Therefore, a clinically relevant concentration of ketamine can inhibit TNF-{alpha} and IL-6 gene expressions in LPS-activated macrophages. The suppressive mechanisms

  7. Alpha 1-Antichymotrypsin, an Inflammatory Protein Overexpressed in the Brains of Patients with Alzheimer's Disease, Induces Tau Hyperphosphorylation through c-Jun N-Terminal Kinase Activation.

    PubMed

    Tyagi, Ethika; Fiorelli, Tina; Norden, Michelle; Padmanabhan, Jaya

    2013-01-01

    The association of inflammatory proteins with neuritic plaques in the brains of Alzheimer's disease (AD) patients has led to the hypothesis that inflammation plays a pivotal role in the development of pathology in AD. Earlier studies have shown that alpha 1-antichymotrypsin (ACT) enhances amyloid beta fibrillization and accelerated plaque formation in APP transgenic mice. Later studies from our laboratory have shown that purified ACT induces tau hyperphosphorylation and degeneration in neurons. In order to understand the mechanisms by which inflammatory proteins enhance tau hyperphosphorylation, we injected interleukin-1 β (IL-1 β ) intracerebroventricularly into mice expressing human ACT, human tau, or both transgenes. It was found that the hyperphosphorylation of tau in ACT and ACT/htau mice after IL-1 β injection correlated with increased phosphorylation of c-Jun N-terminal kinase (JNK). We verified the involvement of JNK in ACT-induced tau phosphorylation by utilizing JNK inhibitors in cultured primary neurons treated with ACT, and we found that the inhibitor showed complete prevention of ACT-induced tau phosphorylation. These results indicate that JNK is one of the major kinases involved in the ACT-mediated tau hyperphosphorylation and suggest that inhibitors of this kinase may protect against inflammation-induced tau hyperphosphorylation and neurodegeneration associated with AD. PMID:24175110

  8. Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling★

    PubMed Central

    Ma, Yinghuan; Bao, Yongxin; Li, Chenghao; Jiao, Fubin; Xin, Hongjie; Yuan, Zhengwei

    2012-01-01

    Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway. PMID:25337099

  9. Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells.

    PubMed

    Li, Qiongshu; Liu, Guomu; Shao, Dan; Wang, Juan; Yuan, Hongyan; Chen, Tanxiu; Zhai, Ruiping; Ni, Weihua; Tai, Guixiang

    2015-02-01

    In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy. PMID:25526895

  10. Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release

    PubMed Central

    Nisticò, Robert; Florenzano, Fulvio; Mango, Dalila; Ferraina, Caterina; Grilli, Massimo; Di Prisco, Silvia; Nobili, Annalisa; Saccucci, Stefania; D'Amelio, Marcello; Morbin, Michela; Marchi, Mario; Mercuri, Nicola B.; Davis, Roger J.; Pittaluga, Anna; Feligioni, Marco

    2015-01-01

    Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a critical step for neuronal death occurring in several neurological conditions. JNKs can be activated via receptor tyrosine kinases, cytokine receptors, G-protein coupled receptors and ligand-gated ion channels, including the NMDA glutamate receptors. While JNK has been generally associated with postsynaptic NMDA receptors, its presynaptic role remains largely unexplored. Here, by means of biochemical, morphological and functional approaches, we demonstrate that JNK and its scaffold protein JIP1 are also expressed at the presynaptic level and that the NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Moreover, using knockout mice for single JNK isoforms, we proved that JNK2 is the essential isoform in mediating this presynaptic event. Overall the present findings unveil a novel JNK2 localization and function, which is likely to play a role in different physiological and pathological conditions. PMID:25762148

  11. Highly Oxygenated Sesquiterpene Lactones from Cousinia aitchisonii and their Cytotoxic Properties: Rhaserolide Induces Apoptosis in Human T Lymphocyte (Jurkat) Cells via the Activation of c-Jun n-terminal Kinase Phosphorylation.

    PubMed

    Iranshahy, Milad; Tayarani-Najaran, Zahra; Kasaian, Jamal; Ghandadi, Morteza; Emami, Seyed Ahmad; Asili, Javad; Chandran, Jima N; Schneider, Bernd; Iranshahi, Mehrdad

    2016-02-01

    Infrared-guided chromatographic fractionation of sesquiterpene lactones from the extracts of Cousinia aitchisonii and Cousinia concolor led to the isolation of five pure compounds. A new sesquiterpene lactone, namely, aitchisonolide, and two known sesquiterpene lactones (desoxyjanerin and rhaserolide) were isolated from C. aitchisonii and two known lignans (arctiin and arctigenin) from C. concolor. The structures of these compounds were elucidated by one-dimensional and two-dimensional nuclear magnetic resonance techniques, as well as high-resolution mass spectrometry. The purified and characterized compounds were subjected to cytotoxicity assay. The sesquiterpene lactones desoxyjanerin and rhaserolide showed significant cytotoxic activities against five different cancer cell lines and the normal human embryonic kidney cell line. Rhaserolide was chosen to evaluate the possible mechanism of action. Western blot analysis revealed that rhaserolide could induce apoptosis in Jurkat cells via the activation of c-Jun n-terminal kinase phosphorylation. PMID:26581585

  12. Role of Akt and c-Jun N-terminal Kinase 2 in Apoptosis Induced by Interleukin-4 Deprivation

    PubMed Central

    Cerezo, Ana; Martínez-A, Carlos; Lanzarot, Diego; Fischer, Siegmund; Franke, Thomas F.; Rebollo, Angelita

    1998-01-01

    We have shown previously that interleukin-4 (IL-4) protects TS1αβ cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1αβ cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1αβ survival is independent of Bcl-2, Bcl-x, or Bax. PMID:9802900

  13. Role for c-jun N-terminal kinase in treatment-refractory acute myeloid leukemia (AML): signaling to multidrug-efflux and hyperproliferation.

    PubMed

    Cripe, L D; Gelfanov, V M; Smith, E A; Spigel, D R; Phillips, C A; Gabig, T G; Jung, S-H; Fyffe, J; Hartman, A D; Kneebone, P; Mercola, D; Burgess, G S; Boswell, H S

    2002-05-01

    A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML. PMID:11986940

  14. Analysis of mitogen-activated protein kinase pathways used by interleukin 1 in tissues in vivo: activation of hepatic c-Jun N-terminal kinases 1 and 2, and mitogen-activated protein kinase kinases 4 and 7.

    PubMed Central

    Finch, A; Davis, W; Carter, W G; Saklatvala, J

    2001-01-01

    The effects of interleukin 1 (IL-1) are mediated by the activation of protein kinase signalling pathways, which have been well characterized in cultured cells. We have investigated the activation of these pathways in rabbit liver and other tissues after the systemic administration of IL-1alpha. In liver there was 30-40-fold activation of c-Jun N-terminal kinase (JNK) and 5-fold activation of both JNK kinases, mitogen-activated protein kinase (MAPK) kinase (MKK)4 and MKK7. IL-1alpha also caused 2-3-fold activation of p38 MAPK and degradation of the inhibitor of nuclear factor kappaB ('IkappaB'), although no activation of extracellular signal-regulated protein kinase (ERK) (p42/44 MAPK) was observed. The use of antibodies against specific JNK isoforms showed that, in liver, short (p46) JNK1 and long (p54) JNK2 are the predominant forms activated, with smaller amounts of long JNK1 and short JNK2. No active JNK3 was detected. A similar pattern of JNK activation was seen in lung, spleen, skeletal muscle and kidney. Significant JNK3 activity was detectable only in the brain, although little activation of the JNK pathway in response to IL-1alpha was observed in this tissue. This distribution of active JNK isoforms probably results from a different expression of JNKs within the tissues, rather than from a selective activation of isoforms. We conclude that IL-1alpha might activate a more restricted set of signalling pathways in tissues in vivo than it does in cultured cells, where ERK and JNK3 activation are often observed. Cultured cells might represent a 'repair' phenotype that undergoes a broader set of responses to the cytokine. PMID:11139391

  15. c-Jun N-terminal Kinase Phosphorylation of Stathmin Confers Protection against Cellular Stress*

    PubMed Central

    Ng, Dominic C. H.; Zhao, Teresa T.; Yeap, Yvonne Y. C.; Ngoei, Kevin R.; Bogoyevitch, Marie A.

    2010-01-01

    The cell stress response encompasses the range of intracellular events required for adaptation to stimuli detrimental to cell survival. Although the c-Jun N-terminal kinase (JNK) is a stress-activated kinase that can promote either cell survival or death in response to detrimental stimuli, the JNK-regulated mechanisms involved in survival are not fully characterized. Here we show that in response to hyperosmotic stress, JNK phosphorylates a key cytoplasmic microtubule regulatory protein, stathmin (STMN), on conserved Ser-25 and Ser-38 residues. In in vitro biochemical studies, we identified STMN Ser-38 as the critical residue required for efficient phosphorylation by JNK and identified a novel kinase interaction domain in STMN required for recognition by JNK. We revealed that JNK was required for microtubule stabilization in response to hyperosmotic stress. Importantly, we also demonstrated a novel cytoprotective function for STMN, as the knockdown of STMN levels by siRNA was sufficient to augment viability in response to hyperosmotic stress. Our findings show that JNK targeting of STMN represents a novel stress-activated cytoprotective mechanism involving microtubule network changes. PMID:20630875

  16. Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

    PubMed

    Garg, Aditya; Zhao, Angela; Erickson, Sarah L; Mukherjee, Subhajit; Lau, Aik Jiang; Alston, Laurie; Chang, Thomas K H; Mani, Sridhar; Hirota, Simon A

    2016-10-01

    The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD. PMID:27440420

  17. Pyridopyrimidinone Derivatives as Potent and Selective c-Jun N-Terminal Kinase (JNK) Inhibitors

    PubMed Central

    2015-01-01

    A novel series of 2-aminopyridopyrimidinone based JNK (c-jun N-terminal kinase) inhibitors were discovered and developed. Structure–activity relationships (SARs) were systematically developed utilizing biochemical and cell based assays and in vitro and in vivo drug metabolism and pharmacokinetic (DMPK) studies. Through the optimization of lead compound 1, several potent and selective JNK inhibitors with high oral bioavailability were developed. Inhibitor 13 was a potent JNK3 inhibitor (IC50 = 15 nM), had high selectivity against p38 (IC50 > 10 μM), had high potency in functional cell based assays, and had high stability in human liver microsome (t1/2 = 76 min), a clean CYP-450 inhibition profile, and excellent oral bioavailability (%F = 87). Moreover, cocrystal structures of compounds 13 and 22 in JNK3 were solved at 2.0 Å. These structures elucidated the binding mode (Type-I binding) and can pave the way for further inhibitor design of this pyridopyrimidinone scaffold for JNK inhibition. PMID:25893042

  18. Reactive oxygen species-mediated activation of AMP-activated protein kinase and c-Jun N-terminal kinase plays a critical role in beta-sitosterol-induced apoptosis in multiple myeloma U266 cells.

    PubMed

    Sook, Song Hyo; Lee, Hyo-Jung; Kim, Ji-Hyun; Sohn, Eun Jung; Jung, Ji Hoon; Kim, Bonglee; Kim, Jin-Hyoung; Jeong, Soo-Jin; Kim, Sung-Hoon

    2014-03-01

    Although beta-sitosterol has been well known to have anti-tumor activity in liver, lung, colon, stomach, breast and prostate cancers via cell cycle arrest and apoptosis induction, the underlying mechanism of anti-cancer effect of beta-sitosterol in multiple myeloma cells was never elucidated until now. Thus, in the present study, the role of reactive oxygen species (ROS) in association with AMP-activated protein kinase (AMPK) and c-Jun N-terminal kinase (JNK) pathways was demonstrated in beta-sitosterol-treated multiple myeloma U266 cells. Beta-sitosterol exerted cytotoxicity, increased sub-G1 apoptotic population and activated caspase-9 and -3, cleaved poly (ADP-ribose) polymerase (PARP) followed by decrease in mitochondrial potential in U266 cells. Beta-sitosterol promoted ROS production, activated AMPK, acetyl-CoA carboxylase (ACC) and JNK in U266 cells. Also, beta-sitosterol attenuated the phosphorylation of AKT, mammalian target of rapamycin and S6K, and the expression of cyclooxygenase-2 and VEGF in U266 cells. Conversely, AMPK inhibitor compound C and JNK inhibitor SP600125 suppressed apoptosis induced by beta-sitosterol in U266 cells. Furthermore, ROS scavenger N-acetyl L-cysteine attenuated beta-sitosterol-mediated sub-G1 accumulation, PARP cleavage, JNK and AMPK activation in U266 cells. Overall, these findings for the first time suggest that ROS-mediated activation of cancer metabolism-related genes such as AMPK and JNK plays an important role in beta-sitosterol-induced apoptosis in U266 multiple myeloma cells. PMID:23640957

  19. Identification and Characterization of a Novel Class of c-Jun N-terminal Kinase Inhibitors

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Khlebnikov, Andrei I.; Hanks, Tracey S.; Kochetkova, Irina; Pascual, David W.; Jutila, Mark A.

    2012-01-01

    In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1β, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC50 values of 0.25 and 0.61 μM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4+ T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs. PMID:22434859

  20. Expression of Ceramide Synthase 6 Transcriptionally Activates Acid Ceramidase in a c-Jun N-terminal Kinase (JNK)-dependent Manner.

    PubMed

    Tirodkar, Tejas S; Lu, Ping; Bai, Aiping; Scheffel, Matthew J; Gencer, Salih; Garrett-Mayer, Elizabeth; Bielawska, Alicja; Ogretmen, Besim; Voelkel-Johnson, Christina

    2015-05-22

    A family of six ceramide synthases with distinct but overlapping substrate specificities is responsible for generation of ceramides with acyl chains ranging from ∼14-26 carbons. Ceramide synthase 6 (CerS6) preferentially generates C14- and C16-ceramides, and we have previously shown that down-regulation of this enzyme decreases apoptotic susceptibility. In this study, we further evaluated how increased CerS6 expression impacts sphingolipid composition and metabolism. Overexpression of CerS6 in HT29 colon cancer cells resulted in increased apoptotic susceptibility and preferential generation of C16-ceramide, which occurred at the expense of very long chain, saturated ceramides. These changes were also reflected in sphingomyelin composition. HT-CerS6 cells had increased intracellular levels of sphingosine, which is generated by ceramidases upon hydrolysis of ceramide. qRT-PCR analysis revealed that only expression of acid ceramidase (ASAH1) was increased. The increase in acid ceramidase was confirmed by expression and activity analyses. Pharmacological inhibition of JNK (SP600125) or curcumin reduced transcriptional up-regulation of acid ceramidase. Using an acid ceramidase promoter driven luciferase reporter plasmid, we demonstrated that CerS1 has no effect on transcriptional activation of acid ceramidase and that CerS2 slightly but significantly decreased the luciferase signal. Similar to CerS6, overexpression of CerS3-5 resulted in an ∼2-fold increase in luciferase reporter gene activity. Exogenous ceramide failed to induce reporter activity, while a CerS inhibitor and a catalytically inactive mutant of CerS6 failed to reduce it. Taken together, these results suggest that increased expression of CerS6 can mediate transcriptional activation of acid ceramidase in a JNK-dependent manner that is independent of CerS6 activity. PMID:25839235

  1. Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

    PubMed

    Parra, Eduardo; Gutierréz, Luís; Ferreira, Jorge

    2016-02-01

    The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells. PMID:26573109

  2. Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7.

    PubMed Central

    Fleming, Y; Armstrong, C G; Morrice, N; Paterson, A; Goedert, M; Cohen, P

    2000-01-01

    Stress-activated protein kinase 1 (SAPK1), also called c-Jun N-terminal kinase (JNK), becomes activated in vivo in response to pro-inflammatory cytokines or cellular stresses. Its full activation requires the phosphorylation of a threonine and a tyrosine residue in a Thr-Pro-Tyr motif, which can be catalysed by the protein kinases mitogen-activated protein kinase kinase (MKK)4 and MKK7. Here we report that MKK4 shows a striking preference for the tyrosine residue (Tyr-185), and MKK7 a striking preference for the threonine residue (Thr-183) in three SAPK1/JNK1 isoforms tested (JNK1 alpha 1, JNK2 alpha 2 and JNK3 alpha 1). For this reason, MKK4 and MKK7 together produce a synergistic increase in the activity of each SAPK1/JNK isoform in vitro. The MKK7 beta variant, which is several hundred-fold more efficient in activating all three SAPK1/JNK isoforms than is MKK7 alpha', is equally specific for Thr-183. MKK7 also phosphorylates JNK2 alpha 2 at Thr-404 and Ser-407 in vitro, Ser-407 being phosphorylated much more rapidly than Thr-183 in vitro. Thr-404/Ser-407 are phosphorylated in unstimulated human KB cells and HEK-293 cells, and phosphorylation is increased in response to an osmotic stress (0.5 M sorbitol). However, in contrast with Thr-183 and Tyr-185, the phosphorylation of Thr-404 and Ser-407 is not increased in response to other agonists that activate MKK7 and SAPK1/JNK, suggesting that phosphorylation of these residues is catalysed by another protein kinase, such as CK2, which also phosphorylates Thr-404 and Ser-407 in vitro. MKK3, MKK4 and MKK6 all show a strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motifs in their known substrates SAPK2a/p38, SAPK3/p38 gamma and SAPK4/p38 delta. MKK7 also phosphorylates SAPK2a/p38 at a low rate (but not SAPK3/p38 gamma or SAPK4/p38 delta), and phosphorylation occurs exclusively at the tyrosine residue, demonstrating that MKK7 is intrinsically a 'dual-specific' protein kinase. PMID:11062067

  3. JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation.

    PubMed Central

    MacFarlane, M; Cohen, G M; Dickens, M

    2000-01-01

    Activation of the stress-activated mitogen-activated protein kinases (MAP kinases), c-Jun N-terminal kinase (JNK) and p38, is necessary for the induction of apoptosis in neuronal cells; however, in other cell types their involvement may be stimulus-dependent. In the present study we investigate the activation of JNK and p38 in a single non-neuronal cell type, undergoing receptor-mediated (tumour necrosis factor-related apoptosis-inducing ligand and CD95) or chemically-induced (lactacystin) apoptosis. In Jurkat T-cells, receptor-mediated and chemically-induced apoptosis resulted in a time-dependent activation of the initiator caspases-8 and -9, respectively. Both types of stimuli resulted in a significant activation of JNK and p38, which closely paralleled the time-dependent induction of apoptosis. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.FMK) inhibited receptor-mediated apoptosis and suppressed JNK and p38 activation. In contrast, inhibition of lactacystin-induced apoptosis with z-VAD.FMK, as assessed by phosphatidylserine exposure and poly(ADP-ribose) polymerase cleavage, did not inhibit activation of JNK or p38, demonstrating that during chemically-induced apoptosis, activation of JNK and p38 is independent of effector caspases. The role of p38 in apoptosis was assessed using the specific p38 inhibitor, SB203580. No effect on the induction of apoptosis or caspase activation was observed, although activation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), an immediate downstream target of p38, was inhibited. Therefore neither p38 activation nor activation of MAPKAPK-2 is critical for induction of either receptor- or chemically-induced apoptosis. Thus, within a single cell type, (1) the mechanism of p38 and JNK activation during apoptosis is stimulus-dependent and (2) activation of the p38 pathway is not required for caspase activation or apoptosis, assessed by phosphatidylserine exposure, but

  4. 1,1-Bis(3′-indolyl)-1-(p-substituted phenyl)methanes inhibit colon cancer cell and tumor growth through activation of c-jun N-terminal kinase

    PubMed Central

    Lei, Ping; Abdelrahim, Maen; Cho, Sung Dae; Liu, Shengxi; Chintharlapalli, Sudhakar; Safe, Stephen

    2008-01-01

    1,1-Bis(3′-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) activate the orphan receptors peroxisome proliferator-activated receptor γ (PPARγ) and Nur77 and induce receptor-dependent and -independent apoptotic pathways in colon and other cancer cells. Structure-activity studies show that the p-bromo (DIM-C-pPhBr) and p-fluoro (DIM-C-pPhF) analogs, which exhibit minimal activation of Nur77 and PPARγ, induce expression of CCAAT/enhancer-binding protein homologous protein (CHOP/GADD153) in colon cancer cells. Moreover, among a series of bromo and fluoro C-DIM analogs, their induction of CHOP was dependent on the position of the phenyl substituents (para ≥ meta ≥ ortho) and required a free indole group. DIM-C-pPhBr and DIM-C-pPhF not only induced CHOP but also activated death receptor 5 (CHOP dependent), cleavage of caspase 8 and poly (ADP ribose) polymerase (PARP) that is consistent with activation of the extrinsic pathway of apoptosis. These responses were associated with the activation of c-jun N-terminal kinase (JNK) pathway since inhibition of JNK inhibited induction of the extrinsic apoptotic pathway by these C-DIMs. However, in contrast to classical inducers of endoplasmic reticulum (ER) stress such as tunicamycin and thapsigargin, the C-DIM compounds did not induce glucose-related protein 78 that is a marker of ER stress. Proapoptotic and anticarcinogenic effects were also observed in athymic nude mice bearing RKO cell xenografts and treated with 30 mg/kg/day DIM-C-pPhBr and this was accompanied by increased JNK phosphorylation in the tumors. Thus, the anticarcinogenic activity of DIM-C-pPhBr in colon cancer cells and tumors is related to a novel ER stress-independent activation of JNK. PMID:18460448

  5. HEPATIC APOPTOSIS POST-BURN IS MEDIATED BY C-JUN N-TERMINAL KINASE-2

    PubMed Central

    Marshall, Alexandra H.; Brooks, Natasha C.; Hiyama, Yaeko; Qa’aty, Nour; Al-mousawi, Ahmed; Finnerty, Celeste C.; Jeschke, Marc G.

    2013-01-01

    The trauma of a severe burn injury induces a hypermetabolic response that increases morbidity and mortality. Previously, our group showed that insulin resistance post-burn injury is associated with endoplasmic reticulum (ER) stress. Evidence suggests that c-jun N-terminal kinase (JNK) -2 may be involved in ER stress-induced apoptosis. Here, we hypothesized that JNK2 contributes to the apoptotic response after burn injury downstream of ER stress. To test this, we compared JNK2 knockout mice (−/−) to wildtype mice after inducing a 30% total body surface area thermal injury. Animals were sacrificed after 1, 3 and 5 days. Inflammatory cytokines in the blood were measured by multiplex analysis. Hepatic ER stress and insulin signaling were assessed by Western Blotting and insulin resistance was measured by a peritoneal glucose tolerance test. Apoptosis in the liver was quantified by TUNEL staining. Liver function was quantified by AST and ALT activity assays. ER stress increased after burn in both JNK2−/− and wildtype mice, indicating that JNK2 activation is downstream of ER stress. Knockout of JNK2 did not affect serum inflammatory cytokines; however, the increase in IL-6 mRNA expression was prevented in the knockouts. Serum insulin did not significantly increase in the JNK2−/− group. On the other hand, insulin signaling (PI3K/Akt pathway) and glucose tolerance tests did not improve in JNK2−/−. As expected, apoptosis in the liver increased after burn injury in wildtype mice but not in JNK2−/−. AST/ALT activity revealed that liver function recovered more quickly in JNK2−/−. This study indicates that JNK2 is a central mediator of hepatic apoptosis after a severe burn. PMID:23324888

  6. The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

    PubMed

    Kim, Jee-Youn; Choi, Ji-Young; Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

    2015-01-01

    The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity. PMID:26375285

  7. Arrestin-3 Binds c-Jun N-terminal Kinase 1 (JNK1) and JNK2 and Facilitates the Activation of These Ubiquitous JNK Isoforms in Cells via Scaffolding*

    PubMed Central

    Kook, Seunghyi; Zhan, Xuanzhi; Kaoud, Tamer S.; Dalby, Kevin N.; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2013-01-01

    Non-visual arrestins scaffold mitogen-activated protein kinase (MAPK) cascades. The c-Jun N-terminal kinases (JNKs) are members of MAPK family. Arrestin-3 has been shown to enhance the activation of JNK3, which is expressed mainly in neurons, heart, and testes, in contrast to ubiquitous JNK1 and JNK2. Although all JNKs are activated by MKK4 and MKK7, both of which bind arrestin-3, the ability of arrestin-3 to facilitate the activation of JNK1 and JNK2 has never been reported. Using purified proteins we found that arrestin-3 directly binds JNK1α1 and JNK2α2, interacting with the latter comparably to JNK3α2. Phosphorylation of purified JNK1α1 and JNK2α2 by MKK4 or MKK7 is increased by arrestin-3. Endogenous arrestin-3 interacted with endogenous JNK1/2 in different cell types. Arrestin-3 also enhanced phosphorylation of endogenous JNK1/2 in intact cells upon expression of upstream kinases ASK1, MKK4, or MKK7. We observed a biphasic effect of arrestin-3 concentrations on phosphorylation of JNK1α1 and JNK2α2 both in vitro and in vivo. Thus, arrestin-3 acts as a scaffold, facilitating JNK1α1 and JNK2α2 phosphorylation by MKK4 and MKK7 via bringing JNKs and their activators together. The data suggest that arrestin-3 modulates the activity of ubiquitous JNK1 and JNK2 in non-neuronal cells, impacting the signaling pathway that regulates their proliferation and survival. PMID:24257757

  8. The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity

    PubMed Central

    Lee, Hyeon-Ju; Byun, Catherine Jeonghae; Park, Jung-Hyun; Park, Jae Hoon; Cho, Ho-Seong; Cho, Sung-Jin; Jo, Sangmee Ahn; Jo, Inho

    2015-01-01

    The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity. PMID:26375285

  9. Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors.

    PubMed

    Humphries, Paul S; Lafontaine, Jennifer A; Agree, Charles S; Alexander, David; Chen, Ping; Do, Quyen-Quyen T; Li, Lilian Y; Lunney, Elizabeth A; Rajapakse, Ranjan J; Siegel, Karen; Timofeevski, Sergei L; Wang, Tianlun; Wilhite, David M

    2009-04-15

    The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported. PMID:19327989

  10. c-Jun N-terminal kinase 1 promotes transforming growth factor-β1-induced epithelial-to-mesenchymal transition via control of linker phosphorylation and transcriptional activity of Smad3.

    PubMed

    Velden, Jos L J van der; Alcorn, John F; Guala, Amy S; Badura, Elsbeth C H L; Janssen-Heininger, Yvonne M W

    2011-04-01

    Transforming growth factor (TGF)-β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1-induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1-induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1-induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor-1, fibronectin-1, high-mobility group A2, CArG box-binding factor-A, and fibroblast-specific protein-1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1-induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1-induced EMT. PMID:20581097

  11. All-trans retinoic acid diminishes collagen production in a hepatic stellate cell line via suppression of active protein-1 and c-Jun N-terminal kinase signal.

    PubMed

    Ye, Yuan; Dan, Zili

    2010-12-01

    Following acute and chronic liver injury, hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content, but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood. The influence of retinoids on HSCs and hepatic fibrosis remains controversial. The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation, mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), fibrolytic genes (MMP-3, MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G). Cell proliferation was evaluated by measuring BrdU incorporation. The mRNA expression levels of collagen genes [procollagen α1 (I), procollagen α1 (III)], profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and fibrolytic genes (MMP-3, MMP-13) were quantitatively detected by using real-time PCR. The mRNA expression of JNK and AP-1 was quantified by RT-PCR. The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (I), procollagen α1 (III)] and profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1. These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal, then decrease the mRNAs expression of profibrogenic genes (TGF-β(1), CTGF, MMP-2, TIMP-1, TIMP-2, PAI-1), and significantly induce the mRNA expression of MMP-3 and MMP-13. PMID:21181362

  12. Pyrazole derivatives as potent inhibitors of c-Jun N-terminal kinase: synthesis and SAR studies.

    PubMed

    Doma, Anuradha; Kulkarni, Ravindra; Palakodety, Radhakrishna; Sastry, G Narahari; Sridhara, Janardhan; Garlapati, Achaiah

    2014-11-01

    Mitogen activated protein kinases including c-Jun N-terminal kinase play an indispensable role in inflammatory diseases. Investigation of reported JNK-1 inhibitors indicated that diverse heterocyclic compounds bearing an amide group rendered potent JNK-1 inhibitory activity which prompted us to synthesize new JNK-1 inhibitors containing a pyrazole heterocyclic group. A DABCO mediated 1,3-dipolar cycloaddition reaction in neat resulted in pyrazole carboxylic acid which was converted to desired amides. Upon confirmation of the structures, all the compounds were screened for JNK-1 inhibitory activity and in vivo anti-inflammatory activity. Several synthesized analogues have exhibited JNK-1 inhibitory activity less than 10 μM, in particular compounds 9 c, 10 a and 10 d were found to be potent among all the compounds. PMID:25261929

  13. Protective role of c-Jun N-terminal kinase 2 in acetaminophen-induced liver injury

    SciTech Connect

    Bourdi, Mohammed Korrapati, Midhun C.; Chakraborty, Mala; Yee, Steven B.; Pohl, Lance R.

    2008-09-12

    Recent studies in mice suggest that stress-activated c-Jun N-terminal protein kinase 2 (JNK2) plays a pathologic role in acetaminophen (APAP)-induced liver injury (AILI), a major cause of acute liver failure (ALF). In contrast, we present evidence that JNK2 can have a protective role against AILI. When male C57BL/6J wild type (WT) and JNK2{sup -/-} mice were treated with 300 mg APAP/kg, 90% of JNK2{sup -/-} mice died of ALF compared to 20% of WT mice within 48 h. The high susceptibility of JNK2{sup -/-} mice to AILI appears to be due in part to deficiencies in hepatocyte proliferation and repair. Therefore, our findings are consistent with JNK2 signaling playing a protective role in AILI and further suggest that the use of JNK inhibitors as a potential treatment for AILI, as has been recommended by other investigators, should be reconsidered.

  14. Multisite phosphorylation of c-Jun at threonine 91/93/95 triggers the onset of c-Jun pro-apoptotic activity in cerebellar granule neurons

    PubMed Central

    Reddy, C E; Albanito, L; De Marco, P; Aiello, D; Maggiolini, M; Napoli, A; Musti, A M

    2013-01-01

    Cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation is a model of election to study the interplay of pro-apoptotic and pro-survival signaling pathways in neuronal cell death. In this model, the c-Jun N-terminal kinase (JNK) induces pro-apoptotic genes through the c-Jun/activator protein 1 (AP-1) transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/AP-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. Here, we used this model system to study the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK sites in neuronal cell death. We found that TK-deprivation led to c-Jun multiphosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of CGCs. Conversely, lithium prevented T91T93 phosphorylation and cell death without affecting the S63 site, suggesting that T91T93 phosphorylation triggers c-Jun pro-apoptotic activity. Accordingly, a c-Jun mutant lacking the T95 priming site for T91/93 phosphorylation protected CGCs from apoptosis, whereas it was able to induce neurite outgrowth in PC12 cells. Vice versa, a c-Jun mutant bearing aspartate substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass spectrometry analysis confirmed multiphosphorylation of c-Jun at T91/T93/T95 in cells. Moreover, JNK phosphorylated recombinant c-Jun at T91/T93 in a T95-dependent manner. On the basis of our results, we propose that T91/T93/T95 multiphosphorylation of c-Jun functions as a

  15. c-Jun N-terminal Kinase (JNK) Signaling as a Therapeutic Target for Alzheimer’s Disease

    PubMed Central

    Yarza, Ramon; Vela, Silvia; Solas, Maite; Ramirez, Maria J.

    2016-01-01

    c-Jun N-terminal kinases (JNKs) are a family of protein kinases that play a central role in stress signaling pathways implicated in gene expression, neuronal plasticity, regeneration, cell death, and regulation of cellular senescence. It has been shown that there is a JNK pathway activation after exposure to different stressing factors, including cytokines, growth factors, oxidative stress, unfolded protein response signals or Aβ peptides. Altogether, JNKs have become a focus of screening strategies searching for new therapeutic approaches to diabetes, cancer or liver diseases. In addition, activation of JNK has been identified as a key element responsible for the regulation of apoptosis signals and therefore, it is critical for pathological cell death associated with neurodegenerative diseases and, among them, with Alzheimer’s disease (AD). In addition, in vitro and in vivo studies have reported alterations of JNK pathways potentially associated with pathogenesis and neuronal death in AD. JNK’s, particularly JNK3, not only enhance Aβ production, moreover it plays a key role in the maturation and development of neurofibrillary tangles. This review aims to explain the rationale behind testing therapies based on inhibition of JNK signaling for AD in terms of current knowledge about the pathophysiology of the disease. Keeping in mind that JNK3 is specifically expressed in the brain and activated by stress-stimuli, it is possible to hypothesize that inhibition of JNK3 might be considered as a potential target for treating neurodegenerative mechanisms associated with AD. PMID:26793112

  16. A Novel c-Jun N-terminal Kinase (JNK) Signaling Complex Involved in Neuronal Migration during Brain Development.

    PubMed

    Zhang, Feng; Yu, Jingwen; Yang, Tao; Xu, Dan; Chi, Zhixia; Xia, Yanheng; Xu, Zhiheng

    2016-05-27

    Disturbance of neuronal migration may cause various neurological disorders. Both the transforming growth factor-β (TGF-β) signaling and microcephaly-associated protein WDR62 are important for neuronal migration during brain development; however, the underlying molecular mechanisms involved remain unclear. We show here that knock-out or knockdown of Tak1 (TGFβ-activated kinase 1) and Jnk2 (c-Jun N-terminal kinase 2) perturbs neuronal migration during cortical development and that the migration defects incurred by knock-out and/or knockdown of Tβr2 (type II TGF-β receptor) or Tak1 can be partially rescued by expression of TAK1 and JNK2, respectively. Furthermore, TAK1 forms a protein complex with RAC1 and two scaffold proteins of the JNK pathway, the microcephaly-associated protein WDR62 and the RAC1-interacting protein POSH (plenty of Src homology). Components of the complex coordinate with each other in the regulation of TAK1 as well as JNK activities. We suggest that unique JNK protein complexes are involved in the diversified biological and pathological functions during brain development and pathogenesis of diseases. PMID:27026702

  17. c-Jun N-terminal kinase-dependent apoptotic photocytotoxicity of solvent exchange-prepared curcumin nanoparticles.

    PubMed

    Paunovic, Verica; Ristic, Biljana; Markovic, Zoran; Todorovic-Markovic, Biljana; Kosic, Milica; Prekodravac, Jovana; Kravic-Stevovic, Tamara; Martinovic, Tamara; Micusik, Matej; Spitalsky, Zdeno; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

    2016-04-01

    Indian spice curcumin is known for its anticancer properties, but the anticancer mechanisms of nanoparticulate curcumin have not been completely elucidated. We here investigated the in vitro anticancer effect of blue light (470 nm, 1 W)-irradiated curcumin nanoparticles prepared by tetrahydrofuran/water solvent exchange, using U251 glioma, B16 melanoma, and H460 lung cancer cells as targets. The size of curcumin nanocrystals was approximately 250 nm, while photoexcitation induced their oxidation and partial agglomeration. Although cell membrane in the absence of light was almost impermeable to curcumin nanoparticles, photoexcitation stimulated their internalization. While irradiation with blue light (1-8 min) or nanocurcumin (1.25-10 μg/ml) alone was only marginally toxic to tumor cells, photoexcited nanocurcumin displayed a significant cytotoxicity depending both on the irradiation time and nanocurcumin concentration. Photoexcited nanocurcumin induced phosphorylation of c-Jun N-terminal kinase (JNK), mitochondrial depolarization, caspase-3 activation, and cleavage of poly (ADP-ribose) polymerase, indicating apoptotic cell death. Accordingly, pharmacologial inhibition of JNK and caspase activity rescued cancer cells from photoexcited nanocurcumin. On the other hand, antioxidant treatment did not reduce photocytotoxicity of nanocurcumin, arguing against the involvement of oxidative stress. By demonstrating the ability of photoexcited nanocurcumin to induce oxidative-stress independent, JNK- and caspase-dependent apoptosis, our results support its further investigation in cancer therapy. PMID:27106025

  18. c-Jun-N-terminal kinase 1 is necessary for nicotine-induced enhancement of contextual fear conditioning.

    PubMed

    Leach, Prescott T; Kenney, Justin W; Gould, Thomas J

    2016-08-01

    Acute nicotine enhances hippocampus-dependent learning. Identifying how acute nicotine improves learning will aid in understanding how nicotine facilitates the development of maladaptive memories that contribute to drug-seeking behaviors, help development of medications to treat disorders associated with cognitive decline, and advance understanding of the neurobiology of learning and memory. The effects of nicotine on learning may involve recruitment of signaling through the c-Jun N-terminal kinase family (JNK 1-3). Learning in the presence of acute nicotine increases the transcription of mitogen-activated protein kinase 8 (MAPK8, also known as JNK1), likely through a CREB-dependent mechanism. The functional significance of JNK1 in the effects of acute nicotine on learning, however, is unknown. The current studies undertook a backward genetic approach to determine the functional contribution JNK1 protein makes to nicotine-enhanced contextual fear conditioning. JNK1 wildtype (WT) and knockout (KO) mice were administered acute nicotine prior to contextual and cued fear conditioning. 24h later, mice were evaluated for hippocampus-dependent (contextual fear conditioning) and hippocampus-independent (cued fear conditioning) memory. Nicotine selectively enhanced contextual conditioning in WT mice, but not in KO mice. Nicotine had no effect on hippocampus-independent learning in either genotype. JNK1 KO and WT mice given saline showed similar levels of learning. These data suggest that JNK1 may be recruited by nicotine and is functionally necessary for the acute effects of nicotine on learning and memory. PMID:27235579

  19. C-Jun N-Terminal Kinase 2 Promotes Graft Injury via the Mitochondrial Permeability Transition After Mouse Liver Transplantation

    PubMed Central

    Theruvath, T. P.; Czerny, C.; Ramshesh, V. K.; Zhong, Z.; Chavin, K. D.; Lemasters, J. J.

    2009-01-01

    The c-Jun N-terminal kinase (JNK) pathway enhances graft injury after liver transplantation (LT). We hypothesized that the JNK2 isoform promotes graft injury via the mitochondrial permeability transition (MPT). Livers of C57BL/6J (wild-type, WT) and JNK2 knockout (KO) mice were transplanted into WT recipients after 30 h of cold storage in UW solution. Injury after implantation was assessed by serum ALT, histological necrosis, TUNEL, Caspase 3 activity, 30-day survival, and cytochrome c and 4-hydroxynonenal immunostaining. Multiphoton microscopy after LT monitored mitochondrial membrane potential in vivo. After LT, ALT increased three times more in WT compared to KO (p < 0.05). Necrosis and TUNEL were more than two times greater in WT than KO (p < 0.05). Immunostaining showed a >80% decrease of mitochondrial cytochrome c release in KO compared to WT (p < 0.01). Lipid peroxidation was similarly decreased. Every KO graft but one survived longer than all WT grafts (p < 0.05, Kaplan-Meier). After LT, depolarization of mitochondria occurred in 73% of WT hepatocytes, which decreased to 28% in KO (p < 0.05). In conclusion, donor JNK2 promotes injury after mouse LT via the MPT. MPT inhibition using specific JNK2 inhibitors may be useful in protecting grafts against adverse outcomes from ischemia/reperfusion injury. PMID:18671679

  20. Effects of sodium selenite on c-Jun N-terminal kinase signalling pathway induced by oxidative stress in human chondrocytes and c-Jun N-terminal kinase expression in patients with Kashin-Beck disease, an endemic osteoarthritis.

    PubMed

    Dai, XiaoXia; Li, YuanYuan; Zhang, RongQiang; Kou, Yan; Mo, XiaoYan; Cao, JunLing; Xiong, YongMin

    2016-05-01

    The c-Jun N-terminal kinases (JNK) are members of the mitogen-activated protein kinase family and are activated by environmental stress. Se plays an important role in the biological pathways by forming selenoprotein. Selenoproteins have been shown to exhibit a variety of biological functions including antioxidant functions and maintaining cellular redox balance, and compromise of such important proteins would lead to oxidative stress and apoptosis. We examined the expression levels of JNK in Kashin-Beck disease (KBD) patients, tested the potential protective effects of sodium selenite on tert-butyl hydroperoxide (tBHP)-induced oxidative injury and apoptosis in human chondrocytes as well as its underlying mechanism in this study. We produced an oxidative damage model induced by tBHP in C28/I2 human chondrocytes to test the essential anti-apoptosis effects of Se in vitro. The results indicated that the expression level of phosphorylated JNK was significantly increased in KBD patients. Cell apoptosis was increased and molecule expressions of the JNK signalling pathway were activated in the tBHP-injured chondrocytes. Na2SeO3 protected against tBHP-induced oxidative stress and apoptosis in cells by increasing cell viability, reducing reactive oxygen species generation, increasing Glutathione peroxidase (GPx) activity and down-regulating the JNK pathway. These results demonstrate that apoptosis induced by tBHP in chondrocytes might be mediated via up-regulation of the JNK pathway; Na2SeO3 has an effect of anti-apoptosis by down-regulating the JNK signalling pathway. PMID:26948765

  1. Critical role of c-jun N-terminal protein kinase in promoting mitochondrial dysfunction and acute liver injury

    PubMed Central

    Jang, Sehwan; Yu, Li-Rong; Abdelmegeed, Mohamed A.; Gao, Yuan; Banerjee, Atrayee; Song, Byoung-Joon

    2015-01-01

    The mechanism by which c-Jun N-terminal protein kinase (JNK) promotes tissue injury is poorly understood. Thus we aimed at studying the roles of JNK and its phospho-target proteins in mouse models of acute liver injury. Young male mice were exposed to a single dose of CCl4 (50 mg/kg, IP) and euthanized at different time points. Liver histology, blood alanine aminotransferase, and other enzyme activities were measured in CCl4-exposed mice without or with the highly-specific JNK inhibitors. Phosphoproteins were purified from control or CCl4-exposed mice and analyzed by differential mass-spectrometry followed by further characterizations of immunoprecipitation and activity measurements. JNK was activated within 1 h while liver damage was maximal at 24 h post-CCl4 injection. Markedly increased phosphorylation of many mitochondrial proteins was observed between 1 and 8 h following CCl4 exposure. Pretreatment with the selective JNK inhibitor SU3327 or the mitochondria-targeted antioxidant mito-TEMPO markedly reduced the levels of p-JNK, mitochondrial phosphoproteins and liver damage in CCl4-exposed mice. Differential proteomic analysis identified many phosphorylated mitochondrial proteins involved in anti-oxidant defense, electron transfer, energy supply, fatty acid oxidation, etc. Aldehyde dehydrogenase, NADH-ubiquinone oxidoreductase, and α-ketoglutarate dehydrogenase were phosphorylated in CCl4-exposed mice but dephosphorylated after SU3327 pretreatment. Consistently, the suppressed activities of these enzymes were restored by SU3327 pretreatment in CCl4-exposed mice. These data provide a novel mechanism by which JNK, rapidly activated by CCl4, promotes mitochondrial dysfunction and acute hepatotoxicity through robust phosphorylation of numerous mitochondrial proteins. PMID:26491845

  2. C-Jun N-Terminal Kinase 2 Promotes Liver Injury via the Mitochondrial Permeability Transition after Hemorrhage and Resuscitation

    PubMed Central

    Czerny, Christoph; Theruvath, Tom P.; Maldonado, Eduardo N.; Lehnert, Mark; Marzi, Ingo; Zhong, Zhi; Lemasters, John J.

    2012-01-01

    Hemorrhagic shock leads to hepatic hypoperfusion and activation of mitogen-activated stress kinases (MAPK) like c-Jun N-terminal kinase (JNK) 1 and 2. Our aim was to determine whether mitochondrial dysfunction leading to hepatic necrosis and apoptosis after hemorrhage/resuscitation (H/R) was dependent on JNK2. Under pentobarbital anesthesia, wildtype (WT) and JNK2 deficient (KO) mice were hemorrhaged to 30 mm Hg for 3 h and then resuscitated with shed blood plus half the volume of lactated Ringer's solution. Serum alanine aminotransferase (ALT), necrosis, apoptosis and oxidative stress were assessed 6 h after resuscitation. Mitochondrial polarization was assessed by intravital microscopy. After H/R, ALT in WT-mice increased from 130 U/L to 4800 U/L. In KO-mice, ALT after H/R was blunted to 1800 U/l (P < 0.05). Necrosis, caspase-3 activity and ROS were all substantially decreased in KO compared to WT mice after H/R. After sham operation, intravital microscopy revealed punctate mitochondrial staining by rhodamine 123 (Rh123), indicating normal mitochondrial polarization. At 4 h after H/R, Rh123 staining became dim and diffuse in 58% of hepatocytes, indicating depolarization and onset of the mitochondrial permeability transition (MPT). By contrast, KO mice displayed less depolarization after H/R (23%, P < 0.05). In conclusion, JNK2 contributes to MPT-mediated liver injury after H/R. PMID:22791932

  3. Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality.

    PubMed

    Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

    2015-10-01

    Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

  4. Specific inhibition of c-Jun N-terminal kinase delays preterm labour and reduces mortality

    PubMed Central

    Pirianov, Grisha; MacIntyre, David A; Lee, Yun; Waddington, Simon N; Terzidou, Vasso; Mehmet, Huseyin; Bennett, Phillip R

    2015-01-01

    Preterm labour (PTL) is commonly associated with infection and/or inflammation. Lipopolysaccharide (LPS) from different bacteria can be used to independently or mutually activate Jun N-terminal kinase (JNK)/AP1- or NF-κB-driven inflammatory pathways that lead to PTL. Previous studies using Salmonella abortus LPS, which activates both JNK/AP-1 and NF-κB, showed that selective inhibition of NF-κB delays labour and improves pup outcome. Where labour is induced using Escherichia coli LPS (O111), which upregulates JNK/AP-1 but not NF-κB, inhibition of JNK/AP-1 activation also delays labour. In this study, to determine the potential role of JNK as a therapeutic target in PTL, we investigated the specific contribution of JNK signalling to S. Abortus LPS-induced PTL in mice. Intrauterine administration of S. Abortus LPS to pregnant mice resulted in the activation of JNK in the maternal uterus and fetal brain, upregulation of pro-inflammatory proteins COX-2, CXCL1, and CCL2, phosphorylation of cPLA2 in myometrium, and induction of PTL. Specific inhibition of JNK by co-administration of specific D-JNK inhibitory peptide (D-JNKI) delayed LPS-induced preterm delivery and reduced fetal mortality. This is associated with inhibition of myometrial cPLA2 phosphorylation and proinflammatory proteins synthesis. In addition, we report that D-JNKI inhibits the activation of JNK/JNK3 and caspase-3, which are important mediators of neural cell death in the neonatal brain. Our data demonstrate that specific inhibition of TLR4-activated JNK signalling pathways has potential as a therapeutic approach in the management of infection/inflammation-associated PTL and prevention of the associated detrimental effects to the neonatal brain. PMID:26183892

  5. Rb binds c-Jun and activates transcription.

    PubMed Central

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

    1998-01-01

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

  6. Radiation-Induced c-Jun Activation Depends on MEK1-ERK1/2 Signaling Pathway in Microglial Cells

    PubMed Central

    Deng, Zhiyong; Sui, Guangchao; Rosa, Paulo Mottin; Zhao, Weiling

    2012-01-01

    Radiation-induced normal brain injury is associated with acute and/or chronic inflammatory responses, and has been a major concern in radiotherapy. Recent studies suggest that microglial activation is a potential contributor to chronic inflammatory responses following irradiation; however, the molecular mechanism underlying the response of microglia to radiation is poorly understood. c-Jun, a component of AP-1 transcription factors, potentially regulates neural cell death and neuroinflammation. We observed a rapid increase in phosphorylation of N-terminal c-Jun (on serine 63 and 73) and MAPK kinases ERK1/2, but not JNKs, in irradiated murine microglial BV2 cells. Radiation-induced c-Jun phosphorylation is dependent on the canonical MEK-ERK signaling pathway and required for both ERK1 and ERK2 function. ERK1/2 directly interact with c-Jun in vitro and in cells; meanwhile, the JNK binding domain on c-Jun is not required for its interaction with ERK kinases. Radiation-induced reactive oxygen species (ROS) potentially contribute to c-Jun phosphorylation through activating the ERK pathway. Radiation stimulates c-Jun transcriptional activity and upregulates c-Jun-regulated proinflammatory genes, such as tumor necrosis factor-α, interleukin-1β, and cyclooxygenase-2. Pharmacologic blockade of the ERK signaling pathway interferes with c-Jun activity and inhibits radiation-stimulated expression of c-Jun target genes. Overall, our study reveals that the MEK-ERK1/2 signaling pathway, but not the JNK pathway, contributes to the c-Jun-dependent microglial inflammatory response following irradiation. PMID:22606284

  7. Bacterial protein AvrA stabilizes intestinal epithelial tight junctions via blockage of the C-Jun N-terminal kinase pathway.

    PubMed

    Zhang, Yongguo; Wu, Shaoping; Ma, Jun; Xia, Yinglin; Ai, Xun; Sun, Jun

    2015-01-01

    The Salmonella type III secretory system secretes virulence proteins, called effectors. Effectors are responsible for the alteration of tight junctions (TJ) and epithelial functions in intestinal infection and inflammation. In a previous study, we have demonstrated that a bacterial effector AvrA plays a role in stabilizing TJs and balancing the opposing action of other bacterial effectors. However, the molecular mechanisms by which AvrA-modulates TJ protein expression remain unknown. AvrA possesses acetyltransferase activity toward specific mitogen-activated protein kinase kinases (MAPKKs) and potently inhibits the c-Jun N-terminal kinase (JNK) pathway in inflammation. Inhibition of the JNK pathway is known to inhibit the TJ protein disassemble. Therefore, we hypothesize that AvrA stabilizes intestinal epithelial TJs via c-Jun and JNK pathway blockage. Using both in vitro and in vivo models, we showed that AvrA targets the c-Jun and JNK pathway that in turn stabilizes TJ protein ZO-1. Inhibition of JNK abolished the effect of AvrA on ZO-1. We further determined that AvrA suppressed the transcription factor activator protein-1, which was regulated by activated JNK. Moreover, we identified the functional domain of AvrA that directly regulated TJs using a series of AvrA mutants. The role of AvrA represents a highly refined bacterial strategy that helps the bacteria survive in the host and dampens the inflammatory response of the host. Our findings have uncovered a novel role of the bacterial protein AvrA in suppressing the inflammatory response of the host through JNK-regulated blockage of epithelial cell barrier function. PMID:25838979

  8. Momordica charantia polysaccharides could protect against cerebral ischemia/reperfusion injury through inhibiting oxidative stress mediated c-Jun N-terminal kinase 3 signaling pathway.

    PubMed

    Gong, Juanjuan; Sun, Fumou; Li, Yihang; Zhou, Xiaoling; Duan, Zhenzhen; Duan, Fugang; Zhao, Lei; Chen, Hansen; Qi, Suhua; Shen, Jiangang

    2015-04-01

    Momordica charantia (MC) is a medicinal plant for stroke treatment in Traditional Chinese Medicine, but its active compounds and molecular targets are unknown yet. M. charantia polysaccharide (MCP) is one of the important bioactive components in MC. In the present study, we tested the hypothesis that MCP has neuroprotective effects against cerebral ischemia/reperfusion injury through scavenging superoxide (O2(-)), nitric oxide (NO) and peroxynitrite (ONOO(-)) and inhibiting c-Jun N-terminal protein kinase (JNK3) signaling cascades. We conducted experiments with in vivo global and focal cerebral ischemia/reperfusion rat models and in vitro oxygen glucose deprivation (OGD) neural cells. The effects of MCP on apoptotic cell death and infarction volume, the bioactivities of scavenging O2(-), NO and ONOO(-), inhibiting lipid peroxidation and modulating JNK3 signaling pathway were investigated. Major results are summarized as below: (1) MCP dose-dependently attenuated apoptotic cell death in neural cells under OGD condition in vitro and reduced infarction volume in ischemic brains in vivo; (2) MCP had directing scavenging effects on NO, O2(-) and ONOO(-) and inhibited lipid peroxidation; (3) MCP inhibited the activations of JNK3/c-Jun/Fas-L and JNK3/cytochrome C/caspases-3 signaling cascades in ischemic brains in vivo. Taken together, we conclude that MCP could be a promising neuroprotective ingredient of M. charantia and its mechanisms could be at least in part attributed to its antioxidant activities and inhibiting JNK3 signaling cascades during cerebral ischemia/reperfusion injury. PMID:25510970

  9. Mitochondrial Permeability Transition in Liver Ischemia and Reperfusion: Role of c-Jun N-Terminal Kinase 2

    PubMed Central

    Theruvath, Tom P.; Snoddy, Mark C.; Zhong, Zhi; Lemasters, John J.

    2009-01-01

    The mitochondrial permeability transition (MPT) mediates hepatic necrosis after ischemia and reperfusion (I/R). Here, we studied the role of c-Jun N-terminal kinase 2 (JNK2) in MPT-induced liver injury. Wildtype (WT) and JNK2 knockout (KO) mice underwent 70% liver ischemia for 1 hr followed by reperfusion for 8 hr, after which hepatocyte injury and animal survival was assessed. Compared with WT, JNK2 KO mice had 38% less alanine transaminase release and 39% less necrosis by histology. Survival out to 14 days was also greater in JNK2 KO mice (57% vs. 11%), and overall Kaplan-Meier survival was improved. No difference in apoptosis was observed. Intravital multiphoton microscopy of potential-indicating rhodamine 123 after reperfusion revealed depolarized mitochondria in 82% of WT hepatocytes, which decreased to 43% in JNK2 KO hepatocytes. In conclusion, JNK2 contributes to hepatocellular injury and death after I/R in association with increased mitochondrial dysfunction via the MPT. PMID:18497693

  10. The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

    SciTech Connect

    Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute; Kieser, Arnd

    2008-02-20

    The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied by a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.

  11. Guggulsterone inhibits tumor cell proliferation, induces S-phase arrest, and promotes apoptosis through activation of c-Jun N-terminal kinase, suppression of Akt pathway, and downregulation of antiapoptotic gene products.

    PubMed

    Shishodia, Shishir; Sethi, Gautam; Ahn, Kwang Seok; Aggarwal, Bharat B

    2007-06-30

    Guggulsterone is a plant polyphenol traditionally used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, possibly through an anti-inflammatory mechanism. Whether this steroid has any role in cancer is not known. In this study, we found that guggulsterone inhibits the proliferation of wide variety of human tumor cell types including leukemia, head and neck carcinoma, multiple myeloma, lung carcinoma, melanoma, breast carcinoma, and ovarian carcinoma. Guggulsterone also inhibited the proliferation of drug-resistant cancer cells (e.g., gleevac-resistant leukemia, dexamethasone-resistant multiple myeloma, and doxorubicin-resistant breast cancer cells). Guggulsterone suppressed the proliferation of cells through inhibition of DNA synthesis, producing cell cycle arrest in S-phase, and this arrest correlated with a decrease in the levels of cyclin D1 and cdc2 and a concomitant increase in the levels of cyclin-dependent kinase inhibitor p21 and p27. Guggulsterone-induced apoptosis as indicated by increase in the number of Annexin V- and TUNEL-positive cells, through the downregulation of anti-apoptototic products. The apoptosis induced by guggulsterone was also indicated by the activation of caspase-8, bid cleavage, cytochrome c release, caspase-9 activation, caspase-3 activation, and PARP cleavage. The apoptotic effects of guggulsterone were preceded by activation of JNK and downregulation of Akt activity. JNK was needed for guggulsterone-induced apoptosis, inasmuch as inhibition of JNK by pharmacological inhibitors or by genetic deletion of MKK4 (activator of JNK) abolished the activity. Overall, our results indicate that guggulsterone can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and downregulation of antiapoptotic protein expression. PMID:17475222

  12. Knockdown of Sec8 enhances the binding affinity of c-Jun N-terminal kinase (JNK)-interacting protein 4 for mitogen-activated protein kinase kinase 4 (MKK4) and suppresses the phosphorylation of MKK4, p38, and JNK, thereby inhibiting apoptosis.

    PubMed

    Tanaka, Toshiaki; Iino, Mitsuyoshi; Goto, Kaoru

    2014-12-01

    The exocyst complex, also called the Sec6/8 complex, is important for targeting exocytic vesicles to specific docking sites on the plasma membrane in yeast and mammalian cells. In addition to these original findings, recent results of studies suggest that Sec8 is also involved in oncogenesis, although the functional implications of Sec8 in cancer cells are not well understood. c-Jun N-terminal kinase-interacting protein 4 (JIP4) is a scaffold protein that plays a crucial role in the regulation of mitogen-activated protein kinase (MAPK) signaling cascades. The present study examined how Sec8 is involved in JIP4-mediated MAPK signaling under apoptotic conditions. It was found that Sec8 binds to and regulates JIP4, and that knockdown of Sec8 enhances the binding of JIP4 to MAPK kinase 4, thereby decreasing the phosphorylation of MAPK kinase 4, JNK, and p38. These results raise the possibility that Sec8 serves as an important regulator of MAPK signaling cascades. PMID:25244576

  13. 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. PMID:26724804

  14. Peripheral KATP activation inhibits pain sensitization induced by skin/muscle incision and retraction via the nuclear factor-κB/c-Jun N-terminal kinase signaling pathway.

    PubMed

    Qian, Li-Ping; Shen, Shi-Ren; Chen, Jun-Jie; Ji, Lu-Lu; Cao, Su

    2016-09-01

    The aim of the current study was to assess the effect of pinacidil activation of ATP‑sensitive potassium (KATP) channels prior to skin/muscle incision and retraction (SMIR) surgery on peripheral and central sensitization, and investigate molecular interferential targets for preventive analgesia. Male Sprague-Dawley rats were randomly assigned to one of the following five groups: Control, incision (sham surgery), incision plus retraction (SMIR) group, SMIR plus pinacidil (pinacidil) group and the SMIR plus pyrrolidine dithiocarbamate (PDTC) group. The rats in the pinacidil and PDTC groups were intraperitoneally injected with pinacidil or PDTC, respectively, prior to the SMIR procedure. The mechanical withdrawal threshold (MWT) was determined. Western blotting was performed to detect the alterations in the subunits of the KATP channels, Kir6.1 and SUR2, levels of nuclear factor‑κB (NF‑κB) in the tissue around the incision and c‑Jun N‑terminal kinase (JNK) in the spinal cord. There was a significant increase observed in the levels of NF‑κB and JNK following SMIR surgery compared with the control group, and a significant reduction in MWT and the levels of Kir6.1 and SUR2. Additionally, intraperitoneal administration of pinacidil inhibited the reduction in MWT, and Kir6.1 and SUR2 levels. SMIR was observed to result in increases in the levels of NF‑κB and JNK. In addition, in the PDTC group, the alterations in MWT, NF‑κB, JNK, Kir6.1 and SUR2 resulting from SMIR were blocked. The results of the current study suggest that the deteriorations in the microenvironment resulting from the SMIR procedure can induce peripheral and central sensitization, and that the activation of peripheral KATP by pinacidil prior to SMIR is able to inhibit peripheral and central sensitization via the NF‑κB/JNK signaling pathway, thus resulting in preventive analgesia. PMID:27484116

  15. c-Jun N-Terminal Kinases Mediate a Wide Range of Targets in the Metastatic Cascade

    PubMed Central

    Ebelt, Nancy D.; Cantrell, Michael A.

    2013-01-01

    Disseminated cancer cells rely on intricate interactions among diverse cell types in the tumor-associated stroma, vasculature, and immune system for survival and growth. Ubiquitous expression of c-Jun N-terminal kinase (jnk) genes in various cell types permits their control of metastasis. In early stages of metastasis, JNKs affect tumor-associated inflammation and angiogenesis as well as tumor cell migration and intravasation. Within the tumor stroma, JNKs are essential for the release of growth factors that promote epithelial-to-mesenchymal transition (EMT) in tumor cells. JNK3, the least ubiquitous isoform, facilitates angiogenesis by increasing endothelial cell migration. Importantly, JNK expression in tumor cells integrates stromal signals to promote tumor cell invasion. However, JNK isoforms differentially regulate migration toward the endothelial barrier. Once tumor cells enter the bloodstream, JNKs increase circulating tumor cell (CTC) survival and homing to tissues. By promoting fibrosis, JNKs improve CTC attachment to the endothelium. Once anchored, JNKs stimulate EMT to facilitate tumor cell extravasation and enhance the secretion of endothelial barrier disrupters. Tumor cells attract barrier-disrupting macrophages by JNK-dependent transcription of macrophage chemoattractant molecules. In the secondary tissue, JNKs are instrumental in the premetastatic niche and stimulate tumor cell proliferation. JNK expression in cancer cells stimulates tissue-remodeling macrophages to improve tumor colonization. However, in T-cells, JNKs alter cytokine production that increases tumor surveillance and inhibits the recruitment of tissue-remodeling macrophages. Therapeutically targeting JNKs for metastatic disease is attractive considering their promotion of metastasis; however, specific JNK tools are needed to determine their definitive actions within the context of the entire metastatic cascade. PMID:24349635

  16. c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

    PubMed Central

    Suzuki, Akiko; Kakisaka, Keisuke; Suzuki, Yuji; Wang, Ting; Takikawa, Yasuhiro

    2016-01-01

    AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed “lipoapoptosis,” in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12 (AML12) cells treated with palmitate (PA) were used as an in vitro model. RESULTS: LC3-II, p62, and Run domain Beclin-1 interacting and cysteine-rich containing (Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase (JNK) inhibition at both the mRNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-II and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown. CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide anti-fibrosis in the NASH liver. PMID:27605885

  17. Myocardial protective effects of a c-Jun N-terminal kinase inhibitor in rats with brain death.

    PubMed

    Guo, Wenzhi; Cao, Shengli; Yan, Bing; Zhang, Gong; Li, Jie; Zhao, Yongfu; Zhang, Shuijun

    2016-07-01

    To investigate whether the mitochondrial apoptotic pathway mediates myocardial cell injuries in rats under brain death (BD), and observe the effects and mechanisms of the c-Jun N-terminal kinase (JNK) inhibitor SP600125 on cell death in the heart. Forty healthy male Sprague-Dawley (SD) rats were randomized into four groups: sham group (dural external catheter with no BD); BD group (maintain the induced BD state for 6 hrs); BD + SP600125 group (intraperitoneal injection of SP600125 10 mg/kg 1 hr before inducing BD, and maintain BD for 6 hrs); and BD + Dimethyl Sulphoxide (DMSO) group (intraperitoneal injection of DMSO 1 hr before inducing BD, and maintain BD for 6 hrs). Real-time quantitative PCR was used to evaluate mRNA levels of Cyt-c and caspase-3. Western blot analysis was performed to examine the levels of mitochondrial apoptosis-related proteins p-JNK, Bcl-2, Bax, Cyt-c and Caspase-3. TUNEL assay was employed to evaluate myocardial apoptosis. Compared with the sham group, the BD group exhibited increased mitochondrial apoptosis-related gene expression, accompanied by the elevation of p-JNK expression and myocardial apoptosis. As the vehicle control, DMSO had no treatment effects. The BD + SP600125 group had decreased p-JNK expression, and reduced mitochondrial apoptosis-related gene expression. Furthermore, the apoptosis rate of myocardial cells was reduced. The JNK inhibitor SP600125 could protect myocardial cells under BD through the inhibition of mitochondrial apoptosis-related pathways. PMID:27072084

  18. Involvement of c-Jun N-terminal kinase in reversal of multidrug resistance of human leukemia cells in hypoxia by 5-bromotetrandrine.

    PubMed

    Zhang, Wei; Chen, Bao-an; Jin, Jun-fei; He, You-ji; Niu, Yi-qi

    2013-11-01

    5-Bromotetrandrine (BrTet), a candidate multidrug resistance (MDR) modulator, is a potential compound for use in cancer therapy when combined with anticancer agents such as daunorubicin (DNR) and paclitaxel. The purposeof this study was to investigate the mechanism of reversal of P-glycoprotein (P-gp)-mediated MDR by BrTet and the involvement of the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway in both adriamycin-sensitive K562 and adriamycin-resistant K562 (KA) leukemia cells in hypoxia. The combination of BrTet and DNR decreased both phosphorylated JNK1/2 and MDR1/P-gp levels under hypoxic conditions. Furthermore, a pharmacological inhibitor of JNK, SP600125, or small interfering RNA (siRNA) oligonucleotides to both JNK1 and JNK2 reversed BrTet- or DNR-induced JNK phosphorylation and MDR1/P-gp levels. We further demonstrated that the decreased JNK phosphorylation and MDR1/P-gp levels were associated with a significant increase in intracellular accumulation of DNR, which dramatically enhanced the sensitivity of drug-resistant KA cells to DNR, and led to cellular apoptosis through activation of the caspase-3 pathway. It is concluded that using BrTet in combination with other chemotherapeutic agents and pharmacological inhibitors of JNK can abrogate the P-gp-induced MDR in adriamycin-resistant K562 cells, which has potential clinical relevance in cancer therapy for chemotherapeutic-resistant human leukemia. PMID:23418897

  19. Defective antiviral CD8 T-cell response and viral clearance in the absence of c-Jun N-terminal kinases.

    PubMed

    Wang, Yong-Qin; Ma, Xi; Lu, Lingling; Zhao, Limei; Zhang, Xiaoqing; Xu, Qingyu; Wang, Yang

    2014-08-01

    The c-Jun N-terminal kinase (JNK) signalling pathway appears to act as a critical intermediate in the regulation of lymphocyte activation and proliferation. The majority of studies on the importance of JNK are focused on its role in T helper responses, with very few reports addressing the mechanisms of JNK in governing CD8 T-cell-mediated immunity. By using a well-defined mousepox model, we demonstrate that JNK is involved in CD8(+) T-cell-mediated antiviral responses. Deficiency of either JNK1 or JNK2 impaired viral clearance, subsequently resulting in an increased susceptibility to ectromelia virus in resistant mice. The impairment of CD8 responses in JNK-deficient mice was not directly due to an inhibition of effector T-cell expansion, as both JNK1 and JNK2 had limited effect on the activation-induced cell death of CD8(+) T cells, and only JNK2-deficient mice exhibited a significant change in CD8(+) T-cell proliferation after acute ectromelia virus infection. However, optimal activation of CD8(+) T cells and their effector functions require signals from both JNK1 and JNK2. Our results suggest that the JNK pathway acts as a critical intermediate in antiviral immunity through regulation of the activation and effector function of CD8(+) T cells rather than by altering their expansion. PMID:24673683

  20. EGCG-targeted p57/KIP2 reduces tumorigenicity of oral carcinoma cells: Role of c-Jun N-terminal kinase

    SciTech Connect

    Yamamoto, Tetsuya; Digumarthi, Hari; Aranbayeva, Zina; Wataha, John; Lewis, Jill; Messer, Regina; Qin, Haiyan; Dickinson, Douglas; Osaki, Tokio; Schuster, George S.; Hsu, Stephen

    2007-11-01

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) regulates gene expression differentially in tumor and normal cells. In normal human primary epidermal keratinocytes (NHEK), one of the key mediators of EGCG action is p57/KIP2, a cyclin-dependent kinase (CDK) inhibitor. EGCG potently induces p57 in NHEK, but not in epithelial cancer cells. In humans, reduced expression of p57 often is associated with advanced tumors, and tumor cells with inactivated p57 undergo apoptosis when exposed to EGCG. The mechanism of p57 induction by EGCG is not well understood. Here, we show that in NHEK, EGCG-induces p57 via the p38 mitogen-activated protein kinase (MAPK) signaling pathway. In p57-negative tumor cells, JNK signaling mediates EGCG-induced apoptosis, and exogenous expression of p57 suppresses EGCG-induced apoptosis via inhibition of c-Jun N-terminal kinase (JNK). We also found that restoration of p57 expression in tumor cells significantly reduced tumorigenicity in athymic mice. These results suggest that p57 expression may be an useful indicator for the clinical course of cancers, and could be potentially useful as a target for cancer therapies.

  1. REG Iα activates c-Jun through MAPK pathways to enhance the radiosensitivity of squamous esophageal cancer cells.

    PubMed

    Wakita, Akiyuki; Motoyama, Satoru; Sato, Yusuke; Koyota, Souichi; Usami, Shuetsu; Yoshino, Kei; Sasaki, Tomohiko; Imai, Kazuhiro; Saito, Hajime; Minamiya, Yoshihiro

    2015-07-01

    Identification of the key molecules that mediate susceptibility to anticancer treatments would be highly desirable. Based on clinical and cell biological studies, we recently proposed that regenerating gene (REG) Iα may be such a molecule. In the present study, we hypothesized that REG Iα increases radiosensitivity through activation of mitogen-activated protein kinase (MAPK) pathways. To test that idea, we transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and examined its involvement in MAPK signaling and its effect on susceptibility to radiotherapy. We found that REG Iα-expressing cells showed increased expression of c-Jun messenger RNA (mRNA) and phospho-c-Jun protein mediated via the c-Jun N-terminal kinase (JNK) pathway and extracellular signal-regulated kinase (ERK) pathway, as well as increased radiosensitivity. Immunohistochemical analysis confirmed the activation of c-Jun in tumors expressing REG Iα. Collectively, these findings suggest that REG Iα activates c-Jun via the JNK and ERK pathway, thereby enhancing radiosensitivity. PMID:25656613

  2. TBP Is Differentially Regulated by c-Jun N-Terminal Kinase 1 (JNK1) and JNK2 through Elk-1, Controlling c-Jun Expression and Cell Proliferation▿

    PubMed Central

    Zhong, Shuping; Fromm, Jody; Johnson, Deborah L.

    2007-01-01

    Emerging evidence supports the idea that the c-Jun N-terminal kinases (JNKs) possess overlapping but distinct functions. The potential roles of the ubiquitously expressed JNK1 and JNK2 in regulating expression of the central transcription initiation factor, TATA-binding protein (TBP), were examined. Relative to wild-type fibroblasts, TBP was decreased in Jnk1−/− cells and increased in Jnk2−/− cells. Similarly, reduction of JNK1 in human hepatoma cells decreased TBP expression, whereas reduction of JNK2 enhanced it. JNK-mediated regulation of TBP expression occurs at the transcriptional level through their ability to target Elk-1, which directly regulates the TBP promoter in response to epidermal growth factor stimulation. JNK1 increases, whereas JNK2 decreases, the phosphorylation state of Elk-1, which differentially affects Elk-1 occupancy at a defined site within the TBP promoter. These JNK-mediated alterations in TBP expression, alone, serve to regulate c-Jun expression and fibroblast proliferation rates. These studies uncovered several new molecular events that distinguish the functions of JNK1 and JNK2 that are critical for their regulation of cellular proliferation. PMID:17074809

  3. The Protein Dredd Is an Essential Component of the c-Jun N-terminal Kinase Pathway in the Drosophila Immune Response*

    PubMed Central

    Guntermann, Silvia; Foley, Edan

    2011-01-01

    The Drosophila immune deficiency (IMD) pathway mobilizes c-Jun N-terminal kinase (JNK), caspase, and nuclear factor-κB (NF-κB) modules to counter infection with Gram-negative bacteria. Dredd is an essential caspase in the IMD pathway, and it is widely established that NF-κB activation depends on Dredd. More recent cell culture studies suggested a role for Dredd in the activation of dJNK (Drosophila JNK). However, there are no epistatic or mechanistic data on the involvement of Dredd in dJNK activation. More importantly, there is no in vivo evidence to demonstrate a physiological requirement for Dredd in the IMD/dJNK pathway. We performed a comprehensive analysis of the role of Dredd in the IMD/dJNK pathway, and we demonstrated that Dredd is essential for the activation of IMD/dJNK in cell culture. We positioned Dredd activity at an early point of the IMD/dJNK pathway and uncovered a series of interactions between Dredd and additional proximal IMD pathway molecules. Mechanistically, we showed that the caspase activity inhibitor p35 blocked dJNK activation and the induction of dJNK-dependent genes in cell culture and in vivo. Most importantly, we demonstrated that dredd mutant flies are completely inhibited in their ability to activate dJNK or express dJNK-responsive target genes after bacterial infection in vivo. In conclusion, we established Dredd as an essential component of the IMD pathway required for the full activation of IMD/dJNK in cell culture and in vivo. Our data enhance our appreciation of Dredd-dependent IMD signal transduction events. PMID:21730059

  4. Colchicine induces apoptosis in HT‑29 human colon cancer cells via the AKT and c-Jun N-terminal kinase signaling pathways.

    PubMed

    Huang, Zhen; Xu, Ye; Peng, Wei

    2015-10-01

    Colchicine is a natural compound, which belongs to the botanical family Colchicaceae and prevents growth of cancer cells via antimitotic activity by interacting with microtubules. Although numerous studies have demonstrated that the effect of colchicine on cell apoptosis is mediated by the activation of caspase‑3, the signaling pathways involved in the process remain unknown. In the current study, evidence is presented regarding the missing information using HT‑29 human colon cancer cells. The effect of colchicine on apoptosis in HT‑29 cells and the apoptosis‑associated signaling pathways were determined using various methods, including cell viability assay, Annexin V/propidium idodide (PI) binding, PI staining, Hoechst 33342 staining, mitochondrial membrane potential (Δψm) assay, reactive oxygen species (ROS) assay and western blot analysis. Colchicine was observed to induce a dose‑dependent reduction in cell viability in HT‑29 cells and early apoptosis occurred when the cells were treated with 1 µg/ml colchicine. Furthermore, colchicine treatment induced a loss of Δψm, increased ROS production, activated caspase‑3, upregulated BAX expression and downregulated Bcl‑2 expression, which evidenced the colchicine activity on apoptosis, potentially by acting via the intrinsic apoptotic signaling pathway. Colchicine increased phosphorylation of p38, although not phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, which indicates that colchicine activates the p38 signaling pathway in order to induce cell apoptosis. Therefore, colchicine exhibited significant growth inhibition of the HT‑29 colon cancer cell line and induced apoptosis in the cells via the mitochondrial pathway, which is regulated by p38 signaling pathways. PMID:26299305

  5. Carnitine palmitoyltransferase 1A prevents fatty acid-induced adipocyte dysfunction through suppression of c-Jun N-terminal kinase.

    PubMed

    Gao, Xuefei; Li, Kuai; Hui, Xiaoyan; Kong, Xiangping; Sweeney, Gary; Wang, Yu; Xu, Aimin; Teng, Maikun; Liu, Pentao; Wu, Donghai

    2011-05-01

    The adipocyte is the principal cell type for fat storage. CPT1 (carnitine palmitoyltransferase-1) is the rate-limiting enzyme for fatty acid β-oxidation, but the physiological role of CPT1 in adipocytes remains unclear. In the present study, we focused on the specific role of CPT1A in the normal functioning of adipocytes. Three 3T3-L1 adipocyte cell lines stably expressing hCPT1A (human CPT1A) cDNA, mouse CPT1A shRNA (short-hairpin RNA) or GFP (green fluorescent protein) were generated and the biological functions of these cell lines were characterized. Alteration in CPT1 activity, either by ectopic overexpression or pharmacological inhibition using etomoxir, did not affect adipocyte differentiation. However, overexpression of hCPT1A significantly reduced the content of intracellular NEFAs (non-esterified fatty acids) compared with the control cells when adipocytes were challenged with fatty acids. The changes were accompanied by an increase in fatty acid uptake and a decrease in fatty acid release. Interestingly, CPT1A protected against fatty acid-induced insulin resistance and expression of pro-inflammatory adipokines such as TNF-α (tumour necrosis factor-α) and IL-6 (interleukin-6) in adipocytes. Further studies demonstrated that JNK (c-Jun N terminal kinase) activity was substantially suppressed upon CPT1A overexpression, whereas knockdown or pharmacological inhibition of CPT1 caused a significant enhancement of JNK activity. The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA- and etomoxir-mediated decrease in CPT1 activity. Moreover, C2C12 myocytes co-cultured with adipocytes pre-treated with fatty acids displayed altered insulin sensitivity. Taken together, our findings have identified a favourable role for CPT1A in adipocytes to attenuate fatty acid-evoked insulin resistance and inflammation via suppression of JNK. PMID:21348853

  6. c-Jun N-terminal kinase modulates oxidant stress and peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen hepatotoxicity

    SciTech Connect

    Saito, Chieko; Lemasters, John J.; Jaeschke, Hartmut

    2010-07-15

    Acetaminophen (APAP) overdose, which causes liver injury in animals and humans, activates c-jun N-terminal kinase (JNK). Although it was shown that the JNK inhibitor SP600125 effectively reduced APAP hepatotoxicity, the mechanisms of protection remain unclear. C57Bl/6 mice were treated with 10 mg/kg SP600125 or vehicle (8% dimethylsulfoxide) 1 h before 600 mg/kg APAP administration. APAP time-dependently induced JNK activation (detected by JNK phosphorylation). SP600125, but not the vehicle, reduced JNK activation, attenuated mitochondrial Bax translocation and prevented the mitochondrial release of apoptosis-inducing factor at 4-12 h. Nuclear DNA fragmentation, nitrotyrosine staining, tissue GSSG levels and liver injury (plasma ALT release and necrosis) were partially attenuated by the vehicle (- 65%) and completely eliminated by SP600125 (- 98%) at 6 and 12 h. Furthermore, SP600125 attenuated the increase of inducible nitric oxide synthase (iNOS) mRNA and protein. However, APAP did not enhance plasma nitrite + nitrate levels (NO formation); SP600125 had no effect on this parameter. The iNOS inhibitor L-NIL did not reduce NO formation or injury after APAP but prevented NO formation caused by endotoxin. Since SP600125 completely eliminated the increase in hepatic GSSG levels, an indicator of mitochondrial oxidant stress, it is concluded that the inhibition of peroxynitrite was mainly caused by reduced superoxide formation. Our data suggest that the JNK inhibitor SP600125 protects against APAP-induced liver injury in part by attenuation of mitochondrial Bax translocation but mainly by preventing mitochondrial oxidant stress and peroxynitrite formation and thereby preventing the mitochondrial permeability transition pore opening, a key event in APAP-induced cell necrosis.

  7. Low humidity environmental challenge causes barrier disruption and cornification of the mouse corneal epithelium via a c-jun N-terminal kinase 2 (JNK2) pathway.

    PubMed

    Pelegrino, F S A; Pflugfelder, S C; De Paiva, C S

    2012-01-01

    Patients with tear dysfunction often experience increased irritation symptoms when subjected to drafty and/or low humidity environmental conditions. The purpose of this study was to investigate the effects of low humidity stress (LHS) on corneal barrier function and expression of cornified envelope (CE) precursor proteins in the epithelium of C57BL/6 and c-jun N-terminal kinase 2 (JNK2) knockout (KO) mice. LHS was induced in both strains by exposure to an air draft for 15 (LHS15D) or 30 days (LHS30D) at a relative humidity <30%RH. Nonstressed (NS) mice were used as controls. Oregon-green-dextran uptake was used to measure corneal barrier function. Levels of small proline-rich protein (SPRR)-2, involucrin, occludin, and MMP-9 were evaluated by immunofluorescent staining in cornea sections. Wholemount corneas immunostained for occludin were used to measure mean apical cell area. Gelatinase activity was evaluated by in situ zymography. Expression of MMP, CE and inflammatory cytokine genes was evaluated by qPCR. C57BL/6 mice exposed to LHS15D showed corneal barrier dysfunction, decreased apical corneal epithelial cell area, higher MMP-9 expression and gelatinase activity and increased involucrin and SPRR-2 immunoreactivity in the corneal epithelium compared to NS mice. JNK2KO mice were resistant to LHS-induced corneal barrier disruption. MMP-3,-9,-13, IL-1α, IL-1β, involucrin and SPRR-2a RNA transcripts were significantly increased in C57BL/6 mice at LHS15D, while no change was noted in JNK2KO mice. LHS is capable of altering corneal barrier function, promoting pathologic alteration of the TJ complex and stimulating production of CE proteins by the corneal epithelium. Activation of the JNK2 signaling pathway contributes to corneal epithelial barrier disruption in LHS. PMID:22166618

  8. Glycogen synthase kinase 3β sustains invasion of glioblastoma via the focal adhesion kinase, Rac1, and c-Jun N-terminal kinase-mediated pathway.

    PubMed

    Chikano, Yuri; Domoto, Takahiro; Furuta, Takuya; Sabit, Hemragul; Kitano-Tamura, Ayako; Pyko, Ilya V; Takino, Takahisa; Sai, Yoshimichi; Hayashi, Yutaka; Sato, Hiroshi; Miyamoto, Ken-ichi; Nakada, Mitsutoshi; Minamoto, Toshinari

    2015-02-01

    The failure of current treatment options for glioblastoma stems from their inability to control tumor cell proliferation and invasion. Biologically targeted therapies offer great hope and one promising target is glycogen synthase kinase-3β (GSK3β), implicated in various diseases, including cancer. We previously reported that inhibition of GSK3β compromises the survival and proliferation of glioblastoma cells, induces their apoptosis, and sensitizes them to temozolomide and radiation. Here, we explore whether GSK3β also contributes to the highly invasive nature of glioblastoma. The effects of GSK3β inhibition on migration and invasion of glioblastoma cells were examined by wound-healing and Transwell assays, as well as in a mouse model of glioblastoma. We also investigated changes in cellular microarchitectures, cytoskeletal components, and proteins responsible for cell motility and invasion. Inhibition of GSK3β attenuated the migration and invasion of glioblastoma cells in vitro and that of tumor cells in a mouse model of glioblastoma. These effects were associated with suppression of the molecular axis involving focal adhesion kinase, guanine nucleotide exchange factors/Rac1 and c-Jun N-terminal kinase. Changes in cellular phenotypes responsible for cell motility and invasion were also observed, including decreased formation of lamellipodia and invadopodium-like microstructures and alterations in the subcellular localization, and activity of Rac1 and F-actin. These changes coincided with decreased expression of matrix metalloproteinases. Our results confirm the potential of GSK3β as an attractive therapeutic target against glioblastoma invasion, thus highlighting a second role in this tumor type in addition to its involvement in chemo- and radioresistance. PMID:25504636

  9. Lower susceptibility of female mice to acetaminophen hepatotoxicity: Role of mitochondrial glutathione, oxidant stress and c-jun N-terminal kinase

    SciTech Connect

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Jaeschke, Hartmut

    2014-11-15

    Acetaminophen (APAP) overdose causes severe hepatotoxicity in animals and humans. However, the mechanisms underlying the gender differences in susceptibility to APAP overdose in mice have not been clarified. In our study, APAP (300 mg/kg) caused severe liver injury in male mice but 69–77% lower injury in females. No gender difference in metabolic activation of APAP was found. Hepatic glutathione (GSH) was rapidly depleted in both genders, while GSH recovery in female mice was 2.6 fold higher in the mitochondria at 4 h, and 2.5 and 3.3 fold higher in the total liver at 4 h and 6 h, respectively. This faster recovery of GSH, which correlated with greater induction of glutamate-cysteine ligase, attenuated mitochondrial oxidative stress in female mice, as suggested by a lower GSSG/GSH ratio at 6 h (3.8% in males vs. 1.4% in females) and minimal centrilobular nitrotyrosine staining. While c-jun N-terminal kinase (JNK) activation was similar at 2 and 4 h post-APAP, it was 3.1 fold lower at 6 h in female mice. However, female mice were still protected by the JNK inhibitor SP600125. 17β-Estradiol pretreatment moderately decreased liver injury and oxidative stress in male mice without affecting GSH recovery. Conclusion: The lower susceptibility of female mice is achieved by the improved detoxification of reactive oxygen due to accelerated recovery of mitochondrial GSH levels, which attenuates late JNK activation and liver injury. However, even the reduced injury in female mice was still dependent on JNK. While 17β-estradiol partially protects male mice, it does not affect hepatic GSH recovery. - Highlights: • Female mice are less susceptible to acetaminophen overdose than males. • GSH depletion and protein adduct formation are similar in both genders. • Recovery of hepatic GSH levels is faster in females and correlates with Gclc. • Reduced oxidant stress in females leads to reduced JNK activation. • JNK activation and mitochondrial translocation are critical

  10. Protocatechuic aldehyde inhibits TNF-α-induced fibronectin expression in human umbilical vein endothelial cells via a c-Jun N-terminal kinase dependent pathway

    PubMed Central

    TONG, YUE-FENG; LIU, YONG; HU, ZHI-XING; LI, ZHE-CHENG; A, AGULA

    2016-01-01

    Fibronectin (FN) is one of the most important extracellular matrix proteins and plays an important role in the pathogenesis of atherosclerosis (AS). The aim of the present study was to evaluate the effect of a potent, water-soluble antioxidant, protocatechuic aldehyde (PA), which is derived from the Chinese herb Salvia miltiorrhiza, on the expression of FN in human umbilical vein endothelial cells (HUVECs) stimulated with tumor necrosis factor-α (TNF-α). The pharmacological effects of PA on the production of FN were investigated using ELISA and western blot analysis. In addition, ELISA and western blot analysis were used to examine the activation and suppression of the mitogen-activated protein kinase (MAPK) pathways and nuclear factor (NF)-κB in TNF-α-stimulated HUVECs, in order to explore the underlying pharmacological mechanism of PA. The inhibitory effect of PA on the total generation of reactive oxygen species (ROS) in TNF-α-stimulated HUVECs was assessed using 2′,7′-dichlorofluorescein diacetate. Pretreatment of HUVECs with PA (0.15, 0.45 and 1.35 mM) for 18 h markedly attenuated the TNF-α-stimulated FN surface expression and secretion in a dose-dependent manner. Intracellular ROS generation and the expression of extracellular signal-regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) were significantly induced by TNF-α (2 ng/ml) in HUVECs. TNF-α-induced ROS generation and JNK activation were inhibited by PA in a concentration-dependent manner. By contrast, ERK1/2 and p38 activation was not significantly affected by PA. Pretreatment of HUVECs with PA for 18 h markedly attenuated TNF-α-stimulated NF-κB activation. In conclusion, the present findings suggest that PA inhibits TNF-α-induced FN expression in HUVECs through a mechanism that involves ROS/JNK and NF-κB. PMID:26889254

  11. Ciliary neurotrophic factor prevents acute lipid-induced insulin resistance by attenuating ceramide accumulation and phosphorylation of c-Jun N-terminal kinase in peripheral tissues.

    PubMed

    Watt, Matthew J; Hevener, Andrea; Lancaster, Graeme I; Febbraio, Mark A

    2006-05-01

    Ciliary neurotrophic factor (CNTF) is a member of the gp130 receptor cytokine family recently identified as an antiobesity agent in rodents and humans by mechanisms that remain unclear. We investigated the impact of acute CNTF treatment on insulin action in the presence of lipid oversupply. To avoid confounding effects of long-term high-fat feeding or genetic manipulation on whole-body insulin sensitivity, we performed a 2-h Intralipid infusion (20% heparinized Intralipid) with or without recombinant CNTF pretreatment (Axokine 0.3 mg/kg), followed by a 2-h hyperinsulinemic-euglycemic clamp (12 mU/kg.min) in fasted, male Wistar rats. Acute Intralipid infusion increased plasma free fatty acid levels from 1.0 +/- 0.1 to 2.5 +/- 0.3 mM, which subsequently caused reductions in skeletal muscle (insulin-stimulated glucose disposal rate) and liver (hepatic glucose production) insulin sensitivity by 30 and 45%, respectively. CNTF pretreatment completely prevented the lipid-mediated reduction in insulin-stimulated glucose disposal rate and the blunted suppression of hepatic glucose production by insulin. Although lipid infusion increased triacylglycerol and ceramide accumulation and phosphorylation of mixed linage kinase 3 and c-Jun N-terminal kinase 1 in skeletal muscle, CNTF pretreatment prevented these lipid-induced effects. Alterations in hepatic and muscle insulin signal transduction as well as phosphorylation of c-Jun N-terminal kinase 1/2 paralleled alterations in insulin sensitivity. These data support the use of CNTF as a potential therapeutic means to combat lipid-induced insulin resistance. PMID:16396984

  12. Ginkgo biloba Extract Individually Inhibits JNK Activation and Induces c-Jun Degradation in Human Chondrocytes: Potential Therapeutics for Osteoarthritis

    PubMed Central

    Ho, Ling-Jun; Hung, Li-Feng; Liu, Feng-Cheng; Hou, Tsung-Yun; Lin, Leou-Chyr; Huang, Chuan-Yueh; Lai, Jenn-Haung

    2013-01-01

    Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation. The ideal anti-OA drug should have immunomodulatory effects while at the same time having limited or no toxicity. We examined the anti-inflammatory effects of Ginkgo biloba extract (EGb) in interleukin-1 (IL-1)-stimulated human chondrocytes. Chondrocytes were prepared from cartilage specimens taken from patients with osteoarthritis who had received total hip or total knee replacement. The concentrations of chemokines and the degree of cell migration were determined by ELISA and chemotaxis assays, respectively. The activation of inducible nitric oxide synthase (iNOS), mitogen-activated protein kinases (MAPKs), activator protein-1 (AP-1), and nuclear factor-kappaB (NF-κB) was determined by immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay. We found that EGb inhibited IL-1-induced production of chemokines, which in turn resulted in attenuation of THP-1 cell migration toward EGb-treated cell culture medium. EGb also suppressed IL-1-stimulated iNOS expression and release of nitric oxide (NO). The EGb-mediated suppression of the iNOS-NO pathway correlated with the attenuation of activator protein-1 (AP-1) but not nuclear factor-kappaB (NF-κB) DNA-binding activity. Of the mitogen-activated protein kinases (MAPKs), EGb inhibited only c-Jun N-terminal kinase (JNK). Unexpectedly, EGb selectively caused degradation of c-Jun protein. Further investigation revealed that EGb-mediated c-Jun degradation was preceded by ubiquitination of c-Jun and could be prevented by the proteosome inhibitor MG-132. The results imply that EGb protects against chondrocyte degeneration by inhibiting JNK activation and inducing ubiquitination-dependent c-Jun degradation. Although additional research is needed, our results suggest that EGb is a potential therapeutic agent for the treatment of OA. PMID:24349175

  13. Inhibition of c-Jun N-terminal Kinase Signaling Pathway Alleviates Lipopolysaccharide-induced Acute Respiratory Distress Syndrome in Rats

    PubMed Central

    Lai, Jian-Bo; Qiu, Chun-Fang; Chen, Chuan-Xi; Chen, Min-Ying; Chen, Juan; Guan, Xiang-Dong; Ouyang, Bin

    2016-01-01

    Background: An acute respiratory distress syndrome (ARDS) is still one of the major challenges in critically ill patients. This study aimed to investigate the effect of inhibiting c-Jun N-terminal kinase (JNK) on ARDS in a lipopolysaccharide (LPS)-induced ARDS rat model. Methods: Thirty-six rats were randomized into three groups: control, LPS, and LPS + JNK inhibitor. Rats were sacrificed 8 h after LPS treatment. The lung edema was observed by measuring the wet-to-dry weight (W/D) ratio of the lung. The severity of pulmonary inflammation was observed by measuring myeloperoxidase (MPO) activity of lung tissue. Moreover, the neutrophils in bronchoalveolar lavage fluid (BALF) were counted to observe the airway inflammation. In addition, lung collagen accumulation was quantified by Sircol Collagen Assay. At the same time, the pulmonary histologic examination was performed, and lung injury score was achieved in all three groups. Results: MPO activity in lung tissue was found increased in rats treated with LPS comparing with that in control (1.26 ± 0.15 U in LPS vs. 0.77 ± 0.27 U in control, P < 0.05). Inhibiting JNK attenuated LPS-induced MPO activity upregulation (0.52 ± 0.12 U in LPS + JNK inhibitor vs. 1.26 ± 0.15 U in LPS, P < 0.05). Neutrophils in BALF were also found to be increased with LPS treatment, and inhibiting JNK attenuated LPS-induced neutrophils increase in BALF (255.0 ± 164.4 in LPS vs. 53 (44.5-103) in control vs. 127.0 ± 44.3 in LPS + JNK inhibitor, P < 0.05). At the same time, the lung injury score showed a reduction in LPS + JNK inhibitor group comparing with that in LPS group (13.42 ± 4.82 vs. 7.00 ± 1.83, P = 0.001). However, the lung W/D ratio and the collagen in BALF did not show any differences between LPS and LPS + JNK inhibitor group. Conclusions: Inhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis. JNK inhibitor might be a potential therapeutic medication in ARDS, in the

  14. c-Jun controls histone modifications, NF-kappaB recruitment, and RNA polymerase II function to activate the ccl2 gene.

    PubMed

    Wolter, Sabine; Doerrie, Anneke; Weber, Axel; Schneider, Heike; Hoffmann, Elke; von der Ohe, Juliane; Bakiri, Latifa; Wagner, Erwin F; Resch, Klaus; Kracht, Michael

    2008-07-01

    Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5' region close to the transcriptional start site and in the far 3' region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-kappaB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-kappaB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-kappaB and HDAC3 at the level of chromatin. PMID:18443042

  15. c-Jun Controls Histone Modifications, NF-κB Recruitment, and RNA Polymerase II Function To Activate the ccl2 Gene▿ †

    PubMed Central

    Wolter, Sabine; Doerrie, Anneke; Weber, Axel; Schneider, Heike; Hoffmann, Elke; von der Ohe, Juliane; Bakiri, Latifa; Wagner, Erwin F.; Resch, Klaus; Kracht, Michael

    2008-01-01

    Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5′ region close to the transcriptional start site and in the far 3′ region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-κB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-κB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-κB and HDAC3 at the level of chromatin. PMID:18443042

  16. Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE) and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK) Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

    PubMed Central

    Zhang, Zhengyu; Sethiel, Mosha Silas; Shen, Weizhi; Liao, Sentai; Zou, Yuxiao

    2013-01-01

    Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs) in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK) activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes. PMID:24252909

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

    PubMed

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

    2003-06-13

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

  18. Glycyrrhetinic acid induces cytoprotective autophagy via the inositol-requiring enzyme 1α-c-Jun N-terminal kinase cascade in non-small cell lung cancer cells

    PubMed Central

    Tang, Zheng-Hai; Zhang, Le-Le; Li, Ting; Lu, Jia-Hong; Ma, Dik-Lung; Leung, Chung-Hang; Chen, Xiu-Ping; Jiang, Hu-Lin; Wang, Yi-Tao; Lu, Jin-Jian

    2015-01-01

    Glycerrhetinic acid (GA), one of the main bioactive constituents of Glycyrrhiza uralensis Fisch, exerts anti-cancer effects on various cancer cells. We confirmed that GA inhibited cell proliferation and induced apoptosis in non-small cell lung cancer A549 and NCI-H1299 cells. GA also induced expression of autophagy marker phosphatidylethanolamine-modified microtubule-associated protein light-chain 3 (LC3-II) and punta formation of green fluorescent protein microtubule-associated protein light-chain 3. We further proved that expression of GA-increased autophagy marker was attributed to activation instead of suppression of autophagic flux. The c-jun N-terminal kinase (JNK) pathway was activated after incubation with GA. Pretreatment with the JNK inhibitor SP600125 or silencing of the JNK pathway by siRNA of JNK or c-jun decreased GA-induced autophagy. The endoplasmic reticulum (ER) stress responses were also apparently stimulated by GA by triggering the inositol-requiring enzyme 1α (IRE1α) pathway. The GA-induced JNK pathway activation and autophagy were decreased by IRE1α knockdown, and inhibition of autophagy or the JNK cascade increased GA-stimulated IRE1α expression. In addition, GA-induced cell proliferative inhibition and apoptosis were increased by inhibition of autophagy or the JNK pathway. Our study was the first to demonstrate that GA induces cytoprotective autophagy in non-small cell lung cancer cells by activating the IRE1α-JNK/c-jun pathway. The combined treatment of autophagy inhibitors markedly enhances the anti-neoplasmic activity of GA. Such combination shows potential as a strategy for GA or GA-contained prescriptions in cancer therapy. PMID:26549806

  19. c-Jun N-terminal kinase 1 (JNK1) is required for coordination of netrin signaling in axon guidance.

    PubMed

    Qu, Chao; Li, Weiquan; Shao, Qiangqiang; Dwyer, Trisha; Huang, Huai; Yang, Tao; Liu, Guofa

    2013-01-18

    The JNK family of MAPKs is involved in a large variety of physiological and pathological processes in brain development, such as neural survival, migration, and polarity as well as axon regeneration. However, whether JNK activation is involved in axon guidance remains unknown. Here, we provide evidence indicating the JNK pathway is required for Netrin signaling in the developing nervous system. Netrin-1 increased JNK1, not JNK2 or JNK3, activity in the presence of deleted in colorectal cancer (DCC) or Down syndrome cell adhesion molecule (DSCAM), and expression of both of them further enhanced Netrin-1-induced JNK1 activity in vitro. Inhibition of JNK signaling either by a JNK inhibitor, SP600125, or expression of a dominant negative form of MKK4, a JNK upstream activator, blocked Netrin-1-induced JNK1 activation in HEK293 cells. Netrin-1 increased endogenous JNK activity in primary neurons. Netrin-1-induced JNK activation was inhibited either by the JNK inhibitor or an anti-DCC function-blocking antibody. Combination of the anti-DCC function-blocking antibody with expression of DSCAM shRNA in primary neurons totally abolished Netrin-1-induced JNK activation, whereas knockdown of DSCAM partially inhibited the Netrin-1 effect. In the developing spinal cord, phospho-JNK was strongly expressed in commissural axons before and as they crossed the floor plate, and Netrin-1 stimulation dramatically increased the level of endogenous phospho-JNK in commissural axon growth cones. Inhibition of JNK signaling either by JNK1 RNA interference (RNAi) or the JNK inhibitor suppressed Netrin-1-induced neurite outgrowth and axon attraction. Knockdown of JNK1 in ovo caused defects in spinal cord commissural axon projection and pathfinding. Our study reveals that JNK1 is important in the coordination of DCC and DSCAM in Netrin-mediated attractive signaling. PMID:23223444

  20. Loss of c-Jun N-terminal kinase-interacting protein-1 does not affect axonal transport of the amyloid precursor protein or Aβ production

    PubMed Central

    Vagnoni, Alessio; Glennon, Elizabeth B.C.; Perkinton, Michael S.; Gray, Emma H.; Noble, Wendy; Miller, Christopher C.J.

    2013-01-01

    Disruption to axonal transport is an early pathological feature in Alzheimer's disease. The amyloid precursor protein (APP) is a key axonal transport cargo in Alzheimer's disease since perturbation of its transport increases APP processing and production of amyloid-β peptide (Aβ) that is deposited in the brains of Alzheimer's disease patients. APP is transported anterogradely through axons on kinesin-1 motors. One favoured route for attachment of APP to kinesin-1 involves the scaffolding protein c-Jun N-terminal kinase-interacting protein-1 (JIP1), which has been shown to bind both APP and kinesin-1 light chain (KLC). However, direct experimental evidence to support a role of JIP1 in APP transport is lacking. Notably, the effect of loss of JIP1 on movement of APP through axons of living neurons, and the impact of such loss on APP processing and Aβ production has not been reported. To address these issues, we monitored how siRNA mediated loss of JIP1 influenced transport of enhanced green fluorescent protein (EGFP)-tagged APP through axons and production of endogenous Aβ in living neurons. Surprisingly, we found that knockdown of JIP1 did not affect either APP transport or Aβ production. These results have important implications for our understanding of APP trafficking in Alzheimer's disease. PMID:23825109

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

    SciTech Connect

    Gao Zhanguo; Ye Jianping

    2008-11-28

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

  2. A novel dual NO-donating oxime and c-Jun N-terminal kinase inhibitor protects against cerebral ischemia-reperfusion injury in mice.

    PubMed

    Atochin, Dmitriy N; Schepetkin, Igor A; Khlebnikov, Andrei I; Seledtsov, Victor I; Swanson, Helen; Quinn, Mark T; Huang, Paul L

    2016-04-01

    The c-Jun N-terminal kinase (JNK) has been shown to be an important regulator of neuronal cell death. Previously, we synthesized the sodium salt of 11H-indeno[1,2-b]quinoxalin-11-one (IQ-1S) and demonstrated that it was a high-affinity inhibitor of the JNK family. In the present work, we found that IQ-1S could release nitric oxide (NO) during its enzymatic metabolism by liver microsomes. Moreover, serum nitrite/nitrate concentration in mice increased after intraperitoneal injection of IQ-1S. Because of these dual actions as JNK inhibitor and NO-donor, the therapeutic potential of IQ-1S was evaluated in an animal stroke model. We subjected wild-type C57BL6 mice to focal ischemia (30min) with subsequent reperfusion (48h). Mice were treated with IQ-1S (25mg/kg) suspended in 10% solutol or with vehicle alone 30min before and 24h after middle cerebral artery (MCA) occlusion (MCAO). Using laser-Doppler flowmetry, we monitored cerebral blood flow (CBF) above the MCA during 30min of MCAO provoked by a filament and during the first 30min of subsequent reperfusion. In mice treated with IQ-1S, ischemic and reperfusion values of CBF were not different from vehicle-treated mice. However, IQ-1S treated mice demonstrated markedly reduced neurological deficit and infarct volumes as compared with vehicle-treated mice after 48h of reperfusion. Our results indicate that the novel JNK inhibitor releases NO during its oxidoreductive bioconversion and improves stroke outcome in a mouse model of cerebral reperfusion. We conclude that IQ-1S is a promising dual functional agent for the treatment of cerebral ischemia and reperfusion injury. PMID:26923672

  3. Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor

    PubMed Central

    Schepetkin, Igor A.; Kirpotina, Liliya N.; Hammaker, Deepa; Kochetkova, Irina; Khlebnikov, Andrei I.; Lyakhov, Sergey A.; Firestein, Gary S.

    2015-01-01

    c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S–treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II–specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II–specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3+CD4+CD25+ regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis. PMID:25784649

  4. Anti-Inflammatory Effects and Joint Protection in Collagen-Induced Arthritis after Treatment with IQ-1S, a Selective c-Jun N-Terminal Kinase Inhibitor.

    PubMed

    Schepetkin, Igor A; Kirpotina, Liliya N; Hammaker, Deepa; Kochetkova, Irina; Khlebnikov, Andrei I; Lyakhov, Sergey A; Firestein, Gary S; Quinn, Mark T

    2015-06-01

    c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1β in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S-treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II-specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II-specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3(+)CD4(+)CD25(+) regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis. PMID:25784649

  5. Activation of c-Jun predicts a poor response to sorafenib in hepatocellular carcinoma: Preliminary Clinical Evidence

    PubMed Central

    Chen, Wei; Xiao, Weikai; Zhang, Kunsong; Yin, Xiaoyu; Lai, Jiaming; Liang, Lijian; Chen, Dong

    2016-01-01

    We determined the mitogen-activated protein kinase (MAPK) gene expression profile of acquired resistance in sorafenib-sensitive hepatocellular carcinoma (HCC) cells and aimed to identify c-Jun as an important molecule mediating the efficacy of sorafenib. Differences in gene expression of the MAPK signaling between untreated and sorafenib-treated HCC cell lines were investigated using real-time polymerase chain reaction array. Western blot and real-time PCR further evaluated the expression of c-Jun. Pathological specimens from 50 patients with advanced HCC were collected to measure p-c-Jun expression. Sorafenib-resistant HCC cells demonstrated greater levels of basal c-Jun mRNA and protein compared with sorafenib-sensitive HCC cells. Sorafenib activated p-c-Jun in a dose- and time-dependent manner in PLC/PRF/5 and MHCC97H cell lines. Decreased expression levels of 6 genes after sorafenib treatment suggested a robust inhibitory impact of sorafenib on MAPK signaling in HCC cells. c-Jun and p-c-Jun expression levels were inversely correlated with the efficacy of sorafenib; a high expression level of p-c-Jun was associated with resistance to sorafenib and poor overall survival in patients with clinical HCC. p-c-Jun may act as a biomarker for predicting responses of sorafenib treatment, thus advocating targeting of JNK/c-Jun signaling as an optimal therapeutic strategy in a subset of HCC. PMID:26964667

  6. c-Jun activation in Schwann cells protects against loss of sensory axons in inherited neuropathy

    PubMed Central

    Hantke, Janina; Carty, Lucy; Wagstaff, Laura J.; Turmaine, Mark; Wilton, Daniel K.; Quintes, Susanne; Koltzenburg, Martin; Baas, Frank; Mirsky, Rhona

    2014-01-01

    Charcot–Marie–Tooth disease type 1A is the most frequent inherited peripheral neuropathy. It is generally due to heterozygous inheritance of a partial chromosomal duplication resulting in over-expression of PMP22. A key feature of Charcot–Marie–Tooth disease type 1A is secondary death of axons. Prevention of axonal loss is therefore an important target of clinical intervention. We have previously identified a signalling mechanism that promotes axon survival and prevents neuron death in mechanically injured peripheral nerves. This work suggested that Schwann cells respond to injury by activating/enhancing trophic support for axons through a mechanism that depends on upregulation of the transcription factor c-Jun in Schwann cells, resulting in the sparing of axons that would otherwise die. As c-Jun orchestrates Schwann cell support for distressed neurons after mechanical injury, we have now asked: do Schwann cells also activate a c-Jun dependent neuron-supportive programme in inherited demyelinating disease? We tested this by using the C3 mouse model of Charcot–Marie–Tooth disease type 1A. In line with our previous findings in humans with Charcot–Marie–Tooth disease type 1A, we found that Schwann cell c-Jun was elevated in (uninjured) nerves of C3 mice. We determined the impact of this c-Jun activation by comparing C3 mice with double mutant mice, namely C3 mice in which c-Jun had been conditionally inactivated in Schwann cells (C3/Schwann cell-c-Jun−/− mice), using sensory-motor tests and electrophysiological measurements, and by counting axons in proximal and distal nerves. The results indicate that c-Jun elevation in the Schwann cells of C3 nerves serves to prevent loss of myelinated sensory axons, particularly in distal nerves, improve behavioural symptoms, and preserve F-wave persistence. This suggests that Schwann cells have two contrasting functions in Charcot–Marie–Tooth disease type 1A: on the one hand they are the genetic source of

  7. The mitochondria of stallion spermatozoa are more sensitive than the plasmalemma to osmotic-induced stress: role of c-Jun N-terminal kinase (JNK) pathway.

    PubMed

    García, Beatriz Macías; Moran, Alvaro Miró; Fernández, Lauro González; Ferrusola, Cristina Ortega; Rodriguez, Antolin Morillo; Bolaños, Juan Maria Gallardo; da Silva, Carolina Maria Balao; Martínez, Heriberto Rodríguez; Tapia, Jose A; Peña, Fernando J

    2012-01-01

    Cryopreservation introduces extreme temperature and osmolality changes that impart lethal and sublethal effects on spermatozoa. Additionally, there is evidence that the osmotic stress induced by cryopreservation causes oxidative stress to spermatozoa. The main sources of reactive oxygen species in mammalian sperm are the mitochondria. In view of this, the aim of our study was to test whether or not osmotic stress was able to induce mitochondrial damage and to explore the osmotic tolerance of the mitochondria of stallion spermatozoa. Ejaculates from 7 stallions were subjected to osmolalities ranging from 75 to 1500 mOsm/kg, and the effect on sperm membrane integrity and mitochondrial membrane potential was studied. Additionally, the effects of changes in osmolality from hyposmotic to isosmotic and from hyperosmotic to isosmotic solutions were studied (osmotic excursions). The cellular volume of stallion spermatozoa under isosmotic conditions was 20.4 ± 0.33 μm(3). When exposed to low osmolality, the stallion spermatozoa behaved like a linear osmometer, whereas exposure to high osmolalities up to 900 mOsm/kg resulted in decreased sperm volume. Although sperm membranes were relatively resistant to changes in osmolality, mitochondrial membrane potential decreased when osmolalities were low or very high (10.7 ± 1.74 and 16.5 ± 1.70 at 75 and 150 mOsm/kg, respectively, and 13.1 ± 1.83 at 1500 mOsm/kg), whereas in isosmolar controls the percentage of stallion sperm mitochondria with a high membrane potential was 41.1 ± 1.69 (P < .01). Osmotic excursions induced greater damage than exposure of spermatozoa to a given nonphysiologic osmolality, and again the mitochondria were more prone to damage induced by osmotic excursions than was the sperm plasma membrane. In search of intracellular components that could mediate these changes, we have detected for the first time the c-Jun N-terminal kinase 1/2 in stallion spermatozoa, which are apparently involved in the

  8. c-Jun induces apoptosis of starved BM2 monoblasts by activating cyclin A-CDK2

    SciTech Connect

    Vanhara, Petr; Bryja, Vitezslav; Horvath, Viktor; Kozubik, Alois; Hampl, Ales; Smarda, Jan . E-mail: smarda@sci.muni.cz

    2007-02-02

    c-Jun is one of the major components of the activating protein-1 (AP-1), the transcription factor that participates in regulation of proliferation, differentiation, and apoptosis. In this study, we explored functional interactions of the c-Jun protein with several regulators of the G1/S transition in serum-deprived v-myb-transformed chicken monoblasts BM2. We show that the c-Jun protein induces expression of cyclin A, thus up-regulating activity of cyclin A-associated cyclin-dependent kinase 2 (CDK2), and causing massive programmed cell death of starved BM2cJUN cells. Specific inhibition of CDK2 suppresses frequency of apoptosis of BM2cJUN cells. We conclude that up-regulation of cyclin A expression and CDK2 activity can represent important link between the c-Jun protein, cell cycle machinery, and programmed cell death pathway in leukemic cells.

  9. Mouse dead end 1-β interacts with c-Jun and stimulates activator protein 1 transactivation

    PubMed Central

    ZHANG, YONG; SU, YAN-LIN; LI, LE-SAI; YANG, ZHI; CHEN, SI; XIONG, JIE; FU, XIAO-HUA; PENG, XIAO-NING

    2015-01-01

    Dead end 1 (DND1), important for maintaining the viability of primordial germ cells, is the first protein containing an RNA recognition motif that has been directly implicated as a heritable cause of spontaneous tumorigenesis. In the present study, c-Jun was identified through yeast two-hybrid screening of a 10.5-day old mouse embryo cDNA library as one of the proteins which interact with DND1-β. The interaction between DND1-β and c-Jun was demonstrated to occur by glutathione S-transferase pull-down and co-immunoprecipitation. Using confocal microscopy, DND1-β was found to be specifically expressed in GC-1 spermatogonia cells, mainly in the nuclei. When transfected into GC-1 cells, DND1-β and c-Jun were demonstrated to be co-localized principally in the nuclei. Furthermore, in a dual luciferase reporter assay, the transcriptional activity of activator protein 1 was demonstrated to be significantly increased by co-transfection with DND1-β and c-Jun plasmids in GC-1 cells. The identification and confirmation of an additional protein interacting with DND1-β facilitates the investigation of the functions and molecular mechanisms of DND1. PMID:25405725

  10. Niclosamide enhances ROS-mediated cell death through c-Jun activation.

    PubMed

    Lee, Sae-lo-oom; Son, A-Rang; Ahn, Jiyeon; Song, Jie-Young

    2014-06-01

    Radiotherapy is an effective treatment modality in the clinical treatment of cancers, and has been combined with chemotherapy in order to improve therapeutic efficacy. Therefore, we aimed to develop small molecules that enhance the cytotoxic effects of radiotherapy. In this study, we provide evidence that niclosamide is an effective radiosensitizer in non-small cell lung cancer cells. Using a cell-based high-throughput viability screen of 1040 compounds in combination with γ-ionizing radiation (IR), we found niclosamide, an FDA-approved antihelminthic agent, had a radiosensitizing effect on H1299 human lung cancer cells. Pretreatment with niclosamide enhanced IR- induced cell death of H1299 in a dose-dependent manner via apoptosis compared with IR or niclosamide alone. The combined treatment induced significantly more phosphorylation of p38 MAPK and c-Jun in H1299 cells than IR or niclosamide alone. Since IR induces apoptosis through generation of reactive oxygen species (ROS), hydrogen peroxide (H2O2) was employed as another ROS generator and we found that niclosamide also sensitized cells to H2O2. Niclosamide pretreatment also induced c-Jun and its phosphorylation in the presence of H2O2, thereby enhancing apoptosis. N-acetyl-L-cysteine (NAC) treatment abolished both cell death and c-Jun activation induced by the combination treatments. Knockdown of c-Jun also decreased PARP cleavage and clonogenic cell survival in niclosamide- and IR-treated H1299 cells. Our findings suggest that niclosamide could be a promising radiosensitizer in lung cancer patients through activation of the p38 MAPK-c-Jun axis. PMID:24750999

  11. Cucurbitacin-I (JSI-124) activates the JNK/c-Jun signaling pathway independent of apoptosis and cell cycle arrest in B Leukemic Cells

    PubMed Central

    2011-01-01

    Background Cucurbitacin-I (JSI-124) is potent inhibitor of JAK/STAT3 signaling pathway and has anti-tumor activity in a variety of cancer including B cell leukemia. However, other molecular targets of JSI-124 beyond the JAK/STAT3 pathway are not fully understood. Methods BJAB, I-83, NALM-6 and primary CLL cells were treated with JSI-124 as indicated. Apoptosis was measured using flow cytometry for accumulation of sub-G1 phase cells (indicator of apoptosis) and Annexin V/PI staining. Cell cycle was analyzed by FACS for DNA content of G1 and G2 phases. Changes in phosphorylation and protein expression of p38, Erk1/2, JNK, c-Jun, and XIAP were detected by Western blot analysis. STAT3 and c-Jun genes were knocked out using siRNA transfection. VEGF expression was determined by mRNA and protein levels by RT-PCR and western blotting. Streptavidin Pull-Down Assay was used to determine c-Jun binding to the AP-1 DNA binding site. Results Herein, we show that JSI-124 activates c-Jun N-terminal kinase (JNK) and increases both the expression and serine phosphorylation of c-Jun protein in the B leukemic cell lines BJAB, I-83 and NALM-6. JSI-124 also activated MAPK p38 and MAPK Erk1/2 albeit at lower levels than JNK activation. Inhibition of the JNK signaling pathway failed to effect cell cycle arrest or apoptosis induced by JSI-124 but repressed JSI-124 induced c-Jun expression in these leukemia cells. The JNK pathway activation c-Jun leads to transcriptional activation of many genes. Treatment of BJAB, I-83, and NALM-6 cells with JSI-124 lead to an increase of Vascular Endothelial Growth Factor (VEGF) at both the mRNA and protein level. Knockdown of c-Jun expression and inhibition of JNK activation significantly blocked JSI-124 induced VEGF expression. Pretreatment with recombinant VEGF reduced JSI-124 induced apoptosis. Conclusions Taken together, our data demonstrates that JSI-124 activates the JNK signaling pathway independent of apoptosis and cell cycle arrest, leading to

  12. Design and characterization of a potent and selective dual ATP- and substrate-competitive sub-nanomolar bi-dentate c-Jun N-terminal Kinase (JNK) inhibitor

    PubMed Central

    Stebbins, John L.; De, Surya K.; Pavlickova, Petra; Chen, Vida; Machleidt, Thomas; Chen, Li-Hsing; Kuntzen, Christian; Kitada, Shinichi; Karin, Michael; Pellecchia, Maurizio

    2011-01-01

    c-Jun N-terminal Kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bi-dentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bi-dentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC50 = 18 nM; Ki = 1.5 nM) and JNK/substrate association in a displacement assay with a substrate peptide (compound 20; IC50 = 46 nM; Ki = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 not only inhibits JNK in a variety of cell-based experiments, but it elicits also in vivo activity where it is shown to improve glucose tolerance in diabetic mice. PMID:21815634

  13. c-Jun N-terminal Kinase-Dependent Endoplasmic Reticulum Stress Pathway is Critically Involved in Arjunic Acid Induced Apoptosis in Non-Small Cell Lung Cancer Cells.

    PubMed

    Joo, HyeEun; Lee, Hyun Joo; Shin, Eun Ah; Kim, Hangil; Seo, Kyeong-Hwa; Baek, Nam-In; Kim, Bonglee; Kim, Sung-Hoon

    2016-04-01

    Though arjunic acid, a triterpene isolated from Terminalia arjuna, was known to have antioxidant, antiinflammatory, and cytotoxic effects, its underlying antitumor mechanism still remains unclear so far. Thus, in the present study, the molecular antitumor mechanism of arjunic acid was examined in A549 and H460 non-small cell lung cancer (NSCLC) cells. Arjunic acid exerted cytotoxicity by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) assay and significantly increased sub-G1 population in A549 and H460 cells by cell cycle analysis. Consistently, arjunic acid cleaved poly (ADP-ribose) polymerase (PARP), activated Bax, and phosphorylation of c-Jun N-terminal kinases (JNK), and also attenuated the expression of pro-caspase-3 and Bcl-2 in A549 and H460 cells. Furthermore, arjunic acid upregulated the expression of endoplasmic reticulum (ER) stress proteins such as IRE1 α, ATF4, p-eIF2α, and C/EBP homologous protein (CHOP) in A549 and H460 cells. Conversely, CHOP depletion attenuated the increase of sub-G1 population by arjunic acid, and also JNK inhibitor SP600125 blocked the cytotoxicity and upregulation of IRE1 α and CHOP induced by arjunic acid in A549 and H460 cells. Overall, our findings suggest that arjunic acid induces apoptosis in NSCLC cells via JNK mediated ER stress pathway as a potent chemotherapeutic agent for NSCLC. PMID:26787261

  14. Inhibition of c-Jun N-terminal kinase sensitizes tumor cells to flavonoid-induced apoptosis through down-regulation of JunD

    SciTech Connect

    Kook, Sung-Ho; Son, Young-Ok; Jang, Yong-Suk; Lee, Kyung-Yeol; Lee, Seung-Ah; Kim, Beom-Soo; Lee, Hyun-Jeong; Lee, Jeong-Chae

    2008-03-15

    Reduction of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun NH{sub 2}-terminal kinase (JNK) has been implicated in stress-induced apoptosis. However, many studies also emphasize the role of JNK on cell survival, although its mechanisms are not completely understood. Previously, we found that inhibition of JNK activity promotes flavonoid-mediated apoptosis of human osteosarcoma cells. We thus determined whether inhibition of JNK sensitizes tumor cells to a bioflavonoid-induced apoptosis, and whether this effect of JNK is a general effect. As the results, quercetin and genistein as well as a flavonoid fraction induced apoptosis of tumor cells, which was further accelerated by specific JNK inhibitor, SP600125 or by small interfering RNA specific to JNK1/2. This effect was specific to types of cells because it was further apparent in tumorigenic cell lines. Inhibition of JNK by SP600125 also reduced flavonoid-stimulated nuclear induction of JunD which was known to have protective role in apoptosis, whereas JNK inhibition alone had little effect on apoptosis. The flavonoid-induced apoptosis of tumor cells was significantly enhanced by transfecting them with antisense JunD oligonucleotides. These results suggest that inhibition of JNK facilitates flavonoid-induced apoptosis through down-regulation of JunD, which is further sensitive to tumor cells. Therefore, combination with a specific JNK inhibitor further enhances the anti-cancer and chemopreventive potential of bio-flavonoids.

  15. Propensity for HBZ-SP1 isoform of HTLV-I to inhibit c-Jun activity correlates with sequestration of c-Jun into nuclear bodies rather than inhibition of its DNA-binding activity

    SciTech Connect

    Clerc, Isabelle; Hivin, Patrick; Rubbo, Pierre-Alain; Lemasson, Isabelle; Barbeau, Benoit; Mesnard, Jean-Michel

    2009-09-01

    HTLV-I bZIP factor (HBZ) contains a C-terminal zipper domain involved in its interaction with c-Jun. This interaction leads to a reduction of c-Jun DNA-binding activity and prevents the protein from activating transcription of AP-1-dependent promoters. However, it remained unclear whether the negative effect of HBZ-SP1 was due to its weak DNA-binding activity or to its capacity to target cellular factors to transcriptionally-inactive nuclear bodies. To answer this question, we produced a mutant in which specific residues present in the modulatory and DNA-binding domain of HBZ-SP1 were substituted for the corresponding c-Fos amino acids to improve the DNA-binding activity of the c-Jun/HBZ-SP1 heterodimer. The stability of the mutant, its interaction with c-Jun, DNA-binding activity of the resulting heterodimer, and its effect on the c-Jun activity were tested. In conclusion, we demonstrate that the repression of c-Jun activity in vivo is mainly due to the HBZ-SP1-mediated sequestration of c-Jun to the HBZ-NBs.

  16. Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury.

    PubMed

    Yoshida, Katsunori; Matsuzaki, Koichi; Mori, Shigeo; Tahashi, Yoshiya; Yamagata, Hideo; Furukawa, Fukiko; Seki, Toshihito; Nishizawa, Mikio; Fujisawa, Junichi; Okazaki, Kazuichi

    2005-04-01

    After liver injury, transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) regulate the activation of hepatic stellate cells (HSCs) and tissue remodeling. Mechanisms of PDGF signaling in the TGF-beta-triggered cascade are not completely understood. TGF-beta signaling involves phosphorylation of Smad2 and Smad3 at linker and C-terminal regions. Using antibodies to distinguish Smad2/3 phosphorylated at linker regions from those phosphorylated at C-terminal regions, we investigated Smad2/3-mediated signaling in rat liver injured by CCl(4) administration and in cultured HSCs. In acute liver injury, Smad2/3 were transiently phosphorylated at both regions. Although linker-phosphorylated Smad2 remained in the cytoplasm of alpha-smooth muscle actin-immunoreactive mesenchymal cells adjacent to necrotic hepatocytes in centrilobular areas, linker-phosphorylated Smad3 accumulated in the nuclei. c-Jun N-terminal kinase (JNK) in the activated HSCs directly phosphorylated Smad2/3 at linker regions. Co-treatment of primary cultured HSCs with TGF-beta and PDGF activated the JNK pathway, subsequently inducing endogenous linker phosphorylation of Smad2/3. The JNK pathway may be involved in migration of resident HSCs within the space of Disse to the sites of tissue damage because the JNK inhibitor SP600125 inhibited HSC migration induced by TGF-beta and PDGF signals. Moreover, treatment of HSCs with both TGF-beta and PDGF increased transcriptional activity of plasminogen activator inhibitor-1 through linker phosphorylation of Smad3. In conclusion, TGF-beta and PDGF activate HSCs by transmitting their signals through JNK-mediated Smad2/3 phosphorylation at linker regions, both in vivo and in vitro. PMID:15793284

  17. Ghrelin protects alveolar macrophages against lipopolysaccharide-induced apoptosis through growth hormone secretagogue receptor 1a-dependent c-Jun N-terminal kinase and Wnt/β-catenin signaling and suppresses lung inflammation.

    PubMed

    Li, Bin; Zeng, Mian; He, Wanmei; Huang, Xubin; Luo, Liang; Zhang, Hongwu; Deng, David Y B

    2015-01-01

    Alveolar macrophages (AMs) undergo increased apoptosis during sepsis-induced acute respiratory distress syndrome (ARDS). Ghrelin exhibits an antiapoptotic effect in several cell types and protects against sepsis-induced ARDS in rats; however, the molecular mechanisms underlying this antiapoptotic effect remain poorly understood. In this study, we first examined the antiapoptotic effect of ghrelin on lipopolysaccharide (LPS)-stimulated AMs in vitro. In AMs, GH secretagogue receptor-1a (GHSR-1a), the ghrelin receptor, was expressed, and treatment of AMs with ghrelin markedly reduced LPS-induced apoptosis, mitochondrial transmembrane potential decrease, and cytochrome c release. These effects of ghrelin were mediated by GHSR-1a because a GHSR-1a-targeting small interfering RNA abolished the antiapoptotic action of ghrelin. LPS treatment activated the c-Jun N-terminal kinase (JNK) signaling pathway but inhibited the Wnt/β-catenin pathway. Interestingly, combined LPS-ghrelin treatment reduced JNK activation and increased Wnt/β-catenin activation. Furthermore, like ghrelin treatment, the addition of the JNK inhibitor SP600125 or the glycogen synthase kinase-3β inhibitor SB216763 rescued AMs from apoptosis. We also demonstrated that ghrelin altered the balance of Bcl-2-family proteins and inhibited caspase-3 activity. Next, we investigated whether ghrelin protected against septic ARDS in vivo. Sepsis was induced in male rats by performing cecal ligation and puncture; administration of ghrelin reduced sepsis-induced AMs apoptosis, pulmonary injury, protein concentrations in the bronchoalveolar lavage fluid, the lung neutrophil infiltration, and wet to dry weight ratio. However, administration of a specific ghrelin-receptor antagonist, [D-Lys-3]-GH-releasing peptide-6, abolished the beneficial effects of ghrelin. Collectively our results suggest that ghrelin exerts an antiapoptotic effect on AMs at least partly by inhibiting JNK and activating the Wnt/β-catenin pathway

  18. c-Jun N-terminal kinase and Akt signalling pathways regulating tumour necrosis factor-α-induced interleukin-32 expression in human lung fibroblasts: implications in airway inflammation

    PubMed Central

    Li, Dagen; Chen, Dapeng; Zhang, Xuemei; Wang, Hong; Song, Zixin; Xu, Wenchun; He, Yujuan; Yin, Yibing; Cao, Ju

    2015-01-01

    Airway inflammatory diseases such as chronic obstructive pulmonary disease (COPD) and asthma are associated with elevated expression of interleukin-32 (IL-32), a recently described cytokine that appears to play a critical role in inflammation. However, so far, the regulation of pulmonary IL-32 production has not been fully established. We examined the expression of IL-32 by tumour necrosis factor-α (TNF-α) in primary human lung fibroblasts. Human lung fibroblasts were cultured in the presence or absence of TNF-α and/or other cytokines/Toll-like receptor (TLR) ligands or various signalling molecule inhibitors to analyse the expression of IL-32 by quantitative RT-PCR and ELISA. Next, activation of Akt and c-Jun N-terminal kinase (JNK) signalling pathways was investigated by Western blot. Interleukin-32 mRNA of four spliced isoforms (α,β,γ and δ) was up-regulated upon TNF-α stimulation, which was associated with a significant IL-32 protein release from TNF-α-activated human lung fibroblasts. The combination of interferon-γ and TNF-α induced enhanced IL-32 release in human lung fibroblasts, whereas IL-4, IL-17A, IL-27 and TLR ligands did not alter IL-32 release in human lung fibroblasts either alone, or in combination with TNF-α. Furthermore, the activation of Akt and JNK pathways regulated TNF-α-induced IL-32 expression in human lung fibroblasts, and inhibition of the Akt and JNK pathways was able to suppress the increased release of IL-32 to nearly the basal level. These data suggest that TNF-α may be involved in airway inflammation via the induction of IL-32 by activating Akt and JNK signalling pathways. Therefore, the TNF-α/IL-32 axis may be a potential therapeutic target for airway inflammatory diseases. PMID:25157456

  19. Development of indole/indazole-aminopyrimidines as inhibitors of c-Jun N-terminal kinase (JNK): optimization for JNK potency and physicochemical properties.

    PubMed

    Gong, Leyi; Han, Xiaochun; Silva, Tania; Tan, Yun-Chou; Goyal, Bindu; Tivitmahaisoon, Parch; Trejo, Alejandra; Palmer, Wylie; Hogg, Heather; Jahagir, Alam; Alam, Muzaffar; Wagner, Paul; Stein, Karin; Filonova, Lubov; Loe, Brad; Makra, Ferenc; Rotstein, David; Rapatova, Lubica; Dunn, James; Zuo, Fengrong; Dal Porto, Joseph; Wong, Brian; Jin, Sue; Chang, Alice; Tran, Patricia; Hsieh, Gary; Niu, Linghao; Shao, Ada; Reuter, Deborah; Hermann, Johaness; Kuglstatter, Andreas; Goldstein, David

    2013-06-15

    A novel series of indole/indazole-aminopyrimidines was designed and synthesized with an aim to achieve optimal potency and selectivity for the c-Jun kinase family or JNKs. Structure guided design was used to optimize the series resulting in a significant potency improvement. The best compound (17) has IC50 of 3 nM for JNK1 and 20 nM for JNK2, with greater than 40-fold selectivity against other kinases with good physicochemical and pharmacokinetic properties. PMID:23664880

  20. β-Amyloid Oligomers Induce Phosphorylation of Tau and Inactivation of Insulin Receptor Substrate via c-Jun N-Terminal Kinase Signaling: Suppression by Omega-3 Fatty Acids and Curcumin

    PubMed Central

    Ma, Qiu-Lan; Yang, Fusheng; Rosario, Emily R.; Ubeda, Oliver J.; Beech, Walter; Gant, Dana J.; Chen, Ping Ping; Hudspeth, Beverly; Chen, Cory; Zhao, Yongle; Vinters, Harry V.; Frautschy, Sally A.

    2009-01-01

    Both insulin resistance (type II diabetes) and β-amyloid (Aβ) oligomers are implicated in Alzheimer's disease (AD). Here, we investigate the role of Aβ oligomer-induced c-Jun N-terminal kinase (JNK) activation leading to phosphorylation and degradation of the adaptor protein insulin receptor substrate-1 (IRS-1). IRS-1 couples insulin and other trophic factor receptors to downstream kinases and neuroprotective signaling. Increased phospho-IRS-1 is found in AD brain and insulin-resistant tissues from diabetics. Here, we report Aβ oligomers significantly increased active JNK and phosphorylation of IRS-1 (Ser616) and tau (Ser422) in cultured hippocampal neurons, whereas JNK inhibition blocked these responses. The omega-3 fatty acid docosahexaenoic acid (DHA) similarly inhibited JNK and the phosphorylation of IRS-1 and tau in cultured hippocampal neurons. Feeding 3xTg-AD transgenic mice a diet high in saturated and omega-6 fat increased active JNK and phosphorylated IRS-1 and tau. Treatment of the 3xTg-AD mice on high-fat diet with fish oil or curcumin or a combination of both for 4 months reduced phosphorylated JNK, IRS-1, and tau and prevented the degradation of total IRS-1. This was accompanied by improvement in Y-maze performance. Mice fed with fish oil and curcumin for 1 month had more significant effects on Y-maze, and the combination showed more significant inhibition of JNK, IRS-1, and tau phosphorylation. These data indicate JNK mediates Aβ oligomer inactivation of IRS-1 and phospho-tau pathology and that dietary treatment with fish oil/DHA, curcumin, or a combination of both has the potential to improve insulin/trophic signaling and cognitive deficits in AD. PMID:19605645

  1. c-Jun N-terminal kinase inhibitor favors transforming growth factor-β to antagonize hepatitis B virus X protein-induced cell growth promotion in hepatocellular carcinoma

    PubMed Central

    WU, YAN-HUI; AI, XI; LIU, FU-YAO; LIANG, HUI-FANG; ZHANG, BI-XIANG; CHEN, XIAO-PING

    2016-01-01

    Transforming growth factor (TGF)-β induces cell growth arrest in well-differentiated hepatocellular carcinoma (HCC) while hepatitis B virus X protein (HBx) minimizes the tumor suppression of TGF-β signaling in early chronic hepatitis B. However, how to reverse the oncogenic effect of HBx and sustain the tumor-suppressive action of TGF-β has yet to be investigated. The present study examined the effect of TGF-β and a c-Jun N-terminal kinase (JNK) inhibitor on cell growth in HCC cells with forced expression of HBx. It was found that HBx promoted cell growth via activation of the JNK/pSMAD3L pathway and inhibition of the transforming growth factor-beta type I receptor (TβRI)/pSMAD3C pathway. pSMAD3L/SMAD4 and pSMAD3C/SMAD4 complexes antagonized each other to regulate c-Myc expression. In the absence of HBx, TGF-β induced cell growth arrest through activation of the TβRI/pSMAD3C pathway in well-differentiated HCC cells. In the presence of HBx, TGF-β had no effect on cell growth. JNK inhibitor SP600125 significantly reversed the oncogenic action of HBx and favored TGF-β to regain the ability to inhibit the cell growth in HBx-expressing well-differentiated HCC cells. In conclusion, targeting JNK signaling favors TGF-β to block HBx-induced cell growth promotion in well-differentiated HCC cells. As an adjunct to anti-viral therapy, the combination of TGF-β and inhibition of JNK signaling is a potential therapy for HBV-infected HCC. PMID:26648552

  2. Retinol-Binding Protein 4 Inhibits Insulin Signaling in Adipocytes by Inducing Proinflammatory Cytokines in Macrophages through a c-Jun N-Terminal Kinase- and Toll-Like Receptor 4-Dependent and Retinol-Independent Mechanism

    PubMed Central

    Norseen, Julie; Hosooka, Tetsuya; Hammarstedt, Ann; Yore, Mark M.; Kant, Shashi; Aryal, Pratik; Kiernan, Urban A.; Phillips, David A.; Maruyama, Hiroshi; Kraus, Bettina J.; Usheva, Anny; Davis, Roger J.; Smith, Ulf

    2012-01-01

    Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1−/− JNK2−/− macrophages and TLR4−/− macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4. PMID:22431523

  3. Role of c-Jun N-terminal kinase in late nerve regeneration monitored by in vivo imaging of thy1-yellow fluorescent protein transgenic mice.

    PubMed

    Tu, Nguyen H; Katano, Tayo; Matsumura, Shinji; Pham, Vuong Minh; Muratani, Tadatoshi; Minami, Toshiaki; Ito, Seiji

    2016-02-01

    The restoration of function to injured peripheral nerves separated by a gap requires regeneration across it and reinnervation to target organs. To elucidate these processes, we have established an in vivo monitoring system of nerve regeneration in thy1-yellow fluorescent protein transgenic mice expressing a fluorescent protein in their nervous system. Here we demonstrated that motor and sensory nerves were regenerated in a coordinated fashion across the gap and that the functional recovery of the response to mechanical stimuli correlated well with sensory innervation to the foot. Among the mitogen-activated protein kinase inhibitors examined, only the c-Jun N-terminal kinase (JNK) inhibitors delayed functional recovery. Although it did not affect the reinnervation of the muscle, the JNK inhibitor delayed sensory nerve innervation to the skin for over 8 weeks and increased the expression of activatng transcription factor 3 (ATF3), a neuronal injury marker, in the dorsal root ganglion over the same time period. Antibodies against nerve growth factor, glia-derived neurotrophic factor, and brain-derived neurotrophic factor applied to the transection site delayed the functional recovery in this order of potency. These neurotrophic factors enhanced neurite outgrowth from cultured dorsal root ganglion neurons, and the JNK inhibitor reversed their stimulatory effects. These results suggest that JNK played roles in nerve regeneration at both early and late phases. Taken together, the present study demonstrated that neurotrophic factors released from the distal nerve may accelerate motor and sensory nerve regeneration across the gap in a coordinated fashion and reinnervation of the target organs independently. The model characterized here has the advantage of in vivo monitoring of the evaluation of morphological and functional recovery in the same mice for a long period of time. PMID:26613205

  4. Redundant roles for cJun-N-terminal kinase 1 and 2 in interleukin-1β–mediated reduction and modification of murine hepatic nuclear retinoid X receptor α

    PubMed Central

    Kosters, Astrid; White, Damara D.; Sun, Hongdan; Thevananther, Sundararajah; Karpen, Saul J.

    2009-01-01

    Background/Aims Retinoid X receptor α (RXRα), the heterodimeric partner for multiple nuclear receptors (NRs), was shown to be an essential target for inflammation-induced cJun-N-terminal kinase (JNK) signaling in vitro. This study aimed to explore the role of hepatic JNK signaling and its effects on nuclear RXRα levels downstream of interleukin-1β (IL-1β) in vivo. Methods Effects of IL-1β on hepatic NR-dependent gene expression, nuclear RXRα levels, and roles for individual JNK isoforms were studied in wild-type, Jnk1−/−, and Jnk2−/− mice and in primary hepatocytes of each genotype. Results IL-1β administration showed a time-dependent reduction in expression of the hepatic NR-dependent genes Ntcp, Cyp7a1, Cyp8b1, Abcg5, Mrp2, and Mrp3. IL-1β treatment for 1 hour activated JNK and resulted in both post-translational modification and reduction of nuclear RXRα. In wild-type primary hepatocytes, IL-1β modified and reduced nuclear RXRα levels time dependently, which was prevented by chemical inhibition of JNK as well as by inhibition of proteasomal degradation. Individual absence of either JNK1 or JNK2 did not significantly influence the reduction or modification of hepatic nuclear RXRα by IL-1β both in vivo and in primary hepatocytes. Conclusions Functional redundancy exists for JNK1 and JNK2 in IL-1β–mediated alterations of hepatic nuclear RXRα levels, stressing the importance of this pathway in mediating the hepatic response to inflammation. PMID:19767119

  5. Combining docking site and phosphosite predictions to find new substrates: Identification of Smoothelin-like-2 (SMTNL2) as a c-Jun N-terminal kinase (JNK) substrate

    PubMed Central

    Gordon, Elizabeth A.; Whisenant, Thomas C.; Zeller, Michael; Kaake, Robyn M.; Gordon, William M.; Krotee, Pascal; Patel, Vishal; Huang, Lan; Baldi, Pierre; Bardwell, Lee

    2014-01-01

    Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site); and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein Smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site (residues 180-193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells. PMID:23981301

  6. P21-activated protein kinase (PAK2)-mediated c-Jun phosphorylation at 5 threonine sites promotes cell transformation

    PubMed Central

    Li, Tingting; Zhang, Jishuai; Zhu, Feng; Wen, Weihong; Zykova, Tatyana; Li, Xiang; Liu, Kangdong; Peng, Cong; Ma, Weiya; Shi, Guozheng; Dong, Ziming; Bode, Ann M.; Dong, Zigang

    2011-01-01

    The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation. PMID:21177766

  7. Loss of E-Cadherin–mediated Cell–Cell Contacts Activates a Novel Mechanism for Up-Regulation of the Proto-Oncogene c-Jun

    PubMed Central

    Knirsh, Revital; Ben-Dror, Iris; Spangler, Barbara; Matthews, Gideon D.; Kuphal, Silke; Bosserhoff, Anja K.

    2009-01-01

    Loss of E-cadherin–mediated cell–cell contacts can elicit a signaling pathway that leads to acquisition of an invasive phenotype. Here, we show that at the receiving end of this pathway is the proto-oncogene c-Jun, a member of the activator protein-1 family of transcription factors that play a key role in stimulation of cell proliferation and tumor promotion. Cell separation or abrogation of E-cadherin–mediated cell–cell contacts both cause a dramatic increase in accumulation of the c-Jun protein. Unlike growth factors that enhance the expression of c-Jun by activating the transcription of the c-jun gene, the cell contact-dependent increase in c-Jun accumulation is not accompanied by a corresponding increase in c-Jun mRNA or c-Jun protein stability but rather in the translatability of the c-Jun transcript. Consistently, the increase in c-Jun accumulation is not dependent on activation of the mitogen-activated protein kinase or β-catenin pathways but is mediated by signals triggered by the restructured cytoskeleton. Depolymerization of the cytoskeleton can mimic the effect of cell separation and cause a dramatic increase in c-Jun accumulation, whereas Taxol inhibits the cell contact-dependent increase. This novel mechanism of c-Jun regulation seems to underlie the robust overexpression of c-Jun in tumor cells of patients with colon carcinoma. PMID:19193763

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

    PubMed

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

    2007-01-12

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

  9. The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival

    PubMed Central

    Chen, Q. Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D.; Zong, Yan; Buti, Wisam; Odom, Kelly R.; Lee, Hannah; Leal, Sandra M.

    2015-01-01

    We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3°L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3°L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3°L and pupal eye imaginal disc development. PMID:25748605

  10. The drosophila T-box transcription factor midline functions within Insulin/Akt and c-Jun-N terminal kinase stress-reactive signaling pathways to regulate interommatial bristle formation and cell survival.

    PubMed

    Chen, Q Brent; Das, Sudeshna; Visic, Petra; Buford, Kendrick D; Zong, Yan; Buti, Wisam; Odom, Kelly R; Lee, Hannah; Leal, Sandra M

    2015-05-01

    We recently reported that the T-box transcription factor midline (mid) functions within the Notch-Delta signaling pathway to specify sensory organ precursor (SOP) cell fates in early-staged pupal eye imaginal discs and to suppress apoptosis (Das et al.). From genetic and allelic modifier screens, we now report that mid interacts with genes downstream of the insulin receptor(InR)/Akt, c-Jun-N-terminal kinase (JNK) and Notch signaling pathways to regulate interommatidial bristle (IOB) formation and cell survival. One of the most significant mid-interacting genes identified from the modifier screen is dFOXO, a transcription factor exhibiting a nucleocytoplasmic subcellular distribution pattern. In common with dFOXO, we show that Mid exhibits a nucleocytoplasmic distribution pattern within WT third-instar larval (3(o)L) tissue homogenates. Because dFOXO is a stress-responsive factor, we assayed the effects of either oxidative or metabolic stress responses on modifying the mid mutant phenotype which is characterized by a 50% loss of IOBs within the adult compound eye. While metabolic starvation stress does not affect the mid mutant phenotype, either 1 mM paraquat or 20% coconut oil, oxidative stress inducers, partially suppresses the mid mutant phenotype resulting in a significant recovery of IOBs. Another significant mid-interacting gene we identified is groucho (gro). Mid and Gro are predicted to act as corepressors of the enhancer-of-split gene complex downstream of Notch. Immunolabeling WT and dFOXO null 3(o)L eye-antennal imaginal discs with anti-Mid and anti-Engrailed (En) antibodies indicate that dFOXO is required to activate Mid and En expression within photoreceptor neurons of the eye disc. Taken together, these studies show that Mid and dFOXO serve as critical effectors of cell fate specification and survival within integrated Notch, InR/dAkt, and JNK signaling pathways during 3(o)L and pupal eye imaginal disc development. PMID:25748605

  11. TGFβ-induced invasion of prostate cancer cells is promoted by c-Jun-dependent transcriptional activation of Snail1

    PubMed Central

    Thakur, Noopur; Gudey, Shyam Kumar; Marcusson, Anders; Fu, Jing Yi; Bergh, Anders; Heldin, Carl-Henrik; Landström, Marene

    2014-01-01

    High levels of transforming growth factor-β (TGFβ) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFβ is a multifunctional cytokine and crucial regulator of cell fate, such as epithelial to mesenchymal transition (EMT), which is implicated in cancer invasion and progression. TGFβ conveys its signals upon binding to type I and type II serine/threonine kinase receptors (TβRI/II); phosphorylation of Smad2 and Smad3 promotes their association with Smad4, which regulates expression of targets genes, such as Smad7, p21, and c-Jun. TGFβ also activates the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6), which associates with TβRI and activates the p38 mitogen-activated protein kinase (MAPK) pathway. Snail1 is a key transcription factor, induced by TGFβ that promotes migration and invasion of cancer cells. In this study, we have identified a novel binding site for c-Jun in the promoter of the Snail1 gene and report that the activation of the TGFβ–TRAF6–p38 MAPK pathway promotes both c-Jun expression and its activation via p38α-dependent phosphorylation of c-Jun at Ser63. The TRAF6-dependent activation of p38 also leads to increased stability of c-Jun, due to p38-dependent inactivation of glycogen synthase kinase (GSK) 3β by phosphorylation at Ser9. Thus, our findings elucidate a novel role for the p38 MAPK pathway in stimulated cells, leading to activation of c-Jun and its binding to the promoter of Snail1, thereby triggering motility and invasiveness of aggressive human prostate cancer cells. PMID:25483191

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

    PubMed Central

    Ferguson, Heather A.; Goodrich, James A.

    2001-01-01

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

  13. Arsenic Trioxide Induces Apoptosis in Human Platelets via C-Jun NH2-Terminal Kinase Activation

    PubMed Central

    Wu, Yicun; Dai, Jin; Zhang, Weilin; Yan, Rong; Zhang, Yiwen; Ruan, Changgeng; Dai, Kesheng

    2014-01-01

    Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. PMID:24466103

  14. Arsenic trioxide induces apoptosis in human platelets via C-Jun NH2-terminal kinase activation.

    PubMed

    Wu, Yicun; Dai, Jin; Zhang, Weilin; Yan, Rong; Zhang, Yiwen; Ruan, Changgeng; Dai, Kesheng

    2014-01-01

    Arsenic trioxide (ATO), one of the oldest drugs in both Western and traditional Chinese medicine, has become an effective anticancer drug, especially in the treatment of acute promyelocytic leukemia (APL). However, thrombocytopenia occurred in most of ATO-treated patients with APL or other malignant diseases, and the pathogenesis remains unclear. Here we show that ATO dose-dependently induces depolarization of mitochondrial inner transmembrane potential (ΔΨm), up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL, caspase-3 activation, and phosphotidylserine (PS) exposure in platelets. ATO did not induce surface expression of P-selectin and PAC-1 binding, whereas, obviously reduced collagen, ADP, and thrombin induced platelet aggregation. ATO dose-dependently induced c-Jun NH2-terminal kinase (JNK) activation, and JNK specific inhibitor dicumarol obviously reduced ATO-induced ΔΨm depolarization in platelets. Clinical therapeutic dosage of ATO was intraperitoneally injected into C57 mice, and the numbers of circulating platelets were significantly reduced after five days of continuous injection. The data demonstrate that ATO induces caspase-dependent apoptosis via JNK activation in platelets. ATO does not incur platelet activation, whereas, it not only impairs platelet function but also reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia in patients treated with ATO. PMID:24466103

  15. Molecular Network Analysis of Endometriosis Reveals a Novel Role for c-Jun Regulated Macrophage Activation

    PubMed Central

    Beste, Michael T.; Pfäffle-Doyle, Nicole; Prentice, Emily A.; Morris, Stephanie N.; Lauffenburger, Douglas A.; Isaacson, Keith B.; Griffith, Linda G.

    2014-01-01

    Clinical management of endometriosis is limited by the complex relationship between symptom severity, heterogeneous surgical presentations, and variability in clinical outcomes. As a complement to visual classification schemes, molecular profiles of disease activity may improve risk stratification to better inform treatment decisions and identify novel approaches to targeted treatment. Here, we employ a network analysis of information flow within and between inflammatory cells to discern consensus behaviors characterizing patient sub-populations. Unsupervised multivariate analysis of cytokine profiles quantified by multiplex immunoassays identified a subset of patients with a shared “consensus signature” of thirteen elevated cytokines that was associated with common clinical features, but was not observed among patient subpopulations defined by morphologic presentation alone. Enrichment analysis of consensus markers reinforced the primacy of peritoneal macrophage infiltration and activation, which was demonstrably elevated in ex vivo cultures. Although familiar targets of the NFκB family emerged among over-represented transcriptional binding sites for consensus markers, our analysis provides evidence for a previously unrecognized contribution from c-Jun, c-Fos, and AP-1 effectors of mitogen associated kinase signaling. Their crucial involvement in propagation of macrophage-driven inflammatory networks was confirmed via targeted inhibition of upstream kinases. Collectively, these analyses provide in vivo validation of a clinically relevant inflammatory network that may serve as an objective measure for guiding treatment decisions for endometriosis management, and in the future may provide a mechanistic endpoint for assessing efficacy of novel agents aimed at curtailing inflammatory mechanisms that drive disease progression. PMID:24500404

  16. MAGE-A1 promotes melanoma proliferation and migration through C-JUN activation.

    PubMed

    Wang, Dong; Wang, Junyun; Ding, Nan; Li, Yongjun; Yang, Yaran; Fang, Xiangdong; Zhao, Hua

    2016-05-13

    MAGE-A1 belongs to the chromosome X-clustered genes of cancer-testis antigen family and is normally expressed in the human germ line but is also overexpressed in various tumors. Previous studies of MAGE-A1 in melanoma mainly focused on methylation changes or its role in immunotherapy, however, its biological functions in melanoma have remained unknown. In order to determine the role of MAGE-A1 in melanoma growth and metastasis, we manipulated melanoma cell lines with overexpression and knockdown of MAGE-A1. Integration of cell proliferation assays, transwell migration and invasion assays, and RNA-Seq analysis revealed that up-regulation of MAGE-A1 dramatically promoted proliferation, migration, and invasion of human melanoma cell lines in vitro, while down-regulation of MAGE-A1 inhibited those characteristics associated with tumor cells. Furthermore, transcriptome sequencing revealed that MAGE-A1 exerts its tumor promoting activity by activating p-C-JUN directly or through ERK-MAPK signaling pathways. Based on our findings, we propose that MAGE-A1 may be a potential therapeutic target for melanoma patients. PMID:27045082

  17. CONTRIBUTION OF INSPIRATORY FLOW TO ACTIVATION OF EGFR, RAS, MAPK, ATF-2 AND C-JUN DURING LUNG STRETCH

    EPA Science Inventory

    Contribution of Inspiratory Flow to Activation of EGFR, Ras, MAPK, ATF-2 and c-Jun during Lung Stretch

    R. Silbajoris 1, Z. Li 2, J. M. Samet 1 and Y. C. Huang 1. 1 NHEERL, ORD, US EPA, RTP, NC and 2 CEMALB, UNC-CH, Chapel Hill, NC .

    Mechanical ventilation with larg...

  18. TLE4 promotes colorectal cancer progression through activation of JNK/c-Jun signaling pathway

    PubMed Central

    Deng, Dan-Ling; Cai, Juan-Juan; Xiao, Zhi-Yuan; He, Liu-Qing; Jiao, Hong-Li; Ye, Ya-Ping; Yang, Run-Wei; Li, Ting-Ting; Liang, Li; Liao, Wen-Ting; Ding, Yan-Qing

    2016-01-01

    The Groucho transcriptional co-repressor TLE4 protein has been shown to be a tumor suppressor in a subset of acute myeloid leukemia. However, little is known about its role in development and progression of solid tumor. In this study, we found that the expression of TLE4 in colorectal cancer (CRC) tissues was significantly higher than that in their matched adjacent intestine epithelial tissues. In addition, high expression of TLE4 was significantly correlated with advanced Dukes stage, lymph node metastasis and poor prognosis of CRC. Moreover, enforced expression of TLE4 in CRC cell lines significantly enhanced proliferation, invasion and tumor growth. On the contrary, knock down of TLE4 repressed cell proliferation, invasion and tumor growth. Furthermore, our study exhibited that the TLE4 promoted cell proliferation and invasion partially via activation of JNK-c-Jun pathway and subsequently increased cyclinD1 and decreased P27Kip1 expression. In conclusion, these results suggested that TLE4, a potential prognostic biomarker for CRC, plays an important role in the development and progression of human CRC. PMID:26701208

  19. SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases (PTK) and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on days...

  20. c-Jun N-terminal kinase 2 prevents luminal cell commitment in normal mammary glands and tumors by inhibiting p53/Notch1 and breast cancer gene 1 expression

    PubMed Central

    Pfefferle, Adam D.; Perou, Charles M.; Van Den Berg, Carla Lynn

    2015-01-01

    Breast cancer is a heterogeneous disease with several subtypes carrying unique prognoses. Patients with differentiated luminal tumors experience better outcomes, while effective treatments are unavailable for poorly differentiated tumors, including the basal-like subtype. Mechanisms governing mammary tumor subtype generation could prove critical to developing better treatments. C-Jun N-terminal kinase 2 (JNK2) is important in mammary tumorigenesis and tumor progression. Using a variety of mouse models, human breast cancer cell lines and tumor expression data, studies herein support that JNK2 inhibits cell differentiation in normal and cancer-derived mammary cells. JNK2 prevents precocious pubertal mammary development and inhibits Notch-dependent expansion of luminal cell populations. Likewise, JNK2 suppresses luminal populations in a p53-competent Polyoma Middle T-antigen tumor model where jnk2 knockout causes p53-dependent upregulation of Notch1 transcription. In a p53 knockout model, JNK2 restricts luminal populations independently of Notch1, by suppressing Brca1 expression and promoting epithelial to mesenchymal transition. JNK2 also inhibits estrogen receptor (ER) expression and confers resistance to fulvestrant, an ER inhibitor, while stimulating tumor progression. These data suggest that therapies inhibiting JNK2 in breast cancer may promote tumor differentiation, improve endocrine therapy response, and inhibit metastasis. PMID:25970777

  1. Ubiquitin D Regulates IRE1α/c-Jun N-terminal Kinase (JNK) Protein-dependent Apoptosis in Pancreatic Beta Cells.

    PubMed

    Brozzi, Flora; Gerlo, Sarah; Grieco, Fabio Arturo; Juusola, Matilda; Balhuizen, Alexander; Lievens, Sam; Gysemans, Conny; Bugliani, Marco; Mathieu, Chantal; Marchetti, Piero; Tavernier, Jan; Eizirik, Décio L

    2016-06-01

    Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells. PMID:27044747

  2. Upregulation of Multidrug Resistance-Associated Protein 1 by Allyl Isothiocyanate in Human Bronchial Epithelial Cell: Involvement of c-Jun N-Terminal Kinase Signaling Pathway

    PubMed Central

    Wang, Shujun; Wang, Shanshan; Wang, Chenyin; Chen, Yajun; Li, Jie; Wang, Xueqi; Wang, Dianlei; Li, Zegeng; Peng, Zhaoliang; Fan, Ling

    2015-01-01

    Multidrug resistance-associated protein 1 (MRP1) plays a protective role in the etiology and progression of chronic obstructive pulmonary disease (COPD) which results from oxidative stress and inflammation of lung injury. The lower functional MRP1 activity is related to COPD development. Our previous study showed that Allyl isothiocyanate (AITC) induced the expression and activity of MRP1 in a dose-dependent manner. However, which signaling pathway contributes to the upregulation of MRP1 by AITC is unclear. In this study, signaling pathway specific inhibitors were used to examine the mechanism of AITC. We found that JNK inhibitor SP600125 treatment decreased MRP1 mRNA expression in 16HBE14o- cells. But the ERK inhibitor U0126 or PI3K/Akt inhibitor LY294002 produced no obvious effect. The AITC-induced increase of MRP1 mRNA expression was abolished by cotreatment of SP600125, while it was not obviously affected by U0126 or LY294002. Furthermore, AITC acivates the JNK signaling pathway in 16HBE14o- cells. Finally, we found that JNK pathway mediated the upregulation of AITC-induced expression and function of MRP1. Taken together, our results indicated that AITC increased the expression and the activity of MRP1 via a JNK-dependent pathway. ERK and PI3K signaling pathway were not involved in the expression of MRP1 mRNA. PMID:26273426

  3. cAMP-dependent Protein Kinase and c-Jun N-terminal Kinase Mediate Stathmin Phosphorylation for the Maintenance of Interphase Microtubules during Osmotic Stress*

    PubMed Central

    Yip, Yan Y.; Yeap, Yvonne Y. C.; Bogoyevitch, Marie A.; Ng, Dominic C. H.

    2014-01-01

    Dynamic microtubule changes after a cell stress challenge are required for cell survival and adaptation. Stathmin (STMN), a cytoplasmic microtubule-destabilizing phosphoprotein, regulates interphase microtubules during cell stress, but the signaling mechanisms involved are poorly defined. In this study ectopic expression of single alanine-substituted phospho-resistant mutants demonstrated that STMN Ser-38 and Ser-63 phosphorylation were specifically required to maintain interphase microtubules during hyperosmotic stress. STMN was phosphorylated on Ser-38 and Ser-63 in response to hyperosmolarity, heat shock, and arsenite treatment but rapidly dephosphorylated after oxidative stress treatment. Two-dimensional PAGE and Phos-tag gel analysis of stress-stimulated STMN phospho-isoforms revealed rapid STMN Ser-38 phosphorylation followed by subsequent Ser-25 and Ser-63 phosphorylation. Previously, we delineated stress-stimulated JNK targeting of STMN. Here, we identified cAMP-dependent protein kinase (PKA) signaling as responsible for stress-induced STMN Ser-63 phosphorylation. Increased cAMP levels induced by cholera toxin triggered potent STMN Ser-63 phosphorylation. Osmotic stress stimulated an increase in PKA activity and elevated STMN Ser-63 and CREB (cAMP-response element-binding protein) Ser-133 phosphorylation that was substantially attenuated by pretreatment with H-89, a PKA inhibitor. Interestingly, PKA activity and subsequent phosphorylation of STMN were augmented in the absence of JNK activation, indicating JNK and PKA pathway cross-talk during stress regulation of STMN. Taken together our study indicates that JNK- and PKA-mediated STMN Ser-38 and Ser-63 phosphorylation are required to preserve interphase microtubules in response to hyperosmotic stress. PMID:24302736

  4. Role of Human CD36 in Bacterial Recognition, Phagocytosis and Pathogen-Induced C-Jun N-Terminal Kinase (JNK) - Mediated Signaling 1

    PubMed Central

    Baranova, Irina N.; Kurlander, Roger; Bocharov, Alexander V.; Vishnyakova, Tatyana G.; Chen, Zhigang; Remaley, Alan T.; Csako, Gyorgy; Patterson, Amy P.; Eggerman, Thomas L.

    2013-01-01

    Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6-signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacteria, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both Gram-positive and Gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36-transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to Gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with Gram-positive bacteria and lipotechoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40%–50% in human fibroblasts induced by both Gram-negative and Gram-positive bacteria as well as LPS. Of all MAP kinase signaling cascade inhibitors tested, only the inhibitor of JNK, a stress activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-κB inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an α-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by Gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner. PMID:18981136

  5. Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

    SciTech Connect

    Waldron, Richard T. . E-mail: rwaldron@mednet.ucla.edu; Whitelegge, Julian P.; Faull, Kym F.; Rozengurt, Enrique

    2007-05-04

    Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic {sup 32}P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate into Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.

  6. PREVENTING POLYGLUTAMINE-INDUCED ACTIVATION OF C-JUN DELAYS NEURONAL DYSFUNCTION IN A MOUSE MODEL OF SCA7 RETINOPATHY

    PubMed Central

    Merienne, Karine; Friedman, James; Akimoto, Masayuki; Abou-Sleymane, Gretta; Weber, Chantal; Swaroop, Anand; Trottier, Yvon

    2007-01-01

    We have approached the role of cellular stress in neurodegenerative diseases caused by polyglutamine expansion (polyQ) in the context of Spinocerebellar ataxia type 7 (SCA7) that includes retinal degeneration. Using the R7E mouse, in which polyQ-ataxin-7 is specifically over-expressed in rod photoreceptors, we previously showed that rod dysfunction correlated to moderate and prolonged activation of the JNK/c-Jun stress pathway. SCA7 retinopathy was also associated with reduced expression of rod-specific genes, including the transcription factor Nrl, which is essential for rod differentiation and function. Here, we report that R7E retinopathy is improved upon breeding with the JunAA knock-in mice, in which JNK-mediated activation of c-Jun is compromised. Expression of Nrl and its downstream targets, which are involved in phototranduction, are partially restored in the JunAA-R7E mice. We further show that c-Jun can directly repress the transcription of Nrl. Our studies suggest that polyQ-induced cellular stress leads to repression of genes necessary for neuronal fate and function. PMID:17189700

  7. Physical interaction of the activator protein-1 factors c-Fos and c-Jun with Cbfa1 for collagenase-3 promoter activation

    NASA Technical Reports Server (NTRS)

    D'Alonzo, Richard C.; Selvamurugan, Nagarajan; Karsenty, Gerard; Partridge, Nicola C.

    2002-01-01

    Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation.

  8. Bile acids-mediated overexpression of MUC4 via FAK-dependent c-Jun activation in pancreatic cancer.

    PubMed

    Joshi, Suhasini; Cruz, Eric; Rachagani, Satyanarayana; Guha, Sushovan; Brand, Randall E; Ponnusamy, Moorthy P; Kumar, Sushil; Batra, Surinder K

    2016-08-01

    The majority of pancreatic cancer (PC) patients are clinically presented with obstructive jaundice with elevated levels of circulatory bilirubin and alkaline phosphatases. In the current study, we examined the implications of bile acids (BA), an important component of bile, on the pathophysiology of PC and investigated their mechanistic association in tumor-promoting functions. Integration of results from PC patient samples and autochthonous mouse models showed an elevated levels of BA (p < 0.05) in serum samples compared to healthy controls. Similarly, an elevated BA levels was observed in pancreatic juice derived from PC patients (p < 0.05) than non-pancreatic non-healthy (NPNH) controls, further establishing the clinical association of BA with the pathogenesis of PC. The tumor-promoting functions of BA were established by observed transcriptional upregulation of oncogenic MUC4 expression. Luciferase reporter assay revealed distal MUC4 promoter as the primary responsive site to BA. In silico analysis recognized two c-Jun binding sites at MUC4 distal promoter, which was biochemically established using ChIP assay. Interestingly, BA treatment led to an increased transcription and activation of c-Jun in a FAK-dependent manner. Additionally, BA receptor, namely FXR, which is also upregulated at transcriptional level in PC patient samples, was demonstrated as an upstream molecule in BA-mediated FAK activation, plausibly by regulating Src activation. Altogether, these results demonstrate that elevated levels of BA increase the tumorigenic potential of PC cells by inducing FXR/FAK/c-Jun axis to upregulate MUC4 expression, which is overexpressed in pancreatic tumors and is known to be associated with progression and metastasis of PC. PMID:27185392

  9. Antiepileptic Effect of Uncaria rhynchophylla and Rhynchophylline Involved in the Initiation of c-Jun N-Terminal Kinase Phosphorylation of MAPK Signal Pathways in Acute Seizures of Kainic Acid-Treated Rats.

    PubMed

    Hsu, Hsin-Cheng; Tang, Nou-Ying; Liu, Chung-Hsiang; Hsieh, Ching-Liang

    2013-01-01

    Seizures cause inflammation of the central nervous system. The extent of the inflammation is related to the severity and recurrence of the seizures. Cell surface receptors are stimulated by stimulators such as kainic acid (KA), which causes intracellular mitogen-activated protein kinase (MAPK) signal pathway transmission to coordinate a response. It is known that Uncaria rhynchophylla (UR) and rhynchophylline (RP) have anticonvulsive effects, although the mechanisms remain unclear. Therefore, the purpose of this study is to develop a novel strategy for treating epilepsy by investigating how UR and RP initiate their anticonvulsive mechanisms. Sprague-Dawley rats were administered KA (12 mg/kg, i.p.) to induce seizure before being sacrificed. The brain was removed 3 h after KA administration. The results indicate that pretreatment with UR (1.0 g/kg), RP (0.25 mg/kg), and valproic acid (VA, 250 mg/kg) for 3 d could reduce epileptic seizures and could also reduce the expression of c-Jun aminoterminal kinase phosphorylation (JNKp) of MAPK signal pathways in the cerebral cortex and hippocampus brain tissues. Proinflammatory cytokines interleukin (IL)-1 β , IL-6, and tumor necrosis factor- α remain unchanged, indicating that the anticonvulsive effect of UR and RP is initially involved in the JNKp MAPK signal pathway during the KA-induced acute seizure period. PMID:24381640

  10. TOPK promotes lung cancer resistance to EGFR tyrosine kinase inhibitors by phosphorylating and activating c-Jun

    PubMed Central

    Wang, Tao; Wang, Ting; Niu, Mengjie; Zhang, Shengli; Jia, Lintao; Li, Shengqing

    2016-01-01

    Tyrosine kinase inhibitors (TKIs) targeting the epidermal growth factor receptor (EGFR) have shown promising clinical efficacy in non-squamous non-small cell lung cancer (NSCLC); however, resistance is frequently observed in malignant cells, operating through a mechanism that remains largely unknown. The present study shows that T-lymphokine-activated killer cell-originated protein kinase (TOPK) is upregulated in NSCLC and excessively activated in TKI-refractory cells. TOPK dictates the responsiveness of lung cancers to the EGFR-targeted TKI gefitinib through the transcription factor AP-1 component c-Jun. TOPK binds directly to and phosphorylates c-Jun, which consequently activates the transcription of AP-1 target genes, including CCND1 and CDC2. TOPK silencing sensitizes EGFR-TKI-resistant lung cancer cells to gefitinib and increases gefitinib efficacy in preclinical lung adenocarcinoma xenograft models. These findings represent a novel mechanism of lung cancer resistance to TKIs and suggest that TOPK may have value both as a predictive biomarker and as a therapeutic target: TOPK-targeted therapy may synergize with EGFR-targeted therapy in lung cancers. PMID:26745678

  11. Mitogen Activated Protein Kinase Family Proteins and c-jun Signaling in Injury-induced Schwann Cell Plasticity.

    PubMed

    Lee, Hye Jeong; Shin, Yoon Kyung; Park, Hwan Tae

    2014-06-01

    Schwann cells (SCs) in the peripheral nerves myelinate axons during postnatal development to allow saltatory conduction of nerve impulses. Well-organized structures of myelin sheathes are maintained throughout life unless nerves are insulted. After peripheral nerve injury, unidentified signals from injured nerves drive SC dedifferentiation into an immature state. Dedifferentiated SCs participate in axonal regeneration by producing neurotrophic factors and removing degenerating nerve debris. In this review, we focus on the role of mitogen activated protein kinase family proteins (MAP kinases) in SC dedifferentiation. In addition, we will highlight neuregulin 1 and the transcription factor c-jun as upstream and downstream signals for MAP kinases in SC responses to nerve injury. PMID:24963277

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

    PubMed

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

    2009-10-01

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

  13. Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

    2001-01-01

    Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

  14. Dual Role of Jun N-Terminal Kinase Activity in Bone Morphogenetic Protein-Mediated Drosophila Ventral Head Development.

    PubMed

    Park, Sung Yeon; Stultz, Brian G; Hursh, Deborah A

    2015-12-01

    The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cis-regulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. PMID:26500262

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

    PubMed Central

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

    2015-01-01

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

  16. Inhibition of spinal c-Jun-NH2-terminal kinase (JNK) improves locomotor activity of spinal cord injured rats.

    PubMed

    Martini, Alessandra C; Forner, Stefânia; Koepp, Janice; Rae, Giles Alexander

    2016-05-16

    Mitogen-activated protein kinases (MAPKs) have been implicated in central nervous system injuries, yet the roles within neurodegeneration following spinal cord injury (SCI) still remain partially elucidated. We aimed to investigate the changes in expression of the three MAPKs following SCI and the role of spinal c-jun-NH2-terminal kinase (JNK) in motor impairment following the lesion. SCI induced at the T9 level resulted in enhanced expression of phosphorylated MAPKs shortly after trauma. SCI increased spinal cord myeloperoxidase levels, indicating a local neutrophil infiltration, and elevated the number of spinal apoptotic cells. Intrathecal administration of a specific inhibitor of JNK phosphorylation, SP600125, given at 1 and 4h after SCI, reduced the p-JNK expression, the number of spinal apoptotic cells and many of the histological signs of spinal injury. Notably, restoration of locomotor performance was clearly ameliorated by SP600125 treatment. Altogether, the results demonstrate that SCI induces activation of spinal MAPKs and that JNK plays a major role in mediating the deleterious consequences of spinal injury, not only at the spinal level, but also those regarding locomotor function. Therefore, inhibition of JNK activation in the spinal cord shortly after trauma might constitute a feasible therapeutic strategy for the functional recovery from SCI. PMID:27080425

  17. Proline-rich tyrosine kinase 2 via enhancing signal transducer and activator of transcription 3-dependent cJun expression mediates retinal neovascularization

    PubMed Central

    Kumar, Raj; Singh, Nikhlesh K.; Rao, Gadiparthi N.

    2016-01-01

    Despite the involvement of proline-rich tyrosine kinase 2 (Pyk2) in endothelial cell angiogenic responses, its role in pathological retinal angiogenesis is not known. In the present study, we show that vascular endothelial growth factor A (VEGFA) induces Pyk2 activation in mediating human retinal microvascular endothelial cell (HRMVEC) migration, sprouting and tube formation. Downstream to Pyk2, VEGFA induced signal transducer and activator of transcription 3 (STAT3) activation and cJun expression in the modulation of HRMVEC migration, sprouting and tube formation. Consistent with these observations, hypoxia induced activation of Pyk2-STAT3-cJun signaling axis and siRNA-mediated downregulation of Pyk2, STAT3 or cJun levels substantially inhibited hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization. Together, these observations suggest that activation of Pyk2-mediated STAT3-cJun signaling is required for VEGFA-induced HRMVEC migration, sprouting and tube formation in vitro and hypoxia-induced retinal endothelial cell proliferation, tip cell formation and neovascularization in vivo. PMID:27210483

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2011-01-01

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

  20. LukS-PV induces differentiation by activating the ERK signaling pathway and c-JUN/c-FOS in human acute myeloid leukemia cells.

    PubMed

    Dai, Chunyang; Zhang, Chengfang; Sun, Xiaoxi; Pan, Qing; Peng, Jing; Shen, Jilong; Ma, Xiaoling

    2016-07-01

    LukS-PV, a component of Panton-Valentine leukocidin, is a pore-forming cytotoxin secreted by Staphylococcus aureus. Here we examined the potential effect of LukS-PV in differentiation of human leukemia cells and the underlying mechanism. We found that LukS-PV could induce differentiation of human acute myeloid leukemia (AML) cells, including AML cell lines and primary AML blasts, as determined by morphological changes, phagocytosis assay and expression of CD14 and CD11b surface antigens. In addition, LukS-PV activated the extracellular signal-regulated kinase (ERK) pathway and significantly upregulated the phosphorylation of c-JUN and c-FOS transcriptional factors in the process of differentiation. Inhibiting ERK pathway activation with U0126 (a MEK1/2 inhibitor) markedly blocked LukS-PV-induced differentiation and decreased the phosphorylation of c-JUN and c-FOS. These findings demonstrate an essential role for the ERK pathway together with c-JUN and c-FOS in the differentiation activity of LukS-PV. Taken together, our data suggest that LukS-PV could be a potential candidate as a differentiation-inducing agent for the therapeutic treatment of AML. PMID:27102414

  1. Induction of apoptosis by the transcription factor c-Jun.

    PubMed Central

    Bossy-Wetzel, E; Bakiri, L; Yaniv, M

    1997-01-01

    c-Jun, a signal-transducing transcription factor of the AP-1 family, normally implicated in cell cycle progression, differentiation and cell transformation, recently has also been linked to apoptosis. To explore further the functional roles of c-Jun, a conditional allele was generated by fusion of c-Jun with the hormone-binding domain of the human estrogen receptor (ER). Here we demonstrate that increased c-Jun activity is sufficient to trigger apoptotic cell death in NIH 3T3 fibroblasts. c-Jun-induced apoptosis is evident at high serum levels, but is enhanced further in factor-deprived fibroblasts. Furthermore, apoptosis by c-Jun is not accompanied by an increase in DNA synthesis. Constitutive overexpression of the apoptosis inhibitor protein Bcl-2 delays the c-Jun-mediated cell death. The regions of c-Jun necessary for apoptosis induction include the amino-terminal transactivation and the carboxy-terminal leucine zipper domain, suggesting that c-Jun may activate cell death by acting as a transcriptional regulator. We further show that alpha-fodrin, a substrate of the interleukin 1beta-converting enzyme (ICE) and CED-3 family of cysteine proteases, becomes proteolytically cleaved in cells undergoing cell death by increased c-Jun activity. Moreover, cell-permeable irreversible peptide inhibitors of the ICE/CED-3 family of cysteine proteases prevented the cell death. PMID:9130714

  2. Salmonella induces SRC protein tyrosine kinase, c-Jun N-terminal kinase (JNK), and NF-kappaBp65 signaling pathways in commercial and wild-type turkey leukocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies comparing signaling in wild-type turkey (WT) leukocytes and commercial turkey (CT) leukocytes found that the activity of protein tyrosine kinases and MAP kinases, ERK 1/2 and p38, were significantly higher in WT leukocytes compared to CT lines upon exposure to both SE and OPSE on d...

  3. Regulation of Lhb and Egr1 Gene Expression by GNRH Pulses in Rat Pituitaries Is Both c-Jun N-Terminal Kinase (JNK)- and Extracellular Signal-Regulated Kinase (ERK)-Dependent1

    PubMed Central

    Burger, Laura L.; Haisenleder, Daniel J.; Aylor, Kevin W.; Marshall, John C.

    2009-01-01

    Pulsatile GNRH regulates the gonadotropin subunit genes in a differential manner, with faster frequencies favoring Lhb gene expression and slower frequencies favoring Fshb. Early growth response 1 (EGR1) is critical for Lhb gene transcription. We examined GNRH regulation of EGR1 and its two corepressors, Ngfi-A-binding proteins 1 and 2 (NAB1 and NAB2), both in vivo and in cultured rat pituitary cells. In rats, fast GNRH pulses (every 30 min) stably induced Egr1 primary transcript (PT) and mRNA 2-fold (P < 0.05) for 1–24 h. In contrast, slow GNRH pulses (every 240 min) increased Egr1 PT at 24 h (6-fold; P < 0.05) but increased Egr1 mRNA 4- to 5-fold between 4 and 24 h. Both GNRH pulse frequencies increased EGR1 protein 3- to 4-fold. In cultured rat pituitary cells, GNRH pulses (every 60 min) increased Egr1 (PT, 2.5- to 3-fold; mRNA, 1.5- to 2-fold; P < 0.05). GNRH pulses had little effect on Nab1/2 PT/mRNAs either in vivo or in vitro. We also examined specific intracellular signaling cascades activated by GNRH. Inhibitors of mitogen-activated protein kinase 8/9 (MAPK8/9 [also known as JNK]; SP600125) and MAP Kinase Kinase 1 (MAP2K1 [also known as MEK1]; PD98059) either blunted or totally suppressed the GNRH induction of Lhb PT and Egr1 PT/mRNA, whereas the MAPK14 (also known as p38) inhibitor SB203580 did not. In summary, pulsatile GNRH stimulates Egr1 gene expression and protein in vivo but not in a frequency-dependent manner. Additionally, GNRH-induced Egr1 gene expression is mediated by MAPK8/9 and MAPK1/3, and both are critical for Lhb gene transcription. PMID:19710510

  4. Rg1 exhibits neuroprotective effects by inhibiting the endoplasmic reticulum stress-mediated c-Jun N-terminal protein kinase apoptotic pathway in a rat model of Alzheimer's disease.

    PubMed

    Mu, Jun-Shan; Lin, Hang; Ye, Jian-Xin; Lin, Min; Cui, Xiao-Ping

    2015-09-01

    . Furthermore, treatment with Rg1 significantly reduced the expression of Grp78, and triggered inositol‑requiring enzyme‑1 (IRE‑1) and phosphorylated protein kinase RNA‑like ER kinase‑associated ER stress. The IRE‑1 UPR pathway downstream gene, tumor necrosis factor receptor‑associated factor 2, was significantly decreased in rats treated with Rg1, compared with untreated AD rats. Furthermore, the activation of p‑JNK was also inhibited when AD rats were treated with Rg1. In conclusion, Rg1 was shown to function as an important factor that inhibits the accumulation of NFTs and Aβ via inhibition of the ER stress‑mediated pathway. Blocking of this pathway was triggered by the IRE‑1 and TRAF2 pathway, as a result of inhibition of the expression of p‑JNK. PMID:26016457

  5. Nucleolin regulates c-Jun/Sp1-dependent transcriptional activation of cPLA2alpha in phorbol ester-treated non-small cell lung cancer A549 cells.

    PubMed

    Tsou, Jen-Hui; Chang, Kwang-Yu; Wang, Wei-Chiao; Tseng, Joseph T; Su, Wu-Chou; Hung, Liang-Yi; Chang, Wen-Chang; Chen, Ben-Kuen

    2008-01-01

    The expression of cPLA2 is critical for transformed growth of non-small cell lung cancer (NSCLC). It is known that phorbol 12-myristate 13-acetate (PMA)-activated signal transduction pathway is thought to be involved in the oncogene action in NSCLC and enzymatic activation of cPLA2. However, the transcriptional regulation of cPLA2alpha in PMA-activated NSCLC is not clear. In this study, we found that PMA induced the mRNA level and protein expression of cPLA2alpha. In addition, two Sp1-binding sites of cPLA2alpha promoter were required for response to PMA and c-Jun overexpression. Small interfering RNA (siRNA) of c-Jun and nucleolin inhibited PMA induced the promoter activity and protein expression of cPLA2alpha. Furthermore, PMA stimulated the formation of c-Jun/Sp1 and c-Jun/nucleolin complexes as well as the binding of these transcription factor complexes to the cPLA2alpha promoter. Although Sp1-binding sites were required for the bindings of Sp1 and nucleolin to the promoter, the binding of nucleolin or Sp1 to the promoter was independent of each other. Our results revealed that c-Jun/nucleolin and c-Jun/Sp1 complexes play an important role in PMA-regulated cPLA2alpha gene expression. It is likely that nucleolin binding at place of Sp1 on gene promoter could also mediate the regulation of c-Jun/Sp1-activated genes. PMID:18025046

  6. Sulforaphane and alpha-lipoic acid upregulate the expression of the pi class of glutathione S-transferase through c-jun and Nrf2 activation.

    PubMed

    Lii, Chong-Kuei; Liu, Kai-Li; Cheng, Yi-Ping; Lin, Ai-Hsuan; Chen, Haw-Wen; Tsai, Chia-Wen

    2010-05-01

    The anticarcinogenic effect of dietary organosulfur compounds has been partly attributed to their modulation of the activity and expression of phase II detoxification enzymes. Our previous studies indicated that garlic allyl sulfides upregulate the expression of the pi class of glutathione S-transferase (GSTP) through the activator protein-1 pathway. Here, we examined the modulatory effect of sulforaphane (SFN) and alpha-lipoic acid (LA) or dihydrolipoic acid (DHLA) on GSTP expression in rat Clone 9 liver cells. Cells were treated with LA or DHLA (50-600 micromol/L) or SFN (0.2-5 micromol/L) for 24 h. Immunoblots and real-time PCR showed that SFN, LA, and DHLA dose dependently induced GSTP protein and mRNA expression. Compared with the induction by the garlic organosulfur compound diallyl trisulfide (DATS), the effectiveness was in the order of SFN > DATS > LA = DHLA. The increase in GSTP enzyme activity in cells treated with 5 micromol/L SFN, 50 micromol/L DATS, and 600 micromol/L LA and DHLA was 172, 75, 122, and 117%, respectively (P < 0.05). A reporter assay showed that the GSTP enhancer I (GPEI) was required for GSTP induction by the organosulfur compounds. Electromobility gel shift assays showed that the DNA binding of GPEI to nuclear proteins reached a maximum at 0.5-1 h after SFN, LA, and DHLA treatment. Super-shift assay revealed that the transcription factors c-jun and nuclear factor erythroid-2 related factor 2 (Nrf2) were bound to GPEI. These results suggest that SFN and LA in either its oxidized or reduced form upregulate the transcription of the GSTP gene by activating c-jun and Nrf2 binding to the enhancer element GPEI. PMID:20237067

  7. Enediyne lidamycin induces apoptosis in human multiple myeloma cells through activation of p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

    PubMed

    Zhen, Yong-Zhan; Lin, Ya-Jun; Shang, Bo-Yang; Zhen, Yong-Su

    2009-07-01

    In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC(50) values of LDM for the U266 and SKO-007 cells were 0.0575 +/- 0.0015 and 0.1585 +/- 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM. PMID:19468799

  8. The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

    SciTech Connect

    Ahn, Jiwon; Choi, Jeong-Hae; Won, Misun; Kang, Chang-Mo; Gyun, Mi-Rang; Park, Hee-Moon; Kim, Chun-Ho; Chung, Kyung-Sook

    2011-06-03

    Highlights: {yields} Regulation of transcriptional activation of RhoB is still unclear. {yields} We examine the effect of p38 MAPK inhibition, and c-Jun and RhoB depletion on UV-induced RhoB expression and apoptosis. {yields} We identify the regions of RhoB promoter necessary to confer UV responsiveness using pRhoB-luciferase reporter assays. {yields} c-Jun, ATF2 and p300 are dominantly associated with NF-Y on the distal CCAAT box. {yields} The activation of p38 MAPK primarily contribute to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins on distal CCAAT box of RhoB promoter. -- Abstract: The Ras-related small GTP-binding protein RhoB is rapidly induced in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter in

  9. 14-3-3σ Gene Loss Leads to Activation of the Epithelial to Mesenchymal Transition Due to the Stabilization of c-Jun Protein.

    PubMed

    Raychaudhuri, Kumarkrishna; Chaudhary, Neelam; Gurjar, Mansa; D'Souza, Roseline; Limzerwala, Jazeel; Maddika, Subbareddy; Dalal, Sorab N

    2016-07-29

    Loss of 14-3-3σ has been observed in multiple tumor types; however, the mechanisms by which 14-3-3σ loss leads to tumor progression are not understood. The experiments in this report demonstrate that loss of 14-3-3σ leads to a decrease in the expression of epithelial markers and an increase in the expression of mesenchymal markers, which is indicative of an induction of the epithelial to mesenchymal transition (EMT). The EMT was accompanied by an increase in migration and invasion in the 14-3-3σ(-/-) cells. 14-3-3σ(-/-) cells show increased stabilization of c-Jun, resulting in an increase in the expression of the EMT transcription factor slug. 14-3-3σ induces the ubiquitination and degradation of c-Jun in an FBW7-dependent manner. c-Jun ubiquitination is dependent on the presence of an intact nuclear export pathway as c-Jun is stabilized and localized to the nucleus in the presence of a nuclear export inhibitor. Furthermore, the absence of 14-3-3σ leads to the nuclear accumulation and stabilization of c-Jun, suggesting that 14-3-3σ regulates the subcellular localization of c-Jun. Our results have identified a novel mechanism by which 14-3-3σ maintains the epithelial phenotype by inhibiting EMT and suggest that this property of 14-3-3σ might contribute to its function as a tumor suppressor gene. PMID:27261462

  10. Antiestrogenic activity of flavnoid phytochemicals mediated via c-Jun N-terminal protein kinase pathway. Cell-type specific regulation of estrogen receptor alpha

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Flavonoid phytochemicals act as both agonists and antagonists of the human estrogen receptors (ERs). While a number of these compounds act by directly binding to the ER, certain phytochemicals, such as the flavonoid compounds chalcone and flavone, elicit antagonistic effects on estrogen signaling in...

  11. N-terminal domain of complexin independently activates calcium-triggered fusion.

    PubMed

    Lai, Ying; Choi, Ucheor B; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A; Wang, Austin L; Diao, Jiajie; Brunger, Axel T

    2016-08-01

    Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release. PMID:27444020

  12. N-terminal domain of complexin independently activates calcium-triggered fusion

    PubMed Central

    Lai, Ying; Choi, Ucheor B.; Zhang, Yunxiang; Zhao, Minglei; Pfuetzner, Richard A.; Wang, Austin L.; Brunger, Axel T.

    2016-01-01

    Complexin activates Ca2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca2+-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca2+-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca2+-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca2+-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca2+-triggered release. PMID:27444020

  13. Protein kinase B/Akt activates c-Jun NH(2)-terminal kinase by increasing NO production in response to shear stress

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Boo, Y. C.; Park, H.; Maland, M. C.; Patel, R.; Pritchard, K. A. Jr; Fujio, Y.; Walsh, K.; Darley-Usmar, V.; Jo, H.

    2001-01-01

    Laminar shear stress activates c-Jun NH(2)-terminal kinase (JNK) by the mechanisms involving both nitric oxide (NO) and phosphatidylinositide 3-kinase (PI3K). Because protein kinase B (Akt), a downstream effector of PI3K, has been shown to phosphorylate and activate endothelial NO synthase, we hypothesized that Akt regulates shear-dependent activation of JNK by stimulating NO production. Here, we examined the role of Akt in shear-dependent NO production and JNK activation by expressing a dominant negative Akt mutant (Akt(AA)) and a constitutively active mutant (Akt(Myr)) in bovine aortic endothelial cells (BAEC). As expected, pretreatment of BAEC with the PI3K inhibitor (wortmannin) prevented shear-dependent stimulation of Akt and NO production. Transient expression of Akt(AA) in BAEC by using a recombinant adenoviral construct inhibited the shear-dependent stimulation of NO production and JNK activation. However, transient expression of Akt(Myr) by using a recombinant adenoviral construct did not induce JNK activation. This is consistent with our previous finding that NO is required, but not sufficient on its own, to activate JNK in response to shear stress. These results and our previous findings strongly suggest that shear stress triggers activation of PI3K, Akt, and endothelial NO synthase, leading to production of NO, which (along with O(2-), which is also produced by shear) activates Ras-JNK pathway. The regulation of Akt, NO, and JNK by shear stress is likely to play a critical role in its antiatherogenic effects.

  14. Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation.

    PubMed

    Hong, Ji-Young; Lebofsky, Margitta; Farhood, Anwar; Jaeschke, Hartmut

    2009-03-01

    Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-Jun NH2-terminal kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma alanine aminotransferase activities increased from 46+/-4 U/l in controls to 955+/-90 U/l at 6 h after diquat treatment. Hematoxylin and eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6 h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP-600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury, but, in combination with phorone and FeSO4, induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation. PMID:19136381

  15. Oxidant stress-induced liver injury in vivo: role of apoptosis, oncotic necrosis, and c-Jun NH2-terminal kinase activation

    PubMed Central

    Hong, Ji-Young; Lebofsky, Margitta; Farhood, Anwar; Jaeschke, Hartmut

    2009-01-01

    Oxidant stress is critically involved in various liver diseases. Superoxide formation causes c-Jun NH2-terminal kinase (JNK)- and caspase-dependent apoptosis in cultured hepatocytes. To verify these findings in vivo, male Fisher rats were treated with diquat and menadione. The oxidant stress induced by both compounds was confirmed by increased formation of glutathione disulfide and 4-hydroxynonenal protein adducts. Plasma alanine aminotransferase activities increased from 46 ± 4 U/l in controls to 955 ± 90 U/l at 6 h after diquat treatment. Hematoxylin and eosin staining of liver sections revealed large areas of necrotic cells at 3 and 6 h. DNA strandbreaks, evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, showed clusters of TUNEL-positive cells, where the staining was predominantly cytosolic and the cells were swollen, indicating oncotic necrosis. There was no significant increase in caspase-3 activities or relevant release of DNA fragments into the cytosol at any time between 0 and 6 h after diquat treatment. Despite the activation of JNK after high doses of diquat, the JNK inhibitor SP-600125 did not protect against diquat-induced necrosis. Menadione alone did not cause liver injury, but, in combination with phorone and FeSO4, induced moderate oncotic necrosis. On the other hand, if animals were treated with galactosamine/endotoxin as positive control for apoptosis, caspase-3 activities were increased by 259%, the number of TUNEL-positive cells with apoptotic morphology was increased 103-fold, and DNA fragmentation was enhanced 6-fold. The data indicate that liver cell death initiated by diquat-induced superoxide formation in vivo is mediated predominantly by oncotic necrosis and is independent of JNK activation. PMID:19136381

  16. Activation of the KCa3.1 channel contributes to traumatic scratch injury-induced reactive astrogliosis through the JNK/c-Jun signaling pathway.

    PubMed

    Yi, Mengni; Dou, Fangfang; Lu, Qin; Yu, Zhihua; Chen, Hongzhuan

    2016-06-15

    Reactive astrogliosis is widely considered to contribute to pathogenic responses to stress and brain injury and to diseases as diverse as ischemia and neurodegeneration. We previously found that expression of the intermediate-conductance calcium-activated potassium channel (KCa3.1) involved in TGF-β-activated astrogliosis. In the present study, we investigated whether migration of cortical astrocytes following mechanical scratch injury involves the KCa3.1 channel, which contributes to Ca(2+)-mediated migration in other cells. We found that scratch injury increased the expression of KCa3.1 protein in reactive astrocytes. Application of the KCa3.1 blocker TRAM-34 decreased glial fibrillary acidic protein (GFAP) expression and slowed migration in a concentration-dependent manner. Application of the Ca(2+) chelators, EGTA and BAPTA-AM, also slowed the migration of astrocytes. Blockade or genetic deletion of KCa3.1 both slowed and dramatically reduced the scratch injuries induced the sharp rise in astrocytes Ca(2+) concentrations. The scratch injury-induced phosphorylation of JNK and c-Jun proteins was also attenuated both by blockade of KCa3.1 with TRAM-34 and in KCa3.1(-/-) astrocytes. Using KCa3.1 knockout mice, we further confirmed that deletion of KCa3.1 reduced expression of GFAP in an in vivo stab wound model. Taken together, our findings highlight a novel role for KCa3.1 in phenotypic modulation of reactive astrocytes and in astrocyte mobilization in response to mechanical stress, providing a potential target for therapeutic intervention in brain injuries. PMID:27163196

  17. Estrogens suppress RANK ligand-induced osteoclast differentiation via a stromal cell independent mechanism involving c-Jun repression

    PubMed Central

    Shevde, Nirupama K.; Bendixen, Amy C.; Dienger, Krista M.; Pike, J. Wesley

    2000-01-01

    Loss of ovarian function following menopause results in a substantial increase in bone turnover and a critical imbalance between bone formation and resorption. This imbalance leads to a progressive loss of trabecular bone mass and eventually osteoporosis, in part the result of increased osteoclastogenesis. Enhanced formation of functional osteoclasts appears to be the result of increased elaboration by support cells of osteoclastogenic cytokines such as IL-1, tumor necrosis factor, and IL-6, all of which are negatively regulated by estrogens. We show here that estrogen can suppress receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)-induced differentiation of myelomonocytic precursors into multinucleated tartrate-resistant acid phosphatase-positive osteoclasts through an estrogen receptor-dependent mechanism that does not require mediation by stromal cells. This suppression is dose-dependent, isomer-specific, and reversed by ICI 182780. Furthermore, the bone-sparing analogues tamoxifen and raloxifene mimic estrogen's effects. Estrogen blocks RANKL/M-CSF-induced activator protein-1-dependent transcription, likely through direct regulation of c-Jun activity. This effect is the result of a classical nuclear activity by estrogen receptor to regulate both c-Jun expression and its phosphorylation by c-Jun N-terminal kinase. Our results suggest that estrogen modulates osteoclast formation both by down-regulating the expression of osteoclastogenic cytokines from supportive cells and by directly suppressing RANKL-induced osteoclast differentiation. PMID:10869427

  18. STRAP regulates c-Jun ubiquitin-mediated proteolysis and cellular proliferation

    SciTech Connect

    Reiner, Jennifer; Ye, Fei; Kashikar, Nilesh D.; Datta, Pran K.

    2011-04-08

    Highlights: {yields} STRAP is specifically correlated with c-Jun expression and activation in fibroblasts. {yields} STRAP inhibits c-Jun ubiquitylation in vivo and prolongs the half-life of c-Jun. {yields} STRAP expression increases expression of the AP-1 target gene, cyclin D1, and promotes cell autonomous growth. -- Abstract: STRAP is a ubiquitous WD40 protein that has been implicated in tumorigenesis. Previous studies suggest that STRAP imparts oncogenic characteristics to cells by promoting ERK and pRb phosphorylation. While these findings suggest that STRAP can activate mitogenic signaling pathways, the effects of STRAP on other MAPK pathways have not been investigated. Herein, we report that STRAP regulates the expression of the c-Jun proto-oncogene in mouse embryonic fibroblasts. Loss of STRAP expression results in reduced phospho-c-Jun and total c-Jun but does not significantly reduce the level of two other early response genes, c-Myc and c-Fos. STRAP knockout also decreases expression of the AP-1 target gene, cyclin D1, which is accompanied by a reduction in cell growth. No significant differences in JNK activity or basal c-Jun mRNA levels were observed between wild type and STRAP null fibroblasts. However, proteasomal inhibition markedly increases c-Jun expression in STRAP knockout MEFs and STRAP over-expression decreases the ubiquitylation of c-Jun in 293T cells. Loss of STRAP accelerates c-Jun turnover in fibroblasts and ectopic over-expression of STRAP in STRAP null fibroblasts increases c-Jun expression. Collectively, our findings indicate that STRAP regulates c-Jun stability by decreasing the ubiquitylation and proteosomal degradation of c-Jun.

  19. Tumor-specific activation of the C-JUN/MELK pathway regulates glioma stem cell growth in a p53-dependent manner.

    PubMed

    Gu, Chunyu; Banasavadi-Siddegowda, Yeshavanth K; Joshi, Kaushal; Nakamura, Yuko; Kurt, Habibe; Gupta, Snehalata; Nakano, Ichiro

    2013-05-01

    Accumulated evidence suggests that glioma stem cells (GSCs) may contribute to therapy resistance in high-grade glioma (HGG). Although recent studies have shown that the serine/threonine kinase maternal embryonic leucine-zipper kinase (MELK) is abundantly expressed in various cancers, the function and mechanism of MELK remain elusive. Here, we demonstrate that MELK depletion by shRNA diminishes the growth of GSC-derived mouse intracranial tumors in vivo, induces glial fibrillary acidic protein (+) glial differentiation of GSCs leading to decreased malignancy of the resulting tumors, and prolongs survival periods of tumor-bearing mice. Tissue microarray analysis with 91 HGG tumors demonstrates that the proportion of MELK (+) cells is a statistically significant indicator of postsurgical survival periods. Mechanistically, MELK is regulated by the c-Jun NH(2)-terminal kinase (JNK) signaling and forms a complex with the oncoprotein c-JUN in GSCs but not in normal progenitors. MELK silencing induces p53 expression, whereas p53 inhibition induces MELK expression, indicating that MELK and p53 expression are mutually exclusive. Additionally, MELK silencing-mediated GSC apoptosis is partially rescued by both pharmacological p53 inhibition and p53 gene silencing, indicating that MELK action in GSCs is p53 dependent. Furthermore, irradiation of GSCs markedly elevates MELK mRNA and protein expression both in vitro and in vivo. Clinically, recurrent HGG tumors following the failure of radiation and chemotherapy exhibit a statistically significant elevation of MELK protein compared with untreated newly diagnosed HGG tumors. Together, our data indicate that GSCs, but not normal cells, depend on JNK-driven MELK/c-JUN signaling to regulate their survival, maintain GSCs in an immature state, and facilitate tumor radioresistance in a p53-dependent manner. PMID:23339114

  20. An alternative model of H ferritin promoter transactivation by c-Jun.

    PubMed Central

    Faniello, Maria C; Chirico, Giuseppa; Quaresima, Barbara; Cuda, Giovanni; Allevato, Giovanna; Bevilacqua, Maria A; Baudi, Francesco; Colantuoni, Vittorio; Cimino, Filiberto; Venuta, Salvatore; Avvedimento, Vittorio E; Costanzo, Francesco

    2002-01-01

    c-Jun is a member of the activator protein 1 family, and its interaction with different nuclear factors generates a wide spectrum of complexes that regulate transcription of different promoters. H ferritin promoter transcription is tightly dependent on nuclear factor Y (NFY). Ferritin transcription is activated by c-Jun, although the promoter does not contain a canonical binding site. NFY, on the other hand, does not bind c-Jun in vitro, whereas in vivo c-Jun is found in the complex containing NFY. Moreover, a c-Jun-GCN4 chimaeric construct containing only the transactivation domain of Jun and the basic-region leucine-zipper domain of GCN4 stimulates the H ferritin promoter. A synthetic GAL4 promoter and the cognate activator, the fusion protein NFY-GAL4, are potently activated by c-Jun. Titration of p300 by co-expressing E1A abolishes the stimulatory effect. Moreover, another p300-dependent promoter, the cAMP-response element, can be superactivated by c-Jun using the same mechanism. These data indicate that c-Jun, when activated or overexpressed, is recruited to the H ferritin promoter by p300, which links NFY, bound to DNA, to the complex. These results add a new level of complexity to transcriptional regulation by c-Jun, which can activate p300-dependent promoters without binding directly to the target DNA. PMID:11903046

  1. COP1 and GSK3β Cooperate to Promote c-Jun Degradation and Inhibit Breast Cancer Cell Tumorigenesis12

    PubMed Central

    Shao, Jing; Teng, Yong; Padia, Ravi; Hong, Sungguan; Noh, Hyangsoon; Xie, Xiayang; Mumm, Jeff S; Dong, Zheng; Ding, Han-Fei; Cowell, John; Kim, Jaejik; Han, Jiahuai; Huang, Shuang

    2013-01-01

    High abundance of c-Jun is detected in invasive breast cancer cells and aggressive breast tumor malignancies. Here, we demonstrate that a major cause of high c-Jun abundance in invasive breast cancer cells is prolonged c-Jun protein stability owing to poor poly-ubiquitination of c-Jun. Among the known c-Jun-targeting E3 ligases, we identified constitutive photomorphogenesis protein 1 (COP1) as an E3 ligase responsible for c-Jun degradation in less invasive breast cancer cells because depletion of COP1 reduced c-Jun poly-ubiquitination leading to the stabilization of c-Jun protein. In a panel of breast cancer cell lines, we observed an inverse association between the levels of COP1 and c-Jun. However, overexpressing COP1 alone was unable to decrease c-Jun level in invasive breast cancer cells, indicating that efficient c-Jun protein degradation necessitates an additional event. Indeed, we found that glycogen synthase kinase 3 (GSK3) inhibitors elevated c-Jun abundance in less invasive breast cancer cells and that GSK3β nonphosphorylable c-Jun-T239A mutant displayed greater protein stability and poorer poly-ubiquitination compared to the wild-type c-Jun. The ability of simultaneously enforced expression of COP1 and constitutively active GSK3β to decrease c-Jun abundance in invasive breast cancer cells allowed us to conclude that c-Jun is negatively regulated through the coordinated action of COP1 and GSK3β. Importantly, co-expressing COP1 and active GSK3β blocked in vitro cell growth/migration and in vivo metastasis of invasive breast cancer cells. Gene expression profiling of breast tumor specimens further revealed that higher COP1 expression correlated with better recurrence-free survival. Our study supports the notion that COP1 is a suppressor of breast cancer progression. PMID:24027432

  2. c-Jun and Ets2 proteins regulate expression of spleen tyrosine kinase in T cells.

    PubMed

    Ghosh, Debjani; Tsokos, George C; Kyttaris, Vasileios C

    2012-04-01

    Effector T cells and T cells from patients with systemic lupus erythematosus (SLE) express increased levels of the spleen tyrosine kinase (Syk). Syk binds to the T cell receptor (TCR)-CD3 complex and transduces the TCR-mediated signal in the cell more efficiently than the canonical CD3ζ chain. The reasons for the increased expression of Syk are unclear. In the present study, we found that Syk is regulated by the transcription factor c-Jun in cooperation with Ets2. c-Jun and Ets2 bound to the SYK promoter in close proximity and increased the promoter activity in a specific manner. Disruption of c-Jun and Ets2 expression by siRNA resulted in decreased expression of Syk. Overexpression of c-Jun but not Ets2 resulted in increase in Syk protein. c-Jun and Ets2 co-immunoprecipitated and had an additive effect on Syk expression. c-Jun-driven SYK promoter activation showed a similar pattern in B cells; however, as expected, basal promoter activity was much higher in B cells as compared with T cells. Overexpression of c-Jun led to increase in intracytoplasmic calcium flux following TCR stimulation. Moreover, we found that SLE T cells had increased levels of c-Jun at baseline and phosphorylated c-Jun upon activation. Finally, disruption of c-Jun and Ets2 in SLE T cells resulted in a decrease in calcium flux upon TCR stimulation. In conclusion, c-Jun in cooperation with Ets2 increases the expression of Syk and contributes to Syk-mediated heightened calcium responses in SLE T cells. PMID:22354960

  3. Rs6295 promoter variants of the serotonin type 1A receptor are differentially activated by c-Jun in vitro and correlate to transcript levels in human epileptic brain tissue.

    PubMed

    Pernhorst, Katharina; van Loo, Karen M J; von Lehe, Marec; Priebe, Lutz; Cichon, Sven; Herms, Stefan; Hoffmann, Per; Helmstaedter, Christoph; Sander, Thomas; Schoch, Susanne; Becker, Albert J

    2013-03-01

    Many brain disorders, including epilepsy, migraine and depression, manifest with episodic symptoms that may last for various time intervals. Transient alterations of neuronal function such as related to serotonin homeostasis generally underlie this phenomenon. Several nucleotide polymorphisms (SNPs) in gene promoters associated with these diseases have been described. For obvious reasons, their regulatory roles on gene expression particularly in human brain tissue remain largely enigmatic. The rs6295 G-/C-allelic variant is located in the promoter region of the human HTR1a gene, encoding the G-protein-coupled receptor for 5-hydroxytryptamine (5HT1AR). In addition to reported transcriptional repressor binding, our bioinformatic analyses predicted a reduced binding affinity of the transcription factor (TF) c-Jun for the G-allele. In vitro luciferase transfection assays revealed c-Jun to (a) activate the rs6295 C- significantly stronger than the G-allelic variant and (b) antagonize efficiently the repressive effect of Hes5 on the promoter. The G-allele of rs6295 is known to be associated with aspects of major depression and migraine. In order to address a potential role of rs6295 variants in human brain tissue, we have isolated DNA and mRNA from fresh frozen hippocampal tissue of pharmacoresistant temporal lobe epilepsy (TLE) patients (n=140) after epilepsy surgery for seizure control. We carried out SNP genotyping studies and mRNA analyses in order to determine HTR1a mRNA expression in human hippocampal samples stratified according to the rs6295 allelic variant. The mRNA expression of HTR1a was significantly more abundant in hippocampal mRNA of TLE patients homozygous for the rs6295 C-allele as compared to those with the GG-genotype. These data may point to a novel, i.e., rs6295 allelic variant and c-Jun dependent transcriptional 5HT1AR 'receptoropathy'. PMID:23333373

  4. Low dose hydroxylated PCB induces c-Jun expression in PC12 cells.

    PubMed

    Shimokawa, Noriaki; Miyazaki, Wataru; Iwasaki, Toshiharu; Koibuchi, Noriyuki

    2006-03-01

    Polychlorinated biphenyls (PCBs) are known as environmental pollutants that may cause adverse health problems. Recently, accumulating evidence shows that PCBs express neurotoxicity through alteration of gene expression and signal transduction. On the other hand, c-Jun, a component of AP-1, is likely to coordinate transcription programs in response to various extracellular signals. However, little is known about the effects of PCBs on c-Jun expression. Here we investigated the expression of c-Jun in response to PCB. PC12 cells were incubated with hydroxylated PCB (4(OH)-2',3,3',4',5'-penta chlorobiphenyl, OH-PCB) at a final concentration from 10(-8) to 10(-5)M. The level of c-Jun expression was increased by OH-PCB at relatively low-dose; concentration of OH-PCB at 10(-8)M and 10(-7)M produced a 2.4- and 3.5-fold increase of c-Jun expression in respectively, compared with the values without OH-PCB treatment. Thyroid hormone (T3) did not induce such c-Jun expression, indicating that the effect of OH-PCB is not mediated through thyroid hormone signaling pathway. OH-PCB also enhanced phosphorylation of c-Jun NH2-terminal kinases. To determine whether the activation of Ca2+ channel is involved in the OH-PCB-induced c-Jun expression, we examined it using a L-type voltage-gated Ca2+ channel blocker nimodipine. Nimodipine partially inhibited OH-PCB-induced c-Jun expression by 50%. Moreover, Na+ channel antagonist tetrodotoxin inhibited OH-PCB-induced c-Jun expression completely. Taken together, our results indicate that exposure to OH-PCB induces c-Jun expression, and the response may be triggered by depolarization of a plasma membrane via Na+ influx, followed by Ca2+ influx partially through voltage-gated Ca2+ channels. PMID:16300829

  5. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation.

    PubMed

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F; Wahl, Markus C

    2015-12-15

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  6. The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

    PubMed Central

    Absmeier, Eva; Wollenhaupt, Jan; Mozaffari-Jovin, Sina; Becke, Christian; Lee, Chung-Tien; Preussner, Marco; Heyd, Florian; Urlaub, Henning; Lührmann, Reinhard; Santos, Karine F.; Wahl, Markus C.

    2015-01-01

    The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation. PMID:26637280

  7. c-Jun regulates adipocyte differentiation via the KLF15-mediated mode.

    PubMed

    Lee, Da Som; Choi, Hyeonjin; Han, Baek Soo; Kim, Won Kon; Lee, Sang Chul; Oh, Kyoung-Jin; Bae, Kwang-Hee

    2016-01-15

    Abnormal adipocyte differentiation is implicated in the development of metabolic disorders such as obesity and type II diabetes. Thus, an in-depth understanding of the molecular mechanisms associated with adipocyte differentiation is the first step in overcoming obesity and its related metabolic diseases. Here, we examined the role of c-Jun as a transcription factor in adipocyte differentiation. c-Jun overexpression in murine 3T3-L1 preadipocytes significantly inhibited adipocyte differentiation. In addition, the expression level of KLF15, an upstream effector of the key adipogenic factors C/EBPα and PPARγ, was decreased upon the ectopic expression of c-Jun. We found that c-Jun inhibited basal and glucocorticoid receptor (GR)-induced promoter activities of KLF15. c-Jun directly bound near the glucocorticoid response element (GRE) sites in the KLF15 promoter and inhibited adjacent promoter occupancies of GR. Furthermore, the restoration of KLF15 expression in 3T3-L1 cells with the stable ectopic expression of c-Jun partially rescued adipocyte differentiation. Our results demonstrate that c-Jun can suppress adipocyte differentiation through the down-regulation of KLF15 at the transcriptional level. This study proposes a novel mechanism by which c-Jun regulates adipocyte differentiation. PMID:26692489

  8. A new tumour suppression mechanism by p27Kip1: EGFR down-regulation mediated by JNK/c-Jun pathway inhibition

    PubMed Central

    Fang, Yong; Wang, Yihong; Wang, Yulei; Meng, Yan; Zhu, Junlan; Jin, Honglei; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wu, Xue-Ru; Huang, Chuanshu

    2014-01-01

    p27Kip1 is a potent inhibitor of cyclin-dependent kinases that drive G1-to-S cell-cycle transition. Reduced p27Kip1 expression is prevalent in a wide range of human tumours; however, the exact mechanism(s) of p27Kip1-mediated tumour suppression remains obscure. In the present study, we identified a close inverse relationship between p27Kip1 and EGFR (epidermal growth factor receptor) expression: the parental T24 human bladder cancer cells had high p27Kip1 expression but low EGFR expression and, in striking contrast, the metastatic derivative of T24 (T24T) had low p27Kip1 expression but high EGFR expression. This relationship was also found in various human cancer tissues, and was not only just correlative but also causal; depletion of p27Kip1 in MEF (mouse embryonic fibroblast) cells resulted in markedly elevated EGFR expression, a result reproducible with an Egfr promoter-luciferase reporter in both T24 and MEF cells, suggesting transcriptional repression of EGFR by p27Kip1. Indeed, p27Kip1 was found to regulate EGFR expression via the JNK (c-Jun N-terminal kinase)/c-Jun transcription factor: p27Kip1 deficiency activated JNK/c-Jun, whereas inhibition of JNK/c-Jun by dominant-negative mutants dramatically repressed Egfr transcription. Furthermore, the proximal promoter of the Egfr gene was crucial for its transcription, where the recruiting activity of c-Jun was much greater in p27Kip1−/− cells than in p27Kip1+/+ cells. Introduction of GFP–p27Kip1 into T24T cells suppressed JNK/c-Jun activation, EGFR expression and anchorage-independent growth. The results of the present study demonstrate that p27Kip1 suppresses JNK/c-Jun activation and EGFR expression in MEFs and human bladder cancer cells, and the results obtained are consistent with those from human cancer specimens. The present study provides new insights into p27Kip1 suppression of cancer cell growth, migration and metastasis. PMID:25121353

  9. The c-Jun-induced transformation process involves complex regulation of tenascin-C expression.

    PubMed Central

    Mettouchi, A; Cabon, F; Montreau, N; Dejong, V; Vernier, P; Gherzi, R; Mercier, G; Binétruy, B

    1997-01-01

    In cooperation with an activated ras oncogene, the site-dependent AP-1 transcription factor c-Jun transforms primary rat embryo fibroblasts (REF). Although signal transduction pathways leading to activation of c-Jun proteins have been extensively studied, little is known about c-Jun cellular targets. We identified c-Jun-upregulated cDNA clones homologous to the tenascin-C gene by differential screening of a cDNA library from REF. This tightly regulated gene encodes a rare extracellular matrix protein involved in cell attachment and migration and in the control of cell growth. Transient overexpression of c-Jun induced tenascin-C expression in primary REF and in FR3T3, an established fibroblast cell line. Surprisingly, tenascin-C synthesis was repressed after stable transformation by c-Jun compared to that in the nontransformed parental cells. As assessed by using the tenascin-C (-220 to +79) promoter fragment cloned in a reporter construct, the c-Jun-induced transient activation is mediated by two binding sites: one GCN4/AP-1-like site, at position -146, and one NF-kappaB site, at position -210. Furthermore, as demonstrated by gel shift experiments and cotransfections of the reporter plasmid and expression vectors encoding the p65 subunit of NF-kappaB and c-Jun, the two transcription factors bind and synergistically transactivate the tenascin-C promoter. We previously described two other extracellular matrix proteins, SPARC and thrombospondin-1, as c-Jun targets. Thus, our results strongly suggest that the regulation of the extracellular matrix composition plays a central role in c-Jun-induced transformation. PMID:9154819

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

    PubMed Central

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

    2007-01-01

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

  11. Synaptonuclear messenger PRR7 inhibits c-Jun ubiquitination and regulates NMDA-mediated excitotoxicity.

    PubMed

    Kravchick, Dana O; Karpova, Anna; Hrdinka, Matous; Lopez-Rojas, Jeffrey; Iacobas, Sanda; Carbonell, Abigail U; Iacobas, Dumitru A; Kreutz, Michael R; Jordan, Bryen A

    2016-09-01

    Elevated c-Jun levels result in apoptosis and are evident in neurodegenerative disorders such as Alzheimer's disease and dementia and after global cerebral insults including stroke and epilepsy. NMDA receptor (NMDAR) antagonists block c-Jun upregulation and prevent neuronal cell death following excitotoxic insults. However, the molecular mechanisms regulating c-Jun abundance in neurons are poorly understood. Here, we show that the synaptic component Proline rich 7 (PRR7) accumulates in the nucleus of hippocampal neurons following NMDAR activity. We find that PRR7 inhibits the ubiquitination of c-Jun by E3 ligase SCF(FBW) (7) (FBW7), increases c-Jun-dependent transcriptional activity, and promotes neuronal death. Microarray assays show that PRR7 abundance is directly correlated with transcripts associated with cellular viability. Moreover, PRR7 knockdown attenuates NMDAR-mediated excitotoxicity in neuronal cultures in a c-Jun-dependent manner. Our results show that PRR7 links NMDAR activity to c-Jun function and provide new insights into the molecular processes that underlie NMDAR-dependent excitotoxicity. PMID:27458189

  12. Design, synthesis and aphicidal activity of N-terminal modified insect kinin analogs.

    PubMed

    Zhang, Chuanliang; Qu, Yanyan; Wu, Xiaoqing; Song, Dunlun; Ling, Yun; Yang, Xinling

    2015-06-01

    The insect kinins are a class of multifunctional insect neuropeptides present in a diverse variety of insects. Insect kinin analogs showed multiple bioactivities, especially, the aphicidal activity. To find a biostable and bioactive insecticide candidate with simplified structure, a series of N-terminal modified insect kinin analogs was designed and synthesized based on the lead compound [Aib]-Phe-Phe-[Aib]-Trp-Gly-NH2. Their aphicidal activity against the soybean aphid Aphis glycines was evaluated. The results showed that all the analogs maintained the aphicidal activity. In particular, the aphicidal activity of the pentapeptide analog X Phe-Phe-[Aib]-Trp-Gly-NH2 (LC50=0.045mmol/L) was similar to the lead compound (LC50=0.048mmol/L). This indicated that the N-terminal protective group may not play an important role in the activity and the analogs structure could be simplified to pentapeptide analogs while retaining good aphicidal activity. The core pentapeptide analog X can be used as the lead compound for further chemical modifications to discover potential insecticides. PMID:25116632

  13. The bZIP repressor proteins, c-Jun dimerization protein 2 and activating transcription factor 3, recruit multiple HDAC members to the ATF3 promoter.

    PubMed

    Darlyuk-Saadon, Ilona; Weidenfeld-Baranboim, Keren; Yokoyama, Kazunari K; Hai, Tsonwin; Aronheim, Ami

    2012-01-01

    JDP2, is a basic leucine zipper (bZIP) protein displaying a high degree of homology with the stress inducible transcription factor, ATF3. Both proteins bind to cAMP and TPA response elements and repress transcription by multiple mechanisms. Histone deacetylases (HDACs) play a key role in gene inactivation by deacetylating lysine residues on histones. Here we describe the association of JDP2 and ATF3 with HDACs 1, 2-6 and 10. Association of HDAC3 and HDAC6 with JDP2 and ATF3 occurs via direct protein-protein interactions. Only part of the N-terminal bZIP motif of JDP2 and ATF3 basic domain is necessary and sufficient for the interaction with HDACs in a manner that is independent of coiled-coil dimerization. Class I HDACs associate with the bZIP repressors via the DAC conserved domain whereas the Class IIb HDAC6 associates through its C-terminal unique binder of ubiquitin Zn finger domain. Both JDP2 and ATF3 are known to bind and repress the ATF3 promoter. MEF cells treated with histone deacetylase inhibitor, trichostatin A (TSA) display enhanced ATF3 transcription. ATF3 enhanced transcription is significantly reduced in MEF cells lacking both ATF3 and JDP2. Collectively, we propose that the recruitment of multiple HDAC members to JDP2 and ATF3 is part of their transcription repression mechanism. PMID:22989952

  14. The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy

    PubMed Central

    Lukey, Michael J.; Greene, Kai Su; Erickson, Jon W.; Wilson, Kristin F.; Cerione, Richard A.

    2016-01-01

    Many transformed cells exhibit altered glucose metabolism and increased utilization of glutamine for anabolic and bioenergetic processes. These metabolic adaptations, which accompany tumorigenesis, are driven by oncogenic signals. Here we report that the transcription factor c-Jun, product of the proto-oncogene JUN, is a key regulator of mitochondrial glutaminase (GLS) levels. Activation of c-Jun downstream of oncogenic Rho GTPase signalling leads to elevated GLS gene expression and glutaminase activity. In human breast cancer cells, GLS protein levels and sensitivity to GLS inhibition correlate strongly with c-Jun levels. We show that c-Jun directly binds to the GLS promoter region, and is sufficient to increase gene expression. Furthermore, ectopic overexpression of c-Jun renders breast cancer cells dependent on GLS activity. These findings reveal a role for c-Jun as a driver of cancer cell metabolic reprogramming, and suggest that cancers overexpressing JUN may be especially sensitive to GLS-targeted therapies. PMID:27089238

  15. N-terminal domain-mediated homodimerization is required for photoreceptor activity of Arabidopsis CRYPTOCHROME 1.

    PubMed

    Sang, Yi; Li, Qing-Hua; Rubio, Vicente; Zhang, Yan-Chun; Mao, Jian; Deng, Xing-Wang; Yang, Hong-Quan

    2005-05-01

    Cryptochromes (CRY) are blue light receptors that share sequence similarity with photolyases, flavoproteins that catalyze the repair of UV light-damaged DNA. Transgenic Arabidopsis thaliana seedlings expressing the C-terminal domains of the Arabidopsis CRY fused to beta-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype, indicating that the signaling mechanism of Arabidopsis CRY is mediated through the C-terminal domain. The role of the Arabidopsis CRY N-terminal photolyase-like domain in CRY action remains poorly understood. Here, we report the essential role of the Arabidopsis CRY1 N-terminal domain (CNT1) in the light activation of CRY1 photoreceptor activity. Yeast two-hybrid assay, in vitro binding, in vivo chemical cross-linking, gel filtration, and coimmunoprecipitation studies indicate that CRY1 homodimerizes in a light-independent manner. Mutagenesis and transgenic studies demonstrate that CNT1-mediated dimerization is required for light activation of the C-terminal domain of CRY1 (CCT1). Transgenic data and native gel electrophoresis studies suggest that multimerization of GUS is both responsible and required for mediating a COP phenotype on fusion to CCT1. These results indicate that the properties of the GUS multimer are analogous to those of the light-modified CNT1 dimer. Irradiation with blue light modifies the properties of the CNT1 dimer, resulting in a change in CCT1, activating CCT1, and eventually triggering the CRY1 signaling pathway. PMID:15805487

  16. Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following excitotoxic damage in vivo.

    PubMed

    Cardoso, A L C; Costa, P; de Almeida, L P; Simões, S; Plesnila, N; Culmsee, C; Wagner, E; de Lima, M C Pedroso

    2010-03-19

    Excitotoxicity is one of the main features responsible for neuronal cell death after acute brain injury and in several neurodegenerative disorders, for which only few therapeutic options are currently available. In this work, RNA interference was employed to identify and validate a potential target for successful treatment of excitotoxic brain injury, the transcription factor c-Jun. The nuclear translocation of c-Jun and its upregulation are early events following glutamate-induced excitotoxic damage in primary neuronal cultures. We present evidence for the efficient knockdown of this transcription factor using a non-viral vector consisting of cationic liposomes associated to transferrin (Tf-lipoplexes). Tf-lipoplexes were able to deliver anti-c-Jun siRNAs to neuronal cells in culture, resulting in efficient silencing of c-Jun mRNA and protein and in a significant decrease of cell death following glutamate-induced damage or oxygen-glucose deprivation. This formulation also leads to a significant c-Jun knockdown in the mouse hippocampus in vivo, resulting in the attenuation of both neuronal death and inflammation following kainic acid-mediated lesion of this region. Furthermore, a strong reduction of seizure activity and cytokine production was observed in animals treated with anti-c-Jun siRNAs. These findings demonstrate the efficient delivery of therapeutic siRNAs to the brain by Tf-lipoplexes and validate c-Jun as a promising therapeutic target in neurodegenerative disorders involving excitotoxic lesions. PMID:19913061

  17. c-Jun NH2-Terminal Kinase Activity in Subcutaneous Adipose Tissue but Not Nuclear Factor-κB Activity in Peripheral Blood Mononuclear Cells Is an Independent Determinant of Insulin Resistance in Healthy Individuals

    PubMed Central

    Sourris, Karly C.; Lyons, Jasmine G.; de Courten, Maximilian P.J.; Dougherty, Sonia L.; Henstridge, Darren C.; Cooper, Mark E.; Hage, Michelle; Dart, Anthony; Kingwell, Bronwyn A.; Forbes, Josephine M.; de Courten, Barbora

    2009-01-01

    OBJECTIVE Chronic low-grade activation of the immune system (CLAIS) predicts type 2 diabetes via a decrease in insulin sensitivity. Our study investigated potential relationships between nuclear factor-κB (NF-κB) and c-Jun NH2-terminal kinase (JNK) pathways—two pathways proposed as the link between CLAIS and insulin resistance. RESEARCH DESIGN AND METHODS Adiposity (dual-energy X-ray absorptiometry), waist-to-hip ratio (WHR), and insulin sensitivity (M, hyperinsulinemic-euglycemic clamp) were measured in 22 healthy nondiabetic volunteers (aged 29 ± 11 years, body fat 28 ± 11%). NF-κB activity (DNA-binding assay) and JNK1/2 activity (phosphorylated JNK) were assessed in biopsies of the vastus lateralis muscle and subcutaneous adipose tissue and in peripheral blood mononuclear cell (PBMC) lysates. RESULTS NF-κB activities in PBMCs and muscle were positively associated with WHR after adjustment for age, sex, and percent body fat (both P < 0.05). NF-κB activity in PBMCs was inversely associated with M after adjustment for age, sex, percent body fat, and WHR (P = 0.02) and explained 16% of the variance of M. There were no significant relationships between NF-κB activity and M in muscle or adipose tissue (both NS). Adipose-derived JNK1/2 activity was not associated with obesity (all P> 0.1), although it was inversely related to M (r = −0.54, P < 0.05) and explained 29% of its variance. When both NF-κB and JNK1/2 were examined statistically, only JNK1/2 activity in adipose tissue was a significant determinant of insulin resistance (P = 0.02). CONCLUSIONS JNK1/2 activity in adipose tissue but not NF-κB activity in PBMCs is an independent determinant of insulin resistance in healthy individuals. PMID:19258436

  18. c-Jun localizes to the nucleus independent of its phosphorylation by and interaction with JNK and vice versa promotes nuclear accumulation of JNK

    SciTech Connect

    Schreck, Ilona; Al-Rawi, Marco; Mingot, Jose-Manuel; Scholl, Christine; Diefenbacher, Markus Elmar; O'Donnell, Paul; Bohmann, Dirk; Weiss, Carsten

    2011-04-22

    Highlights: {yields} HSP70, Ku70 and 80 as well as importin 8 are novel interactors of c-Jun. {yields} Nuclear accumulation of c-Jun does not require its functions as a transcription factor. {yields} Nuclear accumulation of c-Jun does not require the interaction with its kinase JNK. {yields} Nuclear accumulation of JNK is regulated by interaction with c-Jun. -- Abstract: In order to activate gene expression, transcription factors such as c-Jun have to reside in the nucleus. The abundance of c-Jun in the nucleus correlates with the activity of its target genes. As a consequence of excessive c-Jun activation, cells undergo apoptosis or changes in differentiation whereas decreased c-Jun function can reduce proliferation. In the present study we addressed how nuclear accumulation of the transcription factor c-Jun is regulated. First, we analyzed which functions of c-Jun are required for efficient nuclear accumulation. Mutants of c-Jun deficient in dimerization or DNA-binding show no defect in nuclear transport. Furthermore, c-Jun import into the nucleus of living cells occurred when the c-Jun phosphorylation sites were mutated as well in cells that lack the major c-Jun kinase, JNK, suggesting that c-Jun transport into the nucleus does not require JNK signaling. Conversely, however, binding of c-Jun seemed to enhance nuclear accumulation of JNK. In order to identify proteins that might be relevant for the nuclear translocation of c-Jun we searched for novel binding partners by a proteomic approach. In addition to the heat shock protein HSP70 and the DNA damage repair factors Ku70 and 80, we isolated human importin 8 as a novel interactor of c-Jun. Interaction of Imp 8 with c-Jun in human cells was confirmed by co-immunoprecipitation experiments. Nuclear accumulation of c-Jun does not require its functions as a transcription factor or the interaction with its kinase JNK. Interestingly, nuclear accumulation of JNK is regulated by interaction with c-Jun. Unraveling the

  19. Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH{sub 2}-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

    SciTech Connect

    Miyata, Yoshiki; Sato, Takashi . E-mail: satotak@ps.toyaku.ac.jp; Ito, Akira

    2005-11-04

    Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH{sub 2}-terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation.

  20. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    PubMed

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  1. Cellular transformation and malignancy induced by ras require c-jun.

    PubMed Central

    Johnson, R; Spiegelman, B; Hanahan, D; Wisdom, R

    1996-01-01

    ras is an important oncogene in experimental animals and humans. In addition, activated ras proteins are potent inducers of the transcription factor AP-1, which is composed of heterodimeric complexes of Fos and Jun proteins. Together with the fact that deregulated expression of some AP-1 proteins can cause neoplastic transformation, this finding suggests that AP-1 may function as a critical ras effector. We have tested this hypothesis directly by analyzing the response to activated ras in cells that harbor a null mutation in the c-jun gene. The transcriptional response of AP-1-responsive genes to activated ras is severely impaired in c-jun null fibroblasts. Compared with wild-type cells, the c-jun null cells lack many characteristics of ras transformation, including loss of contact inhibition, anchorage independence, and tumorigenicity in nude mice; these properties are restored by forced expression of c-jun. Rare tumorigenic variants of ras-expressing c-jun null fibroblasts do arise. Analysis of these variants reveals a consistent restoration of AP-1 activity. The results provide genetic evidence that c-jun is a crucial effector for transformation by activated ras proteins. PMID:8754851

  2. Persistent induction of c-fos and c-jun expression by asbestos

    SciTech Connect

    Heintz, N.H.; Mossman, B.T. ); Janssen, Y.M. Univ. of Limburg, Maastricht )

    1993-04-15

    To investigate the mechanisms of asbestos-induced carcinogenesis, expression of c-fos and c-jun protooncogenes was examined in rat pleural mesothelial cells and hamster tracheal epithelial cells after exposure to crocidolite or chrysotile asbestos. In contrast to phorbol 12-myristate 13-acetate, which induces rapid and transient increases in c-fos and c-jun mRNA, asbestos causes 2- to 5-fold increases in c-fos and c-jun mRNA that persist for at least 24 hr in mesothelial cells. The induction of c-fos and c-jun mRNA by asbestos in mesothelial cells is dose-dependent and is most pronounced with crocidolite, the type of asbestos most pathogenic in the causation of pleural mesothelioma. Induction of c-jun gene expression by asbestos occurs in tracheal epithelial cells but is not accompanied by a corresponding induction of c-fos gene expression. In both cell types, asbestos induces increases in protein factors that bind specifically to the DNA sites that mediate gene expression by the AP-1 family of transcription factors. The persistent induction of AP-1 transcription factors by asbestos suggests a model of asbestos-induced carcinogenesis involving chronic stimulation of cell proliferation through activation of the early response gene pathway that includes c-jun and/or c-fos. 30 refs., 5 figs.

  3. Activations of c-fos/c-jun signaling are involved in the modulation of hypothalamic superoxide dismutase (SOD) and neuropeptide Y (NPY) gene expression in amphetamine-mediated appetite suppression

    SciTech Connect

    Hsieh, Y.-S.; Yang, S.-F.; Chiou, H.-L.; Kuo, D.-Y. . E-mail: dykuo@csmu.edu.tw

    2006-04-15

    Amphetamine (AMPH) is known as an anorectic agent. The mechanism underlying the anorectic action of AMPH has been attributed to its inhibitory action on hypothalamic neuropeptide Y (NPY), an appetite stimulant in the brain. This study was aimed to examine the molecular mechanisms behind the anorectic effect of AMPH. Results showed that AMPH treatment decreased food intake, which was correlated with changes of NPY mRNA level, but increased c-fos, c-jun and superoxide dismutase (SOD) mRNA levels in hypothalamus. To determine if c-fos or c-jun was involved in the anorectic response of AMPH, infusions of antisense oligonucleotide into the brain were performed at 1 h before daily AMPH treatment in freely moving rats, and the results showed that c-fos or c-jun knockdown could block this anorectic response and restore NPY mRNA level. Moreover, c-fos or c-jun knockdown could partially block SOD mRNA level that might involve in the modulation of NPY gene expression. It was suggested that c-fos/c-jun signaling might involve in the central regulation of AMPH-mediated feeding suppression via the modulation of NPY gene expression.

  4. Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide.

    PubMed

    Coast, Geoffrey M; Zabrocki, Janusz; Nachman, Ronald J

    2002-04-01

    Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH(2)) and N-terminal truncated analogs of 4-14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn(1), but was reduced > 10-fold after the removal of Thr(2). Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys(8)) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg(10), and at 100 microM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg(10) was confirmed by comparing dose-response curves for Musdo-K [6-15] and Acheta kinin-V (AFSHWG-NH(2)) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity. PMID:11897389

  5. Tissue-specific deletion of c-Jun in the pancreas has limited effects on pancreas formation

    SciTech Connect

    Yamamoto, Kaoru; Miyatsuka, Takeshi; Tanaka, Ayako; Toyoda, Shuichi; Kato, Ken; Shiraiwa, Toshihiko; Fujitani, Yoshio; Yamasaki, Yoshimitsu; Hori, Masatsugu; Matsuhisa, Munehide; Matsuoka, Taka-aki; Kaneto, Hideaki

    2007-11-30

    It is well known that activating protein-1 (AP-1) is involved in a variety of cellular functions such as proliferation, differentiation, apoptosis, and oncogenesis. AP-1 is a dimer complex consisting of different subunits, and c-Jun is known to be one of its major components. In addition, it has been shown that mice lacking c-Jun are embryonic lethal and that c-Jun is essential for liver and heart development. However, the role of c-Jun in the pancreas is not well known. The aim of this study was to examine the possible role of c-Jun in the pancreas. First, c-Jun was strongly expressed in pancreatic duct-like structures at an embryonic stage, while a lower level of expression was observed in some part of the adult pancreas, implying that c-Jun might play a role during pancreas development. Second, to address this point, we generated pancreas-specific c-Jun knock-out mice (Ptf1a-Cre; c-Jun{sup flox/flox} mice) by crossing Ptf1a-Cre knock-in mice with c-Jun floxed mice. Ptf1a is a pancreatic transcription factor and its expression is confined to pancreatic stem/progenitor cells, which give rise to all three types of pancreatic tissue: endocrine, exocrine, and duct. Contrary to our expectation, however, there was no morphological difference in the pancreas between Ptf1a-Cre; c-Jun{sup flox/flox} and control mice. In addition, there was no difference in body weight, pancreas weight, and the expression of various pancreas-related factors (insulin, glucagon, cytokeratin, and amylase) between the two groups. Furthermore, there was no difference in glucose tolerance between Ptf1a-Cre; c-Jun{sup flox/flox} and control mice. Taken together, although we cannot exclude the possibility that c-Jun ablation is compensated by some unknown factors, c-Jun appears to be dispensable for pancreas development at least after ptf1a gene promoter is activated.

  6. Interferon-{beta}-induced activation of c-Jun NH{sub 2}-terminal kinase mediates apoptosis through up-regulation of CD95 in CH31 B lymphoma cells

    SciTech Connect

    Takada, Eiko; Shimo, Kuniaki; Hata, Kikumi; Abiake, Maira; Mukai, Yasuo; Moriyama, Masami; Heasley, Lynn; Mizuguchi, Junichiro . E-mail: mizu@tokyo-med.ac.jp

    2005-04-01

    Type I interferon (IFN)-induced antitumor action is due in part to apoptosis, but the molecular mechanisms underlying IFN-induced apoptosis remain largely unresolved. In the present study, we demonstrate that IFN-{beta} induced apoptosis and the loss of mitochondrial membrane potential ({delta}{psi}m) in the murine CH31 B lymphoma cell line, and this was accompanied by the up-regulation of CD95, but not CD95-ligand (CD95-L), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL). Pretreatment with anti-CD95-L mAb partially prevented the IFN-{beta}-induced loss of {delta}{psi}m, suggesting that the interaction of IFN-{beta}-up-regulated CD95 with CD95-L plays a crucial role in the induction of fratricide. IFN-{beta} induced a sustained activation of c-Jun NH{sub 2}-terminal kinase 1 (JNK1), but not extracellular signal-regulated kinases (ERKs). The IFN-{beta}-induced apoptosis and loss of {delta}{psi}m were substantially compromised in cells overexpressing a dominant-negative form of JNK1 (dnJNK1), and it was slightly enhanced in cells carrying a constitutively active JNK construct, MKK7-JNK1 fusion protein. The IFN-{beta}-induced up-regulation of CD95 together with caspase-8 activation was also abrogated in the dnJNK1 cells while it was further enhanced in the MKK7-JNK1 cells. The levels of cellular FLIP (c-FLIP), competitively interacting with caspase-8, were down-regulated by stimulation with IFN-{beta} but were reversed by the proteasome inhibitor lactacystin. Collectively, the IFN-{beta}-induced sustained activation of JNK mediates apoptosis, at least in part, through up-regulation of CD95 protein in combination with down-regulation of c-FLIP protein.

  7. Impact of the N-Terminal Domain of STAT3 in STAT3-Dependent Transcriptional Activity.

    PubMed

    Hu, Tiancen; Yeh, Jennifer E; Pinello, Luca; Jacob, Jaison; Chakravarthy, Srinivas; Yuan, Guo-Cheng; Chopra, Rajiv; Frank, David A

    2015-10-01

    The transcription factor STAT3 is constitutively active in many cancers, where it mediates important biological effects, including cell proliferation, differentiation, survival, and angiogenesis. The N-terminal domain (NTD) of STAT3 performs multiple functions, such as cooperative DNA binding, nuclear translocation, and protein-protein interactions. However, it is unclear which subsets of STAT3 target genes depend on the NTD for transcriptional regulation. To identify such genes, we compared gene expression in STAT3-null mouse embryonic fibroblasts (MEFs) stably expressing wild-type STAT3 or STAT3 from which NTD was deleted. NTD deletion reduced the cytokine-induced expression of specific STAT3 target genes by decreasing STAT3 binding to their regulatory regions. To better understand the potential mechanisms of this effect, we determined the crystal structure of the STAT3 NTD and identified a dimer interface responsible for cooperative DNA binding in vitro. We also observed an Ni(2+)-mediated oligomer with an as yet unknown biological function. Mutations on both dimer and Ni(2+)-mediated interfaces affected the cytokine induction of STAT3 target genes. These studies shed light on the role of the NTD in transcriptional regulation by STAT3 and provide a structural template with which to design STAT3 NTD inhibitors with potential therapeutic value. PMID:26169829

  8. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    SciTech Connect

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  9. Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

    PubMed

    Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

    2014-01-01

    Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS. PMID:25500996

  10. The JNK/c-Jun signaling axis contributes to the TDP-43-induced cell death.

    PubMed

    Suzuki, Hiroaki; Matsuoka, Masaaki

    2013-01-01

    Dysregulation of transactive response DNA-binding protein-43 (TDP-43) is closely linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). The contribution of the upregulation of TDP-43 expression to the pathogenesis has been strongly suggested by the observation that the level of TDP-43 expression is increased in both ALS and FTLD-U patients. We previously found that the low-grade (twice to five times more than the endogenous level) overexpression of TDP-43 induces neuronal cell death through the upregulation of Bim and CHOP expression and the downregulation of Bcl-xL expression. In this study, we further show that the low-grade overexpression of TDP-43 increases the level of phosphorylated c-Jun N-terminal kinase (JNK) and the co-incubation with a JNK inhibitor, the expression of a dominant-negative JNK, or the expression of a dominant-negative c-Jun inhibited the TDP-43-induced death in NSC34 motor neuronal cells. These data together suggest that the JNK/c-Jun signaling axis contributes to the TDP-43-induced cell death. PMID:23001869

  11. c-Jun is required for the specification of joint cell fates.

    PubMed

    Kan, Akinori; Tabin, Clifford J

    2013-03-01

    Joints form within the developing skeleton through the segmentation and cavitation of initially continuous cartilage condensations. However, the molecular pathways controlling joint formation largely remain to be clarified. In particular, while several critical secreted signals have been identified, no transcription factors have yet been described as acting in the early stages of joint formation. Working upstream of the early joint marker Wnt9a, we found that the transcription factor c-Jun plays a pivotal role in specifying joint cell fates. We first identified an enhancer upstream of the Wnt9a gene driving joint-specific expression in transgenic reporter mice. A comprehensive in silico screen suggested c-Jun as a candidate transcription factor activating this Wnt9a enhancer element. c-Jun is specifically expressed in joints during embryonic joint development, and its conditional deletion from early limb bud mesenchyme in mice severely affects both initiation and subsequent differentiation of all limb joints. c-Jun directly regulates Wnt16 as well as Wnt9a during early stages of joint development, causing a decrease of canonical Wnt activity in the joint interzone. Postnatally, c-Jun-deficient mice show a range of joint abnormalities, including cartilaginous continuities between juxtaposed skeletal elements, irregular articular surfaces, and hypoplasia of ligaments. PMID:23475960

  12. Regulation of protein translation and c-Jun expression by prostate tumor overexpressed 1.

    PubMed

    Marqués, N; Sesé, M; Cánovas, V; Valente, F; Bermudo, R; de Torres, I; Fernández, Y; Abasolo, I; Fernández, P L; Contreras, H; Castellón, E; Celià-Terrassa, T; Méndez, R; Ramón Y Cajal, S; Thomson, T M; Paciucci, R

    2014-02-27

    Prostate tumor overexpressed-1 (PTOV1), a modulator of the Mediator transcriptional regulatory complex, is expressed at high levels in prostate cancer and other neoplasias in association with a more aggressive disease. Here we show that PTOV1 interacts directly with receptor of activated protein C kinase 1 (RACK1), a regulator of protein kinase C and Jun signaling and also a component of the 40S ribosome. Consistent with this interaction, PTOV1 was associated with ribosomes and its overexpression promoted global protein synthesis in prostate cancer cells and COS-7 fibroblasts in a mTORC1-dependent manner. Transfection of ectopic PTOV1 enhanced the expression of c-Jun protein without affecting the levels of c-Jun or RACK1 mRNA. Conversely, knockdown of PTOV1 caused significant declines in global protein synthesis and c-Jun protein levels. High levels of PTOV1 stimulated the motility and invasiveness of prostate cancer cells, which required c-Jun, whereas knockdown of PTOV1 strongly inhibited the tumorigenic and metastatic potentials of PC-3 prostate cancer cells. In human prostate cancer samples, the expression of high levels of PTOV1 in primary and metastatic tumors was significantly associated with increased nuclear localization of active c-Jun. These results unveil new functions of PTOV1 in the regulation of protein translation and in the progression of prostate cancer to an invasive and metastatic disease. PMID:23455324

  13. Targeting to Transcriptionally Active Loci by the Hydrophilic N-Terminal Domain of Drosophila DNA Topoisomerase I

    PubMed Central

    Shaiu, Wen-Ling; Hsieh, Tao-shih

    1998-01-01

    DNA topoisomerase I (topo I) from Drosophila melanogaster contains a nonconserved, hydrophilic N-terminal domain of about 430 residues upstream of the conserved core domains. Deletion of this N terminus did not affect the catalytic activity of topo I, while further removal of sequences into the conserved regions inactivated its enzymatic activity. We have investigated the cellular function of the Drosophila topo I N-terminal domain with top1-lacZ transgenes. There was at least one putative nuclear localization signal within the first 315 residues of the N-terminal domain that allows efficient import of the large chimeric proteins into Drosophila nuclei. The top1-lacZ fusion proteins colocalized with RNA polymerase II (pol II) at developmental puffs on the polytene chromosomes. Either topo I or the top1-lacZ fusion protein was colocalized with RNA pol II in some but not all of the nonpuff, interband loci. However, the fusion proteins as well as RNA pol II were recruited to heat shock puffs during heat treatment, and they returned to the developmental puffs after recovery from heat shock. By immunoprecipitation, we showed that two of the largest subunits of RNA pol II coprecipitated with the N-terminal 315-residue fusion protein by using antibodies against β-galactosidase. These data suggest that the topo I fusion protein can be localized to the transcriptional complex on chromatin and that the N-terminal 315 residues were sufficient to respond to cellular processes, especially during the reprogramming of gene expression. PMID:9632819

  14. c-Myc inhibits Ras-mediated differentiation of pheochromocytoma cells by blocking c-Jun up-regulation.

    PubMed

    Vaqué, José P; Fernández-García, Belén; García-Sanz, Pablo; Ferrandiz, Nuria; Bretones, Gabriel; Calvo, Fernando; Crespo, Piero; Marín, María C; León, Javier

    2008-02-01

    Although mutant Ras proteins were originally described as transforming oncoproteins, they induce growth arrest, senescence, and/or differentiation in many cell types. c-Myc is an oncogenic transcription factor that cooperates with Ras in cellular transformation and oncogenesis. However, the Myc-Ras relationship in cellular differentiation is largely unknown. Here, we have analyzed the effects of c-Myc on PC12-derived cells (UR61 cell line), harboring an inducible N-Ras oncogene. In these cells, Ras activation induces neuronal-like differentiation by a process involving c-Jun activation. We found that c-Myc inhibited Ras-mediated differentiation by a mechanism that involves the blockade of c-Jun induction in response to Ras signal. Accordingly, ectopically expressed c-Jun could bypass c-Myc impediment of Ras-induced differentiation and activator protein 1 activation. Interestingly, it did not rescue the proliferative arrest elicited by Ras and did not enhance the differentiation-associated apoptosis. The blockade of Ras-mediated induction of c-Jun takes place at the level of c-Jun proximal promoter. Mutational analysis revealed that c-Myc regions involved in DNA binding and transactivation are required to block differentiation and c-Jun induction. c-Myc does not seem to require Miz-1 to inhibit differentiation and block c-Jun induction. Furthermore, Max is not required for c-Myc activity, as UR61 cells lack a functional Max gene. c-Myc-inhibitory effect on the Ras/c-Jun connection is not restricted to UR61 cells as it can occur in other cell types as K562 or HEK293. In conclusion, we describe a novel interplay between c-Myc and c-Jun that controls the ability of Ras to trigger the differentiation program of pheochromocytoma cells. PMID:18314492

  15. Purification and antimicrobial activity studies of the N-terminal fragment of ubiquitin from human amniotic fluid.

    PubMed

    Kim, Jin-Young; Lee, Sun Young; Park, Seong-Cheol; Shin, Song Yub; Choi, Sang Joon; Park, Yoonkyung; Hahm, Kyung-Soo

    2007-09-01

    A 4.3-kDa antimicrobial peptide was isolated from human amniotic fluid by dialysis, ultrafiltration, and C18 reversed-phase high performance liquid chromatography. This peptide, which we named Amniotic Fluid Peptide-1 (AFP-1), possessed antimicrobial activity but lacked hemolytic activity. In addition, AFP-1 potently inhibited the growth of a variety of bacteria (Escherichia coli, Salmonella typhimurium, Listeria monocytogenes and Staphylococcus aureus), filamentous fungi (Botrytis cinerea, Aspergillus fumigatus, Neurospora crassa and Fusarium oxysporum) and yeast cells (Candida albicans and Cryptococcus neoformans). Automated Edman degradation showed that the N-terminal sequence of AFP-1 was NH(2)-Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-. The partial sequence had 100% homology to the N-terminal sequence of ubiquitin. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the molecular mass of AFP-1 was 4280.2 Da. Our data show an antimicrobial activity of ubiquitin N-terminal derived peptide that makes it suitable for use as an antimicrobial agent. PMID:17669700

  16. Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.

    PubMed Central

    Borel, F; Vincent, C; Leberman, R; Härtlein, M

    1994-01-01

    Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs. Images PMID:8065908

  17. Moracin M inhibits airway inflammation by interrupting the JNK/c-Jun and NF-κB pathways in vitro and in vivo.

    PubMed

    Lee, Ju Hee; Ko, Hae Ju; Woo, Eun-Rhan; Lee, Sang Kook; Moon, Bong Soo; Lee, Chan Woo; Mandava, Suresh; Samala, Mallesham; Lee, Jongkook; Kim, Hyun Pyo

    2016-07-15

    The therapeutic effectiveness of moracins as 2-arylbenzofuran derivatives against airway inflammation was examined. Moracin M, O, and R were isolated from the root barks of Morus alba, and they inhibited interleukin (IL)-6 production from IL-1β-treated lung epithelial cells (A549) at 101-00μM. Among them, moracin M showed the strongest inhibitory effect (IC50=8.1μM). Downregulation of IL-6 expression by moracin M was mediated by interrupting the c-Jun N-terminal kinase (JNK)/c-Jun pathway. Moracin derivatives inhibited inducible nitric oxide synthase (iNOS)-catalyzed NO production from lipopolysaccharide (LPS)-treated alveolar macrophages (MH-S) at 50-100μM. In particular, moracin M inhibited NO production by downregulating iNOS. When orally administered, moracin M (20-60mg/kg) showed comparable inhibitory action with dexamethasone (30mg/kg) against LPS-induced lung inflammation, acute lung injury, in mice with that of dexamethasone (30mg/kg). The action mechanism included interfering with the activation of nuclear transcription factor-κB in inflamed lungs. Therefore, it is concluded that moracin M inhibited airway inflammation in vitro and in vivo, and it has therapeutic potential for treating lung inflammatory disorders. PMID:27138708

  18. Function of steroidogenic factor 1 domains in nuclear localization, transactivation, and interaction with transcription factor TFIIB and c-Jun.

    PubMed

    Li, L A; Chiang, E F; Chen, J C; Hsu, N C; Chen, Y J; Chung, B C

    1999-09-01

    Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation. PMID:10478848

  19. Transcriptional Regulation of PES1 Expression by c-Jun in Colon Cancer

    PubMed Central

    Su, Yahui; Dong, Bin; Wu, Jian; Meng, Lin; Qu, Like; Shou, Chengchao

    2012-01-01

    Pescadillo is a nucleolar protein that has been suggested to be involved in embryonic development and ribosome biogenesis. Deregulated expression of human pescadillo (PES1) was described in some tumors, but its precise roles in tumorigenesis remains unclear. In this study, we generated three monoclonal antibodies recognizing PES1 with high specificity and sensitivity, with which PES1 expression in human colon cancer was analyzed immunohistochemically. Out of 265 colon cancer tissues, 89 (33.6%) showed positive PES1 expression, which was significantly higher than in non-cancerous tissues (P<0.001). Silencing of PES1 in colon cancer cells resulted in decreased proliferation, reduced growth of xenografts, and cell cycle arrest in G1 phase, indicating PES1 functions as an oncogene. We then explored the mechanism by which PES1 expression is controlled in human colon cancers and demonstrated that c-Jun, but not JunB, JunD, c-Fos, or mutant c-Jun, positively regulated PES1 promoter transcription activity. In addition, we mapped −274/−264 region of PES1 promoter as the c-Jun binding sequence, which was validated by chromatin immunoprecipitation and electrophoretic mobility shift assays. Moreover, we demonstrated a positive correlation between c-Jun and PES1 expression in colon cancer cells and colon cancer tissues. Upstream of c-Jun, it was revealed that c-Jun NH2-terminal kinases (JNK) is essential for controlling PES1 expression. Our study, in the first place, uncovers the oncogenic role of PES1 in colon cancer and elucidates the molecular mechanism directing PES1 expression. PMID:22860098

  20. The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

    PubMed Central

    Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A; Fromme, J Christopher

    2016-01-01

    The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth. DOI: http://dx.doi.org/10.7554/eLife.12411.001 PMID:26765562

  1. Effect of N-Terminal Acylation on the Activity of Myostatin Inhibitory Peptides.

    PubMed

    Takayama, Kentaro; Nakamura, Akari; Rentier, Cédric; Mino, Yusaku; Asari, Tomo; Saga, Yusuke; Taguchi, Akihiro; Yakushiji, Fumika; Hayashi, Yoshio

    2016-04-19

    Inhibition of myostatin, which negatively regulates skeletal muscle growth, is a promising strategy for the treatment of muscle atrophic disorders, such as muscular dystrophy, cachexia and sarcopenia. Recently, we identified peptide A (H-WRQNTRYSRIEAIKIQILSKLRL-NH2 ), the 23-amino-acid minimum myostatin inhibitory peptide derived from mouse myostatin prodomain, and highlighted the importance of its N-terminal tryptophan residue for the effective inhibition. In this study, we synthesized a series of acylated peptide derivatives focused on the tryptophan residue to develop potent myostatin inhibitors. As a result of the investigation, a more potent derivative of peptide A was successfully identified in which the N-terminal tryptophan residue is replaced with a 2-naphthyloxyacetyl moiety to give an inhibitory peptide three times (1.19±0.11 μm) more potent than parent peptide A (3.53±0.25 μm). This peptide could prove useful as a new starting point for the development of improved inhibitory peptides. PMID:26954624

  2. c-Jun promotes whereas JunB inhibits epidermal neoplasia.

    PubMed

    Jin, Jane Y; Ke, Hengning; Hall, Russell P; Zhang, Jennifer Y

    2011-05-01

    Deregulation of the activator protein 1 (AP1) family gene regulators has been implicated in a wide range of diseases, including cancer. In this study we report that c-Jun was activated in human squamous cell carcinoma (SCC) and coexpression of c-Jun with oncogenic Ras was sufficient to transform primary human epidermal cells into malignancy in a regenerated human skin grafting model. In contrast, JunB was not induced in a majority of human SCC cells. Moreover, exogenous expression of JunB inhibited tumorigenesis driven by Ras or spontaneous human SCC cells. Conversely, the dominant-negative JunB mutant (DNJunB) promoted tumorigenesis, which is in contrast to the tumor-suppressor function of the corresponding c-Jun mutant. At the cellular level, JunB induced epidermal cell senescence and slowed cell growth in a cell-autonomous manner. Consistently, coexpression of JunB and Ras induced premature epidermal differentiation concomitant with upregulation of p16 and filaggrin and downregulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). These findings indicate that JunB and c-Jun differentially regulate cell growth and differentiation and induce opposite effects on epidermal neoplasia.JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article, please go to http://www.nature.com/jid/journalclub. PMID:21289643

  3. The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen*

    PubMed Central

    Kobayashi, Yuki; Shiga, Takafumi; Shibata, Toshio; Sako, Miyuki; Maenaka, Katsumi; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2014-01-01

    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. PMID:25077965

  4. The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.

    PubMed

    Kobayashi, Yuki; Shiga, Takafumi; Shibata, Toshio; Sako, Miyuki; Maenaka, Katsumi; Koshiba, Takumi; Mizumura, Hikaru; Oda, Toshio; Kawabata, Shun-ichiro

    2014-09-12

    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules. PMID:25077965

  5. Differential Contributions of Tacaribe Arenavirus Nucleoprotein N-Terminal and C-Terminal Residues to Nucleocapsid Functional Activity

    PubMed Central

    D'Antuono, Alejandra; Loureiro, Maria Eugenia; Foscaldi, Sabrina; Marino-Buslje, Cristina

    2014-01-01

    ABSTRACT The arenavirus nucleoprotein (NP) is the main protein component of viral nucleocapsids and is strictly required for viral genome replication mediated by the L polymerase. Homo-oligomerization of NP is presumed to play an important role in nucleocapsid assembly, albeit the underlying mechanism and the relevance of NP-NP interaction in nucleocapsid activity are still poorly understood. Here, we evaluate the contribution of the New World Tacaribe virus (TCRV) NP self-interaction to nucleocapsid functional activity. We show that alanine substitution of N-terminal residues predicted to be available for NP-NP interaction strongly affected NP self-association, as determined by coimmunoprecipitation assays, produced a drastic inhibition of transcription and replication of a TCRV minigenome RNA, and impaired NP binding to RNA. Mutagenesis and functional analysis also revealed that, while dispensable for NP self-interaction, key amino acids at the C-terminal domain were essential for RNA synthesis. Furthermore, mutations at these C-terminal residues rendered NP unable to bind RNA both in vivo and in vitro but had no effect on the interaction with the L polymerase. In addition, while all oligomerization-defective variants tested exhibited unaltered capacities to sustain NP-L interaction, NP deletion mutants were fully incompetent to bind L, suggesting that, whereas NP self-association is dispensable, the integrity of both the N-terminal and C-terminal domains is required for binding the L polymerase. Overall, our results suggest that NP self-interaction mediated by the N-terminal domain may play a critical role in TCRV nucleocapsid assembly and activity and that the C-terminal domain of NP is implicated in RNA binding. IMPORTANCE The mechanism of arenavirus functional nucleocapsid assembly is still poorly understood. No detailed information is available on the nucleocapsid structure, and the regions of full-length NP involved in binding to viral RNA remain to be

  6. The EBNA-2 N-Terminal Transactivation Domain Folds into a Dimeric Structure Required for Target Gene Activation

    PubMed Central

    Hennig, Janosch; Zou, Peijian; Nössner, Elfriede; Ling, Paul D.; Sattler, Michael; Kempkes, Bettina

    2015-01-01

    Epstein-Barr virus (EBV) is a γ-herpesvirus that may cause infectious mononucleosis in young adults. In addition, epidemiological and molecular evidence links EBV to the pathogenesis of lymphoid and epithelial malignancies. EBV has the unique ability to transform resting B cells into permanently proliferating, latently infected lymphoblastoid cell lines. Epstein-Barr virus nuclear antigen 2 (EBNA-2) is a key regulator of viral and cellular gene expression for this transformation process. The N-terminal region of EBNA-2 comprising residues 1-58 appears to mediate multiple molecular functions including self-association and transactivation. However, it remains to be determined if the N-terminus of EBNA-2 directly provides these functions or if these activities merely depend on the dimerization involving the N-terminal domain. To address this issue, we determined the three-dimensional structure of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four β-strands and an α-helix which form a parallel dimer by interaction of two β-strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association in vitro. Importantly, these interface mutants also displayed severely impaired self-association and transactivation in vivo. Moreover, mutations of solvent-exposed residues or deletion of the α-helix do not impair dimerization but strongly affect the functional activity, suggesting that the EBNA-2 dimer presents a surface that mediates functionally important intra- and/or intermolecular interactions. Our study shows that the END domain is a novel dimerization fold that is essential for functional activity. Since this specific fold is a unique feature of EBNA-2 it might provide a novel target for anti-viral therapeutics. PMID:26024477

  7. Characterization of regions within the N-terminal 6-kilodalton domain of phytochrome A that modulate its biological activity.

    PubMed Central

    Jordan, E T; Marita, J M; Clough, R C; Vierstra, R D

    1997-01-01

    Phytochrome A (phyA) is a red/far-red (FR) light photoreceptor responsible for initiating numerous light-mediated plant growth and developmental responses, especially in FR light-enriched environments. We previously showed that the first 70 amino acids of the polypeptide contain at least two regions with potentially opposite functions (E.T. Jordan, J.R. Cherry, J.M. Walker, R.D. Vierstra [1996] Plant J 9: 243-257). One region is required for activity and correct apoprotein/chromophore interactions, whereas the second appears to regulate phytochrome activity. We have further resolved these functional regions by analysis of N-terminal deletion and alanine-scanning mutants of oat (Avena sativa) phyA in transgenic tobacco (Nicotiana tabacum). The results indicate that the region involved in chromophore/apoprotein interactions contains two separate segments (residues 25-33 and 50-62) also required for biological activity. The region that regulates phyA activity requires only five adjacent serines (Sers) (residues 8-12). Removal or alteration of these Sers generates a photoreceptor that increases the sensitivity of transgenic seedlings to red and FR light more than intact phyA. Taken together, these data identify three distinct regions in the N-terminal domain necessary for photoreceptor activity, and further define the Ser-rich region as an important site for phyA regulation. PMID:9342873

  8. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

    PubMed Central

    Ostolaza, Helena

    2013-01-01

    Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically active “soluble AC”. The calpain-mediated ACT processing allows trafficking of the “soluble AC” domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP “pools”, which would play different roles in the cell pathophysiology. PMID:23840759

  9. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    PubMed Central

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  10. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel

    NASA Astrophysics Data System (ADS)

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-06-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

  11. The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster

    PubMed Central

    Landry, Aaron P.

    2014-01-01

    Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1. PMID:25147792

  12. N-terminal tetrapeptide T/SPLH motifs contribute to multimodal activation of human TRPA1 channel.

    PubMed

    Hynkova, Anna; Marsakova, Lenka; Vaskova, Jana; Vlachova, Viktorie

    2016-01-01

    Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence. PMID:27345869

  13. The level of intracellular glutathione is a key regulator for the induction of stress-activated signal transduction pathways including Jun N-terminal protein kinases and p38 kinase by alkylating agents.

    PubMed Central

    Wilhelm, D; Bender, K; Knebel, A; Angel, P

    1997-01-01

    Monofunctional alkylating agents like methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are potent inducers of cellular stress leading to chromosomal aberrations, point mutations, and cell killing. We show that these agents induce a specific cellular stress response program which includes the activation of Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPKs), p38 mitogen-activated protein kinase, and the upstream kinase SEK1/MKK4 and which depends on the reaction mechanism of the alkylating agent in question. Similar to another inducer of cellular stress, UV irradiation, damage of nuclear DNA by alkylation is not involved in the MMS-induced response. However, in contrast to UV and other inducers of the JNK/SAPKs and p38 pathways, activation of growth factor and G-protein-coupled receptors does not play a role in the MMS response. We identified the intracellular glutathione (GSH) level as critical for JNK/SAPK activation by MMS: enhancing the GSH level by pretreatment of the cells with GSH or N-acetylcysteine inhibits, whereas depletion of the cellular GSH pool causes hyperinduction of JNK/SAPK activity by MMS. In light of the JNK/SAPK-dependent induction of c-jun and c-fos transcription, and the Jun/Fos-induced transcription of xenobiotic-metabolizing enzymes, these data provide a potential critical role of JNK/SAPK and p38 in the induction of a cellular defense program against cytotoxic xenobiotics such as MMS. PMID:9234735

  14. The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

    PubMed

    Raynaud, Sandy; Ragel, Paula; Rojas, Tomás; Mérida, Ángel

    2016-05-13

    Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species. PMID:26969163

  15. G12 Signaling through c-Jun NH2-Terminal Kinase Promotes Breast Cancer Cell Invasion

    PubMed Central

    Juneja, Juhi; Cushman, Ian; Casey, Patrick J.

    2011-01-01

    Signaling through the heterotrimeric G protein, G12, via Rho induces a striking increase in breast cancer cell invasion. In this study, evidence is provided that the c-Jun NH2-terminal kinase (JNK) is a key downstream effector of G12 on this pathway. Expression of constitutively-active Gα12 or activation of G12 signaling by thrombin leads to increased JNK and c-Jun phosphorylation. Pharmacologic inhibition of JNK or knockdown of JNK expression by siRNA significantly decreases G12-induced JNK activation as well as the ability of breast cancer cells to invade a reconstituted basement membrane. Furthermore, expression of dominant-negative Rho or treatment of cells with an inhibitor of the Rho kinase, ROCK, reduces G12-induced JNK and c-Jun activation, and ROCK inhibitor treatment also inhibits G12-induced cellular invasion. JNK knockdown or ROCK inhibitor treatment has no effect on activation of Rho by G12. Taken together, our data indicate that JNK activation is required for G12-induced invasion of breast cancer cells and that JNK is downstream of Rho and ROCK on this pathway. This study implicates a G12-stimulated mitogen-activated protein kinase cascade in cancer cell invasion, and supports a role for JNK in cancer progression. PMID:22087220

  16. [Serum determination of N-terminal peptide of type III procollagen as a marker of fibrotic activity].

    PubMed

    García Montes, J M; De Bonilla Blánez, F; Herrerías Gutiérrez, J M

    1989-03-01

    Among the noninvasive methods proposed for the study of collagen metabolism as an of fibrosis and inflammation, the most widely accepted method is quantitation in serum of the N-terminal peptide of type III procollagen (P-III-Ps). We measured this variable in 87 subjects classified into five study groups: 19 controls (C), 18 alcoholics (E), 15 patients diagnosed as liver cirrhosis (CH), 11 chronic liver disease (HC) and 24 pregnant women (EMB). In our environment, the serum level of P-III-P in the healthy population was 9.12-12.8 ng/ml. In 27.77% of the alcoholics studied (5 cases) the mean value exceeded this level, 19.35 +/- 3.05 ng/ml. Forty percent of the cirrhotics (6 cases) presented the highest values, 26.54 +/- 11.45 ng/ml, while 83.33% of the patients with chronic active hepatitis presented a mean value of 18.53 +/- 3.8 ng/ml. Of the 24 pregnant women, 95.83% (23 cases) had higher than normal values, and concentrations roses in the last trimester of gestation with respect to the previous trimesters. Analysis of the correlations of all the biochemical parameters of liver function with P-III-Ps disclosed a relationship between P-III-Ps and alkaline phosphatase in the groups of cirrhotics and chronic persistent hepatitis (p less than 0.05). We conclude that the N-terminal peptide of type III procollagen is a useful marker of active fibrosis. PMID:2734469

  17. Clostridium thermocellum thermostable lichenase with circular permutations and modifications in the N-terminal region retains its activity and thermostability.

    PubMed

    Tyurin, A А; Sadovskaya, N S; Nikiforova, Kh R; Mustafaev, O N; Komakhin, R A; Fadeev, V S; Goldenkova-Pavlova, I V

    2015-01-01

    The Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase) displays a high thermostability and specific activity and has a compact protein molecule, which makes it attractive, in particular, for protein engineering. We have utilized in silico analysis to construct circularly permuted (CP) variants and estimated the retained activity and thermostability. New open termini in the region of residues 53 or 99 in two lichenase CP variants (CN-53 and CN-99) had no effect on their activity and thermal tolerance versus another variant CP variant, CN-140 (cut in the region of residue 140), which displayed a dramatic decrease in the activity and thermostability. Construction and further activity and thermostability testing of the modified lichenase variants (M variants) and CP variants with peptides integrated via insertion fusion have demonstrated that the N-terminal regions in the lichenase catalytic domain (53 and 99 amino acid residues) that permit circular permutations with retention of activity and thermostability of the enzyme as well as the region between the C and N termini of the native lichenase in thermostable and active lichenase variants (CN-53 and CN-99) may be used for integrating small peptides without the loss of activity and thermostability. These findings not only suggest that CP predictions can be used in search for internal integration sites within protein molecule, but also form the background for further enzymatic engineering of the C. thermocellum thermostable lichenase aiming to create new fusion proteins. PMID:25448724

  18. Modulating the activity of short arginine-tryptophan containing antibacterial peptides with N-terminal metallocenoyl groups

    PubMed Central

    Albada, H Bauke; Chiriac, Alina-Iulia; Wenzel, Michaela; Penkova, Maya; Bandow, Julia E; Sahl, Hans-Georg

    2012-01-01

    Summary A series of small synthetic arginine and tryptophan containing peptides was prepared and analyzed for their antibacterial activity. The effect of N-terminal substitution with metallocenoyl groups such as ferrocene (FcCO) and ruthenocene (RcCO) was investigated. Antibacterial activity in different media, growth inhibition, and killing kinetics of the most active peptides were determined. The toxicity of selected derivatives was determined against erythrocytes and three human cancer cell lines. It was shown that the replacement of an N-terminal arginine residue with a metallocenoyl moiety modulates the activity of WRWRW-peptides against Gram-positive and Gram-negative bacteria. MIC values of 2–6 µM for RcCO-W(RW)2 and 1–11 µM for (RW)3 were determined. Interestingly, W(RW)2-peptides derivatized with ferrocene were significantly less active than those derivatized with ruthenocene which have similar structural but different electronic properties, suggesting a major influence of the latter. The high activities observed for the RcCO-W(RW)2- and (RW)3-peptides led to an investigation of the origin of activity of these peptides using several important activity-related parameters. Firstly, killing kinetics of the RcCO-W(RW)2-peptide versus killing kinetics of the (RW)3 derivative showed faster reduction of the colony forming units for the RcCO-W(RW)2-peptide, although MIC values indicated higher activity for the (RW)3-peptide. This was confirmed by growth inhibition studies. Secondly, hemolysis studies revealed that both peptides did not lead to significant destruction of erythrocytes, even up to 500 µg/mL for (RW)3 and 250 µg/mL for RcCO-W(RW)2. In addition, toxicity against three human cancer cell lines (HepG2, HT29, MCF7) showed that the (RW)3-peptide had an IC50 value of ~140 µM and the RcW(RW)2 one of ~90 µM, indicating a potentially interesting therapeutic window. Both the killing kinetics and growth inhibition studies presented in this work point to

  19. Interaction between c-jun and Androgen Receptor Determines the Outcome of Taxane Therapy in Castration Resistant Prostate Cancer

    PubMed Central

    Tinzl, Martina; Chen, Binshen; Chen, Shao-Yong; Semenas, Julius; Abrahamsson, Per-Anders; Dizeyi, Nishtman

    2013-01-01

    Taxane based chemotherapy is the standard of care treatment in castration resistant prostate cancer (CRPC). There is convincing evidence that taxane therapy affects androgen receptor (AR) but the exact mechanisms have to be further elucidated. Our studies identified c-jun as a crucial key player which interacts with AR and thus determines the outcome of the taxane therapy given. Docetaxel (Doc) and paclitaxel (Pac) agents showed different effects on LNCaP and LNb4 evidenced by alteration in the protein and mRNA levels of c-jun, AR and PSA. Docetaxel-induced phophorylation of c-jun occurred before JNK phosphorylation which suggests that c-jun phosphorylation is independent of JNK pathways in prostate cancer cells. A xenograft study showed that mice treated with Pac and bicalutamide showed worse outcome supporting our hypothesis that upregulation of c-jun might act as a potent antiapoptotic factor. We observed in our in vitro studies an inverse regulation of PSA- and AR-mRNA levels in Doc treated LNb4 cells. This was also seen for kallikrein 2 (KLK 2) which followed the same pattern. Given the fact that response to taxane therapy is measured by PSA decrease we have to consider that this might not reflect the true activity of AR in CRPC patients. PMID:24260253

  20. The Expression Patterns of c-Fos and c-Jun Induced by Different Frequencies of Electroacupuncture in the Brain

    PubMed Central

    Qiu, Zheng-Ying; Ding, Yi; Cui, Lu-ying; Hu, Man-Li; Ding, Ming-Xing

    2015-01-01

    To investigate patterns of c-Fos and c-Jun expression induced by different frequencies of electroacupuncture (EA) in the brain, goats were stimulated by EA of 0, 2, 60, or 100 Hz at a set of “Baihui, Santai, Ergen, and Sanyangluo” points for 30 min. The pain threshold was measured using the potassium iontophoresis method. The levels of c-Fos and c-Jun were determined with Streptavidin-Biotin Complex immunohistochemistry. The results showed that the pain threshold induced by 60 Hz was 82.2% higher (P < 0.01) than that by 0, 2, or 100 Hz (6.5%, 35.2%, or 40.9%). EA induced increased c-Fos and c-Jun expression in most analgesia-related nuclei and areas in the brain. Sixty Hz EA increased more c-Fos or c-Jun expression than 2 Hz or 100 Hz EA in all the measured nuclei and areas except for the nucleus accumbens, the area septalis lateralis, the caudate nucleus, the nucleus amygdala basalis, and the locus coeruleus, in which c-Fos or c-Jun expressions induced by 60 Hz EA did not differ from those by 2 Hz or 100 Hz EA. It was suggested that 60 Hz EA activated more extensive neural circuits in goats, which may contribute to optimal analgesic effects. PMID:26491460

  1. Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

    PubMed Central

    Lübker, Carolin; Dove, Stefan; Tang, Wei-Jen; Urbauer, Ramona J. Bieber; Moskovitz, Jackob; Urbauer, Jeffrey L.; Seifert, Roland

    2015-01-01

    Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils. PMID:26184312

  2. Adiponectin exerts antiproliferative effect on human placenta via modulation of the JNK/c-Jun pathway

    PubMed Central

    Chen, Haitian; Chen, Hanqing; Wu, Yanxin; Liu, Bin; Li, Zhuyu; Wang, Zilian

    2014-01-01

    To determine the effects of adiponectin on human placenta during gestational diabetes mellitus (GDM) and on high glucose (HG)-induced BeWo cell proliferation. We examined the expression levels of adiponectin in control and GDM placenta using quantitative real-time PCR, Western blot, and immunohistochemistry (IHC). Cell proliferation and viability were assessed using a colorimetric assay (cell counting kit-8), PCNA immunocytochemical staining, and Western blot analysis of cyclin D1. Transfection of siRNA against c-jun was performed using Lipofectamine 2000. Cell cycle analysis was performed using propidium iodide staining and flow cytometry. Results show a decreased expression of adiponectin and an increased degree of trophoblast cell proliferation in GDM placenta compared to the normal placenta. Similarly, HG can promote BeWo cell proliferation that is associated with adiponectin down-regulation. This proliferation could be depressed by addition of exogenous adiponectin, i.e. adiponectin exerts antiproliferative effects on HG-induced trophoblast cells. Adiponectin suppresses the HG-induced BeWo cell proliferation by inhibiting the activation of JNK/c-jun. In conclusion, adiponectin inhibits HG-induced proliferation of BeWo cells through down-regulation of JNK/c-jun phosphorylation. PMID:25031708

  3. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    SciTech Connect

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  4. N-terminal Serine Dephosphorylation Is Required for KCC3 Cotransporter Full Activation by Cell Swelling*

    PubMed Central

    Melo, Zesergio; de los Heros, Paola; Cruz-Rangel, Silvia; Vázquez, Norma; Bobadilla, Norma A.; Pasantes-Morales, Herminia; Alessi, Dario R.; Mercado, Adriana; Gamba, Gerardo

    2013-01-01

    The K+:Cl− cotransporter (KCC) activity is modulated by phosphorylation/dephosphorylation processes. In isotonic conditions, KCCs are inactive and phosphorylated, whereas hypotonicity promotes their dephosphorylation and activation. Two phosphorylation sites (Thr-991 and Thr-1048) in KCC3 have been found to be critical for its regulation. However, here we show that the double mutant KCC3-T991A/T1048A could be further activated by hypotonicity, suggesting that additional phosphorylation site(s) are involved. We observed that in vitro activated STE20/SPS1-related proline/alanine-rich kinase (SPAK) complexed to its regulatory MO25 subunit phosphorylated KCC3 at Ser-96 and that in Xenopus laevis oocytes Ser-96 of human KCC3 is phosphorylated in isotonic conditions and becomes dephosphorylated during incubation in hypotonicity, leading to a dramatic increase in KCC3 function. Additionally, WNK3, which inhibits the activity of KCC3, promoted phosphorylation of Ser-96 as well as Thr-991 and Thr-1048. These observations were corroborated in HEK293 cells stably transfected with WNK3. Mutation of Ser-96 alone (KCC3-S96A) had no effect on the activity of the cotransporter when compared with wild type KCC3. However, when compared with the double mutant KCC3-T991A/T1048A, the triple mutant KCC3-S96A/T991A/T1048A activity in isotonic conditions was significantly higher, and it was not further increased by hypotonicity or inhibited by WNK3. We conclude that serine residue 96 of human KCC3 is a third site that has to be dephosphorylated for full activation of the cotransporter during hypotonicity. PMID:24043619

  5. Drosophila melanogaster Dis3 N-terminal domains are required for ribonuclease activities, nuclear localization and exosome interactions.

    PubMed

    Mamolen, Megan; Smith, Alexandra; Andrulis, Erik D

    2010-09-01

    Eukaryotic cells use numerous pathways to regulate RNA production, localization and stability. Several of these pathways are controlled by ribonucleases. The essential ribonuclease, Dis3, plays important roles in distinct RNA metabolic pathways. Despite much progress in understanding general characteristics of the Dis3 enzyme in vitro and in vivo, much less is known about the contributions of Dis3 domains to its activities, subcellular localization and protein-protein interactions. To address these gaps, we constructed a set of Drosophila melanogaster Dis3 (dDis3) mutants and assessed their enzymatic activity in vitro and their localizations and interactions in S2 tissue culture cells. We show that the dDis3 N-terminus is sufficient for endoribonuclease activity in vitro and that proper N-terminal domain structure is critical for activity of the full-length polypeptide. We find that the dDis3 N-terminus also contributes to its subcellular distribution, and is necessary and sufficient for interactions with core exosome proteins. Finally, dDis3 interaction with dRrp6 and dImportin-α3 is independent of core interactions and occurs though two different regions. Taken together, our data suggest that the dDis3 N-terminus is a dynamic and complex hub for RNA metabolism and exosome interactions. PMID:20421210

  6. Activation of Histidine Kinase SpaK Is Mediated by the N-Terminal Portion of Subtilin-Like Lantibiotics and Is Independent of Lipid II.

    PubMed

    Spieß, Tobias; Korn, Sophie Marianne; Kötter, Peter; Entian, Karl-Dieter

    2015-08-15

    The biosynthesis of the lantibiotic subtilin is autoinduced in a quorum-sensing mechanism via histidine kinase SpaK. Subtilin-like lantibiotics, such as entianin, ericin S, and subtilin, specifically activated SpaK in a comparable manner, whereas the structurally similar nisin did not provide the signal for SpaK activation at nontoxic concentrations. Surprisingly, nevertheless, nisin if applied together with entianin partly quenched SpaK activation. The N-terminal entianin1-20 fragment (comprising N-terminal amino acids 1 to 20) was sufficient for SpaK activation, although higher concentrations were needed. The N-terminal nisin1-20 fragment also interfered with entianin-mediated activation of SpaK and, remarkably, at extremely high concentrations also activated SpaK. Our data show that the N-terminal entianin1-20 fragment is sufficient for SpaK activation. However, if present, the C-terminal part of the molecule further strongly enhances the activation, possibly by its interference with the cellular membrane. As shown by using lipid II-interfering substances and a lipid II-deficient mutant strain, lipid II is not needed for the sensing mechanism. PMID:26025904

  7. Activation of Histidine Kinase SpaK Is Mediated by the N-Terminal Portion of Subtilin-Like Lantibiotics and Is Independent of Lipid II

    PubMed Central

    Spieß, Tobias; Korn, Sophie Marianne

    2015-01-01

    The biosynthesis of the lantibiotic subtilin is autoinduced in a quorum-sensing mechanism via histidine kinase SpaK. Subtilin-like lantibiotics, such as entianin, ericin S, and subtilin, specifically activated SpaK in a comparable manner, whereas the structurally similar nisin did not provide the signal for SpaK activation at nontoxic concentrations. Surprisingly, nevertheless, nisin if applied together with entianin partly quenched SpaK activation. The N-terminal entianin1–20 fragment (comprising N-terminal amino acids 1 to 20) was sufficient for SpaK activation, although higher concentrations were needed. The N-terminal nisin1–20 fragment also interfered with entianin-mediated activation of SpaK and, remarkably, at extremely high concentrations also activated SpaK. Our data show that the N-terminal entianin1–20 fragment is sufficient for SpaK activation. However, if present, the C-terminal part of the molecule further strongly enhances the activation, possibly by its interference with the cellular membrane. As shown by using lipid II-interfering substances and a lipid II-deficient mutant strain, lipid II is not needed for the sensing mechanism. PMID:26025904

  8. Calmodulin activation of an endoplasmic reticulum-located calcium pump involves an interaction with the N-terminal autoinhibitory domain

    NASA Technical Reports Server (NTRS)

    Hwang, I.; Harper, J. F.; Liang, F.; Sze, H.

    2000-01-01

    To investigate how calmodulin regulates a unique subfamily of Ca(2+) pumps found in plants, we examined the kinetic properties of isoform ACA2 identified in Arabidopsis. A recombinant ACA2 was expressed in a yeast K616 mutant deficient in two endogenous Ca(2+) pumps. Orthovanadate-sensitive (45)Ca(2+) transport into vesicles isolated from transformants demonstrated that ACA2 is a Ca(2+) pump. Ca(2+) pumping by the full-length protein (ACA2-1) was 4- to 10-fold lower than that of the N-terminal truncated ACA2-2 (Delta2-80), indicating that the N-terminal domain normally acts to inhibit the pump. An inhibitory sequence (IC(50) = 4 microM) was localized to a region within valine-20 to leucine-44, because a peptide corresponding to this sequence lowered the V(max) and increased the K(m) for Ca(2+) of the constitutively active ACA2-2 to values comparable to the full-length pump. The peptide also blocked the activity (IC(50) = 7 microM) of a Ca(2+) pump (AtECA1) belonging to a second family of Ca(2+) pumps. This inhibitory sequence appears to overlap with a calmodulin-binding site in ACA2, previously mapped between aspartate-19 and arginine-36 (J.F. Harper, B. Hong, I. Hwang, H.Q. Guo, R. Stoddard, J.F. Huang, M.G. Palmgren, H. Sze inverted question mark1998 J Biol Chem 273: 1099-1106). These results support a model in which the pump is kept "unactivated" by an intramolecular interaction between an autoinhibitory sequence located between residues 20 and 44 and a site in the Ca(2+) pump core that is highly conserved between different Ca(2+) pump families. Results further support a model in which activation occurs as a result of Ca(2+)-induced binding of calmodulin to a site overlapping or immediately adjacent to the autoinhibitory sequence.

  9. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways. PMID:15271990

  10. Longikaurin A, a natural ent-kaurane, induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells.

    PubMed

    Liao, Y-J; Bai, H-Y; Li, Z-H; Zou, J; Chen, J-W; Zheng, F; Zhang, J-X; Mai, S-J; Zeng, M-S; Sun, H-D; Pu, J-X; Xie, D

    2014-01-01

    Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC. PMID:24651440

  11. c-Jun NH2-Terminal Kinase 1/2 and Endoplasmic Reticulum Stress as Interdependent and Reciprocal Causation in Diabetic Embryopathy

    PubMed Central

    Li, Xuezheng; Xu, Cheng; Yang, Peixin

    2013-01-01

    Embryos exposed to high glucose exhibit aberrant maturational and cytoarchitectural cellular changes, implicating cellular organelle stress in diabetic embryopathy. c-Jun-N-terminal kinase 1/2 (JNK1/2) activation is a causal event in maternal diabetes–induced neural tube defects (NTD). However, the relationship between JNK1/2 activation and endoplasmic reticulum (ER) stress in diabetic embryopathy has never been explored. We found that maternal diabetes significantly increased ER stress markers and induced swollen/enlarged ER lumens in embryonic neuroepithelial cells during neurulation. Deletion of either jnk1 or jnk2 gene diminished hyperglycemia-increased ER stress markers and ER chaperone gene expression. In embryos cultured under high-glucose conditions (20 mmol/L), the use of 4-phenylbutyric acid (4-PBA), an ER chemical chaperone, diminished ER stress markers and abolished the activation of JNK1/2 and its downstream transcription factors, caspase 3 and caspase 8, and Sox1 neural progenitor apoptosis. Consequently, both 1 and 2 mmol/L 4-PBA significantly ameliorated high glucose–induced NTD. We conclude that hyperglycemia induces ER stress, which is responsible for the proapoptotic JNK1/2 pathway activation, apoptosis, and NTD induction. Suppressing JNK1/2 activation by either jnk1 or jnk2 gene deletion prevents ER stress. Thus, our study reveals a reciprocal causation of ER stress and JNK1/2 in mediating the teratogenicity of maternal diabetes. PMID:22961085

  12. The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum.

    PubMed

    Mariappan, Malaiyalam; Gande, Santosh Lakshmi; Radhakrishnan, Karthikeyan; Schmidt, Bernhard; Dierks, Thomas; von Figura, Kurt

    2008-04-25

    Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER. PMID:18305113

  13. Assembly of a Functional Beta Interferon Enhanceosome Is Dependent on ATF-2–c-jun Heterodimer Orientation

    PubMed Central

    Falvo, James V.; Parekh, Bhavin S.; Lin, Charles H.; Fraenkel, Ernest; Maniatis, Tom

    2000-01-01

    Heterodimeric transcription factors, including the basic region-leucine zipper (bZIP) protein ATF-2–c-jun, are well-characterized components of an enhanceosome that mediates virus induction of the human beta interferon (IFN-β) gene. Here we report that within the IFN-β enhanceosome the ATF-2–c-jun heterodimer binds in a specific orientation, which is required for assembly of a complex between ATF-2–c-jun and interferon regulatory factor 3 (IRF-3). We demonstrate that correct orientation of the ATF-2–c-jun binding site is required for virus induction of the IFN-β gene and for IRF-3-dependent activation of a composite ATF-2– c-jun–IRF site in the IFN-β promoter. We also show that in vitro the DNA-bound ATF-2–c-jun heterodimer adopts a fixed orientation upon the binding of IRF-3 at an adjacent site in the IFN-β enhancer and that the DNA-binding domain of IRF-3 is sufficient to mediate this effect. In addition, we show that the DNA-binding domain of ATF-2 is necessary and sufficient for selective protein-protein interactions with IRF-3. Strikingly, in vivo chromatin immunoprecipitation experiments with IFN-β reporter constructs reveal that recruitment of IRF-3 to the IFN-β promoter upon virus infection is dependent on the orientation of the ATF-2–c-jun heterodimer binding site. These observations demonstrate functional and physical cooperativity between the bZIP and IRF transcription factor families and illustrate the critical role of heterodimeric transcription factors in formation of the IFN-β enhanceosome. PMID:10848607

  14. Improving the glycosyltransferase activity of Agrobacterium tumefaciens glycogen synthase by fusion of N-terminal starch binding domains (SBDs).

    PubMed

    Martín, Mariana; Wayllace, Nahuel Z; Valdez, Hugo A; Gomez-Casati, Diego F; Busi, María V

    2013-10-01

    Glycogen and starch, the major storage carbohydrate in most living organisms, result mainly from the action of starch or glycogen synthases (SS or GS, respectively, EC 2.4.1.21). SSIII from Arabidopsis thaliana is an SS isoform with a particular modular organization: the C-terminal highly conserved glycosyltransferase domain is preceded by a unique specific region (SSIII-SD) which contains three in tandem starch binding domains (SBDs, named D1, D2 and D3) characteristic of polysaccharide degrading enzymes. N-terminal SBDs have a probed regulatory role in SSIII activity, showing starch binding ability and modulating the catalytic properties of the enzyme. On the other hand, GS from Agrobacterium tumefaciens has a simple primary structure organization, characterized only by the highly conserved glycosyltransferase domain and lacking SBDs. To further investigate the functional role of A. thaliana SSIII-SD, three chimeric proteins were constructed combining the SBDs from A. thaliana with the GS from A. tumefaciens. Recombinant proteins were expressed in and purified to homogeneity from Escherichia coli cells in order to be kinetically characterized. Furthermore, we tested the ability to restore in vivo glycogen biosynthesis in transformed E. coli glgA(-) cells, deficient in GS. Results show that the D3-GS chimeric enzyme showed increased capacity of glycogen synthesis in vivo with minor changes in its kinetics parameters compared to GS. PMID:23796574

  15. Efficient secretion of biologically active Chondroitinase ABC from mammalian cells in the absence of an N-terminal signal peptide.

    PubMed

    Klüppel, Michael

    2011-05-01

    Proteoglycans carrying chondroitin sulfate side chains have been shown to fulfill important biological functions in development, disease, and signaling. One area of considerable interest is the functional importance of chondroitin sulfates as inhibitors of the regeneration of axonal projections in the mammalian central nervous system. In animal models of spinal cord injury, injections of the enzyme Chondroitinase ABC from the bacterium Proteus vulgaris into the lesion site leads to degradation of chondroitin sulfates, and promotes axonal regeneration and significant functional recovery. Here, a mammalian expression system of an epitope-tagged Chondroitinase ABC protein is described. It is demonstrated that the addition of a eukaryotic secretion signal sequence to the N-terminus of the bacterial Chondroitinase ABC sequence allowed secretion, but interfered with function of the secreted enzyme. In contrast, expression of the Chondroitinase ABC gene without N-terminal eukaryotic secretion sequence or bacterial hydrophobic leader sequence led to efficient secretion of a biologically active Chondroitinase ABC protein from both immortalized and primary cells. Moreover, the C-terminal epitope tag could be utilized to follow expression of this protein. This novel Chondroitinase ABC gene is a valuable tool for a better understanding of the in vivo roles of chondroitin sulfates in mammalian development and disease, as well as in gene therapy approaches, including the treatment of spinal chord injuries. PMID:21213020

  16. Jun N-terminal kinase activity is required for invagination but not differentiation of the sea urchin archenteron.

    PubMed

    Long, Jason T; Irwin, Leslie; Enomoto, Addison C; Grow, Zachary; Ranck, Jessica; Peeler, Margaret T

    2015-12-01

    Although sea urchin gastrulation is well described at the cellular level, our understanding of the molecular changes that trigger the coordinated cell movements involved is not complete. Jun N-terminal kinase (JNK) is a component of the planar cell polarity pathway and is required for cell movements during embryonic development in several animal species. To study the role of JNK in sea urchin gastrulation, embryos were treated with JNK inhibitor SP600125 just prior to gastrulation. The inhibitor had a limited and specific effect, blocking invagination of the archenteron. Embryos treated with 2 μM SP600125 formed normal vegetal plates, but did not undergo invagination to form an archenteron. Other types of cell movements, specifically ingression of the skeletogenic mesenchyme, were not affected, although the development and pattern of the skeleton was abnormal in treated embryos. Pigment cells, derived from nonskeletogenic mesenchyme, were also present in SP600125-treated embryos. Despite the lack of a visible archenteron in treated embryos, cells at the original vegetal plate expressed several molecular markers for endoderm differentiation. These results demonstrate that JNK activity is required for invagination of the archenteron but not its differentiation, indicating that in this case, morphogenesis and differentiation are under separate regulation. PMID:26297876

  17. Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity.

    PubMed Central

    Petan, Toni; Krizaj, Igor; Gubensek, Franc; Pungercar, Joze

    2002-01-01

    Ammodytoxins (Atxs) are group II phospholipases A(2) (PLA(2)s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA(2) toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe(24), in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp(24) mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40-80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA(2) activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe(24) also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn(24) mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe(24) in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity. PMID:11931665

  18. Pharmacological activity of the C-terminal and N-terminal domains of secretory leukoprotease inhibitor in vitro.

    PubMed Central

    Masuda, K.; Kamimura, T.; Watanabe, K.; Suga, T.; Kanesaki, M.; Takeuchi, A.; Imaizumi, A.; Suzuki, Y.

    1995-01-01

    1. In order to characterize the physiological functions of the domain structure of secretory leukoprotease inhibitor (SLPI), the biological capacities of half-length SLPIs, (Ser1-Pro54)SLPI and (Asn55-Ala107)SLPI, were investigated and compared with those of full-length SLPI. 2. The activities of these inhibitors against several serine proteases were determined using synthetic chromogenic substrates. The inhibitory capacity of the C-terminal domain, (Asn55-Ala107)SLPI, was as strong as that of full-length SLPI against human neutrophil elastase (NE), cathepsin G and chymotrypsin. It possessed less trypsin inhibitory activity than intact SLPI. For the N-terminal domain of SLPI, (Ser1-Pro54)SLPI, no inhibitory activity could be detected against the serine proteases tested in this study. 3. The inhibitory activity of (Asn55-Ala107)SLPI against the proteolysis of the natural substrates elastin and collagen by NE was comparable with that of full-SLPI (elastin, IC50 = 907 +/- 31 nM for SLPI, 767 +/- 33 nM for (Asn55-Ala107)SLPI; collagen, IC50 = 862 +/- 36 nM for SLPI, 727 +/- 47 nM for (Asn55-Ala107)SLPI). 4. The binding affinities of full- and half-length SLPIs for heparin were measured by affinity column chromatography. Full-length SLPI showed high affinity for heparin while the binding capacities of both half-length SLPIs were lower. (Concentration of NaCl for elution, 0.45 M for SLPI, 0.24 M for (Ser1-Pro54)SLPI, 0.27 M for (Asn55-Ala107)SLPI). 5. The effects of full-SLPI and (Asn55-Ala107)SLPI on blood coagulation were measured using the activated partial thromboplastin time (APTT).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7582515

  19. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity.

    PubMed

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%-25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  20. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity

    PubMed Central

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  1. RhoA regulates invasion of glioma cells via the c-Jun NH2-terminal kinase pathway under hypoxia.

    PubMed

    Tong, Jiao Jian; Yan, Zhang; Jian, Ren; Tao, Huang; Hui, Ouyang Tao; Jian, Chen

    2012-09-01

    The purpose of this study was to investigate the mechanism of glioma cell invasion in hypoxic conditions. We demonstrated that hypoxia increased cell invasion, matrix metalloproteinase-2 (MMP2) activity and time-dependent expression of hypoxia inducible factor-1α (HIF-1α) in human glioma cells. These data suggest that MMP2 may play a significant role in tumor invasion in hypoxic conditions. We investigated the mechanisms involved in the increased MMP2 activity and cell invasion in hypoxic conditions. Increased expression of phospho-Jun NH2-terminal kinase (p-JNK) and phospho-c-Jun (p-c-Jun) in glioma cells induced by hypoxia was detected. Furthermore, this effect may be reduced by inhibiting the JNK signaling pathway. We found that inhibition of RhoA geranylgeranylation by geranylgeranyltransferase inhibitor-2147 (GGTI-2147) or knockdown of RhoA by siRNA against RhoA reduced the expression of p-JNK and p-c-Jun, and decreased MMP2 activity and glioma cell invasion in hypoxic conditions. These data suggest a link among RhoA, JNK, c-Jun and MMP2 activity that is functionally involved in the increased glioma cell invasion induced by hypoxia. PMID:23741249

  2. Synthesis and SAR of novel isoxazoles as potent c-jun N-terminal kinase (JNK) Inhibitors

    PubMed Central

    He, Yuanjun; Duckett, Derek; Chen, Weimin; Ling, Yuan Yuan; Cameron, Michael D.; Lin, Li; Ruiz, Claudia H.; LoGrasso, Philip V.; Kamenecka, Theodore M.; Koenig, Marcel

    2014-01-01

    The design and synthesis of isoxazole 3 is described, a potent JNK inhibitor with two fold selectivity over p38. Optimization of this scaffold led to compounds 27 and 28 which showed greatly improved selectivity over p38 by maintaining the JNK3 potency of compound 3. Extensive SAR studies will be described as well as preliminary in vivo data of the two lead compounds. PMID:24332487

  3. Induction of c-Jun by air particulate matter (PM₁₀) of Mexico city: Participation of polycyclic aromatic hydrocarbons.

    PubMed

    Salcido-Neyoy, Martha Estela; Sánchez-Pérez, Yesennia; Osornio-Vargas, Alvaro Román; Gonsebatt, María Eugenia; Meléndez-Zajgla, Jorge; Morales-Bárcenas, Rocío; Petrosyan, Pavel; Molina-Servin, Edith Danny; Vega, Elizabeth; Manzano-León, Natalia; García-Cuellar, Claudia M

    2015-08-01

    The carcinogenic potential of urban particulate matter (PM) has been partly attributed to polycyclic aromatic hydrocarbons (PAHs) content, which activates the aryl hydrocarbon receptor (AhR). Here we report the effect of PM with an aerodynamic size of 10 μm (PM10) on the induction of AhR pathway in A549 cells, evaluating its downstream targets CYP1B1, IL-6, IL-8 and c-Jun. Significant increases in CYP1B1 protein and enzyme activity; IL-6 and IL-8 secretion and c-Jun protein were found in response to PM10. The formation of PAH-DNA adducts was also detected. The involvement of AhR pathway was confirmed with Resveratrol as AhR antagonist, which reversed CYP1B1 and c-Jun induction. Nevertheless, in IL-6 and IL-8 secretion, the Resveratrol was ineffective, suggesting an effect independent of this pathway. Considering the role of c-Jun in oncogenesis, its induction by PM may be contributing to its carcinogenic potential through induction of AhR pathway by PAHs present in PM10. PMID:25909326

  4. Role of N-terminal methionine residues in the redox activity of copper bound to alpha-synuclein.

    PubMed

    Rodríguez, Esaú E; Arcos-López, Trinidad; Trujano-Ortiz, Lidia G; Fernández, Claudio O; González, Felipe J; Vela, Alberto; Quintanar, Liliana

    2016-09-01

    Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex. PMID:27422629

  5. Modeling the Mechanism of GR/c-Jun/Erg Crosstalk in Apoptosis of Acute Lymphoblastic Leukemia

    PubMed Central

    Chen, Daphne Wei-Chen; Krstic-Demonacos, Marija; Schwartz, Jean-Marc

    2012-01-01

    Acute lymphoblastic leukemia (ALL) is one of the most common forms of malignancy that occurs in lymphoid progenitor cells, particularly in children. Synthetic steroid hormones glucocorticoids (GCs) are widely used as part of the ALL treatment regimens due to their apoptotic function, but their use also brings about various side effects and drug resistance. The identification of the molecular differences between the GCs responsive and resistant cells therefore are essential to decipher such complexity and can be used to improve therapy. However, the emerging picture is complicated as the activities of genes and proteins involved are controlled by multiple factors. By adopting the systems biology framework to address this issue, we here integrated the available knowledge together with experimental data by building a series of mathematical models. This rationale enabled us to unravel molecular interactions involving c-Jun in GC induced apoptosis and identify Ets-related gene (Erg) as potential biomarker of GC resistance. The results revealed an alternative possible mechanism where c-Jun may be an indirect GR target that is controlled via an upstream repressor protein. The models also highlight the importance of Erg for GR function, particularly in GC sensitive C7 cells where Erg directly regulates GR in agreement with our previous experimental results. Our models describe potential GR-controlled molecular mechanisms of c-Jun/Bim and Erg regulation. We also demonstrate the importance of using a systematic approach to translate human disease processes into computational models in order to derive information-driven new hypotheses. PMID:23181019

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

    PubMed

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

    2015-06-01

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

  7. Ulinastatin attenuates LPS-induced human endothelial cells oxidative damage through suppressing JNK/c-Jun signaling pathway.

    PubMed

    Li, Chunping; Ma, Dandan; Chen, Man; Zhang, Linlin; Zhang, Lin; Zhang, Jicheng; Qu, Xin; Wang, Chunting

    2016-06-01

    Lipopolysaccharide (LPS)-induced oxidative stress is a main feature observed in the sepsis by increasing endothelial oxidative damage. Many studies have demonstrated that Ulinastatin (UTI) can inhibit pro-inflammatory proteases, decrease inflammatory cytokine levels and suppress oxidative stress. However, the potential molecular mechanism underlying UTI which exerts its antioxidant effect is not well understood. In this study, we aimed to investigate the effects of UTI on the LPS-induced oxidative stress and the underlying mechanisms using human umbilical vein endothelial cells (HUVECs). After oxidative stress induced By LPS in HUVECs, the cell viability and reactive oxygen species (ROS) in cytoplasm were measured. In addition, superoxide dismutase (SOD) and malondialdehyde (MDA) were examined. We found that LPS resulted in a profound elevation of ROS production and MDA levels. The decrease in Cu/Zn-SOD protein and increased in Mn-SOD protein were observed in a time- and dose-dependent manner. These responses were suppressed by an addition of UTI. The increase in c-Jun N-terminal kinases (JNK) phosphorylation by LPS in HUVECs was markedly blocked by UTI or JNK inhibitor SP600125. Our results suggest that UTI exerts its anti-oxidant effects by decreasing overproduction of ROS induced by LPS via suppressing JNK/c-Jun phosphorylation. Therefore UTI may play a protective role in vascular endothelial injury induced by oxidative stress such as sepsis. This study may provide insight into a possible molecular mechanism by which Ulinastatin inhibits LPS-induced oxidative stress. PMID:27109479

  8. Coupled motions during dynamics reveal a tunnel toward the active site regulated by the N-terminal α-helix in an acylaminoacyl peptidase.

    PubMed

    Papaleo, Elena; Renzetti, Giulia

    2012-09-01

    Acylaminoacyl peptidase (AAP) subfamily belongs to the prolyl oligopeptidase (POP) family of serine-proteases. There is a great interest in the definition of molecular mechanisms related to the activity and substrate recognition of these complex multi-domain enzymes. The active site relies at the interface between the C-terminal catalytic domain and the β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In AAP, the N-terminal extension is characterized by a structurally conserved α1-helix, which is known to affect thermal stability and thermal dependence of the catalytic activity. In the present contribution, results from hundreds nanosecond all-atom molecular dynamics simulations, along with analyses of the networks of cross-correlated motions of a member of the AAP subfamily are discussed. The MD investigation identifies a tunnel that from the surrounding of the N-terminal α1-helix bring to the catalytic site. This cavity seems to be regulated by conformational changes of the α1-helix itself during the dynamics. The evidence here provided can be a useful guide for a better understanding of the mechanistic aspects related to AAP activity, but also for drug design purposes. PMID:23085164

  9. Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans

    PubMed Central

    Ujaoney, Aman K.; Basu, Bhakti; Muniyappa, K.; Apte, Shree K.

    2015-01-01

    Single-stranded DNA binding protein (Ssb) of Deinococcus radiodurans comprises N- and C-terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N-terminal without connector), SsbNC (N-terminal with connector) and SsbC (C-terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single-stranded DNA (ssDNA) binding activity, compared to the full-length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo-dimers while SsbNC/SsbN formed different oligomeric forms. In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase-I activity, but failed to stimulate Deinococcal RecA-promoted DNA strand exchange. The results suggest that the C-terminal OB fold is primarily responsible for ssDNA binding. The N-terminal OB fold binds weakly to ssDNA but is involved in multimerization. PMID:25973364

  10. IL-6 and high glucose synergistically upregulate MMP-1 expression by U937 mononuclear phagocytes via ERK1/2 and JNK pathways and c-Jun.

    PubMed

    Li, Yanchun; Samuvel, Devadoss J; Sundararaj, Kamala P; Lopes-Virella, Maria F; Huang, Yan

    2010-05-01

    Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling and destruction in inflammation-associated diseases such as cardiovascular disease and periodontal disease. Although it is known that interleukin (IL)-6 is a key proinflamatory cytokine, it remains unclear how IL-6 regulates MMP expression by mononuclear phagocytes. Furthermore, it remains undetermined how IL-6 in combination with hyperglycemia affects MMP expression. In the present study, we investigated the regulatory effect of IL-6 alone or in combination with high glucose on MMP-1 expression by U937 mononuclear phagocytes. We found that IL-6 is a powerful stimulator for MMP-1 expression and high glucose further augmented IL-6-stimulated MMP-1 expression. We also found that high glucose, IL-6, and lipopolysaccharide act in concert to stimulate MMP-1 expression. In the studies to elucidate underlying mechanisms, the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways were found to be required for stimulation of MMP-1 by IL-6 and high glucose. We also observed that IL-6 and high glucose stimulated the expression of c-Jun, a key subunit of AP-1 known to be essential for MMP-1 transcription. The role of c-Jun in MMP-1 expression was confirmed by the finding that suppression of c-Jun expression by RNA interference significantly inhibited MMP-1 expression. Finally, we demonstrated that similarly to U937 mononuclear phagocytes, IL-6 and high glucose also stimulated MMP-1 secretion from human primary monocytes. In conclusion, this study demonstrated that IL-6 and high glucose synergistically stimulated MMP-1 expression in mononuclear phagocytes via ERK and JNK cascades and c-Jun upregulation. PMID:20225236

  11. Lys39-Lysophosphatidate Carbonyl Oxygen Interaction Locks LPA1 N-terminal Cap to the Orthosteric Site and partners Arg124 During Receptor Activation

    PubMed Central

    Omotuyi, Olaposi I.; Nagai, Jun; Ueda, Hiroshi

    2015-01-01

    Lysophosphatidic acid (LPA) receptor 1 (LPA1) is a member of the G protein-coupled receptors mediating the biological response to LPA species. Lack of detailed mechanism underlying LPA/LPA1 interaction has hampered the development of specific antagonists. Here, novel N-terminal Lys39 has been identified as a key residue during LPA-type agonist binding and LPA1 activation. Analysis of the molecular dynamics (MD) trajectories showed that LPA-type agonist but not VPC-32183 (antagonist) evolved structures with classical GPCR activation signatures such as reduced cytoplasmic transmembrane (TM) 3/TM6 dynamic network, ruptured ionic lock, and formation of a continuous and highly ordered internal water pathway was also observed. In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39. Site-directed mutagenesis showed complete loss of intracellular calcium mobilization in B103 cells expressing R3.28A and Lys39Ala when treated with LPA-type agonists. Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle. This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1. PMID:26268898

  12. Lys39-Lysophosphatidate Carbonyl Oxygen Interaction Locks LPA1 N-terminal Cap to the Orthosteric Site and partners Arg124 During Receptor Activation.

    PubMed

    Omotuyi, Olaposi I; Nagai, Jun; Ueda, Hiroshi

    2015-01-01

    Lysophosphatidic acid (LPA) receptor 1 (LPA1) is a member of the G protein-coupled receptors mediating the biological response to LPA species. Lack of detailed mechanism underlying LPA/LPA1 interaction has hampered the development of specific antagonists. Here, novel N-terminal Lys39 has been identified as a key residue during LPA-type agonist binding and LPA1 activation. Analysis of the molecular dynamics (MD) trajectories showed that LPA-type agonist but not VPC-32183 (antagonist) evolved structures with classical GPCR activation signatures such as reduced cytoplasmic transmembrane (TM) 3/TM6 dynamic network, ruptured ionic lock, and formation of a continuous and highly ordered internal water pathway was also observed. In activated state, LPA-type agonists interact with Arg124 (R3.28), Gln125 (Q3.29), Lys294 (K7.36) and a novel N-terminal Lys39. Site-directed mutagenesis showed complete loss of intracellular calcium mobilization in B103 cells expressing R3.28A and Lys39Ala when treated with LPA-type agonists. Structurally, LPA-type agonist via Carbonyl-oxygen/Lys39 interaction facilitated the formation of a hypothetical N-terminal cap tightly packed over LPA1 heptahelical bundle. This packing may represent a key mechanism to distinguish an apo-receptor from bound LPA1. PMID:26268898

  13. Cooperative binding of the yeast Spt10p activator to the histone upstream activating sequences is mediated through an N-terminal dimerization domain

    PubMed Central

    Mendiratta, Geetu; Eriksson, Peter R.; Clark, David J.

    2007-01-01

    The yeast Spt10p activator is a putative histone acetyltransferase (HAT) possessing a sequence-specific DNA-binding domain (DBD) which binds to the upstream activation sequences (UAS elements) in the histone gene promoters. Spt10p binds to a pair of histone UAS elements with extreme positive cooperativity. The molecular basis of this cooperativity was addressed. Spt10p (640 residues) is an elongated dimer, but the isolated DBD (residues 283–396) is a monomer and binds non-cooperatively to DNA. A Spt10p fragment comprising the N-terminal domain (NTD), HAT domain and DBD (residues 1–396) binds cooperatively and is a dimer, whereas an overlapping Spt10p fragment comprising the DBD and C-terminal domains (residues 283–640) binds non-cooperatively and is a monomer. These observations imply that cooperative binding requires dimerization. The isolated NTD (residues 1–98) is a dimer and is responsible for dimerization. We propose that cooperativity involves a conformational change in the Spt10p dimer which facilitates the simultaneous recognition of two UAS elements. In vivo, deletion of the NTD results in poor growth, but does not prevent the binding at the HTA1 promoter, suggesting that dimerization is biologically important. Residues 1–396 are sufficient for normal growth, indicating that the critical functions of Spt10p reside in the N-terminal domains. PMID:17202156

  14. The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1*♦

    PubMed Central

    Derler, Isabella; Plenk, Peter; Fahrner, Marc; Muik, Martin; Jardin, Isaac; Schindl, Rainer; Gruber, Hermann J.; Groschner, Klaus; Romanin, Christoph

    2013-01-01

    STIM1 and Orai1 represent the two molecular key components of the Ca2+ release-activated Ca2+ channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73–90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify “hot spot” residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca2+ selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca2+ selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76–90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate. PMID:23943619

  15. Lactobacillus acidophilus stimulates intestinal P-glycoprotein expression via a c-Fos/c-Jun-dependent mechanism in intestinal epithelial cells.

    PubMed

    Priyamvada, Shubha; Anbazhagan, Arivarasu N; Kumar, Anoop; Soni, Vikas; Alrefai, Waddah A; Gill, Ravinder K; Dudeja, Pradeep K; Saksena, Seema

    2016-04-15

    Our previous studies showed that Lactobacillus acidophilus (LA) culture supernatant (CS) increased P-glycoprotein [Pgp/multidrug resistance 1 (MDR1)] function, expression, and promoter activity in Caco-2 cells. The current studies were designed to elucidate the molecular mechanisms mediating the stimulatory effects of LA CS on Pgp promoter activity. Deletion analysis indicated that the LA CS response element(s) is located in the -172/+428-bp region, and sequence analysis of this region revealed three potential binding sites for c-Fos or c-Jun: proximal activating protein (AP) 1a (-119/-98 bp), distal AP1b (-99/-78 bp), and AP1c (+175/+196 bp). LA CS (24 h) showed an approximately twofold increase in the protein expression of c-Fos and c-Jun in Caco-2 cells. Electrophoretic mobility shift assay showed that LA CS markedly increased the binding of Caco-2 nuclear proteins to AP1a and AP1b, but not AP1c. The DNA-protein complex was completely eliminated by c-Fos antibody, while c-Jun antibody partially eliminated the complex. Chromatin immunoprecipitation analysis also showed that LA CS enhanced the association of c-Fos and c-Jun (by ∼4- and 1.5-fold, respectively) with endogenous Pgp promoter in Caco-2 cells (p-172/+1). Interestingly, overexpression of c-Fos or c-Jun activated Pgp promoter by nearly twofold each. This increase was further enhanced (∼14-fold) when c-Fos and c-Jun were simultaneously overexpressed, suggesting that the presence of one of these transcription factors potentiates the effect of the other. These studies, for the first time, provide evidence for the involvement of c-Fos/c-Jun in stimulation of Pgp gene expression by LA CS in the human intestine. PMID:26867563

  16. The red clover necrotic mosaic virus capsid protein N-terminal amino acids possess specific RNA binding activity and are required for stable virion assembly.

    PubMed

    Park, Sang-Ho; Sit, Tim L; Kim, Kook-Hyung; Lommel, Steven A

    2013-09-01

    The red clover necrotic mosaic virus (RCNMV) bipartite RNA genome is packaged into two virion populations containing either RNA-1 and RNA-2 or multiple copies of RNA-2 only. To understand this distinctive packaging scheme, we investigated the RNA-binding properties of the RCNMV capsid protein (CP). Maltose binding protein-CP fusions exhibited the highest binding affinities for RNA probes containing the RNA-2 trans-activator or the 3' non-coding region from RNA-1. Other viral and non-viral RNA probes displayed CP binding but to a much lower degree. Deletion of the highly basic N-terminal 50 residues abolished CP binding to viral RNA transcripts. In planta studies of select CP deletion mutants within this N-terminal region revealed that it was indispensable for stable virion formation and the region spanning CP residues 5-15 is required for systemic movement. Thus, the N-terminal region of the CP is involved in both producing two virion populations due to its RNA binding properties and virion stability. PMID:23747688

  17. Delayed Cell Cycle Progression in Selenoprotein W-depleted Cells Is Regulated by a Mitogen-activated Protein Kinase Kinase 4-p38/c-Jun NH2-terminal Kinase-p53 Pathway*

    PubMed Central

    Hawkes, Wayne Chris; Alkan, Zeynep

    2012-01-01

    Selenoprotein W (SEPW1) is a ubiquitous, highly conserved thioredoxin-like protein whose depletion causes a transient p53- and p21Cip1-dependent G1-phase cell cycle arrest in breast and prostate epithelial cells. SEPW1 depletion increases phosphorylation of Ser-33 in p53, which is associated with decreased p53 ubiquitination and stabilization of p53. We report here that delayed cell cycle progression, Ser-33 phosphorylation, and p53 nuclear accumulation from SEPW1 depletion require mitogen-activated protein kinase kinase 4 (MKK4). Silencing MKK4 rescued G1 arrest, Ser-33 phosphorylation, and nuclear accumulation of p53 induced by SEPW1 depletion, but silencing MKK3, MKK6, or MKK7 did not. SEPW1 silencing did not change the phosphorylation state of MKK4 but increased total MKK4 protein. Silencing p38γ, p38δ, or JNK2 partially rescued G1 arrest from SEPW1 silencing, suggesting they signal downstream from MKK4. These results imply that SEPW1 silencing increases MKK4, which activates p38γ, p38δ, and JNK2 to phosphorylate p53 on Ser-33 and cause a transient G1 arrest. PMID:22730327

  18. Modulation of NifA activity by PII in Azospirillum brasilense: evidence for a regulatory role of the NifA N-terminal domain.

    PubMed Central

    Arsene, F; Kaminski, P A; Elmerich, C

    1996-01-01

    Azospirillum brasilense NifA, which is synthesized under all physiological conditions, exists in an active or inactive from depending on the availability of ammonia. The activity also depends on the presence of PII, as NifA is inactive in a glnB mutant. To investigate further the mechanism that regulates NifA activity, several deletions of the nifA coding sequence covering the amino-terminal domain of NifA were constructed. The ability of these truncated NifA proteins to activate the nifH promoter in the absence or presence of ammonia was assayed in A. brasilense wild-type and mutant strains. Our results suggest that the N-terminal domain is not essential for NifA activity. This domain plays an inhibitory role which prevents NifA activity in the presence of ammonia. The truncated proteins were also able to restore nif gene expression to a glnB mutant, suggesting that PII is required to activate NifA by preventing the inhibitory effect of its N-terminal domain under conditions of nitrogen fixation. Low levels of nitrogenase activity in the presence of ammonia were also observed when the truncated gene was introduced into a strain devoid of the ADP-ribosylation control of nitrogenase. We propose a model for the regulation of NifA activity in A. brasilense. PMID:8759845

  19. Structural Insight into the Critical Role of the N-Terminal Region in the Catalytic Activity of Dual-Specificity Phosphatase 26.

    PubMed

    Won, Eun-Young; Lee, Sang-Ok; Lee, Dong-Hwa; Lee, Daeyoup; Bae, Kwang-Hee; Lee, Sang Chul; Kim, Seung Jun; Chi, Seung-Wook

    2016-01-01

    Human dual-specificity phosphatase 26 (DUSP26) is a novel target for anticancer therapy because its dephosphorylation of the p53 tumor suppressor regulates the apoptosis of cancer cells. DUSP26 inhibition results in neuroblastoma cell cytotoxicity through p53-mediated apoptosis. Despite the previous structural studies of DUSP26 catalytic domain (residues 61-211, DUSP26-C), the high-resolution structure of its catalytically active form has not been resolved. In this study, we determined the crystal structure of a catalytically active form of DUSP26 (residues 39-211, DUSP26-N) with an additional N-terminal region at 2.0 Å resolution. Unlike the C-terminal domain-swapped dimeric structure of DUSP26-C, the DUSP26-N (C152S) monomer adopts a fold-back conformation of the C-terminal α8-helix and has an additional α1-helix in the N-terminal region. Consistent with the canonically active conformation of its protein tyrosine phosphate-binding loop (PTP loop) observed in the structure, the phosphatase assay results demonstrated that DUSP26-N has significantly higher catalytic activity than DUSP26-C. Furthermore, size exclusion chromatography-multiangle laser scattering (SEC-MALS) measurements showed that DUSP26-N (C152S) exists as a monomer in solution. Notably, the crystal structure of DUSP26-N (C152S) revealed that the N-terminal region of DUSP26-N (C152S) serves a scaffolding role by positioning the surrounding α7-α8 loop for interaction with the PTP-loop through formation of an extensive hydrogen bond network, which seems to be critical in making the PTP-loop conformation competent for phosphatase activity. Our study provides the first high-resolution structure of a catalytically active form of DUSP26, which will contribute to the structure-based rational design of novel DUSP26-targeting anticancer therapeutics. PMID:27583453

  20. c-Jun in Schwann cells promotes axonal regeneration and motoneuron survival via paracrine signaling

    PubMed Central

    Fontana, Xavier; Hristova, Mariya; Da Costa, Clive; Patodia, Smriti; Thei, Laura; Makwana, Milan; Spencer-Dene, Bradley; Latouche, Morwena; Mirsky, Rhona; Jessen, Kristjan R.; Klein, Rüdiger

    2012-01-01

    The AP-1 transcription factor c-Jun is a master regulator of the axonal response in neurons. c-Jun also functions as a negative regulator of myelination in Schwann cells (SCs) and is strongly reactivated in SCs upon axonal injury. We demonstrate here that, after injury, the absence of c-Jun specifically in SCs caused impaired axonal regeneration and severely increased neuronal cell death. c-Jun deficiency resulted in decreased expression of several neurotrophic factors, and GDNF and Artemin, both of which encode ligands for the Ret receptor tyrosine kinase, were identified as novel direct c-Jun target genes. Genetic inactivation of Ret specifically in neurons resulted in regeneration defects without affecting motoneuron survival and, conversely, administration of recombinant GDNF and Artemin protein substantially ameliorated impaired regeneration caused by c-Jun deficiency. These results reveal an unexpected function for c-Jun in SCs in response to axonal injury, and identify paracrine Ret signaling as an important mediator of c-Jun function in SCs during regeneration. PMID:22753894

  1. Isolation of a novel cold-active family 11 Xylanase from the filamentous fungus Bispora antennata and deletion of its N-terminal amino acids on thermostability.

    PubMed

    Liu, Qiong; Wang, Yaru; Luo, Huiying; Wang, Liwen; Shi, Pengjun; Huang, Huoqing; Yang, Peilong; Yao, Bin

    2015-01-01

    In the present study, we first reported a cold-active xylanase of glycosyl hydrolase family 11, Xyn11, from the filamentous fungus Bispora antennata. The coding gene (xyn11) was cloned and successfully expressed in Pichia pastoris. Deduced Xyn11 exhibited the highest identity of 65 % with a family 11 endo-β-1,4-xylanase from Alternaria sp. HB186. Recombinant Xyn11 exhibited maximal activity at 35 °C and remained 21 % of the activity at 0 °C. Sequence alignment showed that the N-terminal sequence of Xyn11 is distinct from those of thermophilic xylanases of family 11. To determine its effect on enzyme properties, the Xyn11 mutant without the N-terminal sequence, t-Xyn11, was then constructed, expressed in P. pastoris, and compared with Xyn11. Both enzymes showed optimal activities at 35 °C and pH 5.5 and were stable at pH 2.0-12.0. Compared with truncated mutant t-Xyn11, Xyn11 retained more activity after 20-min incubation at 40 °C (Xyn11:28 % vs. t-Xyn11:4 %) and degraded xylan substrates more completely. Thus, a new factor affecting the thermostability of cold-active xylanase of family 11 was identified. PMID:25351632

  2. Bile acid regulates c-Jun expression through the orphan nuclear receptor SHP induction in gastric cells

    SciTech Connect

    Park, Won Il; Park, Min Jung; An, Jin Kwang; Choi, Yung Hyun; Kim, Hye Young; Cheong, JaeHun Yang, Ung Suk

    2008-05-02

    Bile reflux is considered to be one of the most important causative factors in gastric carcinogenesis, due to the attendant inflammatory changes in the gastric mucosa. In this study, we have assessed the molecular mechanisms inherent to the contribution of bile acid to the transcriptional regulation of inflammatory-related genes. In this study, we demonstrated that bile acid induced the expression of the SHP orphan nuclear receptor at the transcriptional level via c-Jun activation. Bile acid also enhanced the protein interaction of NF-{kappa}B and SHP, thereby resulting in an increase in c-Jun expression and the production of the inflammatory cytokine, TNF{alpha}. These results indicate that bile acid performs a critical function in the regulation of the induction of inflammatory-related genes in gastric cells, and that bile acid-mediated gene expression provides a pre-clue for the development of gastric cellular malformation.

  3. Fibroin and Sericin from Bombyx mori Silk Stimulate Cell Migration through Upregulation and Phosphorylation of c-Jun

    PubMed Central

    García-Vizcaíno, Eva María; Alcaraz, Antonia; Cenis, José Luis; Nicolás, Francisco José

    2012-01-01

    Wound healing is a biological process directed to the restoration of tissue that has suffered an injury. An important phase of wound healing is the generation of a basal epithelium able to wholly replace the epidermis of the wound. A broad range of products derived from fibroin and sericin from Bombyx mori silk are used to stimulate wound healing. However, so far the molecular mechanism underlying this phenomenon has not been elucidated. The aim of this work was to determine the molecular basis underlying wound healing properties of silk proteins using a cell model. For this purpose, we assayed fibroin and sericin in a wound healing scratch assay using MDA-MB-231 and Mv1Lu cells. Both proteins stimulated cell migration. Furthermore, treatment with sericin and fibroin involved key factors of the wound healing process such as upregulation of c-Jun and c-Jun protein phosphorylation. Moreover, fibroin and sericin stimulated the phosphorylation of ERK 1/2 and JNK 1/2 kinases. All these experiments were done in the presence of specific inhibitors for some of the cell signalling pathways referred above. The obtained results revealed that MEK, JNK and PI3K pathways are involved in fibroin and sericin stimulated cells migration. Inhibition of these three kinases prevented c-Jun upregulation and phosphorylation by fibroin or sericin. Fibroin and sericin were tested in the human keratinocyte cell line, HaCaT, with similar results. Altogether, our results showed that fibroin and sericin initiate cell migration by activating the MEK, JNK and PI3K signalling pathways ending in c-Jun activation. PMID:22860103

  4. The N-terminal PIN domain of the exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast core exosome.

    PubMed

    Schneider, Claudia; Leung, Eileen; Brown, Jeremy; Tollervey, David

    2009-03-01

    Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3' exonuclease activity. We report that expression only of Rrp44 lacking 3'-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5'-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3'-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element. PMID:19129231

  5. N-terminal guanidinylation of TIPP (Tyr-Tic-Phe-Phe) peptides results in major changes of the opioid activity profile.

    PubMed

    Weltrowska, Grazyna; Nguyen, Thi M-D; Chung, Nga N; Wilkes, Brian C; Schiller, Peter W

    2013-09-15

    Derivatives of peptides of the TIPP (Tyr-Tic-Phe-Phe; Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) family containing a guanidino (Guan) function in place of the N-terminal amino group were synthesized in an effort to improve their blood-brain barrier permeability. Unexpectedly, N-terminal amidination significantly altered the in vitro opioid activity profiles. Guan-analogues of TIPP-related δ opioid antagonists showed δ partial agonist or mixed δ partial agonist/μ partial agonist activity. Guanidinylation of the mixed μ agonist/δ antagonists H-Dmt-Tic-Phe-Phe-NH2 (DIPP-NH2) and H-Dmt-TicΨ[CH2NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]) converted them to mixed μ agonist/δ agonists. A docking study revealed distinct positioning of DIPP-NH2 and Guan-DIPP-NH2 in the δ receptor binding site. Lys(3)-analogues of DIPP-NH2 and DIPP-NH2[Ψ] (guanidinylated or non-guanidinylated) turned out to be mixed μ/κ agonists with δ antagonist-, δ partial agonist- or δ full agonist activity. Compounds with some of the observed mixed opioid activity profiles have therapeutic potential as analgesics with reduced side effects or for treatment of cocaine addiction. PMID:23932788

  6. Role of TGF-β-induced Claudin-4 expression through c-Jun signaling in non-small cell lung cancer.

    PubMed

    Rachakonda, Girish; Vu, Trung; Jin, Lin; Samanta, Debangshu; Datta, Pran K

    2016-10-01

    Claudin-4 has been identified as an integral member of tight junctions and has been found to be upregulated in various types of cancers especially in metastatic cancers. However, the molecular mechanism of the upregulation of Claudin-4 and its role in lung tumorigenesis are unknown. The aim of the present study is to investigate the role of Claudin-4 on migration and tumorigenicity of lung cancer cells and to examine the regulatory effects of TGF-β on Claudin-4 expression. We have observed that TGF-β induces the expression of Claudin-4 dramatically in lung cell lines in a time dependent manner. TGF-β-induced Smad signaling is important for enhancing Claudin-4 mRNA level through inducing its promoter activity. Treatment with curcumin, a c-Jun inhibitor, or stable knockdown of c-Jun abrogates TGF-β-induced Claudin-4 expression suggesting an involvement of the c-Jun pathway. Notably, TGF-β-induced Claudin-4 expression through c-Jun pathway plays a role in TGF-β-mediated motility and tumorigenicity of these cells. In support of these observations, we have uncovered that Claudin-4 is upregulated in 14 of 24 (58%) lung tumors when compared with normal lung tissue. This is the first study to show how TGF-β regulates the expression of Claudin-4 through c-Jun signaling and how this pathway contributes to the migratory and tumorigenic phenotype of lung tumor cells. PMID:27424491

  7. A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

    PubMed Central

    Sun, J; Fass, D N; Viss, M A; Hummel, A M; Tang, H; Homburger, H A; Specks, U

    1998-01-01

    ANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, δ-rPR3-S176A does not. Culture supernatants of rPR3-S176A and δ-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With δ-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the N-terminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression. PMID:9822293

  8. HDAC inhibitors suppress c-Jun/Fra-1-mediated proliferation through transcriptionally downregulating MKK7 and Raf1 in neuroblastoma cells

    PubMed Central

    Tang, Xiaomei; Xia, Yong; He, Guozhen; Min, Zhiqun; Li, Chun; Xiong, Shiqiu; Shi, Zhi; Lu, Yongjian; Yuan, Zhongmin

    2016-01-01

    Activator protein 1 (AP-1) is a transcriptional factor composed of the dimeric members of bZIP proteins, which are frequently deregulated in human cancer cells. In this study, we aimed to identify an oncogenic AP-1 dimer critical for the proliferation of neuroblastoma cells and to investigate whether histone deacetylase inhibitors (HDACIs), a new generation of anticancer agents, could target the AP-1 dimer. We report here that HDACIs including trichostatin A, suberoylanilidehydroxamic acid, valproic acid and M344 can transcriptionally suppress both c-Jun and Fra-1, preceding their inhibition of cell growth. c-Jun preferentially interacting with Fra-1 as a heterodimer is responsible for AP-1 activity and critical for cell growth. Mechanistically, HDACIs suppress Fra-1 expression through transcriptionally downregulating Raf1 and subsequently decreasing MEK1/2-ERK1/2 activity. Unexpectedly, HDACI treatment caused MKK7 downregulation at both the protein and mRNA levels. Deletion analysis of the 5′-flanking sequence of the MKK7 gene revealed that a major element responsible for the downregulation by HDACI is located at −149 to −3 relative to the transcriptional start site. Knockdown of MKK7 but not MKK4 remarkably decreased JNK/c-Jun activity and proliferation, whereas ectopic MKK7-JNK1 reversed HDACI-induced c-Jun suppression. Furthermore, suppression of both MKK-7/c-Jun and Raf-1/Fra-1 activities was involved in the tumor growth inhibitory effects induced by SAHA in SH-SY5Y xenograft mice. Collectively, these findings demonstrated that c-Jun/Fra-1 dimer is critical for neuroblastoma cell growth and that HDACIs act as effective suppressors of the two oncogenes through transcriptionally downregulating MKK7 and Raf1. PMID:26734995

  9. The N-Terminal Region of IFITM3 Modulates Its Antiviral Activity by Regulating IFITM3 Cellular Localization

    PubMed Central

    Jia, Rui; Pan, Qinghua; Ding, Shilei; Rong, Liwei; Liu, Shan-Lu; Geng, Yunqi

    2012-01-01

    Interferon-inducible transmembrane (IFITM) protein family members IFITM1, -2, and -3 restrict the infection of multiple enveloped viruses. Significant enrichment of a minor IFITM3 allele was recently reported for patients who were hospitalized for seasonal and 2009 H1N1 pandemic flu. This IFITM3 allele lacks the region corresponding to the first amino-terminal 21 amino acids and is unable to inhibit influenza A virus. In this study, we found that deleting this 21-amino-acid region relocates IFITM3 from the endosomal compartments to the cell periphery. This finding likely underlies the lost inhibition of influenza A virus that completes its entry exclusively within endosomes at low pH. Yet, wild-type IFITM3 and the mutant with the 21-amino-acid deletion inhibit HIV-1 replication equally well. Given the pH-independent nature of HIV-1 entry, our results suggest that IFITM3 can inhibit viruses that enter cells via different routes and that its N-terminal region is specifically required for controlling pH-dependent viruses. PMID:23055554

  10. Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

    PubMed

    Conforti, Laura; Wilbrey, Anna; Morreale, Giacomo; Janeckova, Lucie; Beirowski, Bogdan; Adalbert, Robert; Mazzola, Francesca; Di Stefano, Michele; Hartley, Robert; Babetto, Elisabetta; Smith, Trevor; Gilley, Jonathan; Billington, Richard A; Genazzani, Armando A; Ribchester, Richard R; Magni, Giulio; Coleman, Michael

    2009-02-23

    The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration. PMID:19237596

  11. WldS protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice

    PubMed Central

    Conforti, Laura; Wilbrey, Anna; Morreale, Giacomo; Janeckova, Lucie; Beirowski, Bogdan; Adalbert, Robert; Mazzola, Francesca; Di Stefano, Michele; Hartley, Robert; Babetto, Elisabetta; Smith, Trevor; Gilley, Jonathan; Billington, Richard A.; Genazzani, Armando A.; Ribchester, Richard R.; Magni, Giulio

    2009-01-01

    The slow Wallerian degeneration (WldS) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70–amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide–synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of WldS-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the WldS VCP-binding domain with an alternative ataxin-3–derived VCP-binding sequence restores its protective function. Enzyme-dead WldS is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. WldS requires both of its components to protect axons from degeneration. PMID:19237596

  12. Design, synthesis and biological activity of new neurohypophyseal hormones analogues conformationally restricted in the N-terminal part of the molecule. Highly potent OT receptor antagonists.

    PubMed

    Kwiatkowska, Anna; Ptach, Monika; Borovičková, Lenka; Slaninová, Jiřina; Lammek, Bernard; Prahl, Adam

    2012-08-01

    In this study we present the synthesis and some pharmacological properties of fourteen new analogues of neurohypophyseal hormones conformationally restricted in the N-terminal part of the molecule. All new peptides were substituted at position 2 with cis-1-amino-4-phenylcyclohexane-1-carboxylic acid (cis-Apc). Moreover, one of the new analogues: [cis-Apc(2), Val(4)]AVP was also prepared in N-acylated forms with various bulky acyl groups. All the peptides were tested for pressor, antidiuretic, and in vitro uterotonic activities. We also determined the binding affinity of the selected compounds to human OT receptor. Our results showed that introduction of cis -Apc(2) in position 2 of either AVP or OT resulted in analogues with high antioxytocin potency. Two of the new compounds, [Mpa(1),cis-Apc(2)]AVP and [Mpa(1),cis-Apc(2),Val(4)]AVP, were exceptionally potent antiuterotonic agents (pA(2) = 8.46 and 8.40, respectively) and exhibited higher affinities for the human OT receptor than Atosiban (K (i) values 5.4 and 9.1 nM). Moreover, we have demonstrated for the first time that N -terminal acylation of AVP analogue can improve its selectivity. Using this approach, we obtained compound Aba[cis-Apc(2),Val(4)]AVP (XI) which turned out to be a moderately potent and exceptionally selective OT antagonist (pA(2) = 7.26). PMID:22038179

  13. Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-jun

    SciTech Connect

    Kumari, Sangeeta; Saradhi, Mallampati; Rana, Manjul; Chatterjee, Swagata; Aumercier, Marc; Mukhopadhyay, Gauranga; Tyagi, Rakesh K.

    2015-01-15

    Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling. - Highlights: • The study identified cis-regulatory elements in the nuclear receptor PXR promoter. • Several trans-acting factors modulating the PXR-promoter have been identified. • PU.1/Ets-1, Pax5, LEF-1, c-Jun, LyF-VI and NF-1 act as modulators of the PXR-promoter. • Ets-1 in conjunction with LEF-1 and c-Jun exhibit 5-fold activation of the PXR-promoter. • Insights into PXR-regulation have relevance in normal and pathological conditions.

  14. The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation*

    PubMed Central

    Kong, Roy C. K.; Petrie, Emma J.; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C. Y.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  15. The relaxin receptor (RXFP1) utilizes hydrophobic moieties on a signaling surface of its N-terminal low density lipoprotein class A module to mediate receptor activation.

    PubMed

    Kong, Roy C K; Petrie, Emma J; Mohanty, Biswaranjan; Ling, Jason; Lee, Jeremy C Y; Gooley, Paul R; Bathgate, Ross A D

    2013-09-27

    The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true "ligand" of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation. PMID:23926099

  16. Autocrine regulation of growth stimulation in human epithelial ovarian carcinoma by serine-proteinase-catalysed release of the urinary-type-plasminogen-activator N-terminal fragment.

    PubMed Central

    Fishman, D A; Kearns, A; Larsh, S; Enghild, J J; Stack, M S

    1999-01-01

    Ovarian carcinomas secrete single-chain urinary-type plasminogen activator (scuPA) and expression of uPA is up-regulated relative to normal ovarian epithelium, leading to an enhanced proteolytic capacity which may facilitate invasion. Furthermore, the uPA receptor (uPAR) is present on ovarian carcinoma cells and is occupied in tumour tissues. In the present study, incubation of scuPA with serum-free conditioned medium from ovarian carcinoma cells resulted in release of a 14 kDa polypeptide. N-terminal sequence analysis identified this fragment as the uPA N-terminal fragment (NTF), which contains a growth-factor and a kringle domain. NTF generation was abolished by serine-proteinase inhibitors, but not inhibitors of matrix metalloproteinases, and was not enhanced by the addition of plasminogen or plasmin. To determine whether ovarian carcinoma-cell growth is altered by uPA, the effect of exogenous scuPA or NTF on proliferation was analysed. Both NTF and scuPA induced a dose-dependent increase in proliferation, with maximal stimulation obtained at 10-20 nM. Furthermore, blocking the interaction of endogenous uPA with uPAR using anti-NTF antibodies significantly inhibited proliferation. Together these data indicate that, in addition to enhancing the invasive activity of ovarian carcinoma cells via increased pericellular proteolysis, uPA also acts as a mitogen for ovarian carcinoma cells, suggesting a biochemical mechanism whereby uPA may contribute to ovarian carcinoma progression by modulating both cell invasion and proliferation. PMID:10417342

  17. Identifying the activation motif in the N-terminal of rainbow trout and zebrafish melanocortin-2 receptor accessory protein 1 (MRAP1) orthologs.

    PubMed

    Dores, Robert M; Liang, Liang; Hollmann, Rebecca E; Sandhu, Navdeep; Vijayan, Mathilakath M

    2016-08-01

    The activation of mammalian melanocortin-2 receptor (MC2R) orthologs is dependent on a four-amino acid activation motif (LDYL/I) located in the N-terminal of mammalian MRAP1 (melanocortin-2 receptor accessory protein). Previous alanine substitution analysis had shown that the Y residue in this motif appears to be the most important for mediating the activation of mammalian MC2R orthologs. Similar, but not identical amino acid motifs were detected in rainbow trout MRAP1 (YDYL) and zebrafish MRAP1 (YDYV). To determine the importance of these residues in the putative activation motifs, rainbow trout and zebrafish MRAP1 orthologs were individually co-expressed in CHO cells with rainbow trout MC2R, and the activation of this receptor with either the wild-type MRAP1 ortholog or alanine-substituted analogs of the two teleost MRAP1s was analyzed. Alanine substitutions at all four amino acid positions in rainbow trout MRAP1 blocked activation of the rainbow trout MC2R. Single alanine substitutions of the D and Y residues in rainbow trout and zebrafish MRAP1 indicate that these two residues play a significant role in the activation of rainbow trout MC2R. These observations indicate that there are subtle differences in the way that teleost and mammalian MRAPs are involved in the activation of their corresponding MC2R orthologs. PMID:26752246

  18. Parathyroid hormone induces c-fos and c-jun messenger RNA in rat osteoblastic cells

    NASA Technical Reports Server (NTRS)

    Clohisy, J. C.; Scott, D. K.; Brakenhoff, K. D.; Quinn, C. O.; Partridge, N. C.

    1992-01-01

    PTH is a potent regulator of osteoblast gene expression, yet the nuclear events that mediate PTH action are poorly understood. We were interested in identifying immediate early genes which may regulate PTH-altered gene expression in the osteoblast. Therefore, we examined the effects of PTH on c-fos and c-jun gene expression in a rat osteoblastic cell line (UMR 106-01). Under control conditions, c-fos and c-jun mRNAs were present at low basal levels. After PTH treatment, c-fos mRNA abundance dramatically increased, with a maximal and transient response at 30 min. PTH also stimulated an increase in c-jun mRNA, but in a biphasic manner, with maximal levels at 30 min and 2 h. These responses were dose dependent, not altered by cotreatment with the protein synthesis inhibitor cycloheximide, and preceded PTH-induced expression of matrix metallo-proteinase-1 mRNA. Nuclear run-on assays demonstrated an increased rate of c-fos and c-jun transcription after PTH exposure. To determine the signal transduction pathways involved, second messenger analogs were tested for their ability to mimic the effects of PTH. 8-Bromo-cAMP and phorbol 12-myristate 13-acetate (PMA) caused increases in the abundance of c-fos and c-jun transcripts. Ionomycin had no effect on the expression of these genes. Pretreatment of the cells with PMA resulted in a decrease in basal c-jun expression, but did not alter the PTH-mediated increase in c-fos, c-jun, or matrix metalloproteinase-1 mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS).

  19. 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.

  20. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703.

    PubMed

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase's properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca(2+) were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  1. A multilayered regulatory mechanism for the autoinhibition and activation of a plant CC-NB-LRR resistance protein with an extra N-terminal domain.

    PubMed

    Chen, Xiaojiao; Zhu, Min; Jiang, Lei; Zhao, Wenyang; Li, Jia; Wu, Jianyan; Li, Chun; Bai, Baohui; Lu, Gang; Chen, Hongyu; Moffett, Peter; Tao, Xiaorong

    2016-10-01

    The tomato resistance protein Sw-5b differs from the classical coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) resistance proteins by having an extra N-terminal domain (NTD). To understand how NTD, CC and NB-LRR regulate autoinhibition and activation of Sw-5b, we dissected the function(s) of each domain. When viral elicitor was absent, Sw-5b LRR suppressed the central NB-ARC to maintain autoinhibition of the NB-LRR segment. The CC and NTD domains independently and additively enhanced the autoinhibition of NB-LRR. When viral elicitor was present, the NB-LRR segment of Sw-5b was specifically activated to trigger a hypersensitive response. Surprisingly, Sw-5b CC suppressed the activation of NB-LRR, whereas the extra NTD of Sw-5b became a positive regulator and fully activated the resistance protein, probably by relieving the inhibitory effects of the CC. In infection assays of transgenic plants, the NB-LRR segment alone was insufficient to confer resistance against Tomato spotted wilt tospovirus; the layers of NTD and CC regulation on NB-LRR were required for Sw-5b to confer resistance. Based on these findings, we propose that, to counter the negative regulation of the CC on NB-LRR, Sw-5b evolved an extra NTD to coordinate with the CC, thus developing a multilayered regulatory mechanism to control autoinhibition and activation. PMID:27558751

  2. Structure-Activity Relationships of the Antimicrobial Peptide Arasin 1 — And Mode of Action Studies of the N-Terminal, Proline-Rich Region

    PubMed Central

    Paulsen, Victoria S.; Blencke, Hans-Matti; Benincasa, Monica; Haug, Tor; Eksteen, Jacobus J.; Styrvold, Olaf B.; Scocchi, Marco; Stensvåg, Klara

    2013-01-01

    Arasin 1 is a 37 amino acid long proline-rich antimicrobial peptide isolated from the spider crab, Hyas araneus. In this work the active region of arasin 1 was identified through structure-activity studies using different peptide fragments derived from the arasin 1 sequence. The pharmacophore was found to be located in the proline/arginine-rich NH2 terminus of the peptide and the fragment arasin 1(1–23) was almost equally active to the full length peptide. Arasin 1 and its active fragment arasin 1(1–23) were shown to be non-toxic to human red blood cells and arasin 1(1–23) was able to bind chitin, a component of fungal cell walls and the crustacean shell. The mode of action of the fully active N-terminal arasin 1(1–23) was explored through killing kinetic and membrane permeabilization studies. At the minimal inhibitory concentration (MIC), arasin 1(1–23) was not bactericidal and had no membrane disruptive effect. In contrast, at concentrations of 5×MIC and above it was bactericidal and interfered with membrane integrity. We conclude that arasin 1(1–23) has a different mode of action than lytic peptides, like cecropin P1. Thus, we suggest a dual mode of action for arasin 1(1–23) involving membrane disruption at peptide concentrations above MIC, and an alternative mechanism of action, possibly involving intracellular targets, at MIC. PMID:23326415

  3. Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703

    PubMed Central

    Lu, Zhenghui; Wang, Qinhong; Jiang, Sijing; Zhang, Guimin; Ma, Yanhe

    2016-01-01

    High pH condition is of special interest for the potential applications of alkaline α-amylase in textile and detergent industries. Thus, there is a continuous demand to improve the amylase’s properties to meet the requirements set by specific applications. Here we reported the systematic study of modular domain engineering to improve the specific activity and stability of the alkaline α-amylase from Bacillus pseudofirmus 703. The specific activity of the N-terminal domain truncated mutant (N-Amy) increased by ~35-fold with a significantly improved thermo-stability. Kinetic analysis demonstrated that the Kcat and Kcat/Kmof N-Amy were enhanced by 1300-fold and 425.7-fold, respectively, representing the largest catalytic activity improvement of the engineered α-amylases through the methods of domain deletion, fusion or swapping. In addition, different from the wild-type Amy703, no exogenous Ca2+ were required for N-Amy to maintain its full catalytic activity, implying its superior potential for many industrial processes. Circular dichroism analysis and structure modeling revealed that the increased compactness and α-helical content were the main contributors for the improved thermo-stability of N-Amy, while the improved catalytic efficiency was mainly attributed by the increased conformational flexibility around the active center. PMID:26926401

  4. Sequence-specific and general transcriptional activation by the bovine papillomavirus-1 E2 trans-activator require an N-terminal amphipathic helix-containing E2 domain.

    PubMed

    Haugen, T H; Turek, L P; Mercurio, F M; Cripe, T P; Olson, B J; Anderson, R D; Seidl, D; Karin, M; Schiller, J

    1988-12-20

    The sequence-specific trans-activator protein of bovine papillomavirus (BPV)-1, E2, strongly increases transcription at promoters containing papillomaviral ACCG(N)4CGGT (E2P) cis motifs, but can also activate a wide range of co-transfected promoters without E2P cores to a lower extent. Analysis of multiple E2 mutants in transfected cells revealed that the C-terminal DNA binding E2 domain binds to the E2P cis sequences in the form of pre-existing nuclear dimers. The DNA binding function of E2 was required for specific trans-activation of the E2P elements, as well as for the function of the previously described C-terminal 'short E2' transrepressor. In addition to the C terminus, specific trans-activation also required an intact N-terminal half of the E2 protein. When expressed alone, the N-terminal E2 domain was found to activate heterologous promoters without E2P elements to an extent comparable to wild-type E2, and therefore represents the functional transcription activation domain of the E2 factor. In contrast to other DNA-binding activator proteins described to date, the transcriptional activation by the E2 factor can occur without specific DNA binding. Its mechanism may thus involve protein--protein interactions between common transcription factors and the N-terminal E2 domain which contains amphipathic helix motifs. PMID:2854060

  5. Role of a Conserved Salt Bridge between the PAS Core and the N-Terminal Domain in the Activation of the Photoreceptor Photoactive Yellow Protein

    PubMed Central

    Hoersch, Daniel; Otto, Harald; Joshi, Chandra P.; Borucki, Berthold; Cusanovich, Michael A.; Heyn, Maarten P.

    2007-01-01

    The effect of ionic strength on the conformational equilibrium between the I2 intermediate and the signaling state I2′ of the photoreceptor PYP and on the rate of recovery to the dark state were investigated by time-resolved absorption and fluorescence spectroscopy. With increasing salt concentration up to ∼600 mM, the recovery rate k3 decreases and the I2/I2′ equilibrium (K) shifts in the direction of I2′. At higher ionic strength both effects reverse. Experiments with mono-(KCl, NaBr) and divalent (MgCl2, MgSO4) salts show that the low salt effect depends on the ionic strength and not on the cation or anion species. These observations can be described over the entire ionic strength range by considering the activity coefficients of an interdomain salt bridge. At low ionic strength the activity coefficient decreases due to counterion screening whereas at high ionic strength binding of water by the salt leads to an increase in the activity coefficient. From the initial slopes of the plots of log k3 and log K versus the square root of the ionic strength, the product of the charges of the interacting groups was found to be −1.3 ± 0.2, suggesting a monovalent ion pair. The conserved salt bridge K110/E12 connecting the β-sheet of the PAS core and the N-terminal domain is a prime candidate for this ion pair. To test this hypothesis, the mutants K110A and E12A were prepared. In K110A the salt dependence of the I2/I2′ equilibrium was eliminated and of the recovery rate was greatly reduced below ∼600 mM. Moreover, at low salt the recovery rate was six times slower than in wild-type. In E12A significant salt dependence remained, which is attributed to the formation of a novel salt bridge between K110 and E9. At high salt reversal occurs in both mutants suggesting that salting out stabilizes the more compact I2 structure. However, chaotropic anions like SCN shift the I2/I2′ equilibrium toward the partially unfolded I2′ form. The salt linkage K110/E12

  6. The N-terminal propeptide of collagen type III in serum reflects activity and degree of fibrosis in patients with chronic liver disease.

    PubMed

    Frei, A; Zimmermann, A; Weigand, K

    1984-01-01

    To evaluate the diagnostic significance of the collagen Type III (Col 1-3) N-terminal propeptide of procollagen Type III, with respect to activity and degree of liver fibrosis, Col 1-3 serum concentrations were measured in 111 patients with chronic liver diseases and in 60 patients were correlated with liver histology and morphometry. Col 1-3 was measured by a specific radioimmunoassay. Biopsies were read without knowledge of diagnosis. Periportal and intralobular lesions were assessed semiquantitatively by allocating 1 of 4 severity grades to each parameter. All portal areas were measured morphometrically. Compared to 27 normal controls, Col 1-3 concentrations were significantly elevated in patients with untreated chronic active hepatitis, cirrhosis and primary biliary cirrhosis, but not in chronic persistent hepatitis or fatty liver. Morphometrically measured portal tract area significantly correlated with Col 1-3 plasma levels. Among the semiquantitatively measured periportal lesions, the number of fibroblasts exhibited the closest relationship with Col 1-3 levels; there was no relationship between Col 1-3 levels and intralobular lesions. These data suggest that Col 1-3 serum levels reliably reflect the activity and degree of liver fibrosis and are useful along with liver biopsy in follow-up of patients with chronic liver disease. PMID:6479851

  7. A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun.

    PubMed

    Morton, Simon; Davis, Roger J; McLaren, Ann; Cohen, Philip

    2003-08-01

    We have used phospho-specific antibodies to re-examine the multisite phosphorylation of c-Jun in murine RAW macrophages and embryonic fibroblasts. Our results indicate that JNK isoforms are required and sufficient for the phosphorylation of Thr91 and Thr93, as well as the phosphorylation of Ser63 and Ser73, in response to LPS or anisomycin in macrophages and TNFalpha or anisomycin in fibroblasts. However, the phorbol ester (TPA) and EGF-induced phosphorylation of Ser63 and Ser73 is mediated by ERK1/ERK2, as well as JNK1/JNK2, in fibroblasts from wild-type mice and by ERK1/ERK2 alone in fibroblasts from JNK-deficient mice. The phosphorylation of Thr239 is catalysed by GSK3 and the phosphorylation of Ser243 by an as yet unidentified protein kinase. The inhibition of GSK3 is not required for the dephosphorylation of Thr239 in response to LPS, and nor is the phosphorylation of Thr91 and Thr93 required for the TPA- or EGF-induced dephosphorylation of Thr239 in fibroblasts. The agonist-induced dephosphorylation of Thr239 may involve a conformational change that exposes Thr239 to dephosphorylation and/or the activation of a Thr239 phosphatase. PMID:12881422

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

    PubMed

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

    2015-11-01

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

  9. N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin.

    PubMed

    Smith, A Ian; Rajapakse, Niwanthi W; Kleifeld, Oded; Lomonte, Bruno; Sikanyika, Nkumbu L; Spicer, Alexander J; Hodgson, Wayne C; Conroy, Paul J; Small, David H; Kaye, David M; Parkington, Helena C; Whisstock, James C; Kuruppu, Sanjaya

    2016-01-01

    Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer's disease. PMID:26931059

  10. N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin

    PubMed Central

    Smith, A. Ian; Rajapakse, Niwanthi W.; Kleifeld, Oded; Lomonte, Bruno; Sikanyika, Nkumbu L.; Spicer, Alexander J.; Hodgson, Wayne C.; Conroy, Paul J.; Small, David H.; Kaye, David M.; Parkington, Helena C.; Whisstock, James C.; Kuruppu, Sanjaya

    2016-01-01

    Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease. PMID:26931059

  11. A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: design, synthesis, activity against multidrug-resistant bacteria and Candida.

    PubMed

    Mishra, Biswajit; Leishangthem, Geeta Devi; Gill, Kamaldeep; Singh, Abhay K; Das, Swagata; Singh, Kusum; Xess, Immaculata; Dinda, Amit; Kapil, Arti; Patro, Ishan K; Dey, Sharmistha

    2013-02-01

    Lactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8 μg/mL and 6.5 μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800 μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials. PMID:23026014

  12. High Glucose Stimulates Tumorigenesis in Hepatocellular Carcinoma Cells Through AGER-Dependent O-GlcNAcylation of c-Jun.

    PubMed

    Qiao, Yongxia; Zhang, Xiao; Zhang, Yue; Wang, Yulan; Xu, Yanfeng; Liu, Xiangfan; Sun, Fenyong; Wang, Jiayi

    2016-03-01

    Epidemiologic studies suggest that hepatocellular carcinoma (HCC) has a strong relationship with diabetes. However, the underlying molecular mechanisms still remain unclear. Here, we demonstrated that high glucose (HG), one of the main characteristics of diabetes, was capable of accelerating tumorigenesis in HCC cells. Advanced glycosylation end product-specific receptor (AGER) was identified as a stimulator during this process. Mechanistically, AGER activated a hexosamine biosynthetic pathway, leading to enhanced O-GlcNAcylation of target proteins. Notably, AGER was capable of increasing activity and stability of proto-oncoprotein c-Jun via O-GlcNAcylation of this protein at Ser73. Interestingly, c-Jun can conversely enhance AGER transcription. Thereby, a positive autoregulatory feedback loop that stimulates diabetic HCC was established. Finally, we found that AG490, an inhibitor of Janus kinase, has the ability to impair AGER expression and its functions in HCC cells. In conclusion, AGER and its functions to stimulate O-GlcNAcylation are important during liver tumorigenesis, when high blood glucose levels are inadequately controlled. PMID:26825459

  13. Chaperone-like activities of different molecular forms of beta-casein. Importance of polarity of N-terminal hydrophilic domain.

    PubMed

    Yousefi, Reza; Shchutskaya, Yulia Y; Zimny, Jaroslaw; Gaudin, Jean-Charles; Moosavi-Movahedi, Ali A; Muronetz, Vladimir I; Zuev, Yuriy F; Chobert, Jean-Marc; Haertlé, Thomas

    2009-08-01

    As a member of intrinsically unstructured protein family, beta-casein (beta-CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone-like activity in vitro. Like many chaperones, native beta-CN does not contain cysteinyl residues and exhibits strong tendencies for self-association. The chaperone-like activities of three recombinant beta-CNs wild type (WT) beta-CN, C4 beta-CN (with cysteinyl residue in position 4) and C208 beta-CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native beta-CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (beta-CND) of C4-beta-CN and C208 beta-CN were also studied and their chaperone-like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT beta-CN, C208 beta-CN, C4 beta-CN and C4 beta-CND exhibited significantly lower chaperone-like activities than native beta-CN. Dimerization of C208 beta-CN with two distal hydrophilic domains considerably improved its chaperone-like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N-terminal hydrophilic domain as important functional elements in enhancing the chaperone-like activity of native beta-CN. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 623-632, 2009.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com. PMID:19322774

  14. N-terminal modification of VEGF-A C terminus-derived peptides delineates structural features involved in neuropilin-1 binding and functional activity.

    PubMed

    Jia, Haiyan; Aqil, Rehan; Cheng, Lili; Chapman, Chris; Shaikh, Shaheda; Jarvis, Ashley; Chan, A W Edith; Hartzoulakis, Basil; Evans, Ian M; Frolov, Antonina; Martin, John; Frankel, Paul; Djordevic, Snezana; Zachary, Ian C; Selwood, David L

    2014-05-26

    The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect. PMID:24771685

  15. The Unstructured N-terminal Region of Arabidopsis Group 4 Late Embryogenesis Abundant (LEA) Proteins Is Required for Folding and for Chaperone-like Activity under Water Deficit.

    PubMed

    Cuevas-Velazquez, Cesar L; Saab-Rincón, Gloria; Reyes, José Luis; Covarrubias, Alejandra A

    2016-05-13

    Late embryogenesis abundant (LEA) proteins are a conserved group of proteins widely distributed in the plant kingdom that participate in the tolerance to water deficit of different plant species. In silico analyses indicate that most LEA proteins are structurally disordered. The structural plasticity of these proteins opens the question of whether water deficit modulates their conformation and whether these possible changes are related to their function. In this work, we characterized the secondary structure of Arabidopsis group 4 LEA proteins. We found that they are disordered in aqueous solution, with high intrinsic potential to fold into α-helix. We demonstrate that complete dehydration is not required for these proteins to sample ordered structures because milder water deficit and macromolecular crowding induce high α-helix levels in vitro, suggesting that prevalent conditions under water deficit modulate their conformation. We also show that the N-terminal region, conserved across all group 4 LEA proteins, is necessary and sufficient for conformational transitions and that their protective function is confined to this region, suggesting that folding into α-helix is required for chaperone-like activity under water limitation. We propose that these proteins can exist as different conformers, favoring functional diversity, a moonlighting property arising from their structural dynamics. PMID:27006402

  16. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

    PubMed

    Harris, Samantha P; Belknap, Betty; Van Sciver, Robert E; White, Howard D; Galkin, Vitold E

    2016-02-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  17. C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation

    PubMed Central

    Harris, Samantha P.; Belknap, Betty; Van Sciver, Robert E.; White, Howard D.; Galkin, Vitold E.

    2016-01-01

    Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure–function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the “open” structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca2+ have been achieved. We suggest that Ca2+ modulates the interaction of cMyBP-C with the TF in the sarcomere. PMID:26831109

  18. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    SciTech Connect

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam

    2010-07-13

    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC 2.4.1.18, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  19. In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1.

    PubMed

    Shimafuji, Chinatsu; Noguchi, Megumi; Nishie, Mami; Nagao, Jun-Ichi; Shioya, Kouki; Zendo, Takeshi; Nakayama, Jiro; Sonomoto, Kenji

    2015-12-01

    Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively. While the C-terminal domain of NukM is homologous to NisC, the N-terminal domain has no homology with other known proteins. We expressed and characterized the N- and C-terminal domains of NukM, NukMN, and NukMC, separately. In vitro reconstitution revealed that full-length NukM fully modified the substrate peptide NukA. NukMN partially phosphorylated, dehydrated, and cyclized NukA. By contrast, NukMC did not catalyze dehydration, phosphorylation, or cyclization reactions. Interaction studies using surface plasmon resonance analysis indicated that NukM and NukMN can bind NukA with high affinity, whereas NukMC has low substrate-recognition activity. These results suggest that NukMN is mainly responsible for substrate recognition and dehydration and that the whole NukM structure, including the C-terminal domain, is required for the complete modification of NukA. To the best of our knowledge, this is the first report providing insights into the in vitro catalytic activity of individual domains of a LanM-type modification enzyme. PMID:25971839

  20. Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity*

    PubMed Central

    Gagnon, Keith T.; Biswas, Shyamasri; Zhang, Xinxin; Brown, Bernard A.; Wollenzien, Paul; Mattos, Carla; Maxwell, E. Stuart

    2012-01-01

    Box C/D RNA-protein complexes (RNPs) guide the 2′-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C′/D′ RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity. PMID:22496443

  1. Structural insights into the N-terminal GIY-YIG endonuclease activity of "Arabidopsis" glutaredoxin AtGRXS16 in chloroplasts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Glutaredoxins (Grxs) have been identified across taxa as important mediators in various physiological functions. A chloroplastic monothiol glutaredoxin, AtGRXS16 from "Arabidopsis thaliana", comprises two distinct functional domains, an N-terminal domain (NTD) with GlyIleTyr-TyrIleGly (GIY-YIG) endo...

  2. A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells.

    PubMed

    Dias, Juliana Vieira; Benslimane-Ahmim, Zahia; Egot, Marion; Lokajczyk, Anna; Grelac, Françoise; Galy-Fauroux, Isabelle; Juliano, Luiz; Le-Bonniec, Bernard; Takiya, Cristina Maeda; Fischer, Anne-Marie; Blanc-Brude, Olivier; Morandi, Verônica; Boisson-Vidal, Catherine

    2012-10-15

    Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. The TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 μg/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1α. The adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGs) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. PMID:22796565

  3. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules.

    PubMed

    Bruell, Shoni; Kong, Roy C K; Petrie, Emma J; Hoare, Brad; Wade, John D; Scott, Daniel J; Gooley, Paul R; Bathgate, Ross A D

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism. PMID

  4. Chimeric RXFP1 and RXFP2 Receptors Highlight the Similar Mechanism of Activation Utilizing Their N-Terminal Low-Density Lipoprotein Class A Modules

    PubMed Central

    Bruell, Shoni; Kong, Roy C. K.; Petrie, Emma J.; Hoare, Brad; Wade, John D.; Scott, Daniel J.; Gooley, Paul R.; Bathgate, Ross A. D.

    2013-01-01

    Relaxin family peptide (RXFP) receptors 1 and 2 are unique G-protein coupled receptors in that they contain an N-terminal low-density lipoprotein type A (LDLa) module which is necessary for receptor activation. The current hypothesis suggests that upon ligand binding the LDLa module interacts with the transmembrane (TM) domain of a homodimer partner receptor to induce the active receptor conformations. We recently demonstrated that three residues in the N-terminus of the RXFP1 LDLa module are potentially involved in hydrophobic interactions with the receptor to drive activation. RXFP2 shares two out of three of the residues implicated, suggesting that the two LDLa modules could be interchanged without adversely affecting activity. However, in 2007 it was shown that a chimera consisting of the RXFP1 receptor with its LDLa swapped for that of RXFP2 did not signal. We noticed this construct also contained the RXFP2 region linking the LDLa to the leucine-rich repeats. We therefore constructed chimeric RXFP1 and RXFP2 receptors with their LDLa modules swapped immediately C-terminally to the final cysteine residue of the module, retaining the native linker. In addition, we exchanged the TM domains of the chimeras to explore if matching the LDLa module with the TM domain of its native receptor altered activity. All of the chimeras were expressed at the surface of HEK293T cells with ligand binding profiles similar to the wild-type receptors. Importantly, as predicted, ligand binding was able to induce cAMP-based signaling. Chimeras of RXFP1 with the LDLa of RXFP2 demonstrated reduced H2 relaxin potency with the pairing of the RXFP2 TM with the RXFP2 LDLa necessary for full ligand efficacy. In contrast the ligand-mediated potencies and efficacies on the RXFP2 chimeras were similar suggesting the RXFP1 LDLa module has similar efficacy on the RXFP2 TM domain. Our studies demonstrate the LDLa modules of RXFP1 and RXFP2 modulate receptor activation via a similar mechanism. PMID

  5. Basal Cancer Cell Survival Involves JNK2 Suppression of a Novel JNK1/c-Jun/Bcl-3 Apoptotic Network

    PubMed Central

    Ahmed, Shafiq Uddin; Milner, Jo

    2009-01-01

    Background The regulation of apoptosis under basal (non-stress) conditions is crucial for normal mammalian development and also for normal cellular turnover in different tissues throughout life. Deficient regulation of basal apoptosis, or its perturbation, can result in impaired development and/or disease states including cancer. In contrast to stress-induced apoptosis the regulation of apoptosis under basal conditions is poorly understood. To address this issue we have compared basal- and stress-induced apoptosis in human epithelial cells of normal and cancerous origins. For this purpose we focussed our study on the opposing pro-apoptotic JNK/anti-apoptotic NFκB pathways. Methodology/Principal Findings Combinatorial RNAi plus gene knockout were employed to access and map basal regulatory pathways of apoptosis. Follow-on, in-depth analyses included exogenous expression of phosphorylation mutants and chromatin immunoprecipitation. We demonstrate that basal apoptosis is constitutively suppressed by JNK2 in a range of human cancer cell lines. This effect was not observed in non-cancer cells. Silencing JNK2 by RNAi resulted in JNK1-dependent apoptosis of cancer cells via up-regulation of the AP-1 factor c-Jun. Unexpectedly we discovered that JNK1 and c-Jun promote basal apoptosis in the absence of “activating phosphorylations” typically induced by stress. Hypo-phosphorylated c-Jun accumulated to high levels following JNK2 silencing, auto-regulated its own expression and suppressed expression of Bcl-3, an unusual IκB protein and regulator of NFκB. Basal apoptosis was mediated by components of the TNFα response pathway but was mechanistically distinct from TNFα-induced apoptosis. Conclusions/Significance Our results demonstrate that mechanistically distinct pathways operate to regulate apoptosis in mammalian cells under basal (physiological) versus stress-induced conditions. We also describe a novel apoptotic network which governs the basal survival of cancer

  6. Adenovirus E1A downregulates cJun- and JunB-mediated transcription by targeting their coactivator p300.

    PubMed Central

    Lee, J S; See, R H; Deng, T; Shi, Y

    1996-01-01

    Transcription factors and cofactors play critical roles in cell growth and differentiation. Alterations of their activities either through genetic mutations or by viral oncoproteins often result in aberrant cell growth and tumorigenesis. The transcriptional cofactor p300 has recently been shown to be complexed with transcription factors YY1 and CREB. Adenovirus E1A oncoproteins target these transcription complexes via physical interactions with p300, resulting in alterations of transcription mediated by these transcription factors. Here we show that p300 is also critical for repression by E1A of the activities of cJun and JunB, two members of the AP-1 transcriptional complexes. This repressive effect of E1A is dependent on the p300-binding domain of E1A and can be relieved by overexpression of p300. These results suggest that p300 serves as a mediator protein for downregulation of AP-1 activity by E1A. This hypothesis was further supported by the following observations: (i) in the absence of E1A, overexpression of p300 stimulated transcription both through an AP-1 site present in the collagenase promoter and through Jun proteins in GAL4 fusion protein-based assays; and (ii) overexpression of a mutant p300 lacking the E1A-interacting domain reduced the responsiveness of Jun-dependent transcription to E1A repression. As predicted from the functional results, p300 physically interacted with the Jun proteins. These findings thus established that p300 is a cofactor for cJun and JunB. We propose that p300 is a common mediator protein through which E1A gains control over multiple transcriptional regulatory pathways in the host cells. PMID:8754832

  7. Engineering of a disulfide loop instead of a Zn binding loop restores the anti-proliferative, anti-angiogenic and anti-tumor activities of the N-terminal fragment of endostatin: Mechanistic and therapeutic insights.

    PubMed

    Chamani, Reyhane; Asghari, S Mohsen; Alizadeh, Ali Mohammad; Eskandari, Sedigheh; Mansouri, Kamran; Khodarahmi, Reza; Taghdir, Majid; Heidari, Zahra; Gorji, Ali; Aliakbar, Alireza; Ranjbar, Bijan; Khajeh, Khosro

    2015-09-01

    Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and β structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to αVβ3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth. PMID:26187352

  8. Selective participation of c-Jun with Fra-2/c-Fos promotes aggressive tumor phenotypes and poor prognosis in tongue cancer

    PubMed Central

    Gupta, Shilpi; Kumar, Prabhat; Kaur, Harsimrut; Sharma, Nishi; Saluja, Daman; Bharti, Alok C.; Das, Bhudev C.

    2015-01-01

    Tongue squamous cell carcinoma (TSCC) is most aggressive head and neck cancer often associated with HR-HPV infection. The role of AP-1 which is an essential regulator of HPV oncogene expression and tumorigenesis is not reported in tongue cancer. One hundred tongue tissue biopsies comprising precancer, cancer and adjacent controls including two tongue cancer cell lines were employed to study the role of HPV infection and AP-1 family proteins. An exclusive prevalence (28%) of HR-HPV type 16 was observed mainly in well differentiated tongue carcinomas (78.5%). A higher expression and DNA binding activity of AP-1 was observed in tongue tumors and cancer cell lines with c-Fos and Fra-2 as the major binding partners forming the functional AP-1 complex but c-Jun participated only in HPV negative and poorly differentiated carcinoma. Knocking down of Fra-2 responsible for aggressive tongue tumorigenesis led to significant reduction in c-Fos, c-Jun, MMP-9 and HPVE6/E7 expression but Fra-1 and p53 were upregulated. The binding and expression of c-Fos/Fra-2 increased as a function of severity of tongue lesions, yet selective participation of c-Jun appears to promote poor differentiation and aggressive tumorigenesis only in HPV negative cases while HPV infection leads to well differentiation and better prognosis preferably in nonsmokers. PMID:26581505

  9. Protein 4.1N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC.

    PubMed

    Wang, Zi; Ma, Bianyin; Li, Hui; Xiao, Xiaojuan; Zhou, Weihua; Liu, Feng; Zhang, Bin; Zhu, Min; Yang, Qin; Zeng, Yayue; Sun, Yang; Sun, Shuming; Wang, Yanpeng; Zhang, Yibin; Weng, Haibo; Chen, Lixiang; Ye, Mao; An, Xiuli; Liu, Jing

    2016-01-01

    Protein 4.1N is a member of protein 4.1 family and has been recognized as a potential tumor suppressor in solid tumors. Here, we aimed to investigate the role and mechanisms of 4.1N in non-small cell lung cancer (NSCLC). We confirmed that the expression level of 4.1N was inversely correlated with the metastatic properties of NSCLC cell lines and histological grade of clinical NSCLC tissues. Specific knockdown of 4.1N promoted tumor cell proliferation, migration and adhesion in vitro, and tumor growth and metastasis in mouse xenograft models. Furthermore, we identified PP1 as a novel 4.1N-interacting molecule, and the FERM domain of 4.1N mediated the interaction between 4.1N and PP1. Further, ectopic expression of 4.1N could inactivate JNK-c-Jun signaling pathway through enhancing PP1 activity and interaction between PP1 and p-JNK. Correspondingly, expression of potential downstream metastasis targets (ezrin and MMP9) and cell cycle targets (p53, p21 and p19) of JNK-c-Jun pathway were also regulated by 4.1N. Our data suggest that down-regulation of 4.1N expression is a critical step for NSCLC development and that repression of JNK-c-Jun signaling through PP1 is one of the key anti-tumor mechanisms of 4.1N. PMID:26575790

  10. Protein 4.1N acts as a potential tumor suppressor linking PP1 to JNK-c-Jun pathway regulation in NSCLC

    PubMed Central

    Zhou, Weihua; Liu, Feng; Zhang, Bin; Zhu, Min; Yang, Qin; Zeng, Yayue; Sun, Yang; Sun, Shuming; Wang, Yanpeng; Zhang, Yibin; Weng, Haibo; Chen, Lixiang; Ye, Mao; An, Xiuli; Liu, Jing

    2016-01-01

    Protein 4.1N is a member of protein 4.1 family and has been recognized as a potential tumor suppressor in solid tumors. Here, we aimed to investigate the role and mechanisms of 4.1N in non-small cell lung cancer (NSCLC). We confirmed that the expression level of 4.1N was inversely correlated with the metastatic properties of NSCLC cell lines and histological grade of clinical NSCLC tissues. Specific knockdown of 4.1N promoted tumor cell proliferation, migration and adhesion in vitro, and tumor growth and metastasis in mouse xenograft models. Furthermore, we identified PP1 as a novel 4.1N-interacting molecule, and the FERM domain of 4.1N mediated the interaction between 4.1N and PP1. Further, ectopic expression of 4.1N could inactivate JNK-c-Jun signaling pathway through enhancing PP1 activity and interaction between PP1 and p-JNK. Correspondingly, expression of potential downstream metastasis targets (ezrin and MMP9) and cell cycle targets (p53, p21 and p19) of JNK-c-Jun pathway were also regulated by 4.1N. Our data suggest that down-regulation of 4.1N expression is a critical step for NSCLC development and that repression of JNK-c-Jun signaling through PP1 is one of the key anti-tumor mechanisms of 4.1N. PMID:26575790

  11. The requirement of c-Jun N-terminal kinase 2 in regulation of hypoxia-inducing factor-1α mRNA stability.

    PubMed

    Zhang, Dongyun; Li, Jingxia; Zhang, Min; Gao, Guangxun; Zuo, Zhenghong; Yu, Yonghui; Zhu, Linda; Gao, Jimin; Huang, Chuanshu

    2012-10-01

    The mRNA of hif-1α is considered as being constitutively and ubiquitously expressed, regardless of the level of oxygen tension. However many recent reports have showed that hif-1α mRNA could be regulated by natural antisense transcripts, potential microRNAs, and low O(2). In this study, it was found that a deficiency of JNK2 expression reduced HIF-1α protein induction in response to nickel treatment resulting from the impaired expression of hif-1α mRNA. Both the promoter luciferase assay and mRNA degradation assay clearly showed that depletion of JNK2 affected stability of hif-1α mRNA, rather than regulated its transcription. In addition, nucleolin, a classic histone chaperone, was demonstrated to physically bind to hif-1α mRNA and maintain its stability. Further investigation indicated that JNK2 regulated nucleolin expression and might in turn stabilize hif-1α mRNA. Collectively, we provided one more piece of evidence for the oncogenic role of JNK2 and nucleolin in regulating the cancer microenvironments by controlling HIF-1α expression. PMID:22910906

  12. Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

    SciTech Connect

    Rana, Ritu; Jagadish, Nirmala; Garg, Manoj; Mishra, Deepshikha; Dahiya, Neetu; Chaurasiya, Dipak; Suri, Anil . E-mail: anil@nii.res.in

    2006-02-03

    Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.

  13. Localization and regulation of c-fos and c-jun protooncogene induction by systolic wall stress in normal and hypertrophied rat hearts.

    PubMed Central

    Schunkert, H; Jahn, L; Izumo, S; Apstein, C S; Lorell, B H

    1991-01-01

    The effect of changes in left ventricular (LV) systolic force generation on cardiac c-fos and c-jun protooncogene expression was studied by using isolated beating hearts from male Wistar rats. An isovolumic buffer-perfused heart preparation was utilized in which coronary flow and heart rate were held constant and increments in LV balloon volume were used to generate defined levels of LV systolic wall stress. Using Northern and slot-blot analyses, we found that LV tissue from control hearts that generated high levels of LV systolic wall stress expressed 3- to 4.4-fold higher c-fos and c-jun mRNA levels in comparison with tissue from the respective flaccid right ventricles, and in comparison with LV tissue from hearts that generated minimal LV systolic wall stress. To distinguish the role of passive LV diastolic wall stretch from active LV force generation, we found that distension of the LV balloon per se did not have a significant effect on protooncogene induction in hearts perfused with 2,3-butanedione monoxime, which prevents systolic cross-bridge cycling and force generation. In additional hearts studied at a constant LV balloon volume to generate an LV end-diastolic pressure of 10 mm Hg, c-fos mRNA levels were proportional to the magnitude of peak LV systolic wall stress (r = 0.823, P less than 0.05). In these protocols, Fos protein was localized by immunohistochemistry in myocyte nuclei with minimal staining in fibroblasts and vascular smooth muscle. When c-fos and c-jun mRNA expression was compared in hearts with chronic LV hypertrophy due to ascending aortic banding and age-matched control hearts that generated similar incremental levels of LV systolic wall stress, significantly lower levels of c-fos and c-jun mRNA were measured in the hypertrophied hearts. However, there was no difference in protooncogene mRNA expression in response to stimulation by the Ca2+ ionophore A23187. These data suggest that, in this isolated isovolumic beating heart preparation

  14. Protein N-terminal acetyltransferases in cancer.

    PubMed

    Kalvik, T V; Arnesen, T

    2013-01-17

    The human N-terminal acetyltransferases (NATs) catalyze the transfer of acetyl moieties to the N-termini of 80-90% of all human proteins. Six NAT types are present in humans, NatA-NatF, each is composed of specific subunits and each acetylates a set of substrates defined by the N-terminal amino-acid sequence. NATs have been suggested to act as oncoproteins as well as tumor suppressors in human cancers, and NAT expression may be both elevated and decreased in cancer versus non-cancer tissues. Manipulation of NATs in cancer cells induced cell-cycle arrest, apoptosis or autophagy, implying that these enzymes target a variety of pathways. Of particular interest is hNaa10p (human ARD1), the catalytic subunit of the NatA complex, which was coupled to a number of signaling molecules including hypoxia inducible factor-1α, β-catenin/cyclin D1, TSC2/mammalian target of rapamycin, myosin light chain kinase , DNA methyltransferase1/E-cadherin and p21-activated kinase-interacting exchange factors (PIX)/Cdc42/Rac1. The variety of mechanistic links where hNaa10p acts as a NAT, a lysine acetyltransferase or displaying a non-catalytic role, provide insights to how hNaa10p may act as both a tumor suppressor and oncoprotein. PMID:22391571

  15. N-Terminal Region of GbIspH1, Ginkgo biloba IspH Type 1, May Be Involved in the pH-Dependent Regulation of Enzyme Activity

    PubMed Central

    Shin, Bok-Kyu; Ahn, Joong-Hoon; Han, Jaehong

    2015-01-01

    GbIspH1, IspH type 1 in Ginkgo biloba chloroplast, is the Fe/S enzyme catalyzing the reductive dehydroxylation of HMBPP to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) at the final step of methylerythritol phosphate pathway in chloroplast. Compared to the bacterial IspH, plant IspH, including GbIspH1, has an additional polypeptide chain at the N-terminus. Here, biochemical function of the N-terminal region of GbIspH1 was investigated with the N-terminal truncated GbIspH1 (GbIspH1-truncated). Both wild type GbIspH1 (GbIspH1-full) and GbIspH1-truncated were catalytically active and produced IPP and DMAPP in a ratio of 15 : 1. Kinetic parameters of KM (17.3 ± 1.9 and 14.9 ± 2.3 µM) and kcat (369 ± 10 and 347 ± 12 min−1) at pH 8.0 were obtained for GbIspH1-full and GbIspH1-truncated, respectively. Interestingly, GbIspH1-full and GbIspH1-truncated showed significantly different pH-dependent activities, and the maximum enzyme activities were obtained at pH 8.0 and 7.5, respectively. However, catalytic activation energies (Ea) of GbIspH1-full and GbIspH1-truncated were almost the same with 36.5 ± 1.6 and 35.0 ± 1.9 kJ/mol, respectively. It was suggested that the N-terminal region of GbIspH1 is involved in the pH-dependent regulation of enzyme activity during photosynthesis. PMID:25892986

  16. Negative transcriptional regulation of the interferon-gamma promoter by glucocorticoids and dominant negative mutants of c-Jun.

    PubMed

    Cippitelli, M; Sica, A; Viggiano, V; Ye, J; Ghosh, P; Birrer, M J; Young, H A

    1995-05-26

    Interferon-gamma (IFN-gamma) is an immunoregulatory cytokine expressed in large granular lymphocytes and T cells. However, the molecular mechanisms underlying IFN-gamma gene transcription have not been fully defined. Here, we analyze the mechanisms responsible for the inhibition of IFN-gamma promoter activity by the glucocorticoid hormone dexamethasone. Cotransfection assays performed in Jurkat T cells demonstrated that the activity of the initial 108 base pairs of the IFN-gamma promoter was down-regulated in the presence of dexamethasone. Furthermore, utilizing electrophoretic mobility shift analysis, we identified activator protein 1 AP-1-cAMP response element binding protein-activating transcription factor (CREB-ATF) binding elements situated in positions of the IFN-gamma promoter previously identified as essential for promoter activity. Moreover, dominant negative mutants of the c-Jun proto-oncogene were able to mimic the same down-regulatory effect exerted by dexamethasone, and mutations that abolished the binding of the AP-1 CREB-ATF factors were able to block the glucocorticoid effect. These results suggest a model involving the inhibition of IFN-gamma AP-1 CREB-ATF DNA binding complexes as one of the mechanisms involved in the negative regulatory action of glucocorticoids on IFN-gamma gene expression and support the relevance of AP-1 CREB-ATF binding factors during the transcriptional activation of the IFN-gamma promoter in T cells. PMID:7759501

  17. Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH(2)-terminal kinase during human colorectal carcinogenesis.

    PubMed

    Yamagata, Hideo; Matsuzaki, Koichi; Mori, Shigeo; Yoshida, Katsunori; Tahashi, Yoshiya; Furukawa, Fukiko; Sekimoto, Go; Watanabe, Toshihiko; Uemura, Yoshiko; Sakaida, Noriko; Yoshioka, Kazuhiko; Kamiyama, Yasuo; Seki, Toshihito; Okazaki, Kazuichi

    2005-01-01

    Conversion of normal epithelial cells to tumors is associated with a shift in transforming growth factor-beta (TGF-beta) function: reduction of tumor suppressor activity and increase of oncogenic activity. However, specific mechanisms of this functional alteration during human colorectal carcinogenesis remain to be elucidated. TGF-beta signaling involves Smad2/3 phosphorylated at linker regions (pSmad2/3L) and COOH-terminal regions (pSmad2/3C). Using antibodies specific to each phosphorylation site, we herein showed that Smad2 and Smad3 were phosphorylated at COOH-terminal regions but not at linker regions in normal colorectal epithelial cells and that pSmad2/3C were located predominantly in their nuclei. However, the linker regions of Smad2 and Smad3 were phosphorylated in 31 sporadic colorectal adenocarcinomas. In particular, late-stage invasive and metastatic cancers typically showed a high degree of phosphorylation of Smad2/3L. Their extent of phosphorylation in 11 adenomas was intermediate between those in normal epithelial cells and adenocarcinomas. Whereas pSmad2L remained in the cytoplasm, pSmad3L was located exclusively in the nuclei of Ki-67-immunoreactive adenocarcinomas. In contrast, pSmad3C gradually decreased as the tumor stage progressed. Activated c-Jun NH(2)-terminal kinase in cancers could directly phosphorylate Smad2/3L. Although Mad homology 2 region sequencing in the Smad4 gene revealed a G/A substitution at codon 361 in one adenocarcinoma, the mutation did not correlate with phosphorylation. No mutations in the type II TGF-beta receptor and Smad2 genes were observed in the tumors. In conclusion, pSmad3C, which favors tumor suppressor activity of TGF-beta, was found to decrease, whereas c-Jun NH(2)-terminal kinase tended to induce the phosphorylation of Smad2/3L in human colorectal adenoma-carcinoma sequence. PMID:15665291

  18. Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

    PubMed Central

    Gong, Ting; Liao, Yuan; He, Fei; Yang, Yang; Yang, Dan-Dan; Chen, Xiang-Dong

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3Δ rho4Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4. PMID:23264647

  19. Oscillatory Shear Stress Induces Mitochondrial Superoxide Production: Implication of NADPH Oxidase and c-Jun NH2-Terminal Kinase Signaling

    PubMed Central

    Takabe, Wakako; Jen, Nelson; Ai, Lisong; Hamilton, Ryan; Wang, Sky; Holmes, Kristin; Dharbandi, Farhad; Khalsa, Bhavraj; Bressler, Steven; Barr, Mark L.; Li, Rongsong

    2011-01-01

    Abstract Fluid shear stress is intimately linked with vascular oxidative stress and atherosclerosis. We posited that atherogenic oscillatory shear stress (OSS) induced mitochondrial superoxide (mtO2•−) production via NADPH oxidase and c-Jun NH2-terminal kinase (JNK-1 and JNK-2) signaling. In bovine aortic endothelial cells, OSS (±3 dyn/cm2) induced JNK activation, which peaked at 1 h, accompanied by an increase in fluorescein isothiocyanate-conjugated JNK fluorescent and MitoSOX Red (specific for mtO2•− production) intensities. Pretreatment with apocynin (NADPH oxidase inhibitor) or N-acetyl cysteine (antioxidant) significantly attenuated OSS-induced JNK activation. Apocynin further reduced OSS-mediated dihydroethidium and MitoSOX Red intensities specific for cytosolic O2•− and mtO2•− production, respectively. As a corollary, transfecting bovine aortic endothelial cells with JNK siRNA (siJNK) and pretreating with SP600125 (JNK inhibitor) significantly attenuated OSS-mediated mtO2•− production. Immunohistochemistry on explants of human coronary arteries further revealed prominent phosphorylated JNK staining in OSS-exposed regions. These findings indicate that OSS induces mtO2•− production via NADPH oxidase and JNK activation relevant for vascular oxidative stress. Antioxid. Redox Signal. 15, 1379–1388. PMID:20919940

  20. Antimicrobial activity of human α-defensin 5 and its linear analogs: N-terminal fatty acylation results in enhanced antimicrobial activity of the linear analogs.

    PubMed

    Mathew, Basil; Nagaraj, Ramakrishnan

    2015-09-01

    Human α-defensin 5 (HD5) exhibits broad spectrum antimicrobial activity and plays an important role in mucosal immunity of the small intestine. Although there have been several studies, the structural requirements for activity and mechanism of bacterial killing is yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD5 and linear analogs. Cysteine deletions attenuated the antibacterial activity considerably. Candidacidal activity was affected to a lesser extent. Fatty acid conjugated linear analogs showed antimicrobial activity comparable activity to HD5. Effective surface charge neutralization of bacteria was observed for HD5 as compared to the non-fatty acylated linear analogs. Our results show that HD5 and non-fatty acylated linear analogs enter the bacterial cytoplasm without causing damage to the bacterial inner membrane. Although fatty acylated peptides exhibited antimicrobial activity comparable to HD5, their mechanism of action involved permeabilization of the Escherichia coli inner membrane. HD5 and analogs had the ability to bind plasmid DNA. HD5 had greater binding affinity to plasmid DNA as compared to the analogs. The three dimensional structure of HD5 favors greater interaction with the bacterial cell surface and also with DNA. Antibacterial activity of HD5 involves entry into bacterial cytoplasm and binding to DNA which would result in shut down of the bacterial metabolism leading to cell death. We show how a moderately active linear peptide derived from the α-defensin HD5 can be engineered to enhance antimicrobial activity almost comparable to the native peptide. PMID:26206286

  1. Probing the active-site requirements of human intestinal N-terminal maltase glucoamylase: the effect of replacing the sulfate moiety by a methyl ether in ponkoranol, a naturally occurring α-glucosidase inhibitor.

    PubMed

    Eskandari, Razieh; Jones, Kyra; Rose, David R; Pinto, B Mario

    2010-10-01

    Ponkoranol is a naturally occurring glucosidase inhibitor isolated from the plant Salacia reticulata. The compound comprises a sulfonium ion with an internal sulfate counter ion. We report here an efficient synthetic route to 3'-O-methyl ponkoranol to test the hypothesis that occupation of a hydrophobic pocket by a methyl group instead of the polar sulfate ion within the active site of human N-terminal maltase glucoamylase would be beneficial. The synthetic strategy relies on the nucleophilic attack of 2,3,5-tri-O-benzyl-1,4-anhydro-4-thio-D-arabinitol at the C-6 position of benzyl 6-O-p-toluenesulfonyl β-D-glucopyranoside, followed by deprotection using boron trichloride and reduction with sodium borohydride. The target compound inhibited the N-terminal catalytic domain of intestinal human maltase glucoamylase (ntMGAM) with a K(i) value of 0.50 ± 0.04 μM, higher than those of de-O-sulfonated ponkoranol (K(i)=43 ± 3 nM), or its 5'-stereoisomer (K(i)=15 ± 1 nM). We conclude that the interaction of the methyl group with hydrophobic residues in the active site is not as beneficial to inhibition of ntMGAM as the other interactions of the polyhydroxylated chain with active-site residues. PMID:20801033

  2. Effect of vasoactive peptides in Tetrahymena: chemotactic activities of adrenomedullin, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP).

    PubMed

    Kőhidai, László; Tóth, Katalin; Samotik, Paul; Ranganathan, Kiran; Láng, Orsolya; Tóth, Miklós; Ruskoaho, Heikki

    2016-01-01

    Adrenomedullin (AMD), proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) were studied for chemotaxis, chemotactic selection and G-actin/F-actin transition in Tetrahymena. The aim of the experiments was to study the effects of two different peptides encoded by the same gene compared to a peptide related to one of the two, but encoded by a different gene, at a low level of phylogeny. The positive, chemotactic effect of ADM and the strong negative, chemorepellent effect of PAMP suggest that in Tetrahymena, the two peptides elicit their chemotactic effects via different signalling mechanisms. The complexity of swimming behaviour modulated by the three peptides underlines that chemotaxis, chemokinesis and some characteristics of migratory behaviour (velocity, tortuosity) are working as a sub-population level complex functional unit. Chemotactic responsiveness to ADM and CGRP is short-term, in contrast to PAMP, which as a chemorepellent ligand, has the ability to select sub-populations with negative chemotactic responsiveness. The different effects of ADM and PAMP on the polymerization of actin networks show that the microtubular structure of cilia is more essential to chemotactic response than are transitions of the actin network. The results draw attention to the characteristic effects of vasoactive peptides at this low level of phylogeny. PMID:26481478

  3. Multifunctional roles for the N-terminal basic motif of Alfalfa mosaic virus coat protein: nucleolar/cytoplasmic shuttling, modulation of RNA-binding activity, and virion formation.

    PubMed

    Herranz, Mari Carmen; Pallas, Vicente; Aparicio, Frederic

    2012-08-01

    In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation. PMID:22746826

  4. Anti-inflammatory activity of AP-SF, a ginsenoside-enriched fraction, from Korean ginseng

    PubMed Central

    Baek, Kwang-Soo; Hong, Yong Deog; Kim, Yong; Sung, Nak Yoon; Yang, Sungjae; Lee, Kyoung Min; Park, Joo Yong; Park, Jun Seong; Rho, Ho Sik; Shin, Song Seok; Cho, Jae Youl

    2014-01-01

    Background Korean ginseng is an ethnopharmacologically valuable herbal plant with various biological properties including anticancer, antiatherosclerosis, antidiabetic, and anti-inflammatory activities. Since there is currently no drug or therapeutic remedy derived from Korean ginseng, we developed a ginsenoside-enriched fraction (AP-SF) for prevention of various inflammatory symptoms. Methods The anti-inflammatory efficacy of AP-SF was tested under in vitro inflammatory conditions including nitric oxide (NO) production and inflammatory gene expression. The molecular events of inflammatory responses were explored by immunoblot analysis. Results AP-SF led to a significant suppression of NO production compared with a conventional Korean ginseng saponin fraction, induced by both lipopolysaccharide and zymosan A. Interestingly, AP-SF strongly downregulated the mRNA levels of genes for inducible NO synthase, tumor necrosis factor-α, and cyclooxygenase) without affecting cell viability. In agreement with these observations, AP-SF blocked the nuclear translocation of c-Jun at 2 h and also reduced phosphorylation of p38, c-Jun N-terminal kinase, and TAK-1, all of which are important for c-Jun translocation. Conclusion Our results suggest that AP-SF inhibits activation of c-Jun-dependent inflammatory events. Thus, AP-SF may be useful as a novel anti-inflammatory remedy. PMID:26045689

  5. Metformin prevents endoplasmic reticulum stress-induced apoptosis through AMPK-PI3K-c-Jun NH2 pathway

    USGS Publications Warehouse

    Jung, T.W.; Lee, M.W.; Lee, Y.-J.; Kim, S.M.

    2012-01-01

    Type 2 diabetes mellitus is thought to be partially associated with endoplasmic reticulum (ER) stress toxicity on pancreatic beta cells and the result of decreased insulin synthesis and secretion. In this study, we showed that a well-known insulin sensitizer, metformin, directly protects against dysfunction and death of ER stress-induced NIT-1 cells (a mouse pancreatic beta cell line) via AMP-activated protein kinase (AMPK) and phosphatidylinositol-3 (PI3) kinase activation. We also showed that exposure of NIT-1 cells to metformin (5mM) increases cellular resistance against ER stress-induced NIT-1 cell dysfunction and death. AMPK and PI3 kinase inhibitors abolished the effect of metformin on cell function and death. Metformin-mediated protective effects on ER stress-induced apoptosis were not a result of an unfolded protein response or the induced inhibitors of apoptotic proteins. In addition, we showed that exposure of ER stressed-induced NIT-1 cells to metformin decreases the phosphorylation of c-Jun NH(2) terminal kinase (JNK). These data suggest that metformin is an important determinant of ER stress-induced apoptosis in NIT-1 cells and may have implications for ER stress-mediated pancreatic beta cell destruction via regulation of the AMPK-PI3 kinase-JNK pathway.

  6. β1 integrins mediate resistance to ionizing radiation in vivo by inhibiting c-Jun amino terminal kinase 1

    PubMed Central

    Goel, Hira Lal; Sayeed, Aejaz; Breen, Michael; Zarif, Matthew J.; Garlick, David S.; Leav, Irwin; Davis, Roger J.; FitzGerald, Thomas J.; Morrione, Andrea; Hsieh, Chung-Cheng; Liu, Qin; Dicker, Adam P.; Altieri, Dario C.; Languino, Lucia R.

    2013-01-01

    This study was carried out to dissect the mechanism by which β1 integrins promote resistance to radiation. For this purpose, we conditionally ablated β1 integrins in the prostatic epithelium of transgenic adenocarcinoma of mouse prostate (TRAMP) mice. The ability of β1 to promote resistance to radiation was also analyzed by using an inhibitory antibody to β1, AIIB2, in a xenograft model. The role of β1 integrins and of a β1 downstream target, c-Jun amino-terminal kinase 1 (JNK1), in regulating radiation-induced apoptosis in vivo and in vitro was studied. We show that β1 integrins promote prostate cancer (PrCa) progression and resistance to radiation in vivo. Mechanistically, β1 integrins are shown here to suppress activation of JNK1 and, consequently apoptosis, in response to irradiation. Downregulation of JNK1 is necessary to preserve the effect of β1 on resistance to radiation in vitro and in vivo. Finally, given the established cross-talk between β1 integrins and type 1 insulin-like growth factor receptor (IGF-IR), we analyzed the ability of IGF-IR to modulate β1 integrin levels. We report that IGF-IR regulates the expression of β1 integrins, which in turn confer resistance to radiation in PrCa cells. In conclusion, this study demonstrates that β1 integrins mediate resistance to ionizing radiation through inhibition of JNK1 activation. PMID:23359252

  7. COP1D, an alternatively spliced constitutive photomorphogenic-1 (COP1) product, stabilizes UV stress-induced c-Jun through inhibition of full-length COP1.

    PubMed

    Savio, M G; Rotondo, G; Maglie, S; Rossetti, G; Bender, J R; Pardi, R

    2008-04-10

    COP1 is an evolutionarily conserved RING-finger ubiquitin ligase acting within a Cullin-RING ligase (CRL) complex that promotes polyubiquitination of c-Jun and p53. Stability of the above substrates is affected by post-translational changes priming the proteins for polyubiquitination and proteasome-dependent degradation. However, degradation of both substrates is controlled indirectly by signaling pathways affecting the E3 ligases involved in their polyubiquitination. Here, we report the identification of COP1D, a ubiquitously expressed splice variant of COP1 lacking a portion of a coiled-coil region involved in intermolecular associations. While being unable to associate with other components of the CRL complex, COP1D exerts a dominant-negative function over the full-length protein, due to its ability to heterodimerize with COP1 and sequester it from the enzymatically active complex. Ectopic expression of COP1D antagonizes the function of COP1, while its selective downregulation by RNA interference promotes more efficient degradation of c-Jun and p53 by the full-length protein. The COP1/COP1D mRNA ratio is modulated by UV stress and a decreased COP1/COP1D ratio correlates with elevated c-Jun, but not p53 protein levels in invasive ductal breast cancer. Thus, dynamic changes of the COP1/COP1D ratio provide an additional level of regulation of the half-life of the substrates of this E3 ligase under homeostatic or pathological conditions. PMID:17968316

  8. Walleye Dermal Sarcoma Virus: OrfA N-Terminal End Inhibits the Activity of a Reporter Gene Directed by Eukaryotic Promoters and Has a Negative Effect on the Growth of Fish and Mammalian Cells

    PubMed Central

    Zhang, Z.; Martineau, D.

    1999-01-01

    Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis. The WDSV genome contains three short open reading frames designated orfA, orfB, and orfC in addition to the viral structural genes, gag, pol, and env. orfA and orfB transcripts are detected in tumors by reverse transcription-PCR. Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain. We report that expression of the accessory gene orfA inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters. In addition, stable transfection with the wild-type orfA generated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector. An orfA mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects. PMID:10482648

  9. miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis

    PubMed Central

    Xu, Zhenhua; Bu, Yiwen; Chitnis, Nilesh; Koumenis, Costas; Fuchs, Serge Y.; Diehl, J. Alan

    2016-01-01

    The ability of the unfolded protein response, UPR, to regulate cell homeostasis through both gene expression and protein synthesis has been well documented. One primary pro-apoptotic protein that responds to both PERK and Ire1 signalling is the CHOP/GADD153 transcription factor. Although CHOP deficiency delays onset of cell death, questions remain regarding how CHOP regulates apoptosis. Here, we provide evidence demonstrating that CHOP/GADD153-dependent apoptosis reflects expression of micro-RNA, miR-216b. MiR-216b accumulation requires PERK-dependent induction of CHOP/GADD153, which then directly regulates miR-216b expression. As maximal expression of miR-216b is antagonized by Ire1, miR-216b accumulation reflects the convergence of PERK and Ire1 activities. Functionally, miR-216b directly targets c-Jun, thereby reducing AP-1-dependent transcription and sensitizing cells to ER stress-dependent apoptosis. These results provide direct insight into the molecular mechanisms of CHOP/GADD153-dependent cell death. PMID:27173017

  10. miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis.

    PubMed

    Xu, Zhenhua; Bu, Yiwen; Chitnis, Nilesh; Koumenis, Costas; Fuchs, Serge Y; Diehl, J Alan

    2016-01-01

    The ability of the unfolded protein response, UPR, to regulate cell homeostasis through both gene expression and protein synthesis has been well documented. One primary pro-apoptotic protein that responds to both PERK and Ire1 signalling is the CHOP/GADD153 transcription factor. Although CHOP deficiency delays onset of cell death, questions remain regarding how CHOP regulates apoptosis. Here, we provide evidence demonstrating that CHOP/GADD153-dependent apoptosis reflects expression of micro-RNA, miR-216b. MiR-216b accumulation requires PERK-dependent induction of CHOP/GADD153, which then directly regulates miR-216b expression. As maximal expression of miR-216b is antagonized by Ire1, miR-216b accumulation reflects the convergence of PERK and Ire1 activities. Functionally, miR-216b directly targets c-Jun, thereby reducing AP-1-dependent transcription and sensitizing cells to ER stress-dependent apoptosis. These results provide direct insight into the molecular mechanisms of CHOP/GADD153-dependent cell death. PMID:27173017

  11. c-Jun NH2-terminal kinase (JNK)1 and JNK2 have similar and stage-dependent roles in regulating T cell apoptosis and proliferation.

    PubMed

    Sabapathy, K; Kallunki, T; David, J P; Graef, I; Karin, M; Wagner, E F

    2001-02-01

    Apoptotic and mitogenic stimuli activate c-Jun NH2-terminal kinases (JNKs) in T cells. Although T cells express both JNK1 and JNK2 isozymes, the absence of JNK2 alone can result in resistance to anti-CD3-induced thymocyte apoptosis and defective mature T cell proliferation. Similar defects in thymocyte apoptosis and mature T cell proliferation, the latter due to reduced interleukin 2 production, are also caused by JNK1 deficiency. Importantly, T cell function was compromised in Jnk1(+/-)Jnk2(+/-) double heterozygous mice, indicating that JNK1 and JNK2 play similar roles in regulating T cell function. The reduced JNK dose results in defective c-Jun NH2-terminal phosphorylation in thymocytes but not in peripheral T cells, in which nuclear factors of activated T cells (NK-ATs)-DNA binding activity is affected. Thus, JNK1 and JNK2 control similar functions during T cell maturation through differential targeting of distinct substrates. PMID:11157052

  12. The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18.

    PubMed Central

    Le Douarin, B; Zechel, C; Garnier, J M; Lutz, Y; Tora, L; Pierrat, P; Heery, D; Gronemeyer, H; Chambon, P; Losson, R

    1995-01-01

    Nuclear receptors (NRs) bound to response elements mediate the effects of cognate ligands on gene expression. Their ligand-dependent activation function, AF-2, presumably acts on the basal transcription machinery through intermediary proteins/mediators. We have isolated a mouse nuclear protein, TIF1, which enhances RXR and RAR AF-2 in yeast and interacts in a ligand-dependent manner with several NRs in yeast and mammalian cells, as well as in vitro. Remarkably, these interactions require the amino acids constituting the AF-2 activating domain conserved in all active NRs. Moreover, the oestrogen receptor (ER) AF-2 antagonist hydroxytamoxifen cannot promote ER-TIF1 interaction. We propose that TIF1, which contains several conserved domains found in transcriptional regulatory proteins, is a mediator of ligand-dependent AF-2. Interestingly, the TIF1 N-terminal moiety is fused to B-raf in the mouse oncoprotein T18. Images PMID:7744009

  13. Ras association domain family member 10 suppresses gastric cancer growth by cooperating with GSTP1 to regulate JNK/c-Jun/AP-1 pathway.

    PubMed

    Li, X; Liang, Q; Liu, W; Zhang, N; Xu, L; Zhang, X; Zhang, J; Sung, J J Y; Yu, J

    2016-05-12

    The Ras association domain family (RASSF) encodes several members with tumor-suppressive potentials. We aimed to investigate the biological function and clinical implication of RASSF10 in gastric cancer (GC). We found that RASSF10 was silenced in six of seven GC cell lines and in primary GC tissues, but was highly expressed in normal gastric tissues. The silence of RASSAF10 was mediated by promoter methylation as evaluated by bisulfite genomic sequencing. RASSF10 expression could be restored by demethylation treatment. A negative correlation between methylation and mRNA expression of RASSF10 was observed in 223 gastric samples of The Cancer Genome Atlas study (P<0.0001). Re-expression of RASSF10 in GC cell lines (AGS and MKN45) significantly suppressed cell viability, colony formation, migration and invasion, reduced cells in S phase, accumulated cells in G2 phase and induced cell apoptosis in vitro, and inhibited tumorigenicity in nude mice. These were confirmed by decreased expression of proliferation markers (proliferating cell nuclear antigen, p-CDC2 and p-CDC25) and increased apoptotic cascades (cleaved caspases-9, -8, -3 and cleaved poly (ADP-ribose) polymerase). Conversely, RASSF10 knockdown in normal gastric cell line yielded an opposing effect. Co-immunoprecipitation combined with mass spectrometry analyses were performed to reveal the downstream effectors of RASSF10. The result revealed that glutathione S-transferase Pi 1 (GSTP1) was a direct cooperator of RASSF10. The tumor-suppressive effect of RASSF10 was partially mediated by cooperating with GSTP1 to deregulate Jun N-terminal kinase (JNK)/c-Jun/AP-1 pathway. Importantly, RASSF10 methylation was detected in 56.6% (98/173) of primary GCs and is an independent risk factor for poor survival of GC patients (P=0.001). In conclusions, RASSF10 functions as a tumor suppressor by cooperating with GSTP1 to deregulate JNK/c-Jun/AP-1 pathway in GC. Promoter methylation of RASSF10 is associated with poor survival

  14. Biological Activities of 2-Mercaptobenzothiazole Derivatives: A Review

    PubMed Central

    Azam, Mohammed Afzal; Suresh, Bhojraj

    2012-01-01

    2-Mercaptobenzothiazoles are an important class of bioactive and industrially important organic compounds. These compounds are reported for their antimicrobial and antifungal activities, and are subsequently highlighted as a potent mechanism-based inhibitor of several enzymes like acyl coenzyme A cholesterol acyltransferase, monoamine oxidase, heat shock protein 90, cathepsin D, and c-Jun N-terminal kinases. These derivatives are also known to possess antitubercular, anti-inflammatory, antitumor, amoebic, antiparkinsonian, anthelmintic, antihypertensive, antihyperlipidemic, antiulcer, chemoprotective, and selective CCR3 receptor antagonist activity. This present review article focuses on the pharmacological profile of 2-mercaptobenzothiazoles with their potential activities. PMID:23264933

  15. Mutations in N-terminal Flanking Region of Blue Light-sensing Light-Oxygen and Voltage 2 (LOV2) Domain Disrupt Its Repressive Activity on Kinase Domain in the Chlamydomonas Phototropin*

    PubMed Central

    Aihara, Yusuke; Yamamoto, Takaharu; Okajima, Koji; Yamamoto, Kazuhiko; Suzuki, Tomomi; Tokutomi, Satoru; Tanaka, Kazuma; Nagatani, Akira

    2012-01-01

    Phototropin is a light-regulated kinase that mediates a variety of photoresponses such as phototropism, chloroplast positioning, and stomata opening in plants to increase the photosynthetic efficiency. Blue light stimulus first induces local conformational changes in the chromophore-bearing light-oxygen and voltage 2 (LOV2) domain of phototropin, which in turn activates the serine/threonine (Ser/Thr) kinase domain in the C terminus. To examine the kinase activity of full-length phototropin conventionally, we employed the budding yeast Saccharomyces cerevisiae. In this organism, Ser/Thr kinases (Fpk1p and Fpk2p) that show high sequence similarity to the kinase domain of phototropins exist. First, we demonstrated that the phototropin from Chlamydomonas reinhardtii (CrPHOT) could complement loss of Fpk1p and Fpk2p to allow cell growth in yeast. Furthermore, this reaction was blue light-dependent, indicating that CrPHOT was indeed light-activated in yeast cells. We applied this system to a large scale screening for amino acid substitutions in CrPHOT that elevated the kinase activity in darkness. Consequently, we identified a cluster of mutations located in the N-terminal flanking region of LOV2 (R199C, L202L, D203N/G/V, L204P, T207I, and R210H). An in vitro phosphorylation assay confirmed that these mutations substantially reduced the repressive activity of LOV2 on the kinase domain in darkness. Furthermore, biochemical analyses of the representative T207I mutant demonstrated that the mutation affected neither spectral nor multimerization properties of CrPHOT. Hence, the N-terminal flanking region of LOV2, as is the case with the C-terminal flanking Jα region, appears to play a crucial role in the regulation of kinase activity in phototropin. PMID:22291022

  16. The N-terminal domain of the GluN3A subunit determines the efficacy of glycine-activated NMDA receptors.

    PubMed

    Mesic, Ivana; Madry, Christian; Geider, Kirsten; Bernhard, Max; Betz, Heinrich; Laube, Bodo

    2016-06-01

    N-methyl-d-aspartate (NMDA) receptors composed of glycine-binding GluN1 and GluN3 subunits function as excitatory glycine receptors that respond to agonist application only with a very low efficacy. Binding of glycine to the high-affinity GluN3 subunits triggers channel opening, whereas glycine binding to the low-affinity GluN1 subunits causes an auto-inhibition of the maximal glycine-inducible receptor current (Imax). Hence, competitive antagonists of the GluN1 subunit strongly potentiate glycine responses of wild type (wt) GluN1/GluN3 receptors. Here, we show that co-expression of N-terminal domain (NTD) deleted GluN1 (GluN1(ΔNTD)) and GluN3 (GluN3(ΔNTD)) subunits in Xenopus oocytes generates GluN1/GluN3 receptors with a large increase in the glycine-inducible Imax accompanied by a strongly impaired GluN1 antagonist-mediated potentiation. Affinity purification after metabolic or surface labeling revealed no differences in subunit stoichiometry and surface expression between wt GluN1/GluN3A and mutant GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, indicating a specific effect of NTD deletions on the efficacy of receptor opening. Notably, GluN1/GluN3A(ΔNTD) receptors showed a similar increase in Imax and a greatly reduced GluN1 antagonist-mediated current potentiation as GluN1(ΔNTD)/GluN3A(ΔNTD) receptors, whereas the glycine-induced currents of GluN1(ΔNTD)/GluN3A receptors resembled those of wt GluN1/GluN3A receptors. Furthermore, oxidative crosslinking of the homophilic GluN3A NTD intersubunit interface in mutant GluN1/GluN3A(R319C) receptors caused both a decrease in the glycine-induced Imax concomitantly with a marked increase in GluN1 antagonist-mediated current potentiation, whilst mutations within the intrasubunit region linking the GluN3A NTD to the ligand binding domain had opposite effects. Together these results show that the GluN3A NTD constitutes a crucial regulatory determinant of GluN1/GluN3A receptor function. PMID:26777280

  17. Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase pathway

    SciTech Connect

    Zhou, Xiuping; Meng, Qingming; Xu, Xuebin; Zhi, Tongle; Shi, Qiong; Wang, Yong; Yu, Rutong

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer The expression levels of Bex2 markedly increased in glioma tissues. Black-Right-Pointing-Pointer Bex2 over-expression promoted cell proliferation, while its down-regulation inhibited cell growth. Black-Right-Pointing-Pointer Bex2 down-regulation promoted cell apoptosis via JNK/c-Jun signaling pathway. -- Abstract: The function of Bex2, a member of the Brain Expressed X-linked gene family, in glioma is controversial and its mechanism is largely unknown. We report here that Bex2 regulates cell proliferation and apoptosis in malignant glioma cells via the c-Jun NH2-terminal kinase (JNK) pathway. The expression level of Bex2 is markedly increased in glioma tissues. We observed that Bex2 over-expression promotes cell proliferation, while down-regulation of Bex2 inhibits cell growth. Furthermore, Bex2 down-regulation promotes cell apoptosis and activates the JNK pathway; these effects were abolished by administration of the JNK specific inhibitor, (SP600125). Thus, Bex2 may be an important player during the development of glioma.

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

    PubMed

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

    2008-03-11

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

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

    PubMed Central

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

    2013-01-01

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

  20. MITF and c-Jun antagonism interconnects melanoma dedifferentiation with pro-inflammatory cytokine responsiveness and myeloid cell recruitment

    PubMed Central

    Riesenberg, Stefanie; Groetchen, Angela; Siddaway, Robert; Bald, Tobias; Reinhardt, Julia; Smorra, Denise; Kohlmeyer, Judith; Renn, Marcel; Phung, Bengt; Aymans, Pia; Schmidt, Tobias; Hornung, Veit; Davidson, Irwin; Goding, Colin R.; Jönsson, Göran; Landsberg, Jennifer; Tüting, Thomas; Hölzel, Michael

    2015-01-01

    Inflammation promotes phenotypic plasticity in melanoma, a source of non-genetic heterogeneity, but the molecular framework is poorly understood. Here we use functional genomic approaches and identify a reciprocal antagonism between the melanocyte lineage transcription factor MITF and c-Jun, which interconnects inflammation-induced dedifferentiation with pro-inflammatory cytokine responsiveness of melanoma cells favouring myeloid cell recruitment. We show that pro-inflammatory cytokines such as TNF-α instigate gradual suppression of MITF expression through c-Jun. MITF itself binds to the c-Jun regulatory genomic region and its reduction increases c-Jun expression that in turn amplifies TNF-stimulated cytokine expression with further MITF suppression. This feed-forward mechanism turns poor peak-like transcriptional responses to TNF-α into progressive and persistent cytokine and chemokine induction. Consistently, inflammatory MITFlow/c-Junhigh syngeneic mouse melanomas recruit myeloid immune cells into the tumour microenvironment as recapitulated by their human counterparts. Our study suggests myeloid cell-directed therapies may be useful for MITFlow/c-Junhigh melanomas to counteract their growth-promoting and immunosuppressive functions. PMID:26530832

  1. Oxidative Folding and N-terminal Cyclization of Onconase+

    PubMed Central

    Welker, Ervin; Hathaway, Laura; Xu, Guoqiang; Narayan, Mahesh; Pradeep, Lovy; Shin, Hang-Cheol; Scheraga, Harold A.

    2008-01-01

    Cyclization of the N-terminal glutamine residue to pyroglutamic acid in onconase, an anti-cancer chemotherapeutic agent, increases the activity and stability of the protein. Here, we examine the correlated effects of the folding/unfolding process and the formation of this N-terminal pyroglutamic acid. The results in this study indicate that cyclization of the N-terminal glutamine has no significant effect on the rate of either reductive unfolding or oxidative folding of the protein. Both the cyclized and uncyclized proteins seem to follow the same oxidative folding pathways; however, cyclization altered the relative flux of the protein in these two pathways by increasing the rate of formation of a kinetically trapped intermediate. Glutaminyl cyclase (QC) catalyzed the cyclization of the unfolded, reduced protein, but had no effect on the disulfide-intact, uncyclized, folded protein. The structured intermediates of uncyclized onconase were also resistant to QC-catalysis, consistent with their having a native-like fold. These observations suggest that, in vivo, cyclization takes place during the initial stages of oxidative folding, specifically, before the formation of structured intermediates. The competition between oxidative folding and QC-mediated cyclization suggests that QC-catalyzed cyclization of the N-terminal glutamine in onconase occurs in the endoplasmic reticulum, probably co-translationally. PMID:17439243

  2. N-terminal arm of orchardgrass Hsp17.2 (DgHsp17.2) is essential for both in vitro chaperone activity and in vivo thermotolerance in yeast.

    PubMed

    Cha, Joon-Yung; Lee, Sang-Hoon; Seo, Kyung Hye; Choi, Young Jin; Cheong, Mi Sun; Son, Daeyoung

    2016-02-01

    Small heat shock proteins are well-known to function as chaperone in the protection of proteins and subcellular structures against stress-induced denaturation in many cell compartments. Irrespective of such general functional assignment, a proof of function in a living organism is missing. Here, we used heat-induced orchardgrass small Hsp17.2 (DgHsp17.2). Its function in in vitro chaperone properties has shown in protecting the model substrate, malate dehydrogenase (MDH) and citrate synthase (CS). Overexpression of DgHsp17.2 triggering strong chaperone activity enhanced in vivo thermotolerance of yeast cells. To identify the functional domain on DgHsp17.2 and correlationship between in vitro chaperone property and in vivo thermotolerance, we generated truncation mutants of DgHsp17.2 and showed essentiality of the N-terminal arm of DgHsp17.2 for the chaperone function. In addition, beyond for acquisition of thermotolerance irrespective of sequences are diverse among the small Hsps. However, any truncation mutants of DgHsp17.2 did not exhibit strong interaction with orchardgrass heat shock protein 70 (DgHsp70) different from mature DgHsp17.2, indicating that full-length DgHsp17.2 is necessary for cooperating with Hsp70 protein. Our study indicates that the N-terminal arm of DgHsp17.2 is an important region for chaperone activity and thermotolerance. PMID:26724757

  3. c-JUN-like immunoreactivity in the CNS of the adult rat: basal and transynaptically induced expression of an immediate-early gene.

    PubMed

    Herdegen, T; Leah, J D; Manisali, A; Bravo, R; Zimmermann, M

    1991-01-01

    An immunocytochemical study of dorsal root ganglia, spinal cord and medulla oblongata was performed with antisera against the c-jun proto-oncogene encoded protein. The c-JUN-like immunoreactivity was restricted to the cell nucleus. In the CNS of untreated rats a basal c-JUN-like immunoreactivity was present in the nuclei of two types of neurons: motor and autonomic. Labelled nuclei could be seen in many motoneurons of the ventral horn of the entire length of spinal cord and the lower medulla oblongata, as well as in the area of the nucleus hypoglossus, the dorsal motor nucleus of nucleus vagus, nucleus ambiguus, nucleus facialis, nucleus abducens and motor nucleus of nucleus trigeminus. Additionally, labelled nuclei were found in the preganglionic sympathetic and preganglionic parasympathetic cells of the nucleus intermediolateralis and nucleus intercalatus in the spinal cord. In the medulla oblongata we found a cluster of cells with c-JUN-like immunoreactivity in an area between the dorsomedial part of the oral nucleus spinalis trigeminalis and the lateral border of the knee of facial nerve. Additionally, a second cluster of c-JUN-like immunoreactivity cells was visible between the ventromedial part of the oral nucleus spinalis trigeminalis and the lateral border of the rostral nucleus facialis. Examination of the characteristics of all cell groups with a basal c-JUN-like immunoreactivity in the spinal cord and lower brainstem revealed an overlapping distribution with cholinergic cell groups. Basal c-JUN-like immunoreactivity was also seen in the dorsal root ganglion cells. We examined the factors which can effect the expression of the c-JUN protein. Maximal expression of c-JUN-like immunoreactivity was observed after electrical stimulation of primary afferents. Stimulation of sciatic nerve at a strength sufficient to recruit A delta- and C-fibres produced c-JUN-like immunoreactivity in many nuclei of the ipsilateral dorsal horn of the lumbar spinal cord. c-JUN

  4. Expression of active streptolysin O in Escherichia coli as a maltose-binding-protein--streptolysin-O fusion protein. The N-terminal 70 amino acids are not required for hemolytic activity.

    PubMed

    Weller, U; Müller, L; Messner, M; Palmer, M; Valeva, A; Tranum-Jensen, J; Agrawal, P; Biermann, C; Döbereiner, A; Kehoe, M A; Bhakdi, S

    1996-02-15

    Streptolysin 0 (SLO) is the prototype of a family of cytolysins that consists of proteins which bind to cholesterol and form very large transmembrane pores. Structure/function studies on the pore-forming cytolysin SLO have been complicated by the proteolytic inactivation of a substantial portion of recombinant SLO (rSLO) expressed in Escherichia coli. To overcome this problem, translational fusions between the E. coli maltose-binding protein (MBP) gene and SLO were constructed, using the vectors pMAL-p2 and pMAL-c2. MBP-SLO fusion proteins were degraded if secreted into the E. coli periplasm, but intact, soluble MBP-SLO fusion proteins were produced at high levels in the cytoplasm. Active SLO with the expected N-terminus was separated from the MBP carrier by cleavage with factor Xa. Cleavage with plasmin or trypsin also yielded active, but slightly smaller forms of SLO. Surprisingly, uncleaved MBP-SLO was also hemolytic and cytotoxic to human fibroblasts and keratinocytes. The MBP-SLO fusion protein displayed equal activities to SLO. Sucrose density gradient analyses showed that the fusion protein assembled into polymers, and no difference in structure was discerned compared with polymers formed by native SLO. These studies show that the N-terminal 70 residues of mature (secreted) SLO are not required for pore formation and that the N-terminus of the molecule is probably not inserted into the bilayer. In addition, they provide a simple means for producing mutants for structure/function studies and highly purified SLO for use as a permeabilising reagent in cell biology research. PMID:8617283

  5. Ionizing Radiation Induces Macrophage Foam Cell Formation and Aggregation Through JNK-Dependent Activation of CD36 Scavenger Receptors

    SciTech Connect

    Katayama, Ikuo; Hotokezaka, Yuka; Matsuyama, Toshifumi; Sumi, Tadateru; Nakamura, Takashi

    2008-03-01

    Purpose: Irradiated arteries of cancer patients can be associated with atherosclerosis-like lesions containing cholesterol-laden macrophages (foam cells). Endothelial cell damage by irradiation does not completely explain the foam cell formation. We investigated the possible underlying mechanisms for ionizing radiation (IR)-induced foam cell formation. Methods and Materials: Human peripheral blood monocytes were activated by macrophage colony-stimulating factor and then treated with varying doses of IR in vitro in the absence of endothelial cells. Scavenger receptor expression and foam cell formation of IR-treated macrophages were investigated in the presence or absence of oxidized low-density lipoprotein. We also assessed the importance of mitogen-activated protein kinase activity in the macrophage colony-stimulating factor-activated human monocytes (macrophages) for the foam cell formation. Results: We found that IR treatment of macrophage colony-stimulating factor-activated human peripheral blood monocytes resulted in the enhanced expression of CD36 scavenger receptors and that cholesterol accumulated in the irradiated macrophages with resultant foam cell formation in the presence of oxidized low-density lipoprotein. Furthermore, when cultured on collagen gels, human macrophages formed large foam cell aggregates in response to IR. Antibodies against CD36 inhibited the IR-induced foam cell formation and aggregation, indicating that the IR-induced foam cell formation and the subsequent aggregation are dependent on functional CD36. In addition, we found that IR of human macrophages resulted in c-Jun N-terminal kinase activation and that c-Jun N-terminal kinase inhibition suppressed IR-induced CD36 expression and the subsequent foam cell formation and aggregation. Conclusion: Taken together, these results suggest that IR-induced foam cell formation is mediated by c-Jun N-terminal kinase-dependent CD36 activation.

  6. The dark and bright sides of an enzyme: a three dimensional structure of the N-terminal domain of Zophobas morio luciferase-like enzyme, inferences on the biological function and origin of oxygenase/luciferase activity.

    PubMed

    Prado, R A; Santos, C R; Kato, D I; Murakami, M T; Viviani, V R

    2016-05-11

    Beetle luciferases, the enzymes responsible for bioluminescence, are special cases of CoA-ligases which have acquired a novel oxygenase activity, offering elegant models to investigate the structural origin of novel catalytic functions in enzymes. What the original function of their ancestors was, and how the new oxygenase function emerged leading to bioluminescence remains unclear. To address these questions, we solved the crystal structure of a recently cloned Malpighian luciferase-like enzyme of unknown function from Zophobas morio mealworms, which displays weak luminescence with ATP and the xenobiotic firefly d-luciferin. The three dimensional structure of the N-terminal domain showed the expected general fold of CoA-ligases, with a unique carboxylic substrate binding pocket, permitting the binding and CoA-thioesterification activity with a broad range of carboxylic substrates, including short-, medium-chain and aromatic acids, indicating a generalist function consistent with a xenobiotic-ligase. The thioesterification activity with l-luciferin, but not with the d-enantiomer, confirms that the oxygenase activity emerged from a stereoselective impediment of the thioesterification reaction with the latter, favoring the alternative chemiluminescence oxidative reaction. The structure and site-directed mutagenesis support the involvement of the main-chain amide carbonyl of the invariant glycine G323 as the catalytic base for luciferin C4 proton abstraction during the oxygenase activity in this enzyme and in beetle luciferases (G343). PMID:27101527

  7. TNF-α Mediates PKCδ/JNK1/2/c-Jun-Dependent Monocyte Adhesion via ICAM-1 Induction in Human Retinal Pigment Epithelial Cells

    PubMed Central

    Lee, I-Ta; Liu, Shiau-Wen; Chi, Pei-Ling; Lin, Chih-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

    2015-01-01

    Retinal inflammatory diseases induced by cytokines, such as tumor necrosis factor-α (TNF-α) are associated with an up-regulation of intercellular adhesion molecule-1 (ICAM-1) in the retinal pigment epithelial cells (RPECs). Retinal pigment epithelium (RPE) is a monolayer of epithelial cells that forms the outer blood-retinal barrier in the posterior segment of the eye, and is also implicated in the pathology of, such as neovascularization in age-related macular degeneration (AMD). However, the detailed mechanisms of TNF-α-induced ICAM-1 expression are largely unclear in human RPECs. We demonstrated that in RPECs, TNF-α could induce ICAM-1 protein and mRNA expression and promoter activity, and monocyte adhesion. TNF-α-mediated responses were attenuated by pretreatment with the inhibitor of PKCs (Ro318220), PKCδ (Rottlerin), MEK1/2 (U0126), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of TNFR1, TRAF2, JNK2, p42, or c-Jun. We showed that TNF-α could stimulate the TNFR1 and TRAF2 complex formation. TNF-α-stimulated JNK1/2 was also reduced by Rottlerin or SP600125. However, Rottlerin had no effect on TNF-α-induced p42/p44 MAPK phosphorylation. We observed that TNF-α induced c-Jun phosphorylation which was inhibited by Rottlerin or SP600125. On the other hand, TNF-α-stimulated ICAM-1 promoter activity was prominently lost in RPECs transfected with the point-mutated AP-1 ICAM-1 promoter plasmid. These results suggest that TNF-α-induced ICAM-1 expression and monocyte adhesion is mediated through a TNFR1/TRAF2/PKCδ/JNK1/2/c-Jun pathway in RPECs. These findings concerning TNF-α-induced ICAM-1 expression in RPECs imply that TNF-α might play an important role in ocular inflammation and diseases. PMID:25675437

  8. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Saccharomyces cerevisiae shows great potential for development of bioreactor systems geared towards the production of high-value lipids such as polyunsaturated omega-3 fatty acids, the yields of which are largely dependent on the activity of ectopically-expressed enzymes. Here we show that the addit...

  9. N-Terminal Ile-Orn- and Trp-Orn-Motif Repeats Enhance Membrane Interaction and Increase the Antimicrobial Activity of Apidaecins against Pseudomonas aeruginosa

    PubMed Central

    Bluhm, Martina E. C.; Schneider, Viktoria A. F.; Schäfer, Ingo; Piantavigna, Stefania; Goldbach, Tina; Knappe, Daniel; Seibel, Peter; Martin, Lisandra L.; Veldhuizen, Edwin J. A.; Hoffmann, Ralf

    2016-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N′,N′-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8–16 and 8–32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared

  10. N-Terminal Ile-Orn- and Trp-Orn-Motif Repeats Enhance Membrane Interaction and Increase the Antimicrobial Activity of Apidaecins against Pseudomonas aeruginosa.

    PubMed

    Bluhm, Martina E C; Schneider, Viktoria A F; Schäfer, Ingo; Piantavigna, Stefania; Goldbach, Tina; Knappe, Daniel; Seibel, Peter; Martin, Lisandra L; Veldhuizen, Edwin J A; Hoffmann, Ralf

    2016-01-01

    The Gram-negative bacterium Pseudomonas aeruginosa is a life-threatening nosocomial pathogen due to its generally low susceptibility toward antibiotics. Furthermore, many strains have acquired resistance mechanisms requiring new antimicrobials with novel mechanisms to enhance treatment options. Proline-rich antimicrobial peptides, such as the apidaecin analog Api137, are highly efficient against various Enterobacteriaceae infections in mice, but less active against P. aeruginosa in vitro. Here, we extended our recent work by optimizing lead peptides Api755 (gu-OIORPVYOPRPRPPHPRL-OH; gu = N,N,N',N'-tetramethylguanidino, O = L-ornithine) and Api760 (gu-OWORPVYOPRPRPPHPRL-OH) by incorporation of Ile-Orn- and Trp-Orn-motifs, respectively. Api795 (gu-O(IO)2RPVYOPRPRPPHPRL-OH) and Api794 (gu-O(WO)3RPVYOPRPRPPHPRL-OH) were highly active against P. aeruginosa with minimal inhibitory concentrations of 8-16 and 8-32 μg/mL against Escherichia coli and Klebsiella pneumoniae. Assessed using a quartz crystal microbalance, these peptides inserted into a membrane layer and the surface activity increased gradually from Api137, over Api795, to Api794. This mode of action was confirmed by transmission electron microscopy indicating some membrane damage only at the high peptide concentrations. Api794 and Api795 were highly stable against serum proteases (half-life times >5 h) and non-hemolytic to human erythrocytes at peptide concentrations of 0.6 g/L. At this concentration, Api795 reduced the cell viability of HeLa cells only slightly, whereas the IC50 of Api794 was 0.23 ± 0.09 g/L. Confocal fluorescence microscopy revealed no colocalization of 5(6)-carboxyfluorescein-labeled Api794 or Api795 with the mitochondria, excluding interactions with the mitochondrial membrane. Interestingly, Api795 was localized in endosomes, whereas Api794 was present in endosomes and the cytosol. This was verified using flow cytometry showing a 50% higher uptake of Api794 in HeLa cells compared with Api

  11. Vfa1 binds to the N-terminal microtubule-interacting and trafficking (MIT) domain of Vps4 and stimulates its ATPase activity.

    PubMed

    Vild, Cody J; Xu, Zhaohui

    2014-04-11

    The endosomal sorting complexes required for transport (ESCRT) are responsible for multivesicular body biogenesis, membrane abscission during cytokinesis, and retroviral budding. They function as transiently assembled molecular complexes on the membrane, and their disassembly requires the action of the AAA-ATPase Vps4. Vps4 is regulated by a multitude of ESCRT and ESCRT-related proteins. Binding of these proteins to Vps4 is often mediated via the microtubule-interacting and trafficking (MIT) domain of Vps4. Recently, a new Vps4-binding protein Vfa1 was identified in a yeast genetic screen, where overexpression of Vfa1 caused defects in vacuolar morphology. However, the function of Vfa1 and its role in vacuolar biology were largely unknown. Here, we provide the first detailed biochemical and biophysical study of Vps4-Vfa1 interaction. The MIT domain of Vps4 binds to the C-terminal 17 residues of Vfa1. This interaction is of high affinity and greatly stimulates the ATPase activity of Vps4. The crystal structure of the Vps4-Vfa1 complex shows that Vfa1 adopts a canonical MIT-interacting motif 2 structure that has been observed previously in other Vps4-ESCRT interactions. These findings suggest that Vfa1 is a novel positive regulator of Vps4 function. PMID:24567329

  12. Crystal structure of the N-terminal SH3 domain of mouse {beta}PIX, p21-activated kinase-interacting exchange factor

    SciTech Connect

    Li Xiaofeng; Liu Xueqi; Sun Fei; Gao Jia; Zhou Hongwei; Gao, George F.; Bartlam, Mark; Rao Zihe . E-mail: raozh@xtal.tsinghua.edu.cn

    2006-01-06

    The mouse {beta}PIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3{sub 2}21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact {beta}-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse {beta}PIX-SH3 domain binding the way in which the {beta}PIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse {beta}PIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse {beta}PIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of {beta}PIX-SH3 domain resulting in its multiple biological functions.

  13. Identification of N-terminal receptor activity-modifying protein residues important for calcitonin gene-related peptide, adrenomedullin, and amylin receptor function.

    PubMed

    Qi, Tao; Christopoulos, George; Bailey, Richard J; Christopoulos, Arthur; Sexton, Patrick M; Hay, Debbie L

    2008-10-01

    Calcitonin-family receptors comprise calcitonin receptor-like receptor (CL) or calcitonin receptor and receptor activity-modifying protein (RAMP) pairings. Calcitonin gene-related peptide (CGRP) receptors are CL/RAMP1, whereas adrenomedullin (AM) receptors are CL/RAMP2 (AM1 receptor) or CL/RAMP3 (AM2 receptor). Amylin (Amy) receptors are RAMP hetero-oligomers with the calcitonin receptor (AMY1, AMY2, and AMY3, respectively). How RAMPs change G protein-coupled receptor pharmacology is not fully understood. We exploited sequence differences between RAMP1 and RAMP3 to identify individual residues capable of altering receptor pharmacology. Alignment of human RAMPs revealed eight residues that are conserved in RAMP2 and RAMP3 but are different in RAMP1. We hypothesized that residues in RAMP2 and RAMP3, but not RAMP1, are responsible for making CL/RAMP2 and CL/RAMP3 AM receptors. Using site-directed mutagenesis, we introduced individual RAMP3 residues into RAMP1 and vice versa in these eight positions. Mutant or wild-type RAMPs were transfected into Cos7 cells with CL or the insert-negative form of the calcitonin receptor [CT(a)]. Agonist-stimulated cAMP production and cell-surface expression of constructs were measured. Position 74 in RAMP1 and RAMP3 was critical for determining AM potency and affinity, and Phe93 in RAMP1 was an important contributor to alphaCGRP potency at CGRP receptors. Mutant RAMP/CT(a) receptor complexes displayed different phenotypes. It is noteworthy that RAMP1 S103N and W74E mutations led to enhanced rAmy potency, probably related to increased cell-surface expression of these complexes. This differs from the effect on CL-based receptors where expression was unchanged. Targeted substitution has emphasized the importance of position 74 in RAMP1/RAMP3 as a key determinant of AM pharmacology. PMID:18593822

  14. JNK-mediated activation of ATF2 contributes to dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease.

    PubMed

    Huang, Qiaoying; Du, Xiaoxiao; He, Xin; Yu, Qing; Hu, Kunhua; Breitwieser, Wolfgang; Shen, Qingyu; Ma, Shanshan; Li, Mingtao

    2016-03-01

    The c-Jun N-terminal kinase (JNK)/c-Jun pathway is a known critical regulator of dopaminergic neuronal death in Parkinson's disease (PD) and is considered a potential target for neuroprotective therapy. However, whether JNK is activated within dopaminergic neurons remains controversial, and whether JNK acts through downstream effectors other than c-Jun to promote dopaminergic neuronal death remains unclear. In this study, we confirm that JNK but not p38 is activated in dopaminergic neurons after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxication. Furthermore, within the dopaminergic neurons of the substantia nigra in MPTP-treated mice, JNK2/3 phosphorylates threonine 69 (Thr69) of Activating transcription factor-2 (ATF2), a transcription factor of the ATF/CREB family, whereas the phosphorylation of Thr71 is constitutive and remains unchanged. The increased phosphorylation of ATF2 on Thr69 by JNK in the MPTP mouse model suggests a functional relationship between the transcriptional activation of ATF2 and dopaminergic neuron death. By using dopaminergic neuron-specific conditional ATF2 mutant mice, we found that either partial or complete deletion of the ATF2 DNA-binding domain in dopaminergic neurons markedly alleviates the MPTP-induced dopaminergic neurodegeneration, indicating that the activation of ATF2 plays a detrimental role in neuropathogenesis in PD. Taken together, our findings demonstrate that JNK-mediated ATF2 activation contributes to dopaminergic neuronal death in an MPTP model of PD. PMID:26515688

  15. N-terminal nesprin-2 variants regulate β-catenin signalling.

    PubMed

    Zhang, Qiuping; Minaisah, Rose-Marie; Ferraro, Elisa; Li, Chen; Porter, Lauren J; Zhou, Can; Gao, Fang; Zhang, Junyi; Rajgor, Dipen; Autore, Flavia; Shanahan, Catherine M; Warren, Derek T

    2016-07-15

    The spatial compartmentalisation of biochemical signalling pathways is essential for cell function. Nesprins are a multi-isomeric family of proteins that have emerged as signalling scaffolds, herein, we investigate the localisation and function of novel nesprin-2 N-terminal variants. We show that these nesprin-2 variants display cell specific distribution and reside in both the cytoplasm and nucleus. Immunofluorescence microscopy revealed that nesprin-2 N-terminal variants colocalised with β-catenin at cell-cell junctions in U2OS cells. Calcium switch assays demonstrated that nesprin-2 and β-catenin are lost from cell-cell junctions in low calcium conditions whereas emerin localisation at the NE remained unaltered, furthermore, an N-terminal fragment of nesprin-2 was sufficient for cell-cell junction localisation and interacted with β-catenin. Disruption of these N-terminal nesprin-2 variants, using siRNA depletion resulted in loss of β-catenin from cell-cell junctions, nuclear accumulation of active β-catenin and augmented β-catenin transcriptional activity. Importantly, we show that U2OS cells lack nesprin-2 giant, suggesting that the N-terminal nesprin-2 variants regulate β-catenin signalling independently of the NE. Together, these data identify N-terminal nesprin-2 variants as novel regulators of β-catenin signalling that tether β-catenin to cell-cell contacts to inhibit β-catenin transcriptional activity. PMID:27321956

  16. DLK induces developmental neuronal degeneration via selective regulation of proapoptotic JNK activity.

    PubMed

    Ghosh, Arundhati Sengupta; Wang, Bei; Pozniak, Christine D; Chen, Mark; Watts, Ryan J; Lewcock, Joseph W

    2011-09-01

    The c-Jun N-terminal kinase (JNK) signaling pathway is essential for neuronal degeneration in multiple contexts but also regulates neuronal homeostasis. It remains unclear how neurons are able to dissociate proapoptotic JNK signaling from physiological JNK activity. In this paper, we show that the mixed lineage kinase dual leucine zipper kinase (DLK) selectively regulates the JNK-based stress response pathway to mediate axon degeneration and neuronal apoptosis without influencing other aspects of JNK signaling. This specificity is dependent on interaction of DLK with the scaffolding protein JIP3 to form a specialized JNK signaling complex. Local activation of DLK-based signaling in the axon results in phosphorylation of c-Jun and apoptosis after redistribution of JNK to the cell body. In contrast, regulation of axon degeneration by DLK is c-Jun independent and mediated by distinct JNK substrates. DLK-null mice displayed reduced apoptosis in multiple neuronal populations during development, demonstrating that prodegenerative DLK signaling is required in vivo. PMID:21893599

  17. Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

    SciTech Connect

    Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui; Cheng, Tian-Lu; Lin, Shinne-Ren; Chang, Long-Sen

    2015-04-01

    Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressed c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.

  18. β-Catenin Binds to the Activation Function 2 Region of the Androgen Receptor and Modulates the Effects of the N-Terminal Domain and TIF2 on Ligand-Dependent Transcription

    PubMed Central

    Song, Liang-Nian; Herrell, Roger; Byers, Stephen; Shah, Salimuddin; Wilson, Elizabeth M.; Gelmann, Edward P.

    2003-01-01

    β-Catenin is a multifunctional molecule that is activated by signaling through WNT receptors. β-Catenin can also enhance the transcriptional activity of some steroid hormone receptors such as the androgen receptor and retinoic acid receptor α. Androgens can affect nuclear translocation of β-catenin and influence its subcellular distribution. Using mammalian two-hybrid binding assays, analysis of reporter gene transcription, and coimmunoprecipitation, we now show that β-catenin binds to the androgen receptor ligand-binding domain (LBD) and modulates the transcriptional effects of TIF2 and the androgen receptor N-terminal domain (NTD). In functional assays, β-catenin bound to androgen receptor only in the presence of ligand agonists, not antagonists. β-Catenin binding to the androgen receptor LBD was independent of and cooperative with the androgen receptor NTD and the p160 coactivator TIF2, both of which bind to the activation function 2 (AF-2) region of the androgen receptor. Different mutations of androgen receptor helix 3 amino acids disrupted binding of androgen receptor NTD and β-catenin. β-Catenin, androgen receptor NTD, and TIF2 binding to the androgen receptor LBD were affected similarly by a subset of helix 12 mutations, but disruption of two sites on helix 12 affected only binding of β-catenin and not of TIF2 or the androgen receptor NTD. Mutational disruption of each of five LXXLL peptide motifs in the β-catenin armadillo repeats did not disrupt either binding to androgen receptor or transcriptional coactivation. ICAT, an inhibitor of T-cell factor 4 (TCF-4), and E-cadherin binding to β-catenin also blocked binding of the androgen receptor LBD. We also demonstrated cross talk between the WNT and androgen receptor signaling pathways because excess androgen receptor could interfere with WNT signaling and excess TCF-4 inhibited the interaction of β-catenin and androgen receptor. Taken together, the data show that β-catenin can bind to the

  19. Patient derived mutation W257G of PPP2R1A enhances cancer cell migration through SRC-JNK-c-Jun pathway

    PubMed Central

    Jeong, Ae Lee; Han, Sora; Lee, Sunyi; Su Park, Jeong; Lu, Yiling; Yu, Shuangxing; Li, Jane; Chun, Kyung-Hee; Mills, Gordon B.; Yang, Young

    2016-01-01

    Mutation of PPP2R1A has been observed at high frequency in endometrial serous carcinomas but at low frequency in ovarian clear cell carcinoma. However, the biological role of mutation of PPP2R1A in ovarian and endometrial cancer progression remains unclear. In this study, we found that PPP2R1A expression is elevated in high-grade primary tumor patients with papillary serous tumors of the ovary. To determine whether increased levels or mutation of PPP2R1A might contribute to cancer progression, the effects of overexpression or mutation of PPP2R1A on cell proliferation, migration, and PP2A phosphatase activity were investigated using ovarian and endometrial cancer cell lines. Among the mutations, PPP2R1A-W257G enhanced cell migration in vitro through activating SRC-JNK-c-Jun pathway. Overexpression of wild type (WT) PPP2R1A increased its binding ability with B56 regulatory subunits, whereas PPP2R1A-mutations lost the ability to bind to most B56 subunits except B56δ. Total PP2A activity and PPP2R1A-associated PP2Ac activity were significantly increased in cells overexpressing PPP2R1A-WT. In addition, overexpression of PPP2R1A-WT increased cell proliferation in vitro and tumor growth in vivo. PMID:27272709

  20. Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

    PubMed Central

    Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

    2013-01-01

    Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

  1. Targeting Mcl-1 for multiple myeloma (MM) therapy: drug-induced generation of Mcl-1 fragment Mcl-1(128-350) triggers MM cell death via c-Jun upregulation.

    PubMed

    Fan, Fengjuan; Tonon, Giovanni; Bashari, Muhammad Hasan; Vallet, Sonia; Antonini, Elena; Goldschmidt, Hartmut; Schulze-Bergkamen, Henning; Opferman, Joseph T; Sattler, Martin; Anderson, Kenneth C; Jäger, Dirk; Podar, Klaus

    2014-02-28

    Myeloid cell leukemia-1 (Mcl-1, HGNC: 6943), a pro-survival member of the Bcl-2 family, plays a crucial role in Multiple Myeloma (MM) pathogenesis and drug resistance, thus representing a promising therapeutic target in MM. A novel strategy to inhibit Mcl-1 activity is the induction of ubiquitin-independent Mcl-1 degradation. Our own and other previous studies have demonstrated caspase-dependent generation of a 28kDa Mcl-1 fragment, Mcl-1(128-350), which inhibits MM cell proliferation and survival. Here, we show that similar to bortezomib, the novel proteasome inhibitors carfilzomib and ixazomib, as well as staurosporine and adaphostin, induce the generation of Mcl-1(128-350) in MM cells. Next, the molecular sequelae downstream of Mcl-1(128-350), which mediate its pro-apoptotic activity, were delineated. Surprisingly, we observed nuclear accumulation of drug-induced or exogenously overexpressed Mcl-1(128-350), followed by elevated mRNA and protein levels of c-Jun, as well as enhanced AP-1 reporter activity. Moreover, drug-induced AP-1 activity was blocked after introducing a point mutation into the highly conserved Mcl-1 caspase-cleavage site Asp127, but not Asp157. Consequently, drug-triggered cell death was significantly decreased in MM cells transfected with Mcl-1 D127A, but not with Mcl-1 D157A. Consistent with these data, treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1(wt/wt), but not Mcl-1(Δ/null) murine embryonic fibroblasts (MEFs). Transfection of a plasmid carrying Mcl-1(wt) into Mcl-1(Δ/null) MEFs restored bortezomib-induced Mcl-1 fragmentation, c-Jun upregulation and AP-1 reporter activity. Finally, our data indicate that drug-induced generation of a pro-apoptotic Mcl-1 fragment followed by c-Jun upregulation may also be a novel therapeutic approach in other tumor entities. PMID:24120758

  2. Anisomycin and rapamycin define an area upstream of p70/85S6k containing a bifurcation to histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction.

    PubMed Central

    Kardalinou, E; Zhelev, N; Hazzalin, C A; Mahadevan, L C

    1994-01-01

    Anisomycin, a translational inhibitor, synergizes with growth factors and phorbol esters to superinduce c-fos and c-jun by a number mechanisms, one of which is its ability to act as a potent signalling agonist, producing strong, prolonged activation of the same nuclear responses as epidermal growth factor or tetradecanoyl phorbol acetate. These responses include the phosphorylation of pp33, which exists in complexed and chromatin-associated forms, and of histone H3 and an HMG-like protein. By peptide mapping and microsequencing, we show here that pp33 is the phosphoprotein S6, present in ribosomes and in preribosomes in the nucleolus. Ablation of epidermal growth factor-, tetradecanoyl phorbol acetate-, or anisomycin-stimulated S6 phosphorylation by using the p70/85S6k inhibitor rapamycin has no effect on histone H3 and HMG-like protein phosphorylation or on the induction and superinduction of c-fos and c-jun. Further, [35S]methionine-labelling and immunoprecipitation studies show that the ablation of S6 phosphorylation has no discernible effect on translation in general or translation of newly induced c-fos transcripts. Finally, we show that anisomycin augments and prolongs S6 phosphorylation not by blocking S6 phosphatases but by sustained activation of p70/85S6k. These results suggest the possible use of anisomycin and rapamycin to define upstream and downstream boundaries of an area of signalling above p70/85S6k which contains a bifurcation that produces histone H3-HMG-like protein phosphorylation and c-fos-c-jun induction in the nucleus. Images PMID:8289787

  3. c-Jun NH2-terminal kinase promotes apoptosis by down-regulating the transcriptional co-repressor CtBP.

    PubMed

    Wang, Su-Yan; Iordanov, Mihail; Zhang, Qinghong

    2006-11-17

    Genetic knock out of the transcriptional co-repressor carboxyl-terminal-binding protein (CtBP) in mouse embryonic fibroblasts results in up-regulation of several genes involved in apoptosis. We predicted, therefore, that a propensity toward apoptosis might be regulated through changes in cellular CtBP levels. Previously, we have identified the homeodomain-interacting protein kinase 2 as such a regulator and demonstrated that HIPK2 activation causes Ser-422 phosphorylation and degradation of CtBP. In this study, we found that c-Jun NH2-terminal kinase 1 activation triggered CtBP phosphorylation on Ser-422 and subsequent degradation, inducing p53-independent apoptosis in human lung cancer cells. JNK1 has previously been linked to UV-directed apoptosis. Expression of MKK7-JNK1 or exposure to UV irradiation reduced cellular levels of CtBP via a proteasome-mediated pathway. This effect was prevented by JNK1 deficiency. In addition, sustained activation of the JNK1 pathway by cisplatin similarly triggered CtBP degradation. These findings provide a novel target for chemotherapy in cancers lacking p53. PMID:16984892

  4. Naringin Mitigates Cardiac Hypertrophy by Reducing Oxidative Stress and Inactivating c-Jun Nuclear Kinase-1 Protein in Type I Diabetes.

    PubMed

    Adebiyi, A Olubunmi; Adebiyi, Oluwafeysetan O; Owira, Peter M O

    2016-02-01

    Cardiac hypertrophy (CH) in type 1 diabetes mellitus is attributed to increased oxidative stress-associated activation of c-Jun Nuclear Kinase (JNK). We investigated the effects of naringin on hyperglycemia-associated oxidative stress, activation of JNK-1, and CH. Male Sprague-Dawley rats (225-250 g) (n = 7) were divided into 6 groups. Groups I and II were orally treated with distilled water [3.0 mL/kg body weight/day (BW)] and naringin (50 mg/kg BW), respectively. Groups III-VI were rendered diabetic by a single intraperitoneal injection of 65 mg/kg BW of streptozotocin. Groups III, IV, and V were further treated with insulin (4.0 I.U, s.c, twice daily), naringin (50 mg/kg BW), and ramipril (3.0 mg/kg BW), respectively. After 56 days, the animals were sacrificed and then plasma and cardiac tissues obtained for further analysis. Naringin treatment of diabetic rats significantly reversed oxidative stress, lipid peroxidation, proteins oxidation, CH indices, and JNK protein activation compared with untreated diabetic animals. Our results do suggest that naringin mitigates CH by inhibiting oxidative stress leading to inactivation of JNK-1. Naringin supplements could therefore ameliorate CH in diabetic patients. PMID:26421421

  5. miR-138 protects cardiomyocytes from hypoxia-induced apoptosis via MLK3/JNK/c-jun pathway

    SciTech Connect

    He, Siyi; Liu, Peng; Jian, Zhao; Li, Jingwei; Zhu, Yun; Feng, Zezhou; Xiao, Yingbin

    2013-11-29

    Highlights: •First time to find miR-138 is up-regulated in hypoxic cardiomyocytes. •First time to find miR-138 targets MLK3 and regulates JNK/c-jun pathway. •Rare myocardial biopsy of patients with CHD were collected. •Both silence and overexpression of miR-138 were implemented. •Various methods were used to detect cell function. -- Abstract: Cardiomyocytes experience a series of complex endogenous regulatory mechanisms against apoptosis induced by chronic hypoxia. MicroRNAs are a class of endogenous small non-coding RNAs that regulate cellular pathophysiological processes. Recently, microRNA-138 (miR-138) has been found related to hypoxia, and beneficial for cell proliferation. Therefore, we intend to study the role of miR-138 in hypoxic cardiomyocytes and the main mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected to test miR-138 expression. Agomir or antagomir of miR-138 was transfected into H9C2 cells to investigate its effect on cell apoptosis. Higher miR-138 expression was observed in patients with cyanotic CHD, and its expression gradually increased with prolonged hypoxia time in H9C2 cells. Using MTT and LDH assays, cell growth was significantly greater in the agomir group than in the negative control (NC) group, while antagomir decreased cell survival. Dual luciferase reporter gene and Western-blot results confirmed MLK3 was a direct target of miR-138. It was found that miR-138 attenuated hypoxia-induced apoptosis using TUNEL, Hoechst staining and Annexin V-PE/7-AAD flow cytometry analysis. We further detected expression of apoptosis-related proteins. In the agomir group, the level of pro-apoptotic proteins such as cleaved-caspase-3, cleaved-PARP and Bad significantly reduced, while Bcl-2 and Bcl-2/Bax ratio increased. Opposite changes were observed in the antagomir group. Downstream targets of MLK3, JNK and c-jun, were also suppressed by miR-138. Our study demonstrates that up-regulation of miR-138 plays

  6. ANXA11 regulates the tumorigenesis, lymph node metastasis and 5-fluorouracil sensitivity of murine hepatocarcinoma Hca-P cells by targeting c-Jun

    PubMed Central

    Wang, Bo; Qi, Houbao; Sun, Ming-Zhong

    2016-01-01

    Annexin A11 (Anxa11) is associated with various cancers. Using a pair of syngeneic murine hepatocarcinoma cells, Hca-P with ~25% and Hca-F with ~75% lymph node metastatic (LNM) potentials, we demonstrated Anxa11 involvement in hepatocarcinoma lymphatic metastasis. Here, ANXA11 acted as a suppressor for the tumorigenicity, LNM and 5-FU resistance of Hca-P via c-Jun. We constructed monoclonal Hca-P cell line with stable ANXA11 knockdown. Although Bax and Bcl-2 levels increased in shRNA-Anxa11-transfected Hca-P, ANXA11 downregulation showed no clear effect on Hca-P apoptosis. ANXA11 downregulation promoted in vitro migration and invasion capacities of Hca-P. In situ adhesion potential of Hca-P cells toward LN was significantly enhanced following ANXA11 downregulation. Consistently, ANXA11 downregulation promoted the in vivo tumor growth and LNM capacities of Hca-P cells. ANXA11 knockdown enhanced the chemoresistance of Hca-P cells specifically toward 5-FU instead of cisplatin. Its downregulation increased c-Jun (pSer73) and decreased c-Jun (pSer243) levels in Hca-P. c-Jun (pSer243) downregulation seemed to be only correlated with ANXA11 knockdown without the connection to 5-FU treatment. Interestingly, compared with scramble-Hca-P cells, the levels of c-Jun and c-Jun (pSer73) in shRNA-Anxa11-Hca-P cells were upregulated in the presences of 0.1 and 1.0 mg/L 5-FU. The levels changes from c-Jun and c-Jun (pSer73) in Hca-P cells showed a more obvious tendency with the combination of ANXA11 knockdown and 5-FU treatment. ANXA11 level regulates LNM and 5-FU resistance of Hca-P via c-Jun pathway. It might play an important role in hepatocarcinoma cell malignancy and be a therapeutic target for hepatocarcinoma. PMID:26908448

  7. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    SciTech Connect

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

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

    PubMed

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

    2002-09-01

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

  9. 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 Central

    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. PMID:26829121

  10. Involvement of mitogen-activated protein kinase and NF-κB signaling pathways in perfluorooctane sulfonic acid-induced inflammatory reaction in BV2 microglial cells.

    PubMed

    Zhu, Jingying; Qian, Wenyi; Wang, Yixin; Gao, Rong; Wang, Jun; Xiao, Hang

    2015-12-01

    Microglial activation is closely related to the pathogenesis of neurodegenerative diseases by producing proinflammatory cytokines. Perfluorooctane sulfonic acid (PFOS), known as an emerging persistent organic pollutant, is reported to disturb human immune homeostasis; however, whether it affects cytokine production or the immune response in the central nervous system remains unclear. The present study was aimed to explore whether PFOS contributed to inflammatory action and to investigate the corresponding mechanisms in BV2 microglia. PFOS-mediated morphologic changes, cytokine responses and signaling events were examined by light microscopy, real-time polymerase chain reaction, enzyme-linked immunosorbent assay and Western blot assays. Our results indicated that PFOS increased BV2 cells activation and simultaneously increased tumor necrosis factor alpha and interleukin-6 expression. In addition, the c-Jun N-terminal protein kinase inhibitor (SP600125), as well as ERK1/2 blocker (PD98059), transcriptionally at least, displayed anti-inflammatory properties on PFOS-elicited cytokine responses. Moreover, the inflammatory transcription factor NF-κB was specifically activated by PFOS as well. These results, taken together, suggested that PFOS exerts its functional effects on the response of microglial cell activation via, in part, the c-Jun N-terminal protein kinase, ERK and NF-κB signaling pathways with its subsequent influence on proinflammatory action. PMID:25677194

  11. NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells.

    PubMed

    Galardi, Silvia; Mercatelli, Neri; Farace, Maria G; Ciafrè, Silvia A

    2011-05-01

    MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription. PMID:21245048

  12. NF-kB and c-Jun induce the expression of the oncogenic miR-221 and miR-222 in prostate carcinoma and glioblastoma cells

    PubMed Central

    Galardi, Silvia; Mercatelli, Neri; Farace, Maria G.; Ciafrè, Silvia A.

    2011-01-01

    MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in all aspects of cell biology. They finely modulate virtually all physiological pathways in metazoans, and are deeply implicated in all main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription. PMID:21245048

  13. Oral administration of curcumin suppresses production of matrix metalloproteinase (MMP)-1 and MMP-3 to ameliorate collagen-induced arthritis: inhibition of the PKCdelta/JNK/c-Jun pathway.

    PubMed

    Mun, Se Hwan; Kim, Hyuk Soon; Kim, Jie Wan; Ko, Na Young; Kim, Do Kyun; Lee, Beob Yi; Kim, Bokyung; Won, Hyung Sik; Shin, Hwa-Sup; Han, Jeung-Whan; Lee, Hoi Young; Kim, Young Mi; Choi, Wahn Soo

    2009-09-01

    We investigated whether oral administration of curcumin suppressed type II collagen-induced arthritis (CIA) in mice and its effect and mechanism on matrix metalloproteinase (MMP)-1 and MMP-3 production in CIA mice, RA fibroblast-like synoviocytes (FLS), and chondrocytes. CIA in mice was suppressed by oral administration of curcumin in a dose-dependent manner. Macroscopic observations were confirmed by histological examinations. Histological changes including infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw sections were extensively suppressed by curcumin. The histological scores were consistent with clinical arthritis indexes. Production of MMP-1 and MMP-3 were inhibited by curcumin in CIA hind paw sections and tumor necrosis factor (TNF)-alpha-stimulated FLS and chondrocytes in a dose-dependent manner. As for the mechanism, curcumin inhibited activating phosphorylation of protein kinase Cdelta (PKCdelta) in CIA, FLS, and chondrocytes. Curcumin also suppressed the JNK and c-Jun activation in those cells. This study suggests that the suppression of MMP-1 and MMP-3 production by curcumin in CIA is mediated through the inhibition of PKCdelta and the JNK/c-Jun signaling pathway. PMID:19763044

  14. N-Terminal Region of the Catalytic Domain of Human N-Myristoyltransferase 1 Acts as an Inhibitory Module

    PubMed Central

    Kumar, Sujeet; Sharma, Rajendra K.

    2015-01-01

    N-myristoyltransferase (NMT) plays critical roles in the modulation of various signaling molecules, however, the regulation of this enzyme in diverse cellular states remains poorly understood. We provide experimental evidence to show for the first time that for the isoform 1 of human NMT (hNMT1), the regulatory roles extend into the catalytic core. In our present study, we expressed, purified, and characterized a truncation mutant devoid of 28 N-terminal amino acids from the catalytic module (Δ28-hNMT1s) and compared its properties to the full-length catalytic domain of hNMT1. The deletion of the N-terminal peptide had no effect on the enzyme stability. Our findings suggest that the N-terminal region in the catalytic module of hNMT1 functions serves as a regulatory control element. The observations of an ~3 fold increase in enzymatic efficiency following removal of the N-terminal peptide of hNMT1s indicates that N-terminal amino acids acts as an inhibitory segment and negatively regulate the enzyme activity. Our findings that the N-terminal region confers control over activity, taken together with the earlier observations that the N-terminal of hNMT1 is differentially processed in diverse cellular states, suggests that the proteolytic processing of the peptide segment containing the inhibitory region provides a molecular mechanism for physiological up-regulation of myristoyltransferase activity. PMID:26000639

  15. Hind Limb Unloading Model Alters Nuclear Factor kappa B and Activator Protein-1 Signaling in Mouse Brain

    NASA Astrophysics Data System (ADS)

    Ramesh, Govindarajan; Vani, Vani; Renard, Renard; Vera, Vera; Wilosn, Wilosn; Ramesh, Govindarajan

    Microgravity induces inflammatory response and also modulates immune functions, which may increase oxidative stress. Exposure to the microgravity environment induces adverse neurological effects. However, there is little research exploring the etiology of neurological effects of exposure to this environment. To explore this area we evaluated changes in Nuclear Factor kappa B, Activator Protein 1, MAPP kinase and N terminal c-Jun kinase in mouse brain exposed to a simulated microgravity environment using the hindlimb unloading model. BALB/c male mice were randomly assigned to hindlimb unloading group (n=12) and control group (n=12) to simulate a microgravity environment, for 7 days. Changes observed in NF-κB, AP- 1 DNA binding, MAPKK and N terminal c-Jun kinase were measured using electrophoretic mobility shift assay (EMSA) and western blot analysis and compared to unexposed brain regions. Hindlimb unloading exposed mice showed significant increases in generated NF-κB, AP-1, MAPKK and Kinase in all regions of the brain exposed to hindlimb unloading as compared to the control brain regions. Results suggest that exposure to simulated microgravity can induce expression of certain transcription factors and protein kinases. This work was supported by funding from NASA NCC 9-165. 504b030414000600080000002100828abc13fa0000001c020000130000005b436f6e74656e745f54797065735d2e78

  16. An apoptotic signaling pathway in the interferon antiviral response mediated by RNase L and c-Jun NH2-terminal kinase.

    PubMed

    Li, Geqiang; Xiang, Ying; Sabapathy, Kanaga; Silverman, Robert H

    2004-01-01

    Cellular stress responses induced during viral infections are critical to the health and survival of organisms. In higher vertebrates, interferons (IFNs) mediate the innate antiviral response in part through the action of RNase L, a uniquely regulated enzyme. RNase L is activated by 5'-phosphorylated, 2'-5' oligoadenylates (2-5A) produced from IFN-inducible and double stranded RNA-dependent synthetases. We show that viral activation of the c-Jun NH2-terminal kinases (JNK) family of MAP kinases and viral induction of apoptosis are both deficient in mouse cells lacking RNase L. Also, JNK phosphorylation in response to 2-5A was greatly reduced in RNase L-/- mouse cells. In addition, 2-5A treatment of the human ovarian carcinoma cell line, Hey1b, resulted in specific ribosomal RNA cleavage products coinciding with JNK activation. Furthermore, suppression of JNK activity with the chemical inhibitor, SP600125, prevented apoptosis induced by 2-5A. In contrast, inhibition of alternative MAP kinases, p38 and ERK, failed to prevent 2-5A-mediated apoptosis. Short interfering RNA to JNK1/JNK2 mRNAs resulted in JNK ablation while also suppressing 2-5A-mediated apoptosis. Moreover, Jnk1-/- Jnk2-/- cells were highly resistant to the apoptotic effects of IFN and 2-5A. These findings suggest that JNK and RNase L function in an integrated signaling pathway during the IFN response that leads to elimination of virus-infected cells through apoptosis. PMID:14570908

  17. Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of c-jun and c-fos in Human Tissue Culture Cells

    EPA Science Inventory

    Aims: To assess virulence of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. Methods and Results: Aeromonas cells were added to Caco-2 cells at approximately a one to one ratio. After 1, 2 and 3 ...

  18. Augmentation of invadopodia formation in temozolomide-resistant or adopted glioma is regulated by c-Jun terminal kinase-paxillin axis.

    PubMed

    Ueno, Hideaki; Tomiyama, Arata; Yamaguchi, Hideki; Uekita, Takamasa; Shirakihara, Takuya; Nakashima, Katsuhiko; Otani, Naoki; Wada, Kojiro; Sakai, Ryuichi; Arai, Hajime; Mori, Kentaro

    Temozolomide (TMZ) is one of the few effective anticancer agents against gliomas. However, acquisition of TMZ resistance or adaptation by gliomas is currently a crucial problem, especially increased invasiveness which is critical for the determination of clinical prognosis. This study investigated the molecular regulatory mechanisms of TMZ resistance in gliomas involved in invasiveness, particularly invadopodia formation, a molecular complex formed at the invasive front to cause extracellular matrix degradation during cellular local invasion. The TMZ-resistant clone of the U343 MG human glioma cell line (U343-R cells) was established. U343-R cells demonstrated higher invadopodia formation compared with U343 cells without TMZ resistance (U343-Con cells). Immunoblot analysis of DNA damage-related mitogen-activated protein kinase signals found increased phosphorylation of c-Jun terminal kinase (JNK) and higher activation of its downstream signaling in U343-R cells compared with U343-Con cells. Treatment of U343-R cells with specific inhibitors of JNK or siRNA targeting JNK suppressed up-regulation of invadopodia formation. In addition, paxillin, one of the known JNK effectors which is phosphorylated and affects cell migration, was phosphorylated at serine 178 in JNK activity-dependent manner. Expression of paxillin with mutation of the serine 178 phosphorylation site in U343-R cells blocked invadopodia formation. The present findings suggest that increased formation of invadopodia in U343-R cells is mediated by hyperactivation of JNK-paxillin signaling, and both JNK and paxillin might become targets of novel therapies against TMZ-resistant gliomas. PMID:26518652

  19. Overexpression of MAPK15 in gastric cancer is associated with copy number gain and contributes to the stability of c-Jun

    PubMed Central

    Jin, Dong-Hao; Lee, Jeeyun; Kim, Kyoung Mee; Kim, Sung; Kim, Duk-Hwan; Park, Joobae

    2015-01-01

    This study was aimed at understanding the functional and clinicopathological significance of MAPK15 alteration in gastric cancer. Genome-wide copy number alterations (CNAs) were first investigated in 40 gastric cancers using Agilent aCGH-244K or aCGH-400K, and copy number gains of MAPK15 found in aCGH were validated in another set of 48 gastric cancer tissues. The expression of MAPK15 was analyzed using immunohistochemistry in concurrent lesions of normal, adenoma, and carcinoma from additional 45 gastric cancer patients. The effects of MAPK15 on cell cycle, c-Jun phosphorylation, and mRNA stability were analyzed in gastric cancer cells. Copy number gains of MAPK15 were found in 15 (17%) of 88 tumor tissues. The mRNA levels of MAPK15 were relatively high in the gastric cancer tissues and gastric cancer cells with higher copy number gains than those without. Knockdown of MAPK15 using siRNA in gastric cancer cells significantly suppressed cell proliferation and resulted in cell cycle arrest at G1-S phase. Reduced c-Jun phosphorylation and c-Jun half-life were observed in MAPK15-knockdowned cells. In addition, transient transfection of MAPK15 into AGS gastric cancer cells with low copy number resulted in an increase of c-Jun phosphorylation and stability. The overexpression of MAPK15 occurred at a high frequency in carcinomas (37%) compared to concurrent normal tissues (2%) and adenomas (21%). In conclusion, the present study suggests that MAPK15 overexpression may contribute to the malignant transformation of gastric mucosa by prolonging the stability of c-Jun. And, patients with copy number gain of MAPK15 in normal or premalignant tissues of stomach may have a chance to progress to invasive cancer. PMID:26035356

  20. Specific effects of c-Jun NH2-terminal kinase-interacting protein 1 in neuronal axons

    PubMed Central

    Tang, Shu; Wen, Qiang; Zhang, Xiao-jian; Kan, Quan-cheng

    2016-01-01

    c-Jun NH2-terminal kinase (JNK)-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B (TrkB) anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of TrkB anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed TrkB complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of TrkB gradually increased in axon terminals. However, the distribution of TrkB reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of TrkB after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of TrkB in dendrites. These findings confirm that JNK-interacting protein 1 can interact with TrkB in neuronal cells, and can regulate the transport of TrkB in axons, but not in dendrites. PMID:26981098

  1. Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase (hNTAN1)

    PubMed Central

    Cantor, Jason R.; Stone, Everett M.; Georgiou, George

    2011-01-01

    The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein we report the bacterial expression, purification, and biochemical characterization of the human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn, but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the point mutation Cys75Ala. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway. PMID:21375249

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

    PubMed

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

    2015-05-01

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

  3. Nymphaea rubra ameliorates TNF-α-induced insulin resistance via suppression of c-Jun NH2-terminal kinase and nuclear factor-κB in the rat skeletal muscle cells.

    PubMed

    Gautam, Sudeep; Rahuja, Neha; Ishrat, Nayab; Asthana, R K; Mishra, D K; Maurya, Rakesh; Jain, Swatantra Kumar; Srivastava, Arvind Kumar

    2014-12-01

    In this work, we demonstrated insulin signaling and the anti-inflammatory effects by the chloroform fraction of ethanolic extract of Nymphaea rubra flowers in TNF-α-induced insulin resistance in the rat skeletal muscle cell line (L6 myotubes) to dissect out its anti-hyperglycemic mechanism. N. rubra enhances the GLUT4-mediated glucose transport in a dose dependent manner and also increases the tyrosine phosphorylation of both IR-β and IRS-1, and the IRS-1 associated PI-3 kinase activity in TNF-α-treated L6 myotubes. Moreover, N. rubra decreases Ser(307) phosphorylation of IRS-1 by the suppression of JNK and NF-κB activation. In conclusion, N. rubra reverses the insulin resistance by the inhibition of c-Jun NH2-Terminal Kinase and Nuclear-κB. PMID:25234391

  4. Botulinum toxin complex increases paracellular permeability in intestinal epithelial cells via activation of p38 mitogen-activated protein kinase.

    PubMed

    Miyashita, Shin-Ichiro; Sagane, Yoshimasa; Inui, Ken; Hayashi, Shintaro; Miyata, Keita; Suzuki, Tomonori; Ohyama, Tohru; Watanabe, Toshihiro; Niwa, Koichi

    2013-12-30

    Clostridium botulinum produces a large toxin complex (L-TC) that increases paracellular permeability in intestinal epithelial cells by a mechanism that remains unclear. Here, we show that mitogen-activated protein kinases (MAPKs) are involved in this permeability increase. Paracellular permeability was measured by FITC-dextran flux through a monolayer of rat intestinal epithelial IEC-6 cells, and MAPK activation was estimated from western blots. L-TC of C. botulinum serotype D strain 4947 increased paracellular dextran flux and activated extracellular signal-regulated kinase (ERK), p38, but not c-Jun N-terminal kinase (JNK) in IEC-6 cells. The permeability increase induced by L-TC was abrogated by the p38 inhibitor SB203580. These results indicate that L-TC increases paracellular permeability by activating p38, but not JNK and ERK. PMID:23884081

  5. An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds.

    PubMed

    Sutoh, Keita; Washio, Kenji; Imai, Ryozo; Wada, Masamitsu; Nakai, Tomonori; Yamauchi, Daisuke

    2015-01-01

    The expression of the gene for a proteinase (Rep1) is upregulated by gibberellins. The CAACTC regulatory element (CARE) of the Rep1 promoter is involved in the gibberellin response. We isolated a cDNA for a CARE-binding protein containing a Myb domain in its carboxyl-terminal region and designated the gene Carboxyl-terminal Myb1 (CTMyb1). This gene encodes two polypeptides of two distinctive lengths, CTMyb1L and CTMyb1S, which include or exclude 213 N-terminal amino acid residues, respectively. CTMyb1S transactivated the Rep1 promoter in the presence of OsGAMyb, but not CTMyb1L. We observed an interaction between CTMyb1S and the rice prolamin box-binding factor (RPBF). A bimolecular fluorescence complex analysis detected the CTMyb1S and RPBF complex in the nucleus, but not the CTMyb1L and RPBF complex. The results suggest that the arrangement of the transfactors is involved in gibberellin-inducible expression of Rep1. PMID:25559339

  6. Ghrelin Inhibits Oligodendrocyte Cell Death by Attenuating Microglial Activation

    PubMed Central

    Lee, Jee Youn

    2014-01-01

    Background Recently, we reported the antiapoptotic effect of ghrelin in spinal cord injury-induced apoptotic cell death of oligodendrocytes. However, how ghrelin inhibits oligodendrocytes apoptosis, is still unknown. Therefore, in the present study, we examined whether ghrelin inhibits microglia activation and thereby inhibits oligodendrocyte apoptosis. Methods Using total cell extracts prepared from BV-2 cells activated by lipopolysaccharide (LPS) with or without ghrelin, the levels of p-p38 phosphor-p38 mitogen-activated protein kinase (p-p38MAPK), phospho-c-Jun N-terminal kinase (pJNK), p-c-Jun, and pro-nerve growth factor (proNGF) were examined by Western blot analysis. Reactive oxygen species (ROS) production was investigated by using dichlorodihydrofluorescein diacetate. To examine the effect of ghrelin on oligodendrocyte cell death, oligodendrocytes were cocultured in transwell chambers of 24-well plates with LPS-stimulated BV-2 cells. After 48 hours incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and terminal deoxynucleotidyl transferase 2'-deoxyuridine, 5'-triphosphate nick end labeling staining were assessed. Results Ghrelin treatment significantly decreased levels of p-p38MAPK, p-JNK, p-c-Jun, and proNGF in LPS-stimulated BV-2 cells. ROS production increased in LPS-stimulated BV-2 cells was also significantly inhibited by ghrelin treatment. In addition, ghrelin significantly inhibited oligodendrocyte cell death when cocultured with LPS-stimulated BV-2 cells. Conclusion Ghrelin inhibits oligodendrocyte cell death by decreasing proNGF and ROS production as well as p38MAPK and JNK activation in activated microglia as an anti-inflammatory hormone. PMID:25309797

  7. N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy.

    PubMed

    Liu, Xudong; Wang, Chuan-En; Hong, Yan; Zhao, Ting; Wang, Guohao; Gaertig, Marta A; Sun, Miao; Li, Shihua; Li, Xiao-Jiang

    2016-05-01

    The Huntington's disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1-208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD. PMID:27203582

  8. N-terminal Huntingtin Knock-In Mice: Implications of Removing the N-terminal Region of Huntingtin for Therapy

    PubMed Central

    Liu, Xudong; Wang, Chuan-En; Hong, Yan; Zhao, Ting; Wang, Guohao; Gaertig, Marta A.; Sun, Miao; Li, Shihua; Li, Xiao-Jiang

    2016-01-01

    The Huntington’s disease (HD) protein, huntingtin (HTT), is a large protein consisting of 3144 amino acids and has conserved N-terminal sequences that are followed by a polyglutamine (polyQ) repeat. Loss of Htt is known to cause embryonic lethality in mice, whereas polyQ expansion leads to adult neuronal degeneration. Whether N-terminal HTT is essential for neuronal development or contributes only to late-onset neurodegeneration remains unknown. We established HTT knock-in mice (N160Q-KI) expressing the first 208 amino acids of HTT with 160Q, and they show age-dependent HTT aggregates in the brain and neurological phenotypes. Importantly, the N-terminal mutant HTT also preferentially accumulates in the striatum, the brain region most affected in HD, indicating the importance of N-terminal HTT in selective neuropathology. That said, homozygous N160Q-KI mice are also embryonic lethal, suggesting that N-terminal HTT alone is unable to support embryonic development. Using Htt knockout neurons, we found that loss of Htt selectively affects the survival of developing neuronal cells, but not astrocytes, in culture. This neuronal degeneration could be rescued by a truncated HTT lacking the first 237 amino acids, but not by N-terminal HTT (1–208 amino acids). Also, the rescue effect depends on the region in HTT known to be involved in intracellular trafficking. Thus, the N-terminal HTT region may not be essential for the survival of developing neurons, but when carrying a large polyQ repeat, can cause selective neuropathology. These findings imply a possible therapeutic benefit of removing the N-terminal region of HTT containing the polyQ repeat to treat the neurodegeneration in HD. PMID:27203582

  9. Constitutive hypophosphorylation of extracellular signal-regulated kinases-1/2 and down-regulation of c-Jun in human gastric adenocarcinoma

    SciTech Connect

    Wu, William Ka Kei; Sung, Joseph Joe Yiu; Yu Le; Li Zhijie; Chu, Kent Man; Cho, C.H.

    2008-08-22

    Hyperphosphorylation of extracellular signal-regulated protein kinases-1/2 (ERK1/2) is known to promote cancer cell proliferation. We therefore investigated the constitutive phosphorylation levels of ERK1/2 and the expression of its downstream targets c-Fos, c-Jun, and cyclooxygenase-2 (COX-2) in biopsied human gastric cancer tissues. Results showed that ERK1/2 phosphorylation and c-Jun expression were significantly lowered in gastric cancer compared with the non-cancer adjacent tissues. The expression of c-Fos, however, was not altered while COX-2 was significantly up-regulated. To conclude, we demonstrate that hypophosphorylation of ERK1/2 may occur in gastric cancer. Such discovery may have implication in the application of pathway-directed therapy for this malignant disease.

  10. β-glucan reduces exercise-induced stress through downregulation of c-Fos and c-Jun expression in the brains of exhausted rats.

    PubMed

    Hong, Heeok; Kim, Chang-Ju; Kim, Jae-Deung; Seo, Jin-Hee

    2014-05-01

    Immediate-early genes are involved in acute stress responses in the central nervous system. β-glucan stimulates innate immune defenses, exerts an anti-tumor response and increases resistance to a wide variety of types of infection. To date, the effect of β-glucan on the expression of immediate-early genes under stressful conditions has not been elucidated. In the present study, the effects of β-glucan on the expression of the oncogenes c-Fos and c-Jun in the hypothalamus, dentate gyrus and dorsal raphe in rats following exhaustive treadmill running were investigated. Male Sprague Dawley rats were randomly divided into five groups (n=10 in each group) as follows: Control, exercise, exercise and 50 mg/kg β-glucan treatment, exercise and 100 mg/kg β-glucan treatment, and exercise and 200 mg/kg β-glucan treatment. Rats in the β-glucan‑treated groups were administered β-glucan at the respective dose once per day for seven days. Rats in the exercise groups performed treadmill running once per day for six days. On the seventh day of the experiment, the time to exhaustion in response to treadmill running was determined for the exercise groups. The expression of c-Fos and c-Jun in the hypothalamus, dorsal raphe and hippocampus was enhanced by exhaustive treadmill running. Administration of β-glucan resulted in an increase in the time to exhaustion and the suppression of the exercise-induced increment in c-Fos and c-Jun expression. In conclusion, β-glucan may exert an alleviating effect on exercise-induced stress through the suppression of c-Fos and c-Jun expression in the brains of exhausted rats. PMID:24604295

  11. The N-terminal acetyltransferase Naa10 is essential for zebrafish development

    PubMed Central

    Ree, Rasmus; Myklebust, Line M.; Thiel, Puja; Foyn, Håvard; Fladmark, Kari E.; Arnesen, Thomas

    2015-01-01

    N-terminal acetylation, catalysed by N-terminal acetyltransferases (NATs), is among the most common protein modifications in eukaryotes and involves the transfer of an acetyl group from acetyl-CoA to the α-amino group of the first amino acid. Functions of N-terminal acetylation include protein degradation and sub-cellular targeting. Recent findings in humans indicate that a dysfunctional Nα-acetyltransferase (Naa) 10, the catalytic subunit of NatA, the major NAT, is associated with lethality during infancy. In the present study, we identified the Danio rerio orthologue zebrafish Naa 10 (zNaa10). In vitro N-terminal acetylation assays revealed that zNaa10 has NAT activity with substrate specificity highly similar to that of human Naa10. Spatiotemporal expression pattern was determined by in situ hybridization, showing ubiquitous expression with especially strong staining in brain and eye. By morpholino-mediated knockdown, we demonstrated that naa10 morphants displayed increased lethality, growth retardation and developmental abnormalities like bent axis, abnormal eyes and bent tails. In conclusion, we identified the zebrafish Naa10 orthologue and revealed that it is essential for normal development and viability of zebrafish. PMID:26251455

  12. Acidosis Blocks CCAAT/Enhancer-Binding Protein Homologous Protein (CHOP)- and c-Jun-Mediated Induction of p53-Upregulated Mediator of Apoptosis (PUMA) during Amino Acid Starvation

    PubMed Central

    Ryder, Christopher B.; McColl, Karen; Distelhorst, Clark W.

    2012-01-01

    Cancer cells must avoid succumbing to a variety of noxious conditions within their surroundings. Acidosis is one such prominent feature of the tumor microenvironment that surprisingly promotes tumor survival and progression. We recently reported that acidosis prevents apoptosis of starved or stressed lymphoma cells through regulation of several Bcl-2 family members (Ryder et al., JBC, 2012). Mechanistic studies in that work focused on the acid-mediated upregulation of anti-apoptotic Bcl-2 and Bcl-xL, while additionally showing inhibition of glutamine starvation-induced expression of pro-apoptotic PUMA by acidosis. Herein we report that amino acid (AA) starvation elevates PUMA, an effect that is blocked by extracellular acidity. Knockdown studies confirm that PUMA induction during AA starvation requires expression of both CHOP and c-Jun. Interestingly, acidosis strongly attenuates AA starvation-mediated c-Jun expression, which correlates with PUMA repression. As c-Jun exerts a tumor suppressive function in this and other contexts, its inhibition by acidosis has broader implications for survival of cancer cells in the acidic tumor milieu. PMID:23261451

  13. Reduction in bile acid pool causes delayed liver regeneration accompanied by down-regulated expression of FXR and c-Jun mRNA in rats.

    PubMed

    Dong, Xiushan; Zhao, Haoliang; Ma, Xiaoming; Wang, Shiming

    2010-02-01

    The present study attempted to examine the effects of bile acid pool size on liver regeneration after hepatectomy. The rats were fed on 0.2% cholic acid (CA) or 2% cholestyramine for 7 days to induce a change in the bile acid size, and then a partial hepatectomy (PH) was performed. Rats fed on the normal diet served as the controls. Measurements were made on the rate of liver regeneration, the labeling indices of PCNA, the plasma total bile acids (TBA), and the mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), farnesoid X receptor (FXR), and transcription factor c-Jun or c-fos. As compared with the normal and CA groups, the rate of liver regeneration was decreased on the day 3, and 7 after PH; the peak of the labeling indices of PCNA was delayed and the labeling indices were significantly reduced on the day 1; the TBA were also decreased on the day 1; the expression of FXR decreased but that of CYP7A1 increased at any given time; at the 1st, and 3rd h, the expression of c-Jun was declined in the cholestyramine group. The reduction in the bile acid pool size was found to delay the liver regeneration, which may be caused by the down-regulation of FXR and c-Jun expression. PMID:20155456

  14. Analytical cation-exchange chromatography to assess the identity, purity, and N-terminal integrity of human lactoferrin.

    PubMed

    van Veen, Harrie A; Geerts, Marlieke E J; van Berkel, Patrick H C; Nuijens, Jan H

    2002-10-01

    Human lactoferrin (hLF) is an iron-binding glycoprotein involved in the innate host defense. The positively charged N-terminal domain of hLF mediates several of its activities by interacting with ligands such as bacterial lipopolysaccharide (LPS), specific receptors, and other proteins. This cationic domain is highly susceptible to limited proteolysis, which impacts on the affinity of hLF for the ligand. An analytical method, employing cation-exchange chromatography on Mono S, was developed to assess the N-terminal integrity of hLF preparations. The method, which separates N-terminally intact hLF from hLF species lacking two (Gly(1)-Arg(2)) or three (Gly(1)-Arg(2)-Arg(3)) residues, showed that 5-58% of total hLF in commercially obtained preparations was N-terminally degraded. The elution profile of hLF on Mono S unequivocally differed from lactoferrins from other species as well as homologous and other whey proteins. Analysis of fresh human whey samples revealed two variants of N-terminally intact hLF, but not limitedly proteolyzed hLF. Mono S chromatography of 2 out of 26 individual human whey samples showed a rare polymorphic hLF variant with three N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Ser(5)-) instead of the usual variant with four N-terminal arginines (Gly(1)-Arg(2)-Arg(3)-Arg(4)-Arg(5)-Ser(6)-). In conclusion, Mono S cation-exchange chromatography appeared a robust method to assess the identity, purity, N-terminal integrity, and the presence of polymorphic and intact hLF variants. PMID:12381362

  15. Top-down N-terminal sequencing of Immunoglobulin subunits with electrospray ionization time of flight mass spectrometry.

    PubMed

    Ren, Da; Pipes, Gary D; Hambly, David; Bondarenko, Pavel V; Treuheit, Michael J; Gadgil, Himanshu S

    2009-01-01

    An N-terminal top-down sequencing approach was developed for IgG characterization, using high-resolution HPLC separation and collisionally activated dissociation (CAD) on a single-stage LCT Premier time of flight (TOF) mass spectrometer. Fragmentation of the IgG chains on the LCT Premier was optimized by varying the ion guide voltage values. Ion guide 1 voltage had the most significant effect on the fragmentation of the IgG chains. An ion guide 1 voltage value of 100 V was found to be optimum for the N-terminal fragmentation of IgG heavy and light chains, which are approximately 50 and 25 kDa, respectively. The most prominent ion series in this CAD experiment was the terminal b-ion series which allows N-terminal sequencing. Using this technique, we were able to confirm the sequence of up to seven N-terminal residues. Applications of this method for the identification of N-terminal pyroglutamic acid formation will be discussed. The method described could be used as a high-throughput method for the rapid N-terminal sequencing of IgG chains and for the detection of chemical modifications in the terminal residues. PMID:18834850

  16. Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain

    PubMed Central

    Moparthi, Lavanya; Survery, Sabeen; Kreir, Mohamed; Simonsen, Charlotte; Kjellbom, Per; Högestätt, Edward D.; Johanson, Urban; Zygmunt, Peter M.

    2014-01-01

    We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1–688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ9-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1–688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca2+, or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease). PMID:25389312

  17. Astrocytes and microglia but not neurons preferentially generate N-terminally truncated Aβ peptides.

    PubMed

    Oberstein, Timo Jan; Spitzer, Philipp; Klafki, Hans-Wolfgang; Linning, Philipp; Neff, Florian; Knölker, Hans-Joachim; Lewczuk, Piotr; Wiltfang, Jens; Kornhuber, Johannes; Maler, Juan Manuel

    2015-01-01

    The neuropathological hallmarks of Alzheimer's disease include extracellular neuritic plaques and neurofibrillary tangles. The neuritic plaques contain β-amyloid peptides (Aβ peptides) as the major proteinaceous constituent and are surrounded by activated microglia and astrocytes as well as dystrophic neurites. N-terminally truncated forms of Aβ peptides are highly prevalent in neuritic plaques, including Aβ 3-x beginning at Glu eventually modified to pyroglutamate (Aβ N3pE-x), Aβ 2-x, Aβ 4-x, and Aβ 5-x. The precise origin of the different N-terminally modified Aβ peptides currently remains unknown. To assess the contribution of specific cell types to the formation of different N-terminally truncated Aβ peptides, supernatants from serum-free primary cell cultures of chicken neurons, astrocytes, and microglia, as well as human astrocytes, were analyzed by Aβ-ELISA and one- and two-dimensional SDS-urea polyacrylamide gel electrophoresis followed by immunoblot analysis. To evaluate the contribution of β- and γ-secretase to the generation of N-terminally modified Aβ, cultured astrocytes were treated with membrane-anchored "tripartite β-secretase (BACE1) inhibitors" and the γ-secretase inhibitor DAPT. Neurons, astrocytes, and microglia each exhibited cell type-specific patterns of secreted Aβ peptides. Neurons predominantly secreted Aβ peptides that begin at Asp1, whereas those released from astrocytes and microglia included high proportions of N-terminally modified Aβ peptides, presumably including Aβ 2/3-x and 4/5-x. The inhibition of BACE1 reduced the amount of Aβ 1-x in cell culture supernatants but not the amount of Aβ 2-x. PMID:25204716

  18. Site directed spin labeling studies of Escherichia coli dihydroorotate dehydrogenase N-terminal extension

    SciTech Connect

    Couto, Sheila G.; Cristina Nonato, M.

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer EcDHODH is a membrane-associated enzyme and a promising target for drug design. Black-Right-Pointing-Pointer Enzyme's N-terminal extension is responsible for membrane association. Black-Right-Pointing-Pointer N-terminal works as a molecular lid regulating access to the protein interior. -- Abstract: Dihydroorotate dehydrogenases (DHODHs) are enzymes that catalyze the fourth step of the de novo synthesis of pyrimidine nucleotides. In this reaction, DHODH converts dihydroorotate to orotate, using a flavine mononucleotide as a cofactor. Since the synthesis of nucleotides has different pathways in mammals as compared to parasites, DHODH has gained much attention as a promising target for drug design. Escherichia coli DHODH (EcDHODH) is a family 2 DHODH that interacts with cell membranes in order to promote catalysis. The membrane association is supposedly made via an extension found in the enzyme's N-terminal. In the present work, we used site directed spin labeling (SDSL) to specifically place a magnetic probe at positions 2, 5, 19, and 21 within the N-terminal and thus monitor, by using Electron Spin Resonance (ESR), dynamics and structural changes in this region in the presence of a membrane model system. Overall, our ESR spectra show that the N-terminal indeed binds to membranes and that it experiences a somewhat high flexibility that could be related to the role of this region as a molecular lid controlling the entrance of the enzyme's active site and thus allowing the enzyme to give access to quinones that are dispersed in the membrane and that are necessary for the catalysis.

  19. The N-terminal strand modulates immunoglobulin light chain fibrillogenesis

    SciTech Connect

    Pozo-Yauner, Luis del; Wall, Jonathan S.; González Andrade, Martín; Sánchez-López, Rosana; Rodríguez-Ambriz, Sandra L.; Pérez Carreón, Julio I.; and others

    2014-01-10

    Highlights: •We evaluated the impact of mutations in the N-terminal strand of 6aJL2 protein. •Mutations destabilized the protein in a position-dependent manner. •Destabilizing mutations accelerated the fibrillogenesis by shortening the lag time. •The effect on the kinetic of fibril elongation by seeding was of different nature. •The N-terminal strand is buried in the fibrillar state of 6aJL2 protein. -- Abstract: It has been suggested that the N-terminal strand of the light chain variable domain (V{sub L}) protects the molecule from aggregation by hindering spurious intermolecular contacts. We evaluated the impact of mutations in the N-terminal strand on the thermodynamic stability and kinetic of fibrillogenesis of the V{sub L} protein 6aJL2. Mutations in this strand destabilized the protein in a position-dependent manner, accelerating the fibrillogenesis by shortening the lag time; an effect that correlated with the extent of destabilization. In contrast, the effect on the kinetics of fibril elongation, as assessed in seeding experiments was of different nature, as it was not directly dependant on the degree of destabilization. This finding suggests different factors drive the nucleation-dependent and elongation phases of light chain fibrillogenesis. Finally, taking advantage of the dependence of the Trp fluorescence upon environment, four single Trp substitutions were made in the N-terminal strand, and changes in solvent exposure during aggregation were evaluated by acrylamide-quenching. The results suggest that the N-terminal strand is buried in the fibrillar state of 6aJL2 protein. This finding suggest a possible explanation for the modulating effect exerted by the mutations in this strand on the aggregation behavior of 6aJL2 protein.

  20. The basic N-terminal domain of TRF2 limits recombination endonuclease action at human telomeres.

    PubMed

    Saint-Léger, Adélaïde; Koelblen, Melanie; Civitelli, Livia; Bah, Amadou; Djerbi, Nadir; Giraud-Panis, Marie-Josèphe; Londoño-Vallejo, Arturo; Ascenzioni, Fiorentina; Gilson, Eric

    2014-01-01

    The stability of mammalian telomeres depends upon TRF2, which prevents inappropriate repair and checkpoint activation. By using a plasmid integration assay in yeasts carrying humanized telomeres, we demonstrated that TRF2 possesses the intrinsic property to both stimulate initial homologous recombination events and to prevent their resolution via its basic N-terminal domain. In human cells, we further showed that this TRF2 domain prevents telomere shortening mediated by the resolvase-associated protein SLX4 as well as GEN1 and MUS81, 2 different types of endonucleases with resolvase activities. We propose that various types of resolvase activities are kept in check by the basic N-terminal domain of TRF2 in order to favor an accurate repair of the stalled forks that occur during telomere replication. PMID:25483196

  1. ΔPK oncolytic activity includes modulation of the tumour cell milieu.

    PubMed

    Bollino, Dominique; Colunga, Aric; Li, Baiquan; Aurelian, Laure

    2016-02-01

    Oncolytic virotherapy is a unique cancer therapeutic that encompasses tumour cell lysis through both virus replication and programmed cell death (PCD) pathways. Nonetheless, clinical efficacy is relatively modest, likely related to the immunosuppressive tumour milieu. Our studies use the herpes simplex virus type 2 (HSV-2)-based oncolytic virus ΔPK that has documented anti-tumour activity associated with virus replication, PCD and cancer stem cell lysis. They are designed to examine whether ΔPK-mediated oncolysis includes the ability to reverse the immunosuppressive tumour microenvironment by altering the balance of cytokines directly secreted by the melanoma cells and to define its mechanism. Here, we show that melanoma cells secreted the immunosuppressive cytokine IL-10, and that secretion was inhibited by ΔPK through virus replication and c-Jun N-terminal kinase/c-Jun activation. ΔPK-induced IL-10 inhibition upregulated surface expression of MHC class I chain-related protein A, the ligand for the activating NKG2D receptor expressed on NK- and cytotoxic T-cells. Concomitantly, ΔPK also upregulated the secretion of inflammatory cytokines TNF-α, granulocyte macrophage colony-stimulating factor and IL-1β through autophagy-mediated activation of Toll-like receptor 2 pathways and pyroptosis, and it inhibited the expression of the negative immune checkpoint regulator cytotoxic T-lymphocyte antigen 4. Pharmacologic inhibition of these processes significantly reduces the oncolytic activity of ΔPK. PMID:26602205

  2. In vitro phosphorylation of the N-terminal half of hordeivirus movement protein.

    PubMed

    Makarov, V V; Iconnikova, A Y; Guseinov, M A; Vishnichenko, V K; Kalinina, N O

    2012-09-01

    The N-terminal half of TGB1 movement protein of poa semilatent hordeivirus, which forms a ribonucleoprotein complex involved in movement of the viral genome in the plant, and its two domains, NTD and ID, are phosphorylated in vitro by a fraction enriched in cell walls from Nicotiana benthamiana. Using a set of protein kinase inhibitors with different specificities, it was found that enzymes possessing activities of casein kinase 1, protein kinase A, and protein kinase C are involved in phosphorylation. Commercial preparations of protein kinases A and C are able to phosphorylate in vitro recombinant proteins corresponding to the N-terminal half of the protein and its domains NTD and ID. Phosphorylation of the NTD has no effect on the efficiency and character of its binding to RNA. However, phosphorylation of the ID leads to a decrease in its RNA-binding activity and in the ability for homological protein-protein interactions. PMID:23157268

  3. Emerging Functions for N-Terminal Protein Acetylation in Plants.

    PubMed

    Gibbs, Daniel J

    2015-10-01

    N-terminal (Nt-) acetylation is a widespread but poorly understood co-translational protein modification. Two reports now shed light onto the proteome-wide dynamics and protein-specific consequences of Nt-acetylation in relation to plant development, stress-response, and protein stability, identifying this modification as a key regulator of diverse aspects of plant growth and behaviour. PMID:26319188

  4. N-terminal sequence of some ribosome-inactivating proteins.

    PubMed

    Montecucchi, P C; Lazzarini, A M; Barbieri, L; Stirpe, F; Soria, M; Lappi, D

    1989-04-01

    The N-terminal portion of some type 1 ribosome-inactivating proteins (RIPs) isolated from the seeds of Gelonium multiflorum, Momordica charantia, Bryonia dioica, Saponaria officinalis and from the leaves of Saponaria officinalis are reported in the present paper. Their relationship with other RIPs is discussed. PMID:2753596

  5. Myristoylation of the Dual Specificity Phosphatase JSP1 is necessary for its Activation of JNK Signaling and Apoptosis

    PubMed Central

    Schwertassek, Ulla; Buckley, Deirdre A.; Xu, Chong-Feng; Lindsay, Andrew J.; McCaffrey, Mary W.; Neubert, Thomas A.; Tonks, Nicholas K.

    2010-01-01

    Summary Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual specificity phosphatase subfamily of protein tyrosine phosphatases (PTPs). In contrast to other dual specificity phosphatases, which catalyze inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 (and its relative DUSP15) are unique among members of the PTP family in that they contain a potential myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of myristoylation. Using mass spectrometry, we showed that wild type JSP1, but not a JSP1 mutant in which glycine 2 was mutated to alanine (JSP1-G2A), was myristoylated in cells. Abrogation of myristoylation did not impair the intrinsic phosphatase activity of JSP1, but changed the subcellular localization of the enzyme. Compared to wild type, the ability of non-myristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild type JSP1, a subpopulation of cells, with highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the myristoylation mutant was functionally compromised. PMID:20553486

  6. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    PubMed Central

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O’Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-01-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics. PMID:26678960

  7. PRINT: A Protein Bioconjugation Method with Exquisite N-terminal Specificity

    NASA Astrophysics Data System (ADS)

    Sur, Surojit; Qiao, Yuan; Fries, Anja; O'Meally, Robert N.; Cole, Robert N.; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin

    2015-12-01

    Chemical conjugation is commonly used to enhance the pharmacokinetics, biodistribution, and potency of protein therapeutics, but often leads to non-specific modification or loss of bioactivity. Here, we present a simple, versatile and widely applicable method that allows exquisite N-terminal specific modification of proteins. Combining reversible side-chain blocking and protease mediated cleavage of a commonly used HIS tag appended to a protein, we generate with high yield and purity exquisitely site specific and selective bio-conjugates of TNF-α by using amine reactive NHS ester chemistry. We confirm the N terminal selectivity and specificity using mass spectral analyses and show near complete retention of the biological activity of our model protein both in vitro and in vivo murine models. We believe that this methodology would be applicable to a variety of potentially therapeutic proteins and the specificity afforded by this technique would allow for rapid generation of novel biologics.

  8. First Things First: Vital Protein Marks by N-Terminal Acetyltransferases.

    PubMed

    Aksnes, Henriette; Drazic, Adrian; Marie, Michaël; Arnesen, Thomas

    2016-09-01

    N-terminal (Nt) acetylation is known to be a highly abundant co-translational protein modification, but the recent discovery of Golgi- and chloroplast-resident N-terminal acetyltransferases (NATs) revealed that it can also be added post-translationally. Nt-acetylation may act as a degradation signal in a novel branch of the N-end rule pathway, whose functions include the regulation of human blood pressure. Nt-acetylation also modulates protein interactions, targeting, and folding. In plants, Nt-acetylation plays a role in the control of resistance to drought and in regulation of immune responses. Mutations of specific human NATs that decrease their activity can cause either the lethal Ogden syndrome or severe intellectual disability and cardiovascular defects. In sum, recent advances highlight Nt-acetylation as a key factor in many biological pathways. PMID:27498224

  9. Structure of the human histone chaperone FACT Spt16 N-terminal domain.

    PubMed

    Marcianò, G; Huang, D T

    2016-02-01

    The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of human Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding. PMID:26841762

  10. The N-terminal half of membrane CD14 is a functional cellular lipopolysaccharide receptor.

    PubMed Central

    Viriyakosol, S; Kirkland, T N

    1996-01-01

    CD14, a glycosylphosphatidylinositol-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, is a receptor for lipopolysaccharide (LPS). It was recently reported that an N-terminal 152-amino-acid fragment of soluble CD14 was an active soluble lipopolysaccharide receptor (T. S. -C. Juan, M. J. Kelley, D. A. Johnson, L. A. Busse, E. Hailman, S. D. Wright, and H. S. Lichenstein, J. Biol. Chem. 270:1382-1387, 1995). To determine whether the N-terminal half of the membrane CD14 was a functional LPS receptor on the cell membrane, we engineered a chimeric gene coding for amino acids 1 to 151 of CD14 fused to the C-terminal region of decay-accelerating factor and expressed it in Chinese hamster ovary cells and 70Z/3 cells. We found that the chimeric, truncated CD14 is a fully functional LPS receptor in both cell lines. PMID:8550221

  11. Macelignan inhibits histamine release and inflammatory mediator production in activated rat basophilic leukemia mast cells.

    PubMed

    Han, Young Sun; Kim, Myung-Suk; Hwang, Jae-Kwan

    2012-10-01

    Type I allergy is characterized by the release of granule-associated mediators, lipid-derived substances, cytokines, and chemokines by activated mast cells. To evaluate the anti-allergic effects of macelignan isolated from Myristica fragrans Houtt., we determined its ability to inhibit calcium (Ca(2+)) influx, degranulation, and inflammatory mediator production in RBL-2 H3 cells stimulated with A23187 and phorbol 12-myristate 13-acetate. Macelignan inhibited Ca(2+) influx and the secretion of β-hexosaminidase, histamine, prostaglandin E(2), and leukotriene C(4); decreased mRNA levels of cyclooxygenase-2, 5-lipoxygenase, interleukin-4 (IL-4), IL-13, and tumor necrosis factor-α; and attenuated phosphorylation of Akt and the mitogen-activated protein kinases extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase. These results indicate the potential of macelignan as a type I allergy treatment. PMID:22729280

  12. Targeting of TGF-β-activated protein kinase 1 inhibits chemokine (C-C motif) receptor 7 expression, tumor growth and metastasis in breast cancer

    PubMed Central

    Hung, Wen-Chun; Hou, Ming-Feng

    2015-01-01

    TGF-β-activated protein kinase 1 (TAK1) is a critical mediator in inflammation, immune response and cancer development. Our previous study demonstrated that activation of TAK1 increases the expression of chemokine (C-C motif) receptor 7 (CCR7) and promotes lymphatic invasion ability of breast cancer cells. However, the expression and association of activated TAK1 and CCR7 in breast tumor tissues is unknown and the therapeutic effect by targeting TAK1 is also unclear. We showed that activated TAK1 (as indicated by phospho-TAK1) and its binding protein TAB1 are strongly expressed in breast tumor tissues (77% and 74% respectively). In addition, increase of phospho-TAK1 or TAB1 is strongly associated with over-expression of CCR7. TAK1 inhibitor 5Z-7-Oxozeaenol (5Z-O) inhibited TAK1 activity, suppressed downstream signaling pathways including p38, IκB kinase (IKK) and c-Jun N-terminal kinase (JNK) and reduced CCR7 expression in metastatic MDA-MB-231 cells. In addition, 5Z-O repressed NF-κB- and c-JUN-mediated transcription of CCR7 gene. Knockdown of TAB1 attenuated CCR7 expression and tumor growth in an orthotopic animal study. More importantly, lymphatic invasion and lung metastasis were suppressed. Collectively, our results demonstrate that constitutive activation of TAK1 is frequently found in human breast cancer and this kinase is a potential therapeutic target for this cancer. PMID:25557171

  13. Expanding the Phenotype Associated with NAA10-Related N-Terminal Acetylation Deficiency.

    PubMed

    Saunier, Chloé; Støve, Svein Isungset; Popp, Bernt; Gérard, Bénédicte; Blenski, Marina; AhMew, Nicholas; de Bie, Charlotte; Goldenberg, Paula; Isidor, Bertrand; Keren, Boris; Leheup, Bruno; Lampert, Laetitia; Mignot, Cyril; Tezcan, Kamer; Mancini, Grazia M S; Nava, Caroline; Wasserstein, Melissa; Bruel, Ange-Line; Thevenon, Julien; Masurel, Alice; Duffourd, Yannis; Kuentz, Paul; Huet, Frédéric; Rivière, Jean-Baptiste; van Slegtenhorst, Marjon; Faivre, Laurence; Piton, Amélie; Reis, André; Arnesen, Thomas; Thauvin-Robinet, Christel; Zweier, Christiane

    2016-08-01

    N-terminal acetylation is a common protein modification in eukaryotes associated with numerous cellular processes. Inherited mutations in NAA10, encoding the catalytic subunit of the major N-terminal acetylation complex NatA have been associated with diverse, syndromic X-linked recessive disorders, whereas de novo missense mutations have been reported in one male and one female individual with severe intellectual disability but otherwise unspecific phenotypes. Thus, the full genetic and clinical spectrum of NAA10 deficiency is yet to be delineated. We identified three different novel and one known missense mutation in NAA10, de novo in 11 females, and due to maternal germ line mosaicism in another girl and her more severely affected and deceased brother. In vitro enzymatic assays for the novel, recurrent mutations p.(Arg83Cys) and p.(Phe128Leu) revealed reduced catalytic activity. X-inactivation was random in five females. The core phenotype of X-linked NAA10-related N-terminal-acetyltransferase deficiency in both males and females includes developmental delay, severe intellectual disability, postnatal growth failure with severe microcephaly, and skeletal or cardiac anomalies. Genotype-phenotype correlations within and between both genders are complex and may include various factors such as location and nature of mutations, enzymatic stability and activity, and X-inactivation in females. PMID:27094817

  14. Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases.

    PubMed Central

    Sudbeck, B D; Baumann, P; Ryan, G J; Breitkopf, K; Nischt, R; Krieg, T; Mauch, C

    1999-01-01

    Many cell types, including fibroblasts and primary keratinocytes, increase matrix metalloproteinase 1 (MMP-1) production in response to agonists such as growth factors and phorbol esters. However, the spontaneously transformed human keratinocyte cell line HaCaT, although it increases MMP-1 production in response to epidermal growth factor (EGF), does not respond similarly to stimulation with PMA. This phenomenon occurs even though HaCaT cells remain proliferatively responsive to both agonists, suggesting a HaCaT-specific defect in a PMA-mediated signal transduction pathway. Using an inside-out approach to elucidate the source of this defect, we found that EGF, but not PMA, stimulated MMP-1 promoter activity in transiently transfected HaCaT keratinocytes. In addition, an assessment of fibroblast and HaCaT c-fos and c-jun gene expression after exposure to EGF and PMA showed that although both agonists increased the expression of c-fos and c-jun mRNA in fibroblasts, only EGF did so in HaCaT keratinocytes. Finally, we looked at the activation of mitogen-activated protein (MAP) family kinases after stimulation with EGF or PMA and found that both agonists increased the phosphorylation and activation of fibroblast extracellular signal-regulated protein kinase and c-Jun N-terminal kinase, but only EGF activated the same kinase activities in HaCaT cells. Further, the EGF-mediated increase in MMP-1 gene expression was inhibited by the MAP kinase/ERK kinase (MEK)-specific inhibitor PD98059 and the p38 kinase-specific inhibitor SB203580. Our evidence indicates that although HaCaT MAP kinases are functional, they are not properly regulated in response to the activation of protein kinase C, and that the defect that bars HaCaT MMP-1 expression in response to stimulation with PMA lies before MAP kinase activation. PMID:10085241

  15. Disease mutations in the ryanodine receptor N-terminal region couple to a mobile intersubunit interface