Science.gov

Sample records for activate nuclear factor

  1. Arenavirus Nucleoproteins Prevent Activation of Nuclear Factor Kappa B

    PubMed Central

    Rodrigo, W. W. Shanaka I.; Ortiz-Riaño, Emilio; Pythoud, Christelle; Kunz, Stefan

    2012-01-01

    Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728–7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus. PMID:22623788

  2. Arenavirus nucleoproteins prevent activation of nuclear factor kappa B.

    PubMed

    Rodrigo, W W Shanaka I; Ortiz-Riaño, Emilio; Pythoud, Christelle; Kunz, Stefan; de la Torre, Juan C; Martínez-Sobrido, Luis

    2012-08-01

    Arenaviruses include several causative agents of hemorrhagic fever (HF) disease in humans that are associated with high morbidity and significant mortality. Morbidity and lethality associated with HF arenaviruses are believed to involve the dysregulation of the host innate immune and inflammatory responses that leads to impaired development of protective and efficient immunity. The molecular mechanisms underlying this dysregulation are not completely understood, but it is suggested that viral infection leads to disruption of early host defenses and contributes to arenavirus pathogenesis in humans. We demonstrate in the accompanying paper that the prototype member in the family, lymphocytic choriomeningitis virus (LCMV), disables the host innate defense by interfering with type I interferon (IFN-I) production through inhibition of the interferon regulatory factor 3 (IRF3) activation pathway and that the viral nucleoprotein (NP) alone is responsible for this inhibitory effect (C. Pythoud, W. W. Rodrigo, G. Pasqual, S. Rothenberger, L. Martínez-Sobrido, J. C. de la Torre, and S. Kunz, J. Virol. 86:7728-7738, 2012). In this report, we show that LCMV-NP, as well as NPs encoded by representative members of both Old World (OW) and New World (NW) arenaviruses, also inhibits the nuclear translocation and transcriptional activity of the nuclear factor kappa B (NF-κB). Similar to the situation previously reported for IRF3, Tacaribe virus NP (TCRV-NP) does not inhibit NF-κB nuclear translocation and transcriptional activity to levels comparable to those seen with other members in the family. Altogether, our findings demonstrate that arenavirus infection inhibits NF-κB-dependent innate immune and inflammatory responses, possibly playing a key role in the pathogenesis and virulence of arenavirus.

  3. Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity.

    PubMed

    Shen, Zhongliang; Liu, Yanfeng; Luo, Mengjun; Wang, Wei; Liu, Jing; Liu, Wei; Pan, Shaokun; Xie, Youhua

    2016-09-16

    Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses.

  4. Nuclear actin activates human transcription factor genes including the OCT4 gene.

    PubMed

    Yamazaki, Shota; Yamamoto, Koji; Tokunaga, Makio; Sakata-Sogawa, Kumiko; Harata, Masahiko

    2015-01-01

    RNA microarray analyses revealed that nuclear actin activated many human transcription factor genes including OCT4, which is required for gene reprogramming. Oct4 is known to be activated by nuclear actin in Xenopus oocytes. Our findings imply that this process of OCT4 activation is conserved in vertebrates and among cell types and could be used for gene reprogramming of human cells.

  5. Hypoxic preconditioning decreases nuclear factor κB activity via Disrupted in Schizophrenia-1.

    PubMed

    Liu, Jia-Ren; Liu, Qian; Khoury, Joseph; Li, Yue-Jin; Han, Xiao-Hui; Li, Jing; Ibla, Juan C

    2016-01-01

    Nuclear factor κB is a key mediator of inflammation during conditions of hypoxia. Here, we used models of hypoxic pre-conditioning as mechanism to decrease nuclear factor κB activity induced by hypoxia. Our initial studies suggested that Disrupted in Schizophrenia-1 may be induced by hypoxic pre-conditioning and possibly involved in the regulation of nuclear factor κB. In this study we used Disrupted in Schizophrenia-1 exogenous over-expression and knock-down to determine its effect on ataxia telangiectasia mutated--nuclear factor κB activation cascade. Our results demonstrated that hypoxic pre-conditioning significantly increased the expression of Disrupted in Schizophrenia-1 at mRNA and protein levels both in vitro and in vivo. Over-expression of Disrupted in Schizophrenia-1 significantly attenuated the hypoxia-mediated ataxia telangiectasia mutated phosphorylation and prevented its cytoplasm translocation where it functions to activate nuclear factor κB. We further determined that Disrupted in Schizophrenia-1 activated the protein phosphatase 2A, preventing the phosphorylation of ataxia telangiectasia mutated serine-1981, the main regulatory site of ataxia telangiectasia mutated activity. Cellular levels of Disrupted in Schizophrenia-1 protein significantly decreased nuclear factor κB activation profiles and pro-inflammatory gene expression. Taken together, these results demonstrate that hypoxic pre-conditioning decreases the activation of nuclear factor κB through the transcriptional induction of Disrupted in Schizophrenia-1.

  6. Angiotensin II activates the proinflammatory transcription factor nuclear factor-kappaB in human monocytes.

    PubMed

    Kranzhöfer, R; Browatzki, M; Schmidt, J; Kübler, W

    1999-04-21

    The renin-angiotensin system may contribute to the pathogenesis of atherosclerosis. A common feature of all stages of atherosclerosis is inflammation of the vessel wall. The transcription factor nuclear factor-kappaB (NF-kappaB) participates in most signaling pathways involved in inflammation. This study therefore examined the effect of angiotensin (ANG) II on NF-kappaB activation in monocytic cells, a major cellular component of human atheroma, by electrophoretic mobility shift assay. ANG II, like TNFalpha, caused rapid activation of NF-kappaB in human mononuclear cells isolated from peripheral blood by Ficoll density gradient. This ANG II effect was blocked by the angiotensin AT1 receptor antagonist losartan. Specificity of ANG II-induced NF-kappaB activation was ascertained by supershift and competition experiments. Moreover, ANG II stimulated NF-kappaB activation in human monocytes, but not in lymphocytes from the same preparation. Together, the data demonstrate the ability of the vasoactive peptide ANG II to activate inflammatory pathways in human monocytes.

  7. Nuclear Factor of Activated T Cells Transcription Factor Nfatp Controls Superantigen-Induced Lethal Shock

    PubMed Central

    Tsytsykova, Alla V.; Goldfeld, Anne E.

    2000-01-01

    Tumor necrosis factor α (TNF-α) is the key mediator of superantigen-induced T cell lethal shock. Here, we show that nuclear factor of activated T cells transcription factor, NFATp, controls susceptibility to superantigen-induced lethal shock in mice through its activation of TNF-α gene transcription. In NFATp-deficient mice, T cell stimulation leads to delayed induction and attenuation of TNF-α mRNA levels, decreased TNF-α serum levels, and resistance to superantigen-induced lethal shock. By contrast, after lipopolysaccharide (LPS) challenge, serum levels of TNF-α and susceptibility to shock are unaffected. These results demonstrate that NFATp is an essential activator of immediate early TNF-α gene expression in T cells and they present in vivo evidence of the inducer- and cell type–specific regulation of TNF-α gene expression. Furthermore, they suggest NFATp as a potential selective target in the treatment of superantigen-induced lethal shock. PMID:10952728

  8. Activation of nuclear factor-κB in human prostate carcinogenesis and association to biochemical relapse

    PubMed Central

    Domingo-Domenech, J; Mellado, B; Ferrer, B; Truan, D; Codony-Servat, J; Sauleda, S; Alcover, J; Campo, E; Gascon, P; Rovira, A; Ross, J S; Fernández, P L; Albanell, J

    2005-01-01

    Nuclear factor (NF)-κB/p65 regulates the transcription of a wide variety of genes involved in cell survival, invasion and metastasis. We characterised by immunohistochemistry the expression of NF-κB/p65 protein in six histologically normal prostate, 13 high-grade prostatic intraepithelial neoplasia (PIN) and 86 prostate adenocarcinoma specimens. Nuclear localisation of p65 was used as a measure of NF-κB active state. Nuclear localisation of NF-κB was only seen in scattered basal cells in normal prostate glands. Prostatic intraepithelial neoplasias exhibited diffuse and strong cytoplasmic staining but no nuclear staining. In prostate adenocarcinomas, cytoplasmic NF-κB was detected in 57 (66.3%) specimens, and nuclear NF-κB (activated) in 47 (54.7%). Nuclear and cytoplasmic NF-κB staining was not correlated (P=0.19). By univariate analysis, nuclear localisation of NF-κB was associated with biochemical relapse (P=0.0009; log-rank test) while cytoplasmic expression did not. On multivariate analysis, serum preoperative prostate specific antigen (P=0.02), Gleason score (P=0.03) and nuclear NF-κB (P=0.002) were independent predictors of biochemical relapse. These results provide novel evidence for NF-κB/p65 nuclear translocation in the transition from PIN to prostate cancer. Our findings also indicate that nuclear localisation of NF-κB is an independent prognostic factor of biochemical relapse in prostate cancer. PMID:16278667

  9. Rifampicin Attenuated Global Cerebral Ischemia Injury via Activating the Nuclear Factor Erythroid 2-Related Factor Pathway

    PubMed Central

    Chen, Beibei; Cao, Huimin; Chen, Lili; Yang, Xuemei; Tian, Xiaoyan; Li, Rong; Cheng, Oumei

    2016-01-01

    Background: Recent studies have found that rifampicin has neuroprotective properties in neurodegenerative diseases. However, the exact mechanisms of action remain unclear. The nuclear factor erythroid 2-related factor 2 (Nrf2) has been considered a potential target for neuroprotection. In this study, we examined whether rifampicin exhibits beneficial effects mediated by the Nrf2 pathway after global cerebral ischemia (GCI). Methods: Rats were randomly assigned to four groups that included a sham group and three treatment groups with global ischemia-reperfusion [control, rifampicin, and rifampicin plus brusatol (an inhibitor of Nrf2)]. Rats were subjected to transient GCI induced by bilateral common carotid artery occlusion for 20 min with systemic hypotension by blood withdrawal. The Morris water maze test was performed for neurobehavioral testing, whereas the pathological changes were investigated using HE and TUNEL staining. The protein expression of Nrf2, hemeoxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) in the hippocampus were analyzed by Western blotting. The immunofluorescence staining was used to determine the distribution of Nrf2. Results: Rifampicin treatment significantly improved spatial learning ability compared with the control group, which was consistent with the pathological changes. In addition, rifampicin significantly elevated the nuclear expression of Nrf2, Nrf2 downstream anti-oxidant protein, HO-1 compared with the control group, and it simultaneously downregulated the expression of COX-2 in the hippocampus on day 3 after ischemia-reperfusion. Interestingly, the forenamed effects of rifampicin were abolished by pretreatment with brusatol, a specific inhibitor of Nrf2 activation. Conclusions: Rifampicin exerts neuroprotective effects against global cerebral ischemia, which may be attributed to activation of the Nrf2 pathway. PMID:27965540

  10. Activation of the human mitochondrial transcription factor A gene by nuclear respiratory factors: a potential regulatory link between nuclear and mitochondrial gene expression in organelle biogenesis.

    PubMed Central

    Virbasius, J V; Scarpulla, R C

    1994-01-01

    Mitochondrial transcription factor A (mtTFA), the product of a nuclear gene, stimulates transcription from the two divergent mitochondrial promoters and is likely the principal activator of mitochondrial gene expression in vertebrates. Here we establish that the proximal promoter of the human mtTFA gene is highly dependent upon recognition sites for the nuclear respiratory factors, NRF-1 and NRF-2, for activity. These factors have been previously implicated in the activation of numerous nuclear genes that contribute to mitochondrial respiratory function. The affinity-purified factors from HeLa cells specifically bind to the mtTFA NRF-1 and NRF-2 sites through guanine nucleotide contacts that are characteristic for each site. Mutations in these contacts eliminate NRF-1 and NRF-2 binding and also dramatically reduce promoter activity in transfected cells. Although both factors contribute, NRF-1 binding appears to be the major determinant of promoter function. This dependence on NRF-1 activation is confirmed by in vitro transcription using highly purified recombinant proteins that display the same binding specificities as the HeLa cell factors. The activation of the mtTFA promoter by both NRF-1 and NRF-2 therefore provides a link between the expression of nuclear and mitochondrial genes and suggests a mechanism for their coordinate regulation during organelle biogenesis. Images PMID:8108407

  11. Hyperin inhibits nuclear factor kappa B and activates nuclear factor E2-related factor-2 signaling pathways in cisplatin-induced acute kidney injury in mice.

    PubMed

    Chao, Chia-Sheng; Tsai, Chien-Sung; Chang, Yee-Phoung; Chen, Jian-Ming; Chin, Hsien-Kuo; Yang, Shyh-Chyun

    2016-11-01

    Hyperin, a flavonoid compound found in Ericaceae, Guttiferae, and Celastraceae, has been reported to have anti-inflammatory effects. In the present study, we investigated the effects of hyperin on cisplatin-induced acute kidney injury (AKI) in mice. The renal tissue damage induced by cisplatin was detected by H&E staining. Blood urea nitrogen (BUN), creatinine, reactive oxygen species (ROS), and malondialdehyde (MDA) were also detected. Further, the effects of hyperin on cisplatin-induced TNF-α, IL-1β and IL-6 were detected by ELISA. In addition, the phosphorylation of nuclear factor kappa B (NF-κB) and the expression of nuclear factor E2-related factor-2 (Nrf2) and HO-1 were detected by western blot analysis. The results showed that hyperin attenuated histological changes of kidney induced by cisplatin. The levels of BUN, creatinine, ROS, MDA, TNF-α, IL-1β and IL-6 induced by cisplatin were also inhibited by hyperin. Cisplatin-induced NF-κB activation was inhibited by hyperin. Additionally, hyperin was found to up regulate the expression of Nrf2 and HO-1. In conclusion, the results suggest that hyperin protects against cisplatin-induced AKI by inhibiting inflammatory and oxidant response.

  12. Nuclear localization of platelet-activating factor receptor controls retinal neovascularization

    PubMed Central

    K Bhosle, Vikrant; Rivera, José Carlos; Zhou, Tianwei (Ellen); Omri, Samy; Sanchez, Melanie; Hamel, David; Zhu, Tang; Rouget, Raphael; Rabea, Areej Al; Hou, Xin; Lahaie, Isabelle; Ribeiro-da-Silva, Alfredo; Chemtob, Sylvain

    2016-01-01

    Platelet-activating factor (PAF) is a pleiotropic phospholipid with proinflammatory, procoagulant and angiogenic actions on the vasculature. We and others have reported the presence of PAF receptor (Ptafr) at intracellular sites such as the nucleus. However, mechanisms of localization and physiologic functions of intracellular Ptafr remain poorly understood. We hereby identify the importance of C-terminal motif of the receptor and uncover novel roles of Rab11a GTPase and importin-5 in nuclear translocation of Ptafr in primary human retinal microvascular endothelial cells. Nuclear localization of Ptafr is independent of exogenous PAF stimulation as well as intracellular PAF biosynthesis. Moreover, nuclear Ptafr is responsible for the upregulation of unique set of growth factors, including vascular endothelial growth factor, in vitro and ex vivo. We further corroborate the intracrine PAF signaling, resulting in angiogenesis in vivo, using Ptafr antagonists with distinct plasma membrane permeability. Collectively, our findings show that nuclear Ptafr translocates in an agonist-independent manner, and distinctive functions of Ptafr based on its cellular localization point to another dimension needed for pharmacologic selectivity of drugs. PMID:27462464

  13. Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection

    SciTech Connect

    Manley, Kate; O'Hara, Bethany A.; Atwood, Walter J.

    2008-03-01

    Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through {kappa}B sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.

  14. Daily variation of constitutively activated nuclear factor kappa B (NFKB) in rat pineal gland.

    PubMed

    Cecon, Erika; Fernandes, Pedro A; Pinato, Luciana; Ferreira, Zulma S; Markus, Regina P

    2010-01-01

    In mammals, the production of melatonin by the pineal gland is mainly controlled by the suprachiasmatic nuclei (SCN), the master clock of the circadian system. We have previously shown that agents involved in inflammatory responses, such as cytokines and corticosterone, modulate pineal melatonin synthesis. The nuclear transcription factor NFKB, detected by our group in the rat pineal gland, modulates this effect. Here, we evaluated a putative constitutive role for the pineal gland NFKB pathway. Male rats were kept under 12 h:12 h light-dark (LD) cycle or under constant darkness (DD) condition. Nuclear NFKB was quantified by electrophoretic mobility shift assay on pineal glands obtained from animals killed throughout the day at different times. Nuclear content of NFKB presented a daily rhythm only in LD-entrained animals. During the light phase, the amount of NFKB increased continuously, and a sharp drop occurred when lights were turned off. Animals maintained in a constant light environment until ZT 18 showed diurnal levels of nuclear NFKB at ZT15 and ZT18. Propranolol (20 mg/kg, i.p., ZT 11) treatment, which inhibits nocturnal sympathetic input, impaired nocturnal decrease of NFKB only at ZT18. A similar effect was observed in free-running animals, which secreted less nocturnal melatonin. Because melatonin reduces constitutive NFKB activation in cultured pineal glands, we propose that this indolamine regulates this transcription factor pathway in the rat pineal gland, but not at the LD transition. The controversial results regarding the inhibition of pineal function by constant light or blocking sympathetic neurotransmission are discussed according to the hypothesis that the prompt effect of lights-off is not mediated by noradrenaline, which otherwise contributes to maintaining low levels of nuclear NFKB at night. In summary, we report here a novel transcription factor in the pineal gland, which exhibits a constitutive rhythm dependent on environmental photic

  15. Zinc inhibits nuclear factor-kappa B activation and sensitizes prostate cancer cells to cytotoxic agents.

    PubMed

    Uzzo, Robert G; Leavis, Paul; Hatch, William; Gabai, Vladimir L; Dulin, Nickolai; Zvartau, Nadezhda; Kolenko, Vladimir M

    2002-11-01

    Prostate carcinogenesis involves transformation of zinc-accumulating normal epithelial cells to malignant cells, which do not accumulate zinc. In this study, we demonstrate by immunoblotting and immunohistochemistry that physiological levels of zinc inhibit activation of nuclear factor (NF)-kappa B transcription factor in PC-3 and DU-145 human prostate cancer cells, reduce expression of NF-kappa B-controlled antiapoptotic protein c-IAP2, and activate c-Jun NH(2)-terminal kinases. Preincubation of PC-3 cells with physiological concentrations of zinc sensitized tumor cells to tumor necrosis factor (TNF)-alpha, and paclitaxel mediated cell death as defined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. These results suggest one possible mechanism for the inhibitory effect of zinc on the development and progression of prostate malignancy and might have important consequences for the prevention and treatment of prostate cancer.

  16. Moraxella catarrhalis induces mast cell activation and nuclear factor kappa B-dependent cytokine synthesis.

    PubMed

    Krishnaswamy, G; Martin, R; Walker, E; Li, C; Hossler, F; Hall, K; Chi, D S

    2003-01-01

    Human mast cells are often found perivascularly and at mucosal sites and may play crucial roles in the inflammatory response. Recent studies have suggested a prominent role for mast cells in host defense. In this study, we analyzed the effects of a common airway pathogen, Moraxella catarrhalis and a commensal bacterium, Neiserria cinerea, on activation of human mast cells. Human mast cell leukemia cells (HMC-1) were activated with either phorbol myristate acetate (PMA) and calcium ionophore or with varying concentrations of heat-killed suspensions of bacteria. Supernatants were assayed for the cytokines interleukin-4 (IL-4), granulocyte macrophage colony stimulating factor (GM-CSF), IL-6, IL-8, IL-13 and monocyte chemotactic protein-1 (MCP-1). Nuclear proteins were isolated and assayed by electrophoretic mobility shift assay (EMSA) for nuclear factor kappaB (NF-kappaB) nuclear binding activity. In some experiments, NF-kappaB inhibitor, Bay-11 was added to determine functional significance. Both M. catarrhalis and N. cinerea induced mast cell activation and selective secretion of two key inflammatory cytokines, IL-6 and MCP-1. This was accompanied by NF-kappaB activation. Neither spun bacterial supernatants nor bacterial lipopolysaccharide induced cytokine secretion, suggesting need for direct bacterial contact with mast cells. Scanning electron microscopy revealed active aggregation of bacteria over mast cell surfaces. The NF-kappaB inhibitor, Bay-11, inhibited expression of MCP-1. These findings suggest the possibility of direct interactions between human mast cells and common bacteria and provide evidence for a novel role for human mast cells in innate immunity.

  17. Respiratory syncytial virus M2-1 protein induces the activation of nuclear factor kappa B

    SciTech Connect

    Reimers, Kerstin . E-mail: reimers.kerstin@mh-hannover.de; Buchholz, Katja; Werchau, Hermann

    2005-01-20

    Respiratory syncytial virus (RSV) induces the production of a number of cytokines and chemokines by activation of nuclear factor kappa B (NF-{kappa}B). The activation of NF-{kappa}B has been shown to depend on viral replication in the infected cells. In this study, we demonstrate that expression of RSV M2-1 protein, a transcriptional processivity and anti-termination factor, is sufficient to activate NF-{kappa}B in A549 cells. Electromobility shift assays show increased NF-{kappa}B complexes in the nuclei of M2-1-expressing cells. M2-1 protein is found in nuclei of M2-1-expressing cells and in RSV-infected cells. Co-immunoprecipitations of nuclear extracts of M2-1-expressing cells and of RSV-infected cells revealed an association of M2-1 with Rel A protein. Furthermore, the activation of NF-{kappa}B depends on the C-terminus of the RSV M2-1 protein, as shown by NF-{kappa}B-induced gene expression of a reporter gene construct.

  18. Salicylates Inhibit Flavivirus Replication Independently of Blocking Nuclear Factor Kappa B Activation

    PubMed Central

    Liao, Ching-Len; Lin, Yi-Ling; Wu, Bi-Ching; Tsao, Chang-Huei; Wang, Mei-Chuan; Liu, Chiu-I; Huang, Yue-Ling; Chen, Jui-Hui; Wang, Jia-Pey; Chen, Li-Kuang

    2001-01-01

    Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-κB), and salicylates have been shown to inhibit NF-κB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-κB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IκBα-ΔN, a dominant-negative mutant that antagonizes NF-κB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-κB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IκBα-ΔN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-κB pathway. PMID:11483726

  19. Tumor necrosis factor alpha-mediated inhibition of melanogenesis is dependent on nuclear factor kappa B activation.

    PubMed

    Englaro, W; Bahadoran, P; Bertolotto, C; Buscà, R; Dérijard, B; Livolsi, A; Peyron, J F; Ortonne, J P; Ballotti, R

    1999-02-25

    Melanogenesis is a physiological process resulting in the synthesis of melanin pigments which play a crucial protective role against skin photocarcinogenesis. In vivo, solar ultraviolet light triggers the secretion of numerous keratinocyte-derived factors that are implicated in the regulation of melanogenesis. Among these, tumor necrosis factor alpha (TNFalpha), a cytokine implicated in the pro-inflammatory response, down-regulates pigment synthesis in vitro. In this report, we aimed to determine the molecular mechanisms by which this cytokine inhibits melanogenesis in B16 melanoma cells. First, we show that TNFalpha inhibits the activity and protein expression of tyrosinase which is the key enzyme of melanogenesis. Further, we demonstrate that this effect is subsequent to a down-regulation of the tyrosinase promoter activity in both basal and cAMP-induced melanogenesis. Finally, we present evidence indicating that the inhibitory effect of TNFalpha on melanogenesis is dependent on nuclear factor kappa B (NFkappaB) activation. Indeed, overexpression of this transcription factor in B16 cells is sufficient to inhibit tyrosinase promoter activity. Furthermore, a mutant of inhibitory kappa B (IkappaB), that prevents NFkappaB activation, is able to revert the effect of TNFalpha on the tyrosinase promoter activity. Taken together, our results clarify the mechanisms by which TNFalpha inhibits pigmentation and point out the key role of NFkappaB in the regulation of melanogenesis.

  20. Nuclear translocation of phospholipase C-zeta, an egg-activating factor, during early embryonic development

    SciTech Connect

    Sone, Yoshie; Ito, Masahiko; Shirakawa, Hideki; Shikano, Tomohide; Takeuchi, Hiroyuki; Kinoshita, Katsuyuki; Miyazaki, Shunichi . E-mail: shunm@research.twmu.ac.jp

    2005-05-13

    Phospholipase C-zeta (PLC{zeta}), a strong candidate of the egg-activating sperm factor, causes intracellular Ca{sup 2+} oscillations and egg activation, and is subsequently accumulated into the pronucleus (PN), when expressed in mouse eggs by injection of RNA encoding PLC{zeta}. Changes in the localization of expressed PLC{zeta} were investigated by tagging with a fluorescent protein. PLC{zeta} began to translocate into the PN formed at 5-6 h after RNA injection and increased there. Observation in the same embryo revealed that PLC{zeta} in the PN dispersed to the cytoplasm upon nuclear envelope breakdown and translocated again into the nucleus after cleavage. The dynamics was found in the second mitosis as well. When RNA was injected into fertilization-originated 1-cell embryos or blastomere(s) of 2-8-cell embryos, the nuclear localization of expressed PLC{zeta} was recognized in every embryo up to blastocyst. Thus, PLC{zeta} exhibited alternative cytoplasm/nucleus localization during development. This supports the view that the sperm factor could control cell cycle-dependent generation of Ca{sup 2+} oscillations in early embryogenesis.

  1. A Simple and Efficient Method to Detect Nuclear Factor Activation in Human Neutrophils by Flow Cytometry

    PubMed Central

    García-García, Erick; Uribe-Querol, Eileen; Rosales, Carlos

    2013-01-01

    Neutrophils are the most abundant leukocytes in peripheral blood. These cells are the first to appear at sites of inflammation and infection, thus becoming the first line of defense against invading microorganisms. Neutrophils possess important antimicrobial functions such as phagocytosis, release of lytic enzymes, and production of reactive oxygen species. In addition to these important defense functions, neutrophils perform other tasks in response to infection such as production of proinflammatory cytokines and inhibition of apoptosis. Cytokines recruit other leukocytes that help clear the infection, and inhibition of apoptosis allows the neutrophil to live longer at the site of infection. These functions are regulated at the level of transcription. However, because neutrophils are short-lived cells, the study of transcriptionally regulated responses in these cells cannot be performed with conventional reporter gene methods since there are no efficient techniques for neutrophil transfection. Here, we present a simple and efficient method that allows detection and quantification of nuclear factors in isolated and immunolabeled nuclei by flow cytometry. We describe techniques to isolate pure neutrophils from human peripheral blood, stimulate these cells with anti-receptor antibodies, isolate and immunolabel nuclei, and analyze nuclei by flow cytometry. The method has been successfully used to detect NF-κB and Elk-1 nuclear factors in nuclei from neutrophils and other cell types. Thus, this method represents an option for analyzing activation of transcription factors in isolated nuclei from a variety of cell types. PMID:23603868

  2. Nuclear factor E2-related factor-2 (Nrf2) is required for NLRP3 and AIM2 inflammasome activation.

    PubMed

    Zhao, Changcheng; Gillette, Devyn D; Li, Xinghui; Zhang, Zhibin; Wen, Haitao

    2014-06-13

    Despite the number of extensive studies on the immune function and signaling of inflammasomes in various diseases, the activating mechanism of inflammasome, especially the NLRP3 inflammasome, is not fully understood. Nuclear factor E2-related Factor-2 (Nrf2), a key transcription factor that regulates cellular redox homeostasis, has been reported to play both protective and pathogenic roles depending on the disease conditions through undefined mechanism. This study reveals an essential role of Nrf2 in inflammasome activation. LPS stimulation increased Nrf2 protein levels in a Myd88-dependent manner. When compared with wild-type controls, Nrf2-deficient (Nrf2(-/-)) macrophages showed decreased maturation and secretion of caspase-1 and IL-1β and reduced apoptosis-associated speck-like protein containing CARD (ASC) speck formation in response to various NLRP3 and AIM2 inflammasome stimuli. In contrast, NLRC4 inflammasome activation was not controlled by Nrf2. Biochemical analysis revealed that Nrf2 appeared in the ASC-enriched cytosolic compartment after NLRP3 inflammasome activation. Furthermore, mitochondrial reactive oxygen species-induced NLRP3 activation also required Nrf2. Nrf2(-/-) mice showed a dramatic decrease in immune cell recruitment and IL-1β generation in alum-induced peritonitis, which is a typical IL-1 signaling-dependent inflammation animal model. This work discovered a critical proinflammatory effect of Nrf2 by mediating inflammasome activation.

  3. Nuclear Factor E2-related Factor-2 (Nrf2) Is Required for NLRP3 and AIM2 Inflammasome Activation*

    PubMed Central

    Zhao, Changcheng; Gillette, Devyn D.; Li, Xinghui; Zhang, Zhibin; Wen, Haitao

    2014-01-01

    Despite the number of extensive studies on the immune function and signaling of inflammasomes in various diseases, the activating mechanism of inflammasome, especially the NLRP3 inflammasome, is not fully understood. Nuclear factor E2-related Factor-2 (Nrf2), a key transcription factor that regulates cellular redox homeostasis, has been reported to play both protective and pathogenic roles depending on the disease conditions through undefined mechanism. This study reveals an essential role of Nrf2 in inflammasome activation. LPS stimulation increased Nrf2 protein levels in a Myd88-dependent manner. When compared with wild-type controls, Nrf2-deficient (Nrf2−/−) macrophages showed decreased maturation and secretion of caspase-1 and IL-1β and reduced apoptosis-associated speck-like protein containing CARD (ASC) speck formation in response to various NLRP3 and AIM2 inflammasome stimuli. In contrast, NLRC4 inflammasome activation was not controlled by Nrf2. Biochemical analysis revealed that Nrf2 appeared in the ASC-enriched cytosolic compartment after NLRP3 inflammasome activation. Furthermore, mitochondrial reactive oxygen species-induced NLRP3 activation also required Nrf2. Nrf2−/− mice showed a dramatic decrease in immune cell recruitment and IL-1β generation in alum-induced peritonitis, which is a typical IL-1 signaling-dependent inflammation animal model. This work discovered a critical proinflammatory effect of Nrf2 by mediating inflammasome activation. PMID:24798340

  4. RhoA GTPase oxidation stimulates cell proliferation via nuclear factor-κB activation.

    PubMed

    Kim, Jae-Gyu; Kwon, Hyung-Joo; Wu, Guang; Park, Yohan; Lee, Jae-Yong; Kim, Jaebong; Kim, Sung-Chan; Choe, Myoen; Kang, Seung Goo; Seo, Goo-Young; Kim, Pyeung-Hyeun; Park, Jae-Bong

    2017-02-01

    Reactive oxygen species (ROS) produced by many kinds of stimuli are essential for cellular signaling including cell proliferation. The dysregulation of ROS, therefore, is related to a variety of diseases including cancer. However, it was not clearly elucidated how ROS regulate cell proliferation and tumorigenesis. In this study, we investigated a mechanism by which the oxidation of RhoA GTPase regulates nuclear factor-κB (NF-κB) and cell proliferation. Hydrogen peroxide activated NF-κB and RhoA GTPase, but did not activate RhoA C16/20A mutant, an oxidation-resistant form. Remarkably, the oxidation of RhoA reduced its affinity towards RhoGDI, leading to the dissociation of RhoA-RhoGDI complex. Si-Vav2, a guanine nucleotide exchange factor (GEF), inhibited RhoA activation upon hydrogen peroxide. The oxidized RhoA (oxRhoA)-GTP was readily bound to IκB kinase γ (IKKγ), whereas oxidized RhoGDI did not bind to IKKγ. The oxRhoA-GTP bound to IKKγ activated IKKβ, leading to IκB phosphorylation and degradation, consequently NF-κB activation. Hydrogen peroxide induced cell proliferation, but RhoA C16/20A mutant suppressed cell proliferation and tumorigenesis. Conclusively, RhoA oxidation at Cys16/20 is critically involved in cell proliferation and tumorigenesis through NF-κB activation in response to ROS.

  5. Nuclear factor of activated T cells (NFAT) signaling regulates PTEN expression and intestinal cell differentiation

    PubMed Central

    Wang, Qingding; Zhou, Yuning; Jackson, Lindsey N.; Johnson, Sara M.; Chow, Chi-Wing; Evers, B. Mark

    2011-01-01

    The nuclear factor of activated T cell (NFAT) proteins are a family of transcription factors (NFATc1–c4) involved in the regulation of cell differentiation and adaptation. Previously we demonstrated that inhibition of phosphatidylinositol 3-kinase or overexpression of PTEN enhanced intestinal cell differentiation. Here we show that treatment of intestinal-derived cells with the differentiating agent sodium butyrate (NaBT) increased PTEN expression, NFAT binding activity, and NFAT mRNA expression, whereas pretreatment with the NFAT signaling inhibitor cyclosporine A (CsA) blocked NaBT-mediated PTEN induction. Moreover, knockdown of NFATc1 or NFATc4, but not NFATc2 or NFATc3, attenuated NaBT-induced PTEN expression. Knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt and downstream targets Foxo1 and GSK-3α/β. Furthermore, overexpression of NFATc1 or the NFATc4 active mutant increased PTEN and p27kip1 expression and decreased Akt phosphorylation. In addition, pretreatment with CsA blocked NaBT-mediated induction of intestinal alkaline phosphatase (IAP) activity and villin and p27kip1 expression; knockdown of either NFATc1 or NFATc4 attenuated NaBT-induced IAP activity. We provide evidence showing that NFATc1 and NFATc4 are regulators of PTEN expression. Importantly, our results suggest that NFATc1 and NFATc4 regulation of intestinal cell differentiation may be through PTEN regulation. PMID:21148296

  6. Activated nuclear factor kappa B and airway inflammation after smoke inhalation and burn injury in sheep.

    PubMed

    Cox, Robert A; Burke, Ann S; Jacob, Sam; Oliveras, Gloria; Murakami, Kazunori; Shimoda, Katsumi; Enkhbaatar, Perenlei; Traber, Lillian D; Herndon, David N; Traber, Daniel L; Hawkins, Hal K

    2009-01-01

    In a recent study, we have shown a rapid inflammatory cell influx across the glandular epithelium and strong proinflammatory cytokine expression at 4 hours after inhalation injury. Studies have demonstrated a significant role of nuclear factor kappa B in proinflammatory cytokine gene transcription. This study examines the acute airway inflammatory response and immunohistochemical detection of p65, a marker of nuclear factor kappa B activation, in sheep after smoke inhalation and burn injury. Pulmonary tissue from uninjured sheep and sheep at 4, 8, 12, 24, and 48 hours after inhalation and burn injury was included in the study. Following immunostaining for p65 and myeloperoxidase, the cell types and the percentage of bronchial submucosal gland cells staining for p65 and the extent of myeloperoxidase stained neutrophils in the bronchial submucosa were determined. Results indicate absence of detection of P65 before 12 hours after injury. At 12 hours after injury, strong perinuclear staining for p65 was evident in bronchial gland epithelial cells, macrophages, and endothelial cells. Bronchial submucosal gland cells showed a significant increase in the percentage of cells stained for p65 compared with uninjured animals and earlier times after injury, P < .05. At 24 and 48 hours after injury, p65 expression was evident in the bronchiolar epithelium, Type II pneumocytes, macrophages, and endothelial cells. Quantitation of the neutrophil influx into the bronchial submucosa showed a significant increase compared with uninjured tissue at 24 and 48 hours after injury, P < .05. In conclusion, immunohistochemical detection of activated p65 preceded the overall inflammatory response measured in the lamina propria. However, detection of p65 did not correlate with a recent study showing rapid emigration of neutrophils at 4 hours postinjury. Together, these results suggest that p65 immunostaining may identify cells that are activated to produce proinflammatory cytokines after

  7. Anandamide inhibits nuclear factor-kappaB activation through a cannabinoid receptor-independent pathway.

    PubMed

    Sancho, Rocío; Calzado, Marco A; Di Marzo, Vincenzo; Appendino, Giovanni; Muñoz, Eduardo

    2003-02-01

    Anandamide (arachidonoylethanolamine, AEA), an endogenous agonist for both the cannabinoid CB(1) receptor and the vanilloid VR1 receptor, elicits neurobehavioral, anti-inflammatory, immunomodulatory, and proapoptotic effects. Because of the central role of nuclear factor-kappaB (NF-kappaB) in the inflammatory process and the immune response, we postulated that AEA might owe some of its effects to the suppression of NF-kappaB. This study shows that AEA inhibits tumor necrosis factor-alpha (TNFalpha)-induced NF-kappaB activation by direct inhibition of the IkappaB kinase (IKK)beta and, to a lesser extent, the IKKalpha subunits of kappaB inhibitor (IkappaB) kinase complex, and that IKKs inhibition by AEA correlates with inhibition of IkappaBalpha degradation, NF-kappaB binding to DNA, and NF-kappaB-dependent transcription in TNFalpha-stimulated cells. AEA also prevents NF-kappaB-dependent reporter gene expression induced by mitogen-activated protein kinase kinase kinase and NF-kappaB-inducing kinase. The NF-kappaB inhibitory activity of AEA was independent of CB(1) and CB(2) activation in TNFalpha-stimulated 5.1 and A549 cell lines, which do not express vanilloid receptor 1, and was not mediated by hydrolytic products formed through the activity of the enzyme fatty acid amide hydrolase. Chemical modification markedly affected AEA inhibitory activity on NF-kappaB, suggesting rather narrow structure-activity relationships and the specific interaction with a molecular target. Substitution of the alkyl moiety with less saturated fatty acids generally reduced or abolished activity. However, replacement of the ethanolamine "head" with a vanillyl group led to potent inhibition of TNFalpha-induced NF-kappaB-dependent transcription. These findings provide new mechanistic insights into the anti-inflammatory and proapoptotic activities of AEA, and should foster the synthesis of improved analogs amenable to pharmaceutical development as anti-inflammatory agents.

  8. Opioid treatment of experimental pain activates nuclear factor-κB

    PubMed Central

    Compton, Peggy; Griffis, Charles; Breen, Elizabeth Crabb; Torrington, Matthew; Sadakane, Ryan; Tefera, Eshetu; Irwin, Michael R.

    2015-01-01

    Objective To determine the independent and combined effects of pain and opioids on the activation of an early marker of inflammation, nuclear factor-κB (NF-κB). Design NF-κB activation was compared within-subjects following four randomly ordered experimental sessions of opioid-only (intravenous fentanyl 1 μg/kg), pain-only (cold-pressor), opioid + pain, and a resting condition. Setting University General Clinical Research Center. Participants Twenty-one (11 female) healthy controls. Interventions Following exposure to treatment (fentanyl administration and/or cold-pressor pain), blood samples for NF-kB analysis were obtained. Main outcome measures Intracellular levels of activated NF-κB, in unstimulated and stimulated peripheral blood mononuclear cells at 15 and 30 minutes. Results Neither pain nor opioid administration alone effected NF-κB levels in cell populations; however, the combination of treatments induced significant increases of NF-κB in stimulated peripheral blood mononuclear cell, lymphocytes, and monocytes. Conclusions The combination of acute pain with opioids, as occurs in clinical situations, activates a key transcription factor involved in proinflammatory responses. PMID:25901477

  9. Caffeic acid phenethyl ester inhibits T-cell activation by targeting both nuclear factor of activated T-cells and NF-kappaB transcription factors.

    PubMed

    Márquez, Nieves; Sancho, Rocío; Macho, Antonio; Calzado, Marco A; Fiebich, Bernd L; Muñoz, Eduardo

    2004-03-01

    Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been shown to reveal anti-inflammatory properties. Since T-cells play a key role in the onset of several inflammatory diseases, we have evaluated the immunosuppressive activity of CAPE in human T-cells, discovering that this phenolic compound is a potent inhibitor of early and late events in T-cell receptor-mediated T-cell activation. Moreover, we found that CAPE specifically inhibited both interleukin (IL)-2 gene transcription and IL-2 synthesis in stimulated T-cells. To further characterize the inhibitory mechanisms of CAPE at the transcriptional level, we examined the DNA binding and transcriptional activities of nuclear factor (NF)-kappaB, nuclear factor of activated cells (NFAT), and activator protein-1 (AP-1) transcription factors in Jurkat cells. We found that CAPE inhibited NF-kappaB-dependent transcriptional activity without affecting the degradation of the cytoplasmic NF-kappaB inhibitory protein, IkappaBalpha. However, both NF-kappaB binding to DNA and transcriptional activity of a Gal4-p65 hybrid protein were clearly prevented in CAPE-treated Jurkat cells. Moreover, CAPE inhibited both the DNA-binding and transcriptional activity of NFAT, a result that correlated with its ability to inhibit phorbol 12-myristate 13-acetate plus ionomycin-induced NFAT1 dephosphorylation. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory activities of this natural compound.

  10. Copper-dependent inflammation and nuclear factor-kappaB activation by particulate air pollution.

    PubMed

    Kennedy, T; Ghio, A J; Reed, W; Samet, J; Zagorski, J; Quay, J; Carter, J; Dailey, L; Hoidal, J R; Devlin, R B

    1998-09-01

    Particulate air pollution causes increased cardiopulmonary morbidity and mortality, but the chemical determinants responsible for its biologic effects are not understood. We studied the effect of total suspended particulates collected in Provo, Utah, an area where an increase in respiratory symptoms in relation to levels of particulate pollution has been well documented. Provo particulates caused cytokine-induced neutrophil chemoattractant-dependent inflammation of rat lungs. Provo particulates stimulated interleukin-6 (IL-6) and IL-8 production, increased IL-8 messenger RNA (mRNA) and enhanced expression of intercellular adhesion molecule-1 (ICAM-1) in cultured BEAS-2B cells, and stimulated IL-8 secretion in primary cultures of human bronchial epithelium. Cytokine secretion was preceded by activation of the transcription factor nuclear factor-kappaB (NF-kappaB) and was reduced by treatment of cultures with superoxide dismutase, deferoxamine, or N-acetylcysteine. These biologic effects were replicated by culturing BEAS cells with quantities of Cu2+ found in Provo extract. IL-8 secretion by BEAS cells could be modified by addition of normal constituents of airway lining fluid to the culture medium. Mucin significantly reduced IL-8 secretion, and ceruloplasmin significantly increased IL-8 secretion and activation of NF-kappaB. These findings suggest that copper ions may cause some of the biologic effects of inhaled particulate air pollution in the Provo region of the United States, and may provide an explanation for the sensitivity of asthmatic individuals to Provo particulates that has been observed in epidemiologic studies.

  11. Reactive Oxygen Species and Nuclear Factor Erythroid 2-Related Factor 2 Activation in Diabetic Nephropathy: A Hidden Target

    PubMed Central

    Abdo, Shaaban; Zhang, Shao-Ling; Chan, John S.D.

    2015-01-01

    Hyperglycemia, oxidative stress and renin-angiotensin system (RAS) dysfunction have been implicated in diabetic nephropathy (DN) progression, but the underlying molecular mechanisms are far from being fully understood. In addition to the systemic RAS, the existence of a local intrarenal RAS in renal proximal tubular cells has been recognized. Angiotensinogen is the sole precursor of all angiotensins (Ang). Intrarenal reactive oxygen species (ROS) generation, Ang II level and RAS gene expression are up-regulated in diabetes, indicating that intrarenal ROS and RAS activation play an important role in DN. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is one of the major protective processes that occurs in response to intracellular oxidative stress. Nrf2 stimulates an array of antioxidant enzymes that convert excessive ROS to less reactive or less damaging forms. Recent studies have, however, revealed that Nrf2 activation might have other undesirable effects in diabetic animals and in diabetic patients with chronic kidney disease. This mini-review summarizes current knowledge of the relationship between ROS, Nrf2 and intra renal RAS activation in DN progression as well as possible novel target(s) for DN treatment. PMID:26213634

  12. Inhibition of nuclear factor kappa B activation reduces Coxsackievirus B3 replication in lymphoid cells.

    PubMed

    Sobotta, Katharina; Wilsky, Steffi; Althof, Nadine; Wiesener, Nadine; Wutzler, Peter; Henke, Andreas

    2012-02-01

    Interactions between viral replication machineries and host cell metabolism display interesting information how certain viruses capitalize cellular pathways to support progeny production. Among those pathogens, Coxsackievirus B3 (CVB3) has been identified to manipulate intracellular signaling very comprehensively. Next to others, this human pathogenic virus causes acute and chronic forms of myocarditis, pancreatitis, and meningitis. Here, activation of nuclear factor kappa B (NFκB) signaling appears to be involved in successful infection. Viral replication is not restricted to solid organs but involves susceptible immune cells as well. In the present study, p65 phosphorylation as one aspect of NFκB activation and inhibition via BAY 11-7085 administration was analyzed in the context of CVB3 replication in lymphoid cells. During CVB3 infection, an up-regulation of p65 translation is detectable, which is accompanied by noticeable phosphorylation. Inhibition of NFκB signaling reduces viral replication in a dose- and time-dependent manner. Taken together, these results indicate that during CVB3 replication in human and murine lymphoid cells, NFκB signaling is activated and facilitates viral replication. Therefore, antiviral strategies to target such central cellular signaling pathways may represent potential possibilities for the development of new virostatica.

  13. Structure activity relationship of phenolic diterpenes from Salvia officinalis as activators of the nuclear factor E2-related factor 2 pathway.

    PubMed

    Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A; Johnson, Jeffrey A

    2013-05-01

    Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate cytoprotective genes which may be useful in the treatment of neurodegenerative disease. In order to better understand the structure activity relationship of phenolic diterpenes from Salvia officinalis L., we isolated carnosic acid, carnosol, epirosmanol, rosmanol, 12-methoxy-carnosic acid, sageone, and carnosaldehyde using polyamide column, centrifugal partition chromatography, and semi-preparative high performance liquid chromatography. Isolated compounds were screened in vitro for their ability to active the Nrf2 and general cellular toxicity using mouse primary cortical cultures. All compounds except 12-methoxy-carnosic acid were able to activate the antioxidant response element. Furthermore both carnosol and carnoasldehyde were able to induce Nrf2-dependent gene expression as well as protect mouse primary cortical neuronal cultures from H(2)O(2) induced cell death.

  14. Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

    PubMed Central

    Martínez-Martínez, S; Gómez del Arco, P; Armesilla, A L; Aramburu, J; Luo, C; Rao, A; Redondo, J M

    1997-01-01

    Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants. PMID:9343406

  15. Growth Factor Midkine Promotes T-Cell Activation through Nuclear Factor of Activated T Cells Signaling and Th1 Cell Differentiation in Lupus Nephritis.

    PubMed

    Masuda, Tomohiro; Maeda, Kayaho; Sato, Waichi; Kosugi, Tomoki; Sato, Yuka; Kojima, Hiroshi; Kato, Noritoshi; Ishimoto, Takuji; Tsuboi, Naotake; Uchimura, Kenji; Yuzawa, Yukio; Maruyama, Shoichi; Kadomatsu, Kenji

    2017-04-01

    Activated T cells play crucial roles in the pathogenesis of autoimmune diseases, including lupus nephritis (LN). The activation of calcineurin/nuclear factor of activated T cells (NFAT) and STAT4 signaling is essential for T cells to perform various effector functions. Here, we identified the growth factor midkine (MK; gene name, Mdk) as a novel regulator in the pathogenesis of 2,6,10,14-tetramethylpentadecane-induced LN via activation of NFAT and IL-12/STAT4 signaling. Wild-type (Mdk(+/+)) mice showed more severe glomerular injury than MK-deficient (Mdk(-/-)) mice, as demonstrated by mesangial hypercellularity and matrix expansion, and glomerular capillary loops with immune-complex deposition. Compared with Mdk(-/-) mice, the frequency of splenic CD69(+) T cells and T helper (Th) 1 cells, but not of regulatory T cells, was augmented in Mdk(+/+) mice in proportion to LN disease activity, and was accompanied by skewed cytokine production. MK expression was also enhanced in activated CD4(+) T cells in vivo and in vitro. MK induced activated CD4(+) T cells expressing CD69 through nuclear activation of NFAT transcription and selectively increased in vitro differentiation of naive CD4(+) T cells into Th1 cells by promoting IL-12/STAT4 signaling. These results suggest that MK serves an indispensable role in the NFAT-regulated activation of CD4(+) T cells and Th1 cell differentiation, eventually leading to the exacerbation of LN.

  16. Sesamin attenuates allergic airway inflammation through the suppression of nuclear factor-kappa B activation.

    PubMed

    Li, Liangchang; Piao, Hongmei; Zheng, Mingyu; Jin, Zhewu; Zhao, Liguang; Yan, Guanghai

    2016-12-01

    The aim of the present study is to determine the role of sesamin, the most abundant lignan in sesame seed oil, on the regulation of allergic airway inflammation in a murine asthma model. A BALB/c mouse model with allergic asthma was used to evaluate the effects of sesamin on nuclear factor-kappa B (NF-κB) activation. An enzyme-linked immunosorbent assay was used to determine protein expression in bronchoalveolar lavage (BAL) fluids. Hematoxylin and eosin staining was performed to examine histological changes. Moreover, western blot analysis was used to detect the expression of proteins in tissues. Prior to administering sesamin, the mice developed the following pathophysiological features of asthma: An increase in the number of inflammatory cells, increased levels of interleukin (IL)-4, IL-5 and IL-13, decreased levels of interferon-γ in BAL fluids and lung tissues, increased immunoglobulin E (IgE) levels in the serum and an increased activation of NF-κB in lung tissues. Following treatment with sesamin, the mice had evidently reduced peribronchiolar inflammation and airway inflammatory cell recruitment, inhibited production of several cytokines in BAL fluids and lung tissues, and decreased IgE levels. Following inhalation of ovalbumin, the administration of sesamin also inhibited the activation of NF-κB. In addition, sesamin administration reduced the phosphorylation of p38 mitogen-activated protein kinases (MAPKs). The present study demonstrates that sesamin decreases the activation of NF-κB in order to attenuate allergic airway inflammation in a murine model of asthma, possibly via the regulation of phosphorylation of p38 MAPK. These observations provide an important molecular mechanism for the potential use of sesamin in preventing and/or treating asthma, as well as other airway inflammatory disorders.

  17. Sesamin attenuates allergic airway inflammation through the suppression of nuclear factor-kappa B activation

    PubMed Central

    Li, Liangchang; Piao, Hongmei; Zheng, Mingyu; Jin, Zhewu; Zhao, Liguang; Yan, Guanghai

    2016-01-01

    The aim of the present study is to determine the role of sesamin, the most abundant lignan in sesame seed oil, on the regulation of allergic airway inflammation in a murine asthma model. A BALB/c mouse model with allergic asthma was used to evaluate the effects of sesamin on nuclear factor-kappa B (NF-κB) activation. An enzyme-linked immunosorbent assay was used to determine protein expression in bronchoalveolar lavage (BAL) fluids. Hematoxylin and eosin staining was performed to examine histological changes. Moreover, western blot analysis was used to detect the expression of proteins in tissues. Prior to administering sesamin, the mice developed the following pathophysiological features of asthma: An increase in the number of inflammatory cells, increased levels of interleukin (IL)-4, IL-5 and IL-13, decreased levels of interferon-γ in BAL fluids and lung tissues, increased immunoglobulin E (IgE) levels in the serum and an increased activation of NF-κB in lung tissues. Following treatment with sesamin, the mice had evidently reduced peribronchiolar inflammation and airway inflammatory cell recruitment, inhibited production of several cytokines in BAL fluids and lung tissues, and decreased IgE levels. Following inhalation of ovalbumin, the administration of sesamin also inhibited the activation of NF-κB. In addition, sesamin administration reduced the phosphorylation of p38 mitogen-activated protein kinases (MAPKs). The present study demonstrates that sesamin decreases the activation of NF-κB in order to attenuate allergic airway inflammation in a murine model of asthma, possibly via the regulation of phosphorylation of p38 MAPK. These observations provide an important molecular mechanism for the potential use of sesamin in preventing and/or treating asthma, as well as other airway inflammatory disorders. PMID:28105144

  18. Melatonin overcomes gemcitabine resistance in pancreatic ductal adenocarcinoma by abrogating nuclear factor-κB activation.

    PubMed

    Ju, Huai-Qiang; Li, Hao; Tian, Tian; Lu, Yun-Xin; Bai, Long; Chen, Le-Zong; Sheng, Hui; Mo, Hai-Yu; Zeng, Jun-Bo; Deng, Wuguo; Chiao, Paul J; Xu, Rui-Hua

    2016-01-01

    Constitutive activation and gemcitabine induction of nuclear factor-κB (NF-κB) contribute to the aggressive behavior and chemotherapeutic resistance of pancreatic ductal adenocarcinoma (PDAC). Thus, targeting the NF-κB pathway has proven an insurmountable challenge for PDAC therapy. In this study, we investigated whether the inhibition of NF-κB signaling pathway by melatonin might lead to tumor suppression and overcome gemcitabine resistance in pancreatic tumors. Our results showed that melatonin inhibited activities of NF-κB by suppressing IκBα phosphorylation and decreased the expression of NF-κB response genes in MiaPaCa-2, AsPc-1, Panc-28 cells and gemcitabine resistance MiaPaCa-2/GR cells. Moreover, melatonin not only inhibited cell proliferation and invasion in a receptor-independent manner, but also enhanced gemcitabine cytotoxicity at pharmacologic concentrations in these PDAC cells. In vivo, the mice treated with both agents experienced a larger reduction in tumor burden than the single drug-treated groups in an orthotopic xenograft mouse model. Taken together, these results indicate that melatonin inhibits proliferation and invasion of PDAC cells and overcomes gemcitabine resistance of pancreatic tumors through NF-κB inhibition. Our findings therefore provide novel preclinical knowledge about melatonin inhibition of NF-κB in PDAC and suggest that melatonin should be investigated clinically, alone or in combination with gemcitabine for PDAC treatment.

  19. Lycopene activates antioxidant enzymes and nuclear transcription factor systems in heat-stressed broilers.

    PubMed

    Sahin, K; Orhan, C; Tuzcu, M; Sahin, N; Hayirli, A; Bilgili, S; Kucuk, O

    2016-05-01

    This study was conducted to evaluate the effects of dietary lycopene supplementation on growth performance, antioxidant status, and muscle nuclear transcription factor [Kelch like-ECH-associated protein 1 (Keap1) and (erythroid-derived 2)-like 2 (Nrf2)] expressions in broiler chickens exposed to heat stress (HS). A total of 180 one-day-old male broiler chicks (Ross 308) were assigned randomly to one of 2×3 factorially arranged treatments: two housing temperatures (22°C for 24 h/d; thermoneutral, TN or 34°C for 8 h/d HS) and three dietary lycopene levels (0, 200, or 400 mg/kg). Each treatment consisted of three replicates of 10 birds. Birds were reared to 42 d of age. Heat stress caused reductions in feed intake and weight gain by 12.2 and 20.7% and increased feed efficiency by 10.8% (P<0.0001 for all). Increasing dietary lycopene level improved performance in both environments. Birds reared under the HS environment had lower serum and muscle lycopene concentration (0.34 vs. 0.50 μg/mL and 2.80 vs. 2.13 μg/g), activities of superoxide dismutase (151 vs. 126 U/mL and 131 vs. 155 U/mg protein), glutathione peroxidase (184 vs. 154 U/mL and 1.39 vs. 1.74 U/mg protein), and higher malondialdehyde (MDA) concentration (0.53 vs. 0.83 μg/mL and 0.78 vs. 0.45 μg/ mg protein) than birds reared under the TN environment. Changes in levels of lycopene and MDA and activities of enzymes in serum and muscle varied by the environmental temperature as dietary lycopene level increased. Moreover, increasing dietary lycopene level suppressed muscle Keap1 expression and enhanced muscle Nrf2 expression, which had increased by 150% and decreased by 40%, respectively in response to HS. In conclusion, lycopene supplementation alleviates adverse effects of HS on performance through modulating expressions of stress-related nuclear transcription factors.

  20. Signal Transducer and Activator of Transcription (STAT)-3 Activates Nuclear Factor (NF)-κB in Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Liu, Zhiming; Hazan-Halevy, Inbal; Harris, David M.; Li, Ping; Ferrajoli, Alessandra; Faderl, Stefan; Keating, Michael J.; Estrov, Zeev

    2014-01-01

    Nuclear factor (NF)-κB plays a major role in the pathogenesis of B-cell neoplasms. A broad array of mostly extracellular stimuli has been reported to activate NF-κB, to various degrees, in chronic lymphocytic leukemia (CLL) cells. Because CLL cells harbor high levels of unphosphorylated (U) signal transducer and activator of transcription (STAT)-3 protein and U-STAT3 was reported to activate NF-κB, we sought to determine whether U-STAT3 activates NF-κB in CLL. Using the electrophoretic mobility shift assay (EMSA) we studied peripheral blood low-density cells from 15 patients with CLL and found that CLL cell nuclear extracts from all the samples bound to an NF-κB DNA probe, suggesting that NF-κB is constitutively activated in CLL. Immunoprecipitation studies showed that STAT3 bound NF-κB p65, and confocal microscopy studies detected U-STAT3/NF-κB complexes in the nuclei of CLL cells, thereby confirming these findings. Furthermore, infection of CLL cells with retroviral STAT3-shRNA attenuated the binding of NF-κB to DNA, as assessed by EMSA, and downregulated mRNA levels of NF-κB-regulated genes, as assessed by quantitative polymerase chain reaction. Taken together, our data suggest that U-STAT3 binds to the NF-κB p50/p65 dimers and that the U-STAT3/NF-κB complexes bind to DNA and activate NF-κB-regulated genes in CLL cells. PMID:21364020

  1. Early nuclear exclusion of the transcription factor max is associated with retinal ganglion cell death independent of caspase activity.

    PubMed

    Petrs-Silva, Hilda; de Freitas, Fabíola G; Linden, Rafael; Chiarini, Luciana B

    2004-02-01

    We examined the behavior of the transcription factor Max during retrograde neuronal degeneration of retinal ganglion cells. Using immunohistochemistry, we found a progressive redistribution of full-length Max from the nucleus to the cytoplasm and dendrites of the ganglion cells following axon damage. Then, the axotomized cells lose all their content of Max, while undergoing nuclear pyknosis and apoptotic cell death. After treatment of retinal explants with either anisomycin or thapsigargin, the rate of nuclear exclusion of Max accompanied the rate of cell death as modulated by either drug. Treatment with a pan-caspase inhibitor abolished both TUNEL staining and immunoreactivity for activated caspase-3, but did not affect the subcellular redistribution of Max immunoreactivity after axotomy. The data show that nuclear exclusion of the transcription factor Max is an early event, which precedes and is independent of the activation of caspases, during apoptotic cell death in the central nervous system.

  2. Glycosylated human oxyhaemoglobin activates nuclear factor-κB and activator protein-1 in cultured human aortic smooth muscle

    PubMed Central

    Peiró, Concepción; Matesanz, Nuria; Nevado, Julián; Lafuente, Nuria; Cercas, Elena; Azcutia, Verónica; Vallejo, Susana; Rodríguez-Mañas, Leocadio; Sánchez-Ferrer, Carlos F

    2003-01-01

    Diabetic vessels undergo structural changes that are linked to a high incidence of cardiovascular diseases. Reactive oxygen species (ROS) mediate cell signalling in the vasculature, where they can promote cell growth and activate redox-regulated transcription factors, like activator protein-1 (AP-1) or nuclear factor-κB (NF-κB), which are involved in remodelling and inflammation processes. Amadori adducts, formed through nonenzymatic glycosylation, can contribute to ROS formation in diabetes. In this study, we analysed whether Amadori-modified human oxyhaemoglobin, glycosylated at either normal (N-Hb) or elevated (E-Hb) levels, can induce cell growth and activate AP-1 and NF-κB in cultured human aortic smooth muscle cells (HASMC). E-Hb (1 nM–1 μM), but not N-Hb, promoted a concentration-dependent increase in cell size from nanomolar concentrations, although it failed to stimulate HASMC proliferation. At 10 nM, E-Hb stimulated both AP-1 and NF-κB activity, as assessed by transient transfection, electromobility shift assays or immunofluorescence staining. The effects of E-Hb resembled those of the proinflammatory cytokine tumour necrosis factor-α (TNF-α). E-Hb enhanced intracellular superoxide anions content and its effects on HASMC were abolished by different ROS scavengers. In conclusion, E-Hb stimulates growth and activates AP-1 and NF-κB in human vascular smooth muscle by redox-sensitive pathways, thus suggesting a possible direct role for Amadori adducts in diabetic vasculopathy. PMID:14504138

  3. Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

    PubMed Central

    Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

    2016-01-01

    Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation

  4. Jianpi Qingchang decoction alleviates ulcerative colitis by inhibiting nuclear factor-κB activation

    PubMed Central

    Zheng, Lie; Zhang, Ya-Li; Dai, Yan-Cheng; Chen, Xuan; Chen, De-Liang; Dai, Yue-Ting; Tang, Zhi-Peng

    2017-01-01

    AIM To investigate the therapeutic effect of Jianpi Qingchang decoction (JPQCD) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. METHODS C57BL/c mice were injected intragastrically with 5% DSS instead of drinking water for 7 d, and their body weight, diarrhea severity and fecal bleeding were monitored, while the mice in the control group were treated with standard drinking water, without DSS. After 7 d, the DSS drinking water was changed to normal water and the DSS group continued with DSS water. The control and DSS groups were given normal saline by intragastric injection. The 5-aminosalicylic acid (5-ASA) group was treated orally with 5-ASA at a dose of 100 mg/kg daily. The JPQCD group was treated orally with JPQCD at a dose of 17.1 g/kg daily. On day 14, the colon length was measured, the colorectal histopathological damage score was assessed, and protein levels of interleukin (IL)-1β, IL-8 and tumor necrosis factor-alpha (TNF-α) in colon supernatants were measured by enzyme-linked immunosorbent assay. mRNA expression of IL-1β, IL-8, TNF-α and nuclear factor-kappa B (NF-κB) was detected by real-time quantitative polymerase chain reaction. Western blotting was used to detect the protein expression of NF-κB and inhibitor of kappa B. RESULTS Acute inflammation occurred in the mice administered DSS, including the symptoms of losing body weight, loose feces/watery diarrhea and presence of fecal blood; all these symptoms worsened at 7 d. The colons of mice treated with DSS were assessed by histological examination, and the results confirmed that acute inflammation had occurred, as evidenced by loss of colonic mucosa and chronic inflammatory cell infiltration, and these features extended into the deeper layer of the colon walls. The expression levels of IL-1β, IL-8 and TNF-α in the DSS group were higher than those in the control group (P < 0.05), and the expression levels of IL-1β, IL-8 and TNF-α in the JPQCD and 5-ASA groups were

  5. Hydrogen Sulfide Levels and Nuclear Factor-Erythroid 2-Related Factor 2 (NRF2) Activity Are Attenuated in the Setting of Critical Limb Ischemia (CLI)

    PubMed Central

    Islam, Kazi N; Polhemus, David J; Donnarumma, Erminia; Brewster, Luke P; Lefer, David J

    2015-01-01

    Background Cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase are endogenous enzymatic sources of hydrogen sulfide (H2S). Functions of H2S are mediated by several targets including ion channels and signaling proteins. Nuclear factor-erythroid 2-related factor 2 is responsible for the expression of antioxidant response element–regulated genes and is known to be upregulated by H2S. We examined the levels of H2S, H2S-producing enzymes, and nuclear factor-erythroid 2-related factor 2 activation status in skeletal muscle obtained from critical limb ischemia (CLI) patients. Methods and Results Gastrocnemius tissues were attained postamputation from human CLI and healthy control patients. We found mRNA and protein levels of cystathionine γ-lyase, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase were significantly decreased in skeletal muscle of CLI patients as compared to control. H2S and sulfane sulfur levels were significantly decreased in skeletal muscle of CLI patients. We also observed significant reductions in nuclear factor-erythroid 2-related factor 2 activation as well as antioxidant proteins, such as Cu, Zn-superoxide dismutase, catalase, and glutathione peroxidase in skeletal muscle of CLI patients. Biomarkers of oxidative stress, such as malondialdehyde and protein carbonyl formation, were significantly increased in skeletal muscle of CLI patients as compared to healthy controls. Conclusions The data demonstrate that H2S bioavailability and nuclear factor-erythroid 2-related factor 2 activation are both attenuated in CLI tissues concomitant with significantly increased oxidative stress. Reductions in the activity of H2S-producing enzymes may contribute to the pathogenesis of CLI. PMID:25977470

  6. Cell-specific Activation of Nuclear Factor-κB by the Parasite Trypanosoma cruzi Promotes Resistance to Intracellular Infection

    PubMed Central

    Hall, Belinda S.; Tam, Winnie; Sen, Ranjan; Pereira, Miercio E. A.

    2000-01-01

    The transcription factor nuclear factor-κB (NF-κB) is central to the innate and acquired immune response to microbial pathogens, coordinating cellular responses to the presence of infection. Here we demonstrate a direct role for NF-κB activation in controlling intracellular infection in nonimmune cells. Trypanosoma cruzi is an intracellular parasite of mammalian cells with a marked preference for infection of myocytes. The molecular basis for this tissue tropism is unknown. Trypomastigotes, the infectious stage of T. cruzi, activate nuclear translocation and DNA binding of NF-κB p65 subunit and NF-κB-dependent gene expression in epithelial cells, endothelial cells, and fibroblasts. Inactivation of epithelial cell NF-κB signaling by inducible expression of the inhibitory mutant IκBaM significantly enhances parasite invasion. T. cruzi do not activate NF-κB in cells derived from skeletal, smooth, or cardiac muscle, despite the ability of these cells to respond to tumor necrosis factor-α with NF-κB activation. The in vitro infection level in these muscle-derived cells is more than double that seen in the other cell types tested. Therefore, the ability of T. cruzi to activate NF-κB correlates inversely with susceptibility to infection, suggesting that NF-κB activation is a determinant of the intracellular survival and tissue tropism of T. cruzi. PMID:10637298

  7. Nuclear respiratory factors 1 and 2 utilize similar glutamine-containing clusters of hydrophobic residues to activate transcription.

    PubMed Central

    Gugneja, S; Virbasius, C M; Scarpulla, R C

    1996-01-01

    Nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) are ubiquitous transcription factors that have been implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. Recently, both factors have been found to be major transcriptional determinants for a subset of these genes that define a class of simple promoters involved in respiratory chain expression. Here, functional domains required for transactivation by NRF-1 have been defined. An atypical nuclear localization signal resides in a conserved amino-terminal region adjacent to the DNA binding domain and consists of functionally redundant clusters of basic residues. A second domain in the carboxy-terminal half of the molecule is necessary for transcriptional activation. The activation domains of both NRF-1 and NRF-2 were extensively characterized by both deletion and alanine substitution mutagenesis. The results show that these domains do not fall into known classes defined by a preponderance of amino acid residues, including glutamines, prolines, or isoleucines, as found in other eukaryotic activators. Rather, in both factors, a series of tandemly arranged clusters of hydrophobic amino acids were required for activation. Although all of the functional clusters contain glutamines, the glutamines differ from the hydrophobic residues in that they are inconsequential for activation. Unlike the NRF-2 domain, which contains its essential hydrophobic motifs within 40 residues, the NRF-1 domain spans about 40% of the molecule and appears to have a bipartite structure. The findings indicate that NRF-1 and NRF-2 utilize similar hydrophobic structural motifs for activating transcription. PMID:8816484

  8. Phytol metabolites are circulating dietary factors that activate the nuclear receptor RXR.

    PubMed Central

    Kitareewan, S; Burka, L T; Tomer, K B; Parker, C E; Deterding, L J; Stevens, R D; Forman, B M; Mais, D E; Heyman, R A; McMorris, T; Weinberger, C

    1996-01-01

    RXR is a nuclear receptor that plays a central role in cell signaling by pairing with a host of other receptors. Previously, 9-cis-retinoic acid (9cRA) was defined as a potent RXR activator. Here we describe a unique RXR effector identified from organic extracts of bovine serum by following RXR-dependent transcriptional activity. Structural analyses of material in active fractions pointed to the saturated diterpenoid phytanic acid, which induced RXR-dependent transcription at concentrations between 4 and 64 microM. Although 200 times more potent than phytanic acid, 9cRA was undetectable in equivalent amounts of extract and cannot be present at a concentration that could account for the activity. Phytanic acid, another phytol metabolite, was synthesized and stimulated RXR with a potency and efficacy similar to phytanic acid. These metabolites specifically displaced [3H]-9cRA from RXR with Ki values of 4 microM, indicating that their transcriptional effects are mediated by direct receptor interactions. Phytol metabolites are compelling candidates for physiological effectors, because their RXR binding affinities and activation potencies match their micromolar circulating concentrations. Given their exclusive dietary origin, these chlorophyll metabolites may represent essential nutrients that coordinate cellular metabolism through RXR-dependent signaling pathways. PMID:8856661

  9. Plasma corticosterone elevation inhibits the activation of nuclear factor kappa B (NFKB) in the Syrian hamster pineal gland.

    PubMed

    Ferreira, Z S; Bothorel, B; Markus, R P; Simonneaux, V

    2012-05-01

    We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

  10. Overexpression of an enzymically inactive interleukin-1-receptor-associated kinase activates nuclear factor-kappaB.

    PubMed Central

    Maschera, B; Ray, K; Burns, K; Volpe, F

    1999-01-01

    Upon interleukin 1 (IL-1) stimulation, the IL-1-receptor (IL-1R)-associated kinase (IRAK) is rapidly recruited to the IL-1R complex and undergoes phosphorylation. Here we demonstrate that recombinant wild-type IRAK (IRAK-WT), but not a kinase-defective mutant with Asp340 replaced by an asparagine residue (IRAK-Asp340Asn), is highly phosphorylated and is capable of auto-phosphorylation in vitro. Overexpression of both IRAK-WT and IRAK-Asp340Asn caused activation of nuclear factor kappaB, suggesting that the kinase activity of IRAK is not required outside of the IL-1R complex. PMID:10191251

  11. The immunosuppressive effect of Gamisanghyulyunbueum through inhibition of mitogen-activated protein kinase and nuclear factor activation in MOLT-4 cells.

    PubMed

    Shin, Hye-Young; Jeong, Hyun-Ja; Na, Ho-Jeong; Kim, Hong-Joon; Moon, Goo; Shin, Tae-Yong; Yang, Deok-Chun; Hong, Seung-Heon; Kim, Hyung-Min

    2005-07-01

    Gamisanghyulyunbueum (GSHYBE) has been used clinically to treat skin related disease in South Korea. We investigated GSHYBE-mediated changes in downstream T cell signal transduction. To determine the mechanism of inhibition, we have studied many of the major pathways in phytohemagglutinin (PHA)-activated T cell. We show that among the mitogen-activated protein kinase family activation of phosphorylation of extra cellular signal-regulated kinase 1/2 (ERK1/2, p44/42) and p38, but not c-jun NH2-terminal kinase is inhibited. In activated MOLT-4 cells, the nuclear localization of nuclear factor of activated T cells (NFATc) was blocked by GSHYBE (1 mg/ml). Also, degradation of inhibitor kappaB-alpha and transactivation by nuclear factor-kappaB (NF-kappaB)/Rel A were impaired by GSHYBE (1 mg/ml). Furthermore, interlukin (IL)-2, IL-4 and Interferen (IFN)-gamma secretion by PHA activated MOLT-4 cells and peripheral blood mononuclear cells (PBMC) were significantly diminishes following GSHYBE treatment (1 mg/ml). Also, oral administration of GSHYBE inhibited IL-2 secretion in skin allergic reaction. In conclusion, our data indicate that GSHYBE treatment of T cells inhibits ERK1/2 and p38 activation and nuclear translocation of NFATc, NF-kappaB, resulting in diminished secretion of IL-2.

  12. Chlorogenic acid inhibits osteoclast differentiation and bone resorption by down-regulation of receptor activator of nuclear factor kappa-B ligand-induced nuclear factor of activated T cells c1 expression.

    PubMed

    Kwak, Sung Chul; Lee, Cheol; Kim, Ju-Young; Oh, Hyun Mee; So, Hong-Seob; Lee, Myeung Su; Rho, Mun Chual; Oh, Jaemin

    2013-01-01

    Excessive osteoclastic bone resorption plays a critical role in inflammation-induced bone loss such as rheumatoid arthritis and periodontal bone erosion. Therefore, identification of osteoclast targeted-agents may be a therapeutic approach to the treatment of pathological bone loss. In this study, we isolated chlorogenic acid (CGA) from fructus of Gardenia jasminoides to discover anti-bone resorptive agents. CGA is a polyphenol with anti-inflammatory and anti-oxidant activities, however, its effects on osteoclast differentiation is unknown. Thus, we investigated the effect of CGA in receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL)-induced osteoclast differentiation and RANKL signaling. CGA dose-dependently inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without any evidence of cytotoxicity. CGA inhibited the phosphorylation of p38, Akt, extracellular signal-regulated kinase (ERK), and inhibitor of nuclear factor-kappa B (IκB), and IκB degradation by RANKL treatment. CGA suppressed the mRNA expression of nuclear factor of activated T cells c1 (NFATc1), TRAP and OSCAR in RANKL-treated bone marrow macrophages (BMMs). Also, overexpression of NFATc1 in BMMs blocked the inhibitory effect of CGA on RANKL-mediated osteoclast differentiation. Furthermore, to evaluate the effects of CGA in vivo, lipopolysaccharide (LPS)-induced bone erosion study was carried out. CGA remarkably attenuated LPS-induced bone loss based on micro-computed tomography and histologic analysis of femurs. Taken together, our findings suggest that CGA may be a potential treatment option for osteoclast-related diseases with inflammatory bone destruction.

  13. Chronic intermittent hypoxia activates nuclear factor-{kappa}B in cardiovascular tissues in vivo

    SciTech Connect

    Greenberg, Harly; Ye Xiaobing; Wilson, David; Htoo, Aung K.; Hendersen, Todd; Liu Shufang . E-mail: sliu@lij.edu

    2006-05-05

    Obstructive sleep apnea (OSA) is an important risk factor for cardiovascular morbidity and mortality. The mechanisms through which OSA promotes the development of cardiovascular disease are poorly understood. In this study, we tested the hypotheses that chronic exposure to intermittent hypoxia and reoxygenation (CIH) is a major pathologic factor causing cardiovascular inflammation, and that CIH-induces cardiovascular inflammation and pathology by activating the NF-{kappa}B pathway. We demonstrated that exposure of mice to CIH activated NF-{kappa}B in cardiovascular tissues, and that OSA patients had markedly elevated monocyte NF-{kappa}B activity, which was significantly decreased when obstructive apneas and their resultant CIH were eliminated by nocturnal CPAP therapy. The elevated NF-{kappa}B activity induced by CIH is accompanied by and temporally correlated to the increased expression of iNOS protein, a putative and important NF-{kappa}B-dependent gene product. Thus, CIH-mediated NF-{kappa}B activation may be a molecular mechanism linking OSA and cardiovascular pathologies seen in OSA patients.

  14. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

    PubMed

    Sharma, Sonia; Findlay, Gregory M; Bandukwala, Hozefa S; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G; Sacks, David B; Rao, Anjana

    2011-07-12

    Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.

  15. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex

    PubMed Central

    Sharma, Sonia; Findlay, Gregory M.; Bandukwala, Hozefa S.; Oberdoerffer, Shalini; Baust, Beate; Li, Zhigang; Schmidt, Valentina; Hogan, Patrick G.; Sacks, David B.; Rao, Anjana

    2011-01-01

    Nuclear factor of activated T cells (NFAT) proteins are Ca2+-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca2+, NFAT proteins are dephosphorylated by the Ca2+/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function. PMID:21709260

  16. Controls of Nuclear Factor-Kappa B Signaling Activity by 5’-AMP-Activated Protein Kinase Activation With Examples in Human Bladder Cancer Cells

    PubMed Central

    Kim, Jin

    2016-01-01

    Generally, both lipopolysaccharide (LPS)- and hypoxia-induced nuclear factor kappa B (NF-κB) effects are alleviated through differential posttranslational modification of NF-κB phosphorylation after pretreatment with 5´-AMP-activated protein kinase (AMPK) activators such as 5´-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or the hypoglycemic agent metformin. We found that AICAR or metformin acts as a regulator of LPS/NF-κB-or hypoxia/NF-κB-mediated cyclooxygenase induction by an AMPK-dependent mechanism with interactions between p65-NF-κB phosphorylation and acetylation, including in a human bladder cancer cell line (T24). In summary, we highlighted the regulatory interactions of AMPK activity on NF-κB induction, particularly in posttranslational phosphorylation and acetylation of NF-κB under inflammatory conditions or hypoxia environment. PMID:27706018

  17. Nuclear factors that bind two regions important to transcriptional activity of the simian immunodeficiency virus long terminal repeat.

    PubMed Central

    Winandy, S; Renjifo, B; Li, Y; Hopkins, N

    1992-01-01

    Previous studies identified two regions in the U3 region of a molecular clone of simian immunodeficiency virus, SIVmac142, that are important to transcriptional activity under conditions of induction as well as basal-level expression (B. Renjifo, N. A. Speck, S. Winandy, N. Hopkins, and Y. Li, J. Virol. 64:3130-3134, 1990). One region includes the NF-kappa B binding site, while the other lies just 5' of this site between nucleotides -162 and -114 (the -162 to -114 region). The fact that the NF-kappa B site mutation attenuated transcriptional activity in uninduced T cells and fibroblasts where activated NF-kappa B would not be present suggested that a factor(s) other than NF-kappa B could be acting through this site. In this study, we have identified a factor which binds to a cis element overlapping the NF-kappa B site. This factor, which we call simian factor 3 (SF3), would play a role in regulation under conditions of basal level expression, whereas under conditions of induction, NF-kappa B would act via this region. SF3 may also bind to an element in the -162 to -114 region. In addition, we have identified two other factors that bind the -162 to -114 region. One, which we designated SF1, is a ubiquitous basal factor, and the other, SF2, is a T-cell-predominant phorbol myristate acetate-inducible factor. Through identification of nuclear factors that interact with the U3 region of the SIVmac142 long terminal repeat, we can gain insight into how this virus is transcriptionally regulated under conditions of basal-level expression as well as conditions of T-cell activation. Images PMID:1501272

  18. The immunosuppressive effect of Buchang-tang through inhibition of mitogen-activated protein kinase and nuclear factor activation in MOLT-4 cells.

    PubMed

    Shin, Hye-Young; Shin, Tae-Yong; An, Nyeon-Hyoung; Kim, Hyung-Ryong; Chae, Han-Jung; Kim, Yun-Kyung; Um, Jae-Young; Hong, Seung-Heon; Kim, Hyung-Min

    2005-10-31

    Buchang-tang (BCT) has been known to suppress inflammatory and autoimmune responses. Accordingly, BCT has been clinically used in Korea as an immunomodulatory oriental medicine. Here, we report on the mechanism of action of BCT in activated MOLT-4 cells by determining the affected signaling pathways. BCT inhibits extracellular signal-regulated kinases (ERK)l/2 and p38 activation but does not interfere with phosphorylation of other mitogen-activated protein kinases, c-Jun NH2-terminal kinases 1/2 in MOLT-4 cells. The nuclear localization of nuclear factor of activated T cells 2 (NFATc) was blocked by BCT. Also, degradation of inhibitor kappaB-alpha and transactivation by nuclear factor-kappa B (NF-kappaB)/Rel A were impaired. Furthermore, interlukin (IL)-2 mRNA and protein levels were significantly diminished by BCT treatment. Our data indicate that BCT inhibits ERK1/2, p38 activation, nuclear translocation of NFATc, and NF-kappaB, resulting in diminished secretion of IL-2.

  19. The hepatitis B virus X protein activates nuclear factor of activated T cells (NF-AT) by a cyclosporin A-sensitive pathway.

    PubMed Central

    Lara-Pezzi, E; Armesilla, A L; Majano, P L; Redondo, J M; López-Cabrera, M

    1998-01-01

    The X gene product of the human hepatitis B virus (HBx) is a transcriptional activator of various viral and cellular genes. We recently have determined that the production of tumor necrosis factor-alpha (TNF-alpha) by HBV-infected hepatocytes is transcriptionally up-regulated by HBx, involving nuclear factor of activated T cells (NF-AT)-dependent activation of the TNF-alpha gene promoter. Here we show that HBx activates NF-AT by a cyclosporin A-sensitive mechanism involving dephosphorylation and nuclear translocation of the transcription factor. Luciferase gene expression assays demonstrated that HBx transactivates transcription through NF-AT-binding sites and activates a Gal4-NF-AT chimeric protein. DNA-protein interaction assays revealed that HBx induces the formation of NF-AT-containing DNA-binding complexes. Immunofluorescence analysis demonstrated that HBx induces the nuclear translocation of NF-AT, which can be blocked by the immunosuppressive drug cyclosporin A. Furthermore, immunoblot analysis showed that the HBx-induced activation and translocation of NF-AT are associated with its dephosphorylation. Thus, HBx may play a relevant role in the intrahepatic inflammatory processes by inducing locally the expression of cytokines that are regulated by NF-AT. PMID:9843511

  20. Esculetin attenuates receptor activator of nuclear factor kappa-B ligand-mediated osteoclast differentiation through c-Fos/nuclear factor of activated T-cells c1 signaling pathway

    SciTech Connect

    Baek, Jong Min; Park, Sun-Hyang; Cheon, Yoon-Hee; Ahn, Sung-Jun; Lee, Myeung Su; Oh, Jaemin; Kim, Ju-Young

    2015-05-29

    Esculetin exerts various biological effects on anti-oxidation, anti-tumors, and anti-inflammation. However, the involvement of esculetin in the bone metabolism process, particularly osteoclast differentiation has not yet been investigated. In the present study, we first confirmed the inhibitory effect of esculetin on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. We then revealed the relationship between esculetin and the expression of osteoclast-specific molecules to elucidate its underlying mechanisms. Esculetin interfered with the expression of c-Fos and nuclear factor of activated T cell c1 (NFATc1) both at the mRNA and protein level with no involvement in osteoclast-associated early signaling pathways, suppressing the expression of various transcription factors exclusively expressed in osteoclasts such as tartrate-resistant acid phosphatase (Trap), osteoclast-associated receptor (Oscar), dendritic cell-specific transmembrane protein (Dcstamp), osteoclast stimulatory transmembrane protein (Ocstamp), cathepsin K, αvβ3 integrin, and calcitonin receptor (Ctr). Additionally, esculetin inhibited the formation of filamentous actin (F-actin) ring-positive osteoclasts during osteoclast differentiation. However, the development of F-actin structures and subsequent bone resorbing activity of mature osteoclasts, which are observed in osteoclast/osteoblast co-culture systems were not affected by esculetin. Taken together, our results indicate for the first time that esculetin inhibits RANKL-mediated osteoclastogenesis via direct suppression of c-Fos and NFATc1 expression and exerts an inhibitory effect on actin ring formation during osteoclastogenesis. - Highlights: • We first investigated the effects of esculetin on osteoclast differentiation and function. • Our data demonstrate for the first time that esculetin can suppress osteoclastogenesis in vitro. • Esculetin acts as an inhibitor of c-Fos and NFATc1 activation.

  1. Porcine circovirus type 2 induces the activation of nuclear factor kappa B by I{kappa}B{alpha} degradation

    SciTech Connect

    Wei Li; Kwang, Jimmy; Wang Jin; Shi Lei; Yang Bing; Li Yongqing; Liu Jue

    2008-08-15

    The transcription factor NF-{kappa}B is commonly activated upon virus infection and a key player in the induction and regulation of the host immune response. The present study demonstrated for the first time that porcine circovirus type 2 (PCV2), which is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome, can activate NF-{kappa}B in PCV2-infected PK15 cells. In PCV2-infected cells, NF-{kappa}B was activated concomitantly with viral replication, which was characterized by increased DNA binding activity, translocation of NF-{kappa}B p65 from the cytoplasm to the nucleus, as well as degradation and phosphorylation of I{kappa}B{alpha} protein. We further demonstrated PCV2-induced activation of NF-{kappa}B and colocalization of p65 nuclear translocation with virus replication in cultured cells. Treatment of cells with CAPE, a selective inhibitor of NF-{kappa}B activation, reduced virus protein expression and progeny production followed by decreasing PCV2-induced apoptotic caspase activity, indicating the involvement of this transcription factor in induction of cell death. Taken together, these data suggest that NF-{kappa}B activation is important for PCV2 replication and contributes to virus-mediated changes in host cells. The results presented here provide a basis for understanding molecular mechanism of PCV2 infection.

  2. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2.

    PubMed

    Vető, Borbála; Bojcsuk, Dóra; Bacquet, Caroline; Kiss, Judit; Sipeki, Szabolcs; Martin, Ludovic; Buday, László; Bálint, Bálint L; Arányi, Tamás

    2017-01-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression.

  3. The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

    PubMed Central

    Bacquet, Caroline; Kiss, Judit; Sipeki, Szabolcs; Martin, Ludovic; Buday, László; Bálint, Bálint L.; Arányi, Tamás

    2017-01-01

    Hepatocyte nuclear factor 4 alpha (HNF4α) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4α is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4α. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4α. However, based on our previous results we hypothesized that HNF4α is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4α in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4α leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4α. Accordingly, we have observed decreasing but not disappearing binding of HNF4α to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4α chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4α-dependent hepatic gene expression. PMID:28196117

  4. Activation of nuclear factor κB in colonic mucosa from patients with collagenous and ulcerative colitis

    PubMed Central

    Andresen, L; Jørgensen, V L; Perner, A; Hansen, A; Eugen-Olsen, J; Rask-Madsen, J

    2005-01-01

    Background and aims: Expression of inducible nitric oxide synthase (iNOS) is greatly upregulated in the colonic mucosa of patients with collagenous and ulcerative colitis. As the transcription factor nuclear factor κB (NFκB) is a major inducer of iNOS gene expression, we compared activation and transcriptional activity of NFκB in colonic mucosal biopsies from these patients. Patients: Eight patients with collagenous colitis, six with relapsing ulcerative colitis, and eight with uninflamed bowel were studied. Methods: NFκB DNA binding activity was assessed by electrophoretic mobility shift assay and inhibitor of NFκB (IκB) kinase (IKK) activity by immunocomplex kinase assay. In vivo recruitment of NFκB to the iNOS promoter was determined by chromatin immunoprecipitation analysis and transcriptional activity by NFκB gene expression profiling arrays. Cells showing NFκB activation were identified by immunohistochemistry. Results: In collagenous and ulcerative colitis, as opposed to uninflamed bowel, IKKβ activity and strong NFκB DNA binding gave rise to activation of identical NFκB subunits and recruitment of transcriptionally active p65 to the iNOS promoter. In collagenous colitis, activated NFκB was observed only in epithelial cells while up to 10% of lamina propria macrophages showed activation in ulcerative colitis. Conclusions: In collagenous and ulcerative colitis, colonic mucosal NFκB is activated and recruited to the iNOS promoter in vivo via an IKKβ mediated pathway. As collagenous colitis is not associated with tissue injury, these data challenge the prevailing view that activation of NFκB per se mediates tissue injury. Our results suggest that downstream inflammatory reactions leading to tissue damage originate in lamina propria immune cells, as increased NFκB activity in collagenous colitis was localised solely in epithelial cells, but present also in macrophages in ulcerative colitis. PMID:15753535

  5. Selective inhibition of nuclear factor-kappaB activation after hypoxia/ischemia in neonatal rats is not neuroprotective.

    PubMed

    van den Tweel, Evelyn R W; Kavelaars, Annemieke; Lombardi, Maria S; Groenendaal, Floris; May, Michael; Heijnen, Cobi J; van Bel, Frank

    2006-02-01

    Activated nuclear factor-kappaB (NFkappaB) has been shown to increase transcription of several genes that could potentially contribute to neuronal damage, such as proinflammatory cytokines, chemokines, and inducible nitric oxide synthase. The aim of our study was to investigate whether inhibition of NFkappaB activation could prevent hypoxia/ischemia (HI)-induced cerebral damage in neonatal rats. We used a cell permeable peptide (NEMO binding domain [NBD] peptide) that is known to prevent the association of the regulatory protein NEMO with IKK, the kinase that activates NFkappaB. Via this mechanism, the NBD peptide can specifically block the activation of NFkappaB, without inhibiting basal NFkappaB activity. Cerebral HI was induced in neonatal rats by occlusion of the right carotid artery followed by 90 min of hypoxia (Fio(2) = 0.08). Immediately upon reoxygenation, as well as 6 and 12 h later, rats were treated with vehicle or NBD peptide (20 mg/kg i.p.). Histologic analysis of brain damage was performed at 6 wk after HI. To assess NFkappaB activation, electromobility shift assays (EMSAs) were performed on brain nuclear extracts obtained 6 h after reoxygenation. Increased NFkappaB activity could be shown at 6 h after HI in both hemispheres. Peripheral administration of NBD peptide prevented this HI-induced increase in NFkappaB activity in both hemispheres. Histologic analysis of long-term cerebral damage revealed that inhibition of NFkappaB activation by administration of NBD peptide at 0, 6, and 12 h after HI resulted in an increment of neuronal damage. In conclusion, our data suggest that inhibition of NFkappaB activation using NBD peptide early after HI increases brain damage in neonatal rats.

  6. Suppression of tumor necrosis factor-α-induced nuclear factor κB activation and aromatase activity by capsaicin and its analog capsazepine.

    PubMed

    Luqman, Suaib; Meena, Abha; Marler, Laura E; Kondratyuk, Tamara P; Pezzuto, John M

    2011-11-01

    Target-specific drugs, including natural products, offer promise for the amelioration of cancer and other human ailments. Capsaicin, the pungent ingredient present in chilies (Capsicum annuum L.), and capsazepine, a synthetic analog of capsaicin (collectively referred to as vanilloids), are known to possess a variety of pharmacological and physiological properties. In our continuous effort to discover and characterize cancer chemopreventive agents from natural products, we investigated the effect of vanilloids on nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activation using stably transfected 293/NFκB-Luc human embryonic kidney cells induced by treatment with tumor necrosis factor-α (TNFα) and on aromatase activity. Capsaicin and capsazepine blocked TNFα-induced NFκB activation in a dose-dependent manner with 50% inhibitory concentration (IC(50)) values of 0.68 and 4.2 μM, respectively. No significant cytotoxicity was observed at the highest concentrations tested (53.1 μM for capsazepine and 65.5 μM for capsaicin). In addition, these vanilloids inhibited aromatase activity with IC(50) values of 13.6 and 8.8 μM, respectively. Computer-aided molecular docking studies showed docking scores indicative of good binding affinity of vanilloids with aromatase and NFκB. The highly conserved residues for capsaicin and capsazepine binding with NFκB p50 were Ser299 and Ile278 (H-bond 2.81Å) and with NFκB p100 were Ser6, Arg82, Val86, Arg90 (H-bond 2.89Å), Gly4, and Ser2 (H-bond 2.81Å). The amino acids Trp224, Arg435, and Val373 (H-bond 2.80Å) were found to be important for the binding of capsaicin and capsazepine with aromatase. Based on these findings, aromatase and NFκB are suggested as valid targets for these compounds; additional investigation of chemopreventive or chemotherapeutic potential is required.

  7. Monitoring international nuclear activity

    SciTech Connect

    Firestone, R.B.

    2006-05-19

    The LBNL Table of Isotopes website provides primary nuclearinformation to>150,000 different users annually. We have developedthe covert technology to identify users by IP address and country todetermine the kinds of nuclear information they are retrieving. Wepropose to develop pattern recognition software to provide an earlywarning system to identify Unusual nuclear activity by country or regionSpecific nuclear/radioactive material interests We have monitored nuclearinformation for over two years and provide this information to the FBIand LLNL. Intelligence is gleaned from the website log files. Thisproposal would expand our reporting capabilities.

  8. Improved efficacy of soluble human receptor activator of nuclear factor kappa B (RANK) fusion protein by site-directed mutagenesis.

    PubMed

    Son, Young Jun; Han, Jihye; Lee, Jae Yeon; Kim, HaHyung; Chun, Taehoon

    2015-06-01

    Soluble human receptor activator of nuclear factor kappa B fusion immunoglobulin (hRANK-Ig) has been considered as one of the therapeutic agents to treat osteoporosis or diseases associated with bone destruction by blocking the interaction between RANK and the receptor activator of nuclear factor kappa B ligand (RANKL). However, no scientific record showing critical amino acid residues within the structural interface between the human RANKL and RANK complex is yet available. In this study, we produced several mutants of hRANK-Ig by replacement of amino acid residue(s) and tested whether the mutants had increased binding affinity to human RANKL. Based on the results from flow cytometry and surface plasmon resonance analyses, the replacement of E(125) with D(125), or E(125) and C(127) with D(125) and F(127) within loop 3 of cysteine-rich domain 3 of hRANK-Ig increases binding affinity to human RANKL over the wild-type hRANK-Ig. This result may provide the first example of improvement in the efficacy of hRANK-Ig by protein engineering and may give additional information to understand a more defined structural interface between hRANK and RANKL.

  9. Cynaropicrin from Cynara scolymus L. suppresses photoaging of skin by inhibiting the transcription activity of nuclear factor-kappa B.

    PubMed

    Tanaka, Yuka Tsuda; Tanaka, Kiyotaka; Kojima, Hiroyuki; Hamada, Tomoji; Masutani, Teruaki; Tsuboi, Makoto; Akao, Yukihiro

    2013-01-15

    Aging of skin is characterized by skin wrinkling, laxity, and pigmentation induced by several environmental stress factors. Histological changes during the photoaging of skin include hyperproliferation of keratinocytes and melanocytes causing skin wrinkles and pigmentation. Nuclear factor kappa B (NF-κB) is one of the representative transcription factors active in conjunction with inflammation. NF-κB is activated by stimulation such as ultraviolet rays and inflammatory cytokines and induces the expression of various genes such as those of basic fibroblast growth factor (bFGF) and matrix metalloprotease-1 (MMP-1). We screened several plant extracts for their possible inhibitory effect on the transcriptional activity of NF-κB. One of them, an extract from Cynara scolymus L., showed a greatest effect on the suppression of NF-κB transactivation. As a result, we found that cynaropicrin, which is a sesquiterpene lactone, inhibited the NF-κB-mediated transactivation of bFGF and MMP-1. Furthermore, it was confirmed that in an in vivo mouse model cynaropicrin prevented skin photoaging processes leading to the hyperproliferation of keratinocytes and melanocytes. These findings taken together indicate that cynaropicrin is an effective antiphotoaging agent that acts by inhibiting NF-κB-mediated transactivation.

  10. Induction of hepatic cyclooxygenase-2 by hyperhomocysteinemia via nuclear factor-kappaB activation.

    PubMed

    Wu, Nan; Siow, Yaw L; O, Karmin

    2009-10-01

    Hyperhomocysteinemia, an elevation of blood homocysteine (Hcy), is a metabolic disorder associated with dysfunction of multiple organs. Apart from endothelial dysfunction, Hcy can cause hepatic lipid accumulation and liver injury. However, the mechanism responsible for Hcy-induced liver injury is poorly understood. The aim of this study was to investigate the regulation of cyclooxygenase-2 (COX-2), a proinflammatory factor, expression in the liver during the initial phase of hyperhomocysteinemia. Sprague-Dawley rats were fed a high-methionine diet for 1 or 4 wk. Serum and liver concentrations of Hcy were significantly elevated after 1 or 4 wk of dietary treatment. COX-2 mRNA and protein levels were significantly elevated in the liver of hyperhomocysteinemic rats. The induction of COX-2 expression was more prominent in 1-wk hyperhomocysteinemic rats than that in the 4-wk group. EMSA revealed an activation of NF-kappaB in the same liver tissue in which COX-2 was induced. Administration of a NF-kappaB inhibitor to hyperhomocysteinemic rats effectively abolished hepatic COX-2 expression, inhibited the formation of inflammatory foci, and improved liver function. Further investigation revealed that oxidative stress due to increased superoxide generation was responsible for increased phosphorylation and degradation of IkappaBalpha leading to NF-kappaB activation in the liver. Administration of 4-hydroxy-tetramethyl-piperidine-1-oxyl, an SOD mimetic, to hyperhomocysteinemic rats not only inhibited NF-kappaB activation but also prevented hepatic COX-2 induction and improved liver function. These results suggest that hyperhomocysteinemia-induced COX-2 expression is mediated via NF-kappaB activation. Increased oxidative stress and inflammatory response may contribute to liver injury associated with hyperhomocysteinemia.

  11. Identification of Novel Small Molecule Activators of Nuclear Factor-κB With Neuroprotective Action Via High-Throughput Screening

    PubMed Central

    Manuvakhova, Marina S.; Johnson, Guyla G.; White, Misti C.; Ananthan, Subramaniam; Sosa, Melinda; Maddox, Clinton; McKellip, Sara; Rasmussen, Lynn; Wennerberg, Krister; Hobrath, Judith V.; White, E. Lucile; Maddry, Joseph A.; Grimaldi, Maurizio

    2012-01-01

    Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity. PMID:21046675

  12. Activation of Keap1/Nrf2 signaling pathway by nuclear epidermal growth factor receptor in cancer cells

    PubMed Central

    Huo, Longfei; Li, Chia-Wei; Huang, Tzu-Hsuan; Lam, Yung Carmen; Xia, Weiya; Tu, Chun; Chang, Wei-Chao; Hsu, Jennifer L; Lee, Dung-Fang; Nie, Lei; Yamaguchi, Hirohito; Wang, Yan; Lang, Jingyu; Li, Long-Yuan; Chen, Chung-Hsuan; Mishra, Lopa; Hung, Mien-Chie

    2014-01-01

    Nuclear translocation of EGFR has been shown to be important for tumor cell growth, survival, and therapeutic resistance. Previously, we detected the association of EGFR with Keap1 in the nucleus. Keap1 is a Kelch-like ECH-associated protein, which plays an important role in cellular response to chemical and oxidative stress by regulating Nrf2 protein stability and nuclear translocation. In this study, we investigate the role of EGFR in regulating Keap1/Nrf2 cascade in the nucleus and provide evidence to show that nuclear EGFR interacts with and phosphorylates nuclear Keap1 to reduce its nuclear protein level. The reduction of nuclear Keap1 consequently stabilizes nuclear Nrf2 and increases its transcriptional activity in cancer cells, which contributes to tumor cell resistance to chemotherapy. PMID:25628777

  13. Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-kB, Calcium Signaling, and Calcineurin

    PubMed Central

    Jin, Shunqian; Orabi, Abrahim I.; Le, Tianming; Javed, Tanveer A.; Sah, Swati; Eisses, John F.; Bottino, Rita; Molkentin, Jeffery D.; Husain, Sohail Z.

    2015-01-01

    Background & Aims Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice. Methods We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque) and measured intracellular release of Ca2+, calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ−/− mice were also used. This experiment was also performed in mice given infusions of AAV6-NF-κB-luciferase, to assess activation of this transcription factor in vivo. Results Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca2+ signaling, along with activation of the transcription factors NF-κB and NFAT. Suppressing Ca2+ signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice; this was observed by live imaging of mice given infusions of AAV6- NF-kB-luciferase. Conclusions Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca2+ signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis

  14. Antioxidant alpha-lipoic acid inhibits osteoclast differentiation by reducing nuclear factor-kappaB DNA binding and prevents in vivo bone resorption induced by receptor activator of nuclear factor-kappaB ligand and tumor necrosis factor-alpha.

    PubMed

    Kim, Hyon Jong; Chang, Eun-Ju; Kim, Hyun-Man; Lee, Seung Bok; Kim, Hyun-Duck; Su Kim, Ghi; Kim, Hong-Hee

    2006-05-01

    The relationship between oxidative stress and bone mineral density or osteoporosis has recently been reported. As bone loss occurring in osteoporosis and inflammatory diseases is primarily due to increases in osteoclast number, reactive oxygen species (ROS) may be relevant to osteoclast differentiation, which requires receptor activator of nuclear factor-kappaB ligand (RANKL). Tumor necrosis factor-alpha (TNF-alpha) frequently present in inflammatory conditions has a profound synergy with RANKL in osteoclastogenesis. In this study, we investigated the effects of alpha-lipoic acid (alpha-LA), a strong antioxidant clinically used for some time, on osteoclast differentiation and bone resorption. At concentrations showing no growth inhibition, alpha-LA potently suppressed osteoclastogenesis from bone marrow-derived precursor cells driven either by a high-dose RANKL alone or by a low-dose RANKL plus TNF-alpha (RANKL/TNF-alpha). alpha-LA abolished ROS elevation by RANKL or RANKL/TNF-alpha and inhibited NF-kappaB activation in osteoclast precursor cells. Specifically, alpha-LA reduced DNA binding of NF-kappaB but did not inhibit IKK activation. Furthermore, alpha-LA greatly suppressed in vivo bone loss induced by RANKL or TNF-alpha in a calvarial remodeling model. Therefore, our data provide evidence that ROS plays an important role in osteoclast differentiation through NF-kappaB regulation and the antioxidant alpha-lipoic acid has a therapeutic potential for bone erosive diseases.

  15. Hepatocyte nuclear factor 1α downregulates HBV gene expression and replication by activating the NF-κB signaling pathway

    PubMed Central

    Shen, Zhongliang; Liu, Yanfeng; Wang, Wei; Tao, Shuai; Cui, Xiaoxian; Liu, Jing

    2017-01-01

    The role of hepatocyte nuclear factor 1α (HNF1α) in the regulation of gene expression and replication of hepatitis B virus (HBV) is not fully understood. Previous reports have documented the induction of the expression of viral large surface protein (LHBs) by HNF1α through activating viral Sp1 promoter. Large amount of LHBs can block the secretion of hepatitis B surface antigen (HBsAg). Here we found that HNF1α overexpression inhibited HBV gene expression and replication in Huh7 cells, resulting in marked decreases in HBsAg, hepatitis B e antigen (HBeAg) and virion productions. In contrast, knockdown of endogenous HNF1α expression enhanced viral gene expression and replication. This HNF1α-mediated inhibition did not depend on LHBs. Instead, HNF1α promoted the expression of NF-κB p65 and slowed p65 protein degradation, leading to nuclear accumulation of p65 and activation of the NF-κB signaling, which in turn inhibited HBV gene expression and replication. The inhibitor of the NF-κB signaling, IκBα-SR, could abrogate this HNF1α-mediated inhibition. While the dimerization domain of HNF1α was dispensable for the induction of LHBs expression, all the domains of HNF1α was required for the inhibition of HBV gene expression. Our findings identify a novel role of HNF1α in the regulation of HBV gene expression and replication. PMID:28319127

  16. Tyrosol ameliorates lipopolysaccharide-induced ocular inflammation in rats via inhibition of nuclear factor (NF)-κB activation

    PubMed Central

    SATO, Kazuaki; MIHARA, Yuko; KANAI, Kazutaka; YAMASHITA, Yohei; KIMURA, Yuya; ITOH, Naoyuki

    2016-01-01

    We evaluated the anti-inflammatory effect of tyrosol (Tyr) on endotoxin-induced uveitis (EIU) in rats. EIU was induced in male Lewis rats by subcutaneous injection of lipopolysaccharide (LPS). Tyr (10, 50 or 100 mg/kg) was intravenously injected 2 hr before, simultaneously and 2 hr after LPS injection. The aqueous humor (AqH) was collected 24 hr after LPS injection; the infiltrating cell number, protein concentration, and tumor necrosis factor (TNF)-α, prostaglandin (PG)-E2 and nitric oxide (NO) levels were determined. Histopathologic examination and immunohistochemical studies for nuclear factor (NF)-κB, inhibitor of κB (IκB)-α, cyclooxygenase (COX)-2 and inducible NO synthase (iNOS) in the iris–ciliary body (ICB) were performed at 3 or 24 hr after LPS injection. To further clarify the anti-inflammatory effects, RAW264.7 macrophages were stimulated with LPS in the presence or absence of Tyr. Tyr reduced, in a dose-dependent manner, the infiltrating cell number, protein concentration, and TNF-α, PGE2 and NO levels in AqH and improved histopathologic scores of EIU. Tyr also inhibited LPS-induced COX-2 and iNOS expression, IκB-α degradation and nuclear translocation of activated NF-κB in ICB. Tyr significantly suppressed inflammatory mediator production in the culture medium and COX-2 and iNOS expression and activated NF-κB translocation in LPS-stimulated RAW264.7 cells. These results suggest that Tyr suppresses ocular inflammation of EIU by inhibiting NF-κB activation and subsequent proinflammatory mediator production. PMID:27238160

  17. Pterostilbene attenuates high glucose-induced oxidative injury in hippocampal neuronal cells by activating nuclear factor erythroid 2-related factor 2.

    PubMed

    Yang, Yang; Fan, Chongxi; Wang, Bodong; Ma, Zhiqiang; Wang, Dongjin; Gong, Bing; Di, Shouyin; Jiang, Shuai; Li, Yue; Li, Tian; Yang, Zhi; Luo, Erping

    2017-04-01

    In the present study, neuroblastoma (SH-SY5Y) cells were used to investigate the mechanisms mediating the potential protective effects of pterostilbene (PTE) against mitochondrial metabolic impairment and oxidative stress induced by hyperglycemia for mimicking the diabetic encephalopathy. High glucose medium (100mM) decreased cellular viability after 24h incubation which was evidenced by: (i) reduced mitochondrial complex I and III activities; (ii) reduced mitochondrial cytochrome C; (iii) increased reactive oxygen species (ROS) generation; (iv) decreased mitochondrial membrane potential (ΔΨm); and (v) increased lactate dehydrogenase (LDH) levels. PTE (2.5, 5, and 10μM for 24h) was nontoxic and induced the nuclear transition of Nrf2. Pretreatment of PTE (2.5, 5, and 10μM for 2h) displayed a dose-dependently neuroprotective effect, as indicated by significantly prevented high glucose-induced loss of cellular viability, generation of ROS, reduced mitochondrial complex I and III activities, reduced mitochondrial cytochrome C, decreased ΔΨm, and increased LDH levels. Moreover, the levels of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and glutathione S-transferase (GST) were elevated after PTE treatment. In addition, the elevation of nuclear Nrf2 by PTE treatment (10μM for 2h) was abolished by Nrf2 siRNA. Importantly, Nrf2 siRNA induced the opposite changes in mitochondrial complex I and III activities, mitochondrial cytochrome C, reactive species generation, ΔΨm, and LDH. Overall, the present findings were the first to show that pterostilbene attenuated high glucose-induced central nervous system injury in vitro through the activation of Nrf2 signaling, displaying protective effects against mitochondrial dysfunction-derived oxidative stress.

  18. Factors Limiting Microbial Growth and Activity at a Proposed High-Level Nuclear Repository, Yucca Mountain, Nevada

    PubMed Central

    Kieft, T. L.; Kovacik, W. P.; Ringelberg, D. B.; White, D. C.; Haldeman, D. L.; Amy, P. S.; Hersman, L. E.

    1997-01-01

    As part of the characterization of Yucca Mountain, Nev., as a potential repository for high-level nuclear waste, volcanic tuff was analyzed for microbial abundance and activity. Tuff was collected aseptically from nine sites along a tunnel in Yucca Mountain. Microbial abundance was generally low: direct microscopic cell counts were near detection limits at all sites (3.2 x 10(sup4) to 2.0 x 10(sup5) cells g(sup-1) [dry weight]); plate counts of aerobic heterotrophs ranged from 1.0 x 10(sup1) to 3.2 x 10(sup3) CFU g(sup-1) (dry weight). Phospholipid fatty acid concentrations (0.1 to 3.7 pmol g(sup-1)) also indicated low microbial biomasses; diglyceride fatty acid concentrations, indicative of dead cells, were in a similar range (0.2 to 2.3 pmol g(sup-1)). Potential microbial activity was quantified as (sup14)CO(inf2) production in microcosms containing radiolabeled substrates (glucose, acetate, and glutamic acid); amendments with water and nutrient solutions (N and P) were used to test factors potentially limiting this activity. Similarly, the potential for microbial growth and the factors limiting growth were determined by performing plate counts before and after incubating volcanic tuff samples for 24 h under various conditions: ambient moisture, water-amended, and amended with various nutrient solutions (N, P, and organic C). A high potential for microbial activity was demonstrated by high rates of substrate mineralization (as much as 70% of added organic C in 3 weeks). Water was the major limiting factor to growth and microbial activity, while amendments with N and P resulted in little further stimulation. Organic C amendments stimulated growth more than water alone. PMID:16535670

  19. Imaging of nuclear factor κB activation induced by ionizing radiation in human embryonic kidney (HEK) cells.

    PubMed

    Chishti, Arif Ali; Baumstark-Khan, Christa; Hellweg, Christine E; Reitz, Günther

    2014-08-01

    Ionizing radiation modulates several signaling pathways resulting in transcription factor activation. Nuclear factor kappa B (NF-κB) is one of the most important transcription factors that respond to changes in the environment of a mammalian cell. NF-κB plays a key role not only in inflammation and immune regulation but also in cellular radiation response. In response to DNA damage, NF-κB might inhibit apoptosis and promote carcinogenesis. Our previous studies showed that ionizing radiation is very effective in inducing biological damages. Therefore, it is important to understand the radiation-induced NF-κB signaling cascade. The current study aims to improve existing mammalian cell-based reporter assays for NF-κB activation by the use of DD-tdTomato which is a destabilized variant of red fluorescent protein tdTomato. It is demonstrated that exposure of recombinant human embryonic kidney cells (HEK/293 transfected with a reporter constructs containing NF-κB binding sites in its promoter) to ionizing radiation induces NF-κB-dependent DD-tdTomato expression. Using this reporter assays, NF-κB signaling in mammalian cells was monitored by flow cytometry and fluorescence microscopy. Activation of NF-κB by the canonical pathway was found to be quicker than by the genotoxin- and stress-induced pathway. X-rays activate NF-κB in HEK cells in a dose-dependent manner, and the extent of NF-κB activation is higher as compared to camptothecin.

  20. Stably transfected human cell lines as fluorescent screening assay for nuclear factor KB activation dependent gene expression

    NASA Astrophysics Data System (ADS)

    Hellweg, Christine E.; Baumstark-Khan, Christa; Horneck, Gerda

    2004-06-01

    Activation of the Nuclear Factor kappaB (NF-kappaB) pathway as a possible antiapoptotic route represents one important cellular stress response. For identifying conditions which are capable to modify this pathway, a screening assay for detection of NF-kappaB-dependent gene activation using the reporter proteins Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) has been developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing four copies of the NF-kappaB response element. Treatment with tumor necrosis factor alpha (TNF-alpha) gave rise to substantial EGFP / d2EGFP expression in up to 90 % of the cells and was therefore used to screen different stably transfected clones for induction of NF-kappaB dependent gene expression. The time course of d2EGFP expression after treatment with TNF-alpha or phorbol ester was measured using flow cytometry. Cellular response to TNF-alpha was faster than to phorbol ester. Treatment of cells with TNF-alpha and DMSO revealed antagonistic interactions of these substances in the activation NF-kappaB dependent gene expression. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening.

  1. Oxidative stress causes hypertension and activation of nuclear factor-κB after high-fructose and salt treatments

    PubMed Central

    Dornas, Waleska C.; Cardoso, Leonardo M.; Silva, Maísa; Machado, Natália L. S.; Chianca-Jr., Deoclécio A.; Alzamora, Andréia C.; Lima, Wanderson G.; Lagente, Vincent; Silva, Marcelo E.

    2017-01-01

    There is evidence that diets rich in salt or simple sugars as fructose are associated with abnormalities in blood pressure regulation. However, the mechanisms underlying pathogenesis of salt- and fructose-induced kidney damage and/or consequent hypertension yet remain largely unexplored. Here, we tested the role of oxidative state as an essential factor along with high salt and fructose treatment in causing hypertension. Fischer male rats were supplemented with a high-fructose diet (20% in water) for 20 weeks and maintained on high-salt diet (8%) associate in the last 10 weeks. Fructose-fed rats exhibited a salt-dependent hypertension accompanied by decrease in renal superoxide dismutase activity, which is the first footprint of antioxidant inactivation by reactive oxygen species (ROS). Metabolic changes and the hypertensive effect of the combined fructose-salt diet (20 weeks) were markedly reversed by a superoxide scavenger, Tempol (10 mg/kg, gavage); moreover, Tempol (50 mM) potentially reduced ROS production and abolished nuclear factor-kappa B (NF-κB) activation in human embryonic kidney HEK293 cells incubated with L-fructose (30 mM) and NaCl (500 mosmol/kg added). Taken together, our data suggested a possible role of oxygen radicals and ROS-induced activation of NF-κB in the fructose- and salt-induced hypertension associated with the progression of the renal disease.

  2. Nuclear factor κB–inducing kinase activation as a mechanism of pancreatic β cell failure in obesity

    PubMed Central

    Malle, Elisabeth K.; Zammit, Nathan W.; Walters, Stacey N.; Koay, Yen Chin; Wu, Jianmin; Tan, Bernice M.; Villanueva, Jeanette E.; Brink, Robert; Loudovaris, Tom; Cantley, James; McAlpine, Shelli R.; Hesselson, Daniel

    2015-01-01

    The nuclear factor κB (NF-κB) pathway is a master regulator of inflammatory processes and is implicated in insulin resistance and pancreatic β cell dysfunction in the metabolic syndrome. Whereas canonical NF-κB signaling is well studied, there is little information on the divergent noncanonical NF-κB pathway in the context of pancreatic islet dysfunction. Here, we demonstrate that pharmacological activation of the noncanonical NF-κB–inducing kinase (NIK) disrupts glucose homeostasis in zebrafish in vivo. We identify NIK as a critical negative regulator of β cell function, as pharmacological NIK activation results in impaired glucose-stimulated insulin secretion in mouse and human islets. NIK levels are elevated in pancreatic islets isolated from diet-induced obese (DIO) mice, which exhibit increased processing of noncanonical NF-κB components p100 to p52, and accumulation of RelB. TNF and receptor activator of NF-κB ligand (RANKL), two ligands associated with diabetes, induce NIK in islets. Mice with constitutive β cell–intrinsic NIK activation present impaired insulin secretion with DIO. NIK activation triggers the noncanonical NF-κB transcriptional network to induce genes identified in human type 2 diabetes genome-wide association studies linked to β cell failure. These studies reveal that NIK contributes a central mechanism for β cell failure in diet-induced obesity. PMID:26122662

  3. The role of constitutive nitric-oxide synthase in ultraviolet B light-induced nuclear factor κB activity.

    PubMed

    Tong, Lingying; Wu, Shiyong

    2014-09-19

    NF-κB is a transcription factor involved in many signaling pathways that also plays an important role in UV-induced skin tumorigenesis. UV radiation can activate NF-κB, but the detailed mechanism remains unclear. In this study, we provided evidence that the activation of constitutive nitric-oxide synthase plays a role in regulation of IκB reduction and NF-κB activation in human keratinocyte HaCaT cells in early phase (within 6 h) post-UVB. Treating the cells with l-NAME, a selective inhibitor of constitutive nitric-oxide synthase (cNOS), can partially reverse the IκB reduction and inhibit the DNA binding activity as well as nuclear translocation of NF-κB after UVB radiation. A luciferase reporter assay indicates that UVB-induced NF-κB activation is totally diminished in cNOS null cells. The cNOS-mediated reduction of IκB is likely due to the imbalance of nitric oxide/peroxynitrite because treating the cells with lower (50 μm), but not higher (100-500 μm), concentration of S-nitroso-N-acetylpenicillamine (SNAP) can reverse the effect of l-NAME in partial restore IκB level post-UVB. Our data also showed that NF-κB activity was required for maintaining a stable IκB kinase α subunit (IKKα) level because treating the cells with NF-κB or cNOS inhibitors could reduce IKKα level upon UVB radiation. In addition, our data demonstrated that although NF-κB protects cells from UVB-induced death, its pro-survival activity was likely neutralized by the pro-death activity of peroxynitrite after UVB radiation.

  4. Activation of nuclear transcription factor-kappaB in mouse brain induced by a simulated microgravity environment

    NASA Technical Reports Server (NTRS)

    Wise, Kimberly C.; Manna, Sunil K.; Yamauchi, Keiko; Ramesh, Vani; Wilson, Bobby L.; Thomas, Renard L.; Sarkar, Shubhashish; Kulkarni, Anil D.; Pellis, Neil R.; Ramesh, Govindarajan T.

    2005-01-01

    Microgravity induces inflammatory responses and modulates immune functions that may increase oxidative stress. Exposure to a microgravity environment induces adverse neurological effects; however, there is little research exploring the etiology of these effects resulting from exposure to such an environment. It is also known that spaceflight is associated with increase in oxidative stress; however, this phenomenon has not been reproduced in land-based simulated microgravity models. In this study, an attempt has been made to show the induction of reactive oxygen species (ROS) in mice brain, using ground-based microgravity simulator. Increased ROS was observed in brain stem and frontal cortex with concomitant decrease in glutathione, on exposing mice to simulated microgravity for 7 d. Oxidative stress-induced activation of nuclear factor-kappaB was observed in all the regions of the brain. Moreover, mitogen-activated protein kinase kinase was phosphorylated equally in all regions of the brain exposed to simulated microgravity. These results suggest that exposure of brain to simulated microgravity can induce expression of certain transcription factors, and these have been earlier argued to be oxidative stress dependent.

  5. Time dependency and topography of hepatic nuclear factor κB activation after hemorrhagic shock and resuscitation in mice.

    PubMed

    Korff, Sebastian; Falsafi, Reza; Czerny, Christoph; Jobin, Christian; Nau, Christoph; Jakob, Heike; Marzi, Ingo; Lehnert, Mark

    2012-11-01

    The leading causes of death in people aged 1 to 44 years are unintentional injuries with associated hemorrhagic shock. Hemorrhagic shock followed by resuscitation (H/R) activates the nuclear factor κB (NF-κB) pathway. To further address the association between liver damage and NF-κB activation, we analyzed the H/R-induced activation of NF-κB using cis-NF-κB reporter gene mice. In these mice, the expression of green fluorescent protein (GFP) is linked to the activation of NF-κB, and therefore tracing of GFP colocalizes NF-κB activation. Mice were hemorrhaged to a mean arterial blood pressure of 30mmHg for 90 min, followed by resuscitation. Six, 14, or 24 h after resuscitation, mice were killed. Compared with sham-operated mice, H/R led to a profound hepatic and cellular damage as measured by aspartate aminotransferase, creatine kinase, and lactate dehydrogenase levels, which was accompanied by an elevation in interleukin 6 levels and hepatic leukocyte infiltration. Interleukin 10 levels in plasma were elevated 6 h after H/R. Using serial liver sections, we found an association between necrotic areas, oxidative stress, and enhanced GFP-positive cells. Furthermore, enhanced GFP-positive cells surrounded areas of necrotic liver tissue, predominantly in a penumbra-like-shape pericentrally. These results elucidate spatial relationship between oxidative stress, liver necrosis, and NF-κB activation, using an in vivo approach and therefore might help to further analyze mechanisms of NF-κB activation after resuscitated blood loss.

  6. Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotrophin-mediated gene expression.

    PubMed

    Groth, Rachel D; Mermelstein, Paul G

    2003-09-03

    A member of the neurotrophin family, brain-derived neurotrophic factor (BDNF) regulates neuronal survival and differentiation during development. Within the adult brain, BDNF is also important in neuronal adaptive processes, such as the activity-dependent plasticity that underlies learning and memory. These long-term changes in synaptic strength are mediated through alterations in gene expression. However, many of the mechanisms by which BDNF is linked to transcriptional and translational regulation remain unknown. Recently, the transcription factor NFATc4 (nuclear factor of activated T-cells isoform 4) was discovered in neurons, where it is believed to play an important role in long-term changes in neuronal function. Interestingly, NFATc4 is particularly sensitive to the second messenger systems activated by BDNF. Thus, we hypothesized that NFAT-dependent transcription may be an important mediator of BDNF-induced plasticity. In cultured rat CA3-CA1 hippocampal neurons, BDNF activated NFAT-dependent transcription via TrkB receptors. Inhibition of calcineurin blocked BDNF-induced nuclear translocation of NFATc4, thus preventing transcription. Further, phospholipase C was a critical signaling intermediate between BDNF activation of TrkB and the initiation of NFAT-dependent transcription. Both inositol 1,4,5-triphosphate (IP3)-mediated release of calcium from intracellular stores and activation of protein kinase C were required for BDNF-induced NFAT-dependent transcription. Finally, increased expression of IP3 receptor 1 and BDNF after neuronal exposure to BDNF was linked to NFAT-dependent transcription. These results suggest that NFATc4 plays a crucial role in neurotrophin-mediated synaptic plasticity.

  7. Nuclear factor-κB activity in T cells from patients with rheumatic diseases: A preliminary report

    PubMed Central

    Collantes, E.; Blazquez, M; Mazorra, V.; Macho, A.; Aranda, E.; Munoz, E.

    1998-01-01

    OBJECTIVE—The NF-κB/Rel family of transcription factors regulates the expression of many genes involved in the immune or inflammatory response at the transcriptional level. The aim of this study was to determine whether distinctive patterns of NF-kB activation are seen in different forms of joint disease.
METHODS—The DNA binding activity of these nucleoproteins was examined in purified synovial and peripheral T cells from patients with various chronic rheumatic diseases (12: four with rheumatoid arthritis; five with spondyloarthropathies; and three with osteoarthritis).
RESULTS—Electrophoretic mobility shift assays disclosed two specific complexes bound to a NF-κB specific 32P-labelled oligonucleotide in nucleoproteins extracted from purified T cells isolated from synovial fluid and peripheral blood of patients with rheumatoid arthritis. The complexes consisted of p50/p50 homodimers and p50/p65 heterodimers. Increased NF-kB binding to DNA in synovial T cells was observed relative to peripheral T cells. In non-rheumatoid arthritis, binding of NF-κB in synovial T cells was exclusively mediated by p50/p50 homodimers.
CONCLUSION—Overall, the results suggest that NF-κB may play a central part in the activation of infiltrating T cells in chronic rheumatoid arthritis. The activation of this nuclear factor is qualitatively different in rheumatoid synovial T cells to that in other forms of non-rheumatoid arthritis (for example, osteoarthritis, spondyloarthropathies).

 Keywords: NF-κB; synovial T cells; rheumatoid arthritis; spondyloarthropathy; osteoarthritis PMID:10070274

  8. Nuclear Factor Erythroid 2-Related Factor 2 Drives Podocyte-Specific Expression of Peroxisome Proliferator-Activated Receptor γ Essential for Resistance to Crescentic GN

    PubMed Central

    Bollee, Guillaume; Lenoir, Olivia; Dhaun, Neeraj; Camus, Marine; Chipont, Anna; Flosseau, Kathleen; Mandet, Chantal; Yamamoto, Masayuki; Karras, Alexandre; Thervet, Eric; Bruneval, Patrick; Nochy, Dominique; Mesnard, Laurent

    2016-01-01

    Necrotizing and crescentic rapidly progressive GN (RPGN) is a life-threatening syndrome characterized by a rapid loss of renal function. Evidence suggests that podocyte expression of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) may prevent podocyte injury, but the function of glomerular PPARγ in acute, severe inflammatory GN is unknown. Here, we observed marked loss of PPARγ abundance and transcriptional activity in glomerular podocytes in experimental RPGN. Blunted expression of PPARγ in podocyte nuclei was also found in kidneys from patients diagnosed with crescentic GN. Podocyte-specific Pparγ gene targeting accentuated glomerular damage, with increased urinary loss of albumin and severe kidney failure. Furthermore, a PPARγ gain-of-function approach achieved by systemic administration of thiazolidinedione (TZD) failed to prevent severe RPGN in mice with podocyte-specific Pparγ gene deficiency. In nuclear factor erythroid 2-related factor 2 (NRF2)–deficient mice, loss of podocyte PPARγ was observed at baseline. NRF2 deficiency markedly aggravated the course of RPGN, an effect that was partially prevented by TZD administration. Furthermore, delayed administration of TZD, initiated after the onset of RPGN, still alleviated the severity of experimental RPGN. These findings establish a requirement for the NRF2–PPARγ cascade in podocytes, and we suggest that these transcription factors have a role in augmenting the tolerance of glomeruli to severe immune-complex mediated injury. The NRF2–PPARγ pathway may be a therapeutic target for RPGN. PMID:25999406

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

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  10. Phytochemicals of Aristolochia tagala and Curcuma caesia exert anticancer effect by tumor necrosis factor-α-mediated decrease in nuclear factor kappaB binding activity

    PubMed Central

    Hadem, Khetbadei Lysinia Hynniewta; Sharan, Rajeshwar Nath; Kma, Lakhan

    2015-01-01

    Rationale: The active compounds or metabolites of herbal plants exert a definite physiological action on the human body and thus are widely used in human therapy for various diseases including cancer. Previous studies by our group have reported the anticarcinogenic properties of the two herbal plants extracts (HPE) of Aristolochia tagala (AT) Cham. and Curcuma caesia (CC) Roxb. in diethylnitrosamine-induced mouse liver cancer in vivo. The anticarcinogenic properties of these extracts may be due to the active compounds present in them. Objectives: Our objective was to analyze the phytochemical constituents present in AT and CC, to assay their antioxidant properties and to determine their role in a possible intervention on tumor progression. Materials and Methods: Qualitative and quantitative analysis of constituent with anticancer properties present in the crude methanol extract of the two plants CC and AT was carried out following standard methods. Separation of the phytochemical compounds was done by open column chromatography. The extracts were eluted out with gradients of chloroform-methanol solvents. Ultraviolet-visible spectra of individual fractions were recorded, and the fractions were combined based on their λmax. The free radical scavenging activity of crude extracts and fractions obtained was also determined; the radical scavenging activity was expressed as IC50. High-performance thin layer chromatography (HPTLC) analysis of fractionated compounds was carried out to identify partially the phytochemical compounds. The anti-inflammatory and anticancer activity of AT and CC extracts was studied in DEN induced BALB/c mice by analyzing the tumor necrosis factor-α (TNF-α) levels in serum and the nuclear factor kappaB (NF-κB) binding activity in nuclear extracts of the liver. Results: It was observed that both AT and CC contained compounds such as phenolics, tannins, flavonoids, terpenoids, etc., and both extracts exhibited antioxidant capacity. HPTLC

  11. A Systems Biology Approach to Link Nuclear Factor Kappa B Activation with Lethal Prostate Cancer

    DTIC Science & Technology

    2012-05-01

    progression of prostate cancer to a lethal disease . We aim to identify patients with lethal prostate cancer using a systems biology approach focused on...activation which are associated with lethal disease . (Months 1 to 18) Task 1A: Perform gene profiling of tumors and determine whether a set of genes and...panel to be assessed for correlation with lethal disease . (Month 1 to 18) Accomplishments: In the first 12 months of the grant we have (i

  12. Nuclear Factor-Kappa B Activity in the Host-Tumor Microenvironment of Ovarian Cancer

    DTIC Science & Technology

    2012-08-01

    Thymoquinone, macrophages 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF RESPONSIBLE PERSON USAMRMC...from ascites fluid or peritoneal lavages. Quantification of raw NF-B reporter activity indicated that there was a time-dependent increase (Fig 5D...relative proportion of M2 to M1 macrophages changes in response to tumor cells. We would anticipate such an increase, but are currently limited by

  13. Expression of receptor activator of nuclear factor-κB ligand by B cells in response to oral bacteria

    PubMed Central

    Han, X.; Lin, X.; Seliger, A. R.; Eastcott, J.; Kawai, T.; Taubman, M. A.

    2009-01-01

    Introduction We investigated receptor activator of nuclear factor-κB ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Methods Expression of messenger RNA transcripts (tumor necrosis factor-α, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription–polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. Results The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans-immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans-immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. Conclusion This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease. PMID

  14. Antrodia camphorata suppresses lipopolysaccharide-induced nuclear factor-kappaB activation in transgenic mice evaluated by bioluminescence imaging.

    PubMed

    Hseu, You-Cheng; Huang, Hui-Chi; Hsiang, Chien-Yun

    2010-01-01

    In an earlier study, we found that Antrodia camphorata inhibited the production of lipopolysaccharide (LPS)-induced cytokines, inducible nitric oxide synthase, and cyclooxygenase-2 by blocking nuclear factor-kappaB (NF-kappaB) activation in cultured RAW 264.7 macrophages. This study was aimed at evaluating the inhibitory effects of the fermented culture broth of A. camphorata in terms of LPS-induced NF-kappaB activation in transgenic mice by using a non-invasive, real-time NF-kappaB bioluminescence imaging technique. Transgenic mice carrying the luciferase gene under the control of NF-kappaB were given A. camphorata (570 mg/kg, p.o.) for three consecutive days and then injected with LPS (4 mg/kg, i.p.). In vivo imaging showed that treatment with LPS increased the luminescent signal, whereas A. camphorata suppressed the LPS-induced inflammatory response significantly. Ex vivo imaging showed that A. camphorata suppressed LPS-induced NF-kappaB activity in the small intestine, mesenteric lymph nodes, liver, spleen, and kidney. Immunohistochemical staining revealed that A. camphorata suppressed production of the LPS-induced tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and NF-kappaB p65 subunit in these organs. Furthermore, A. camphorata attenuated the productions of LPS-induced TNF-alpha and IL-1beta in serum from transgenic mice. We report the first confirmation of the anti-inflammatory action in vivo of this potentially beneficial mushroom.

  15. MicroRNA-200a mediates nasopharyngeal carcinoma cell proliferation through the activation of nuclear factor-κB.

    PubMed

    Shi, Zhuliang; Hu, Zhiqiang; Chen, Delu; Huang, Jie; Fan, Jie; Zhou, Subo; Wang, Xin; Hu, Jiandao; Huang, Fei

    2016-02-01

    In nasopharyngeal carcinoma (NPC), the nuclear factor-κB (NF-κB) signaling pathway is highly active. The constitutive activation of NF-κB prompts malignant cell proliferation, and microRNAs are considered an important mediator in regulating the NF-κB signaling pathway. The current study investigated the effect of microRNA-200a (miR-200a) on NF-κB activation. Reverse transcription-quantitative polymerase chain reaction was used to quantify the relative level of miR-200a in NPC tissue samples and CNE2 cells. An MTT assay was used to investigate the effect of miR-200a on cell proliferation. To investigate the activation of NF-κB, western blotting was used to measure the protein levels of NF-κB and its downstream targets. To identify the target genes of miR-200a, a luciferase reporter assay was used. The current study demonstrated that miR-200a was upregulated in NPC tissue samples and cell lines. Overexpression of miR-200a resulted in the proliferation of CNE2 cells. Western blot analysis indicated that the protein levels of p65 increased when CNE2 cells were transfected with miR-200a mimics. Additionally, the downstream targets of miR-200a were upregulated, including vascular cell adhesion molecule, intercellular adhesion molecule and monocyte chemoattractant protein-1. The luciferase assay indicated that IκBα was the target gene of miR-200a. In conclusion, miR-200a was demonstrated to enhance NPC cell proliferation by activating the NF-κB signaling pathway.

  16. Ghrelin ameliorates intestinal barrier dysfunction in experimental colitis by inhibiting the activation of nuclear factor-kappa B

    SciTech Connect

    Cheng, Jian; Zhang, Lin; Dai, Weiqi; Mao, Yuqing; Li, Sainan; Wang, Jingjie; Li, Huanqing; Guo, Chuanyong; Fan, Xiaoming

    2015-02-27

    Aim: This study aimed to investigate the effect and underlying mechanism of ghrelin on intestinal barrier dysfunction in dextran sulfate sodium (DSS)-induced colitis. Methods and results: Acute colitis was induced in C57BL/6J mice by administering 2.5% DSS. Saline or 25, 125, 250 μg/kg ghrelin was administrated intraperitoneally (IP) to mice 1 day before colitis induction and on days 4, 5, and 6 after DSS administration. IP injection of a ghrelin receptor antagonist, [D-lys{sup 3}]-GHRP-6, was performed immediately prior to ghrelin injection. Ghrelin (125 or 250 μg/kg) could reduce the disease activity index, histological score, and myeloperoxidase activities in experimental colitis, and also prevented shortening of the colon. Ghrelin could prevent the reduction of transepithelial electrical resistance and tight junction expression, and bolstered tight junction structural integrity and regulated cytokine secretion. Ultimately, ghrelin inhibited nuclear factor kappa B (NF-κB), inhibitory κB-α, myosin light chain kinase, and phosphorylated myosin light chain 2 activation. Conclusions: Ghrelin prevented the breakdown of intestinal barrier function in DSS-induced colitis. The protective effects of ghrelin on intestinal barrier function were mediated by its receptor GHSR-1a. The inhibition of NF-κB activation might be part of the mechanism underlying the effects of ghrelin that protect against barrier dysfunction. - Highlights: • Ghrelin ameliorates intestinal barrier dysfunction in experimental colitis. • The effect of ghrelin is mediated by GHSR-1a. • Inhibition of NF-κB activation.

  17. Nuclear Factor-Kappa B Activity in the Host-Tumor Microenvironment of Ovarian Cancer

    DTIC Science & Technology

    2013-08-01

    between any time points in mice receiving 8 only vehicle treatment. It is possible that the sensitivity of detection of reporter activity in...following injection of ID8-NGL cells into BL/6 mice. Control groups used were vehicle only (PBS) and mice receiving empty liposomes (EL). As shown in Figs...treatment for 4 weeks in WT mice injected with ID8- NGL cells in the Progress Report for Year 1. Mice were treated with Vehicle or TQ from day 30-60

  18. Co-Activation of Nuclear Factor-κB and Myocardin/Serum Response Factor Conveys the Hypertrophy Signal of High Insulin Levels in Cardiac Myoblasts*

    PubMed Central

    Madonna, Rosalinda; Geng, Yong-Jian; Bolli, Roberto; Rokosh, Gregg; Ferdinandy, Peter; Patterson, Cam; De Caterina, Raffaele

    2014-01-01

    Hyperinsulinemia contributes to cardiac hypertrophy and heart failure in patients with the metabolic syndrome and type 2 diabetes. Here, high circulating levels of tumor necrosis factor (TNF)-α may synergize with insulin in signaling inflammation and cardiac hypertrophy. We tested whether high insulin affects activation of TNF-α-induced NF-κB and myocardin/serum response factor (SRF) to convey hypertrophy signaling in cardiac myoblasts. In canine cardiac myoblasts, treatment with high insulin (10−8 to 10−7 m) for 0–24 h increased insulin receptor substrate (IRS)-1 phosphorylation at Ser-307, decreased protein levels of chaperone-associated ubiquitin (Ub) E3 ligase C terminus of heat shock protein 70-interacting protein (CHIP), increased SRF activity, as well as β-myosin heavy chain (MHC) and myocardin expressions. Here siRNAs to myocardin or NF-κB, as well as CHIP overexpression prevented (while siRNA-mediated CHIP disruption potentiated) high insulin-induced SR element (SRE) activation and β-MHC expression. Insulin markedly potentiated TNF-α-induced NF-κB activation. Compared with insulin alone, insulin+TNF-α increased SRF/SRE binding and β-MHC expression, which was reversed by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and by NF-κB silencing. In the hearts of db/db diabetic mice, in which Akt phosphorylation was decreased, p38MAPK, Akt1, and IRS-1 phosphorylation at Ser-307 were increased, together with myocardin expression as well as SRE and NF-κB activities. In response to high insulin, cardiac myoblasts increase the expression or the promyogenic transcription factors myocardin/SRF in a CHIP-dependent manner. Insulin potentiates TNF-α in inducing NF-κB and SRF/SRE activities. In hyperinsulinemic states, myocardin may act as a nuclear effector of insulin, promoting cardiac hypertrophy. PMID:24855642

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

    PubMed Central

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

    2017-01-01

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

  20. Bridelia ferruginea Produces Antineuroinflammatory Activity through Inhibition of Nuclear Factor-kappa B and p38 MAPK Signalling

    PubMed Central

    Olajide, Olumayokun A.; Aderogba, Mutalib A.; Okorji, Uchechukwu P.; Fiebich, Bernd L.

    2012-01-01

    Bridelia ferruginea is commonly used in traditional African medicine (TAM) for treating various inflammatory conditions. Extracts from the plant have been shown to exhibit anti-inflammatory property in a number of in vivo models. In this study the influence of B. ferruginea (BFE) on the production of PGE2, nitrite, and proinflammatory cytokines from LPS-stimulated BV-2 microglia was investigated. The effects of BFE on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions were evaluated in LPS-activated rat primary microglia. The roles of NF-κB and MAPK signalling in the actions of BFE were also investigated. BFE (25–200 μg) inhibited the production of PGE2, nitrite, tumour necrosis factor-α (TNFα), and interleukin-6 (IL-6) as well as COX-2 and iNOS protein expressions in LPS-activated microglial cells. Further studies to elucidate the mechanism of anti-inflammatory action of BFE revealed interference with nuclear translocation of NF-κBp65 through mechanisms involving inhibition of IκB degradation. BFE prevented phosphorylation of p38, but not p42/44 or JNK MAPK. It is suggested that Bridelia ferruginea produces anti-inflammatory action through mechanisms involving p38 MAPK and NF-κB signalling. PMID:23320030

  1. Physiological role of receptor activator nuclear factor-kB (RANK) in denervation-induced muscle atrophy and dysfunction

    PubMed Central

    Dufresne, Sébastien S.; Boulanger-Piette, Antoine; Bossé, Sabrina; Frenette, Jérôme

    2016-01-01

    The bone remodeling and homeostasis are mainly controlled by the receptor-activator of nuclear factor kB (RANK), its ligand RANKL, and the soluble decoy receptor osteoprotegerin (OPG) pathway. While there is a strong association between osteoporosis and skeletal muscle dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology remains elusive. Our recent article published in the American Journal of Physiology (Cell Physiology) showed that RANK is also expressed in fully differentiated C2C12 myotubes and skeletal muscles. We used the Cre-Lox approach to inactivate muscle RANK (RANKmko) and showed that RANK deletion preserves the force of denervated fast-twitch EDL muscles. However, RANK deletion had no positive impact on slow-twitch Sol muscles. In addition, denervating RANKmko EDL muscles induced an increase in the total calcium concentration ([CaT]), which was associated with a surprising decrease in SERCA activity. Interestingly, the levels of STIM-1, which mediates Ca2+ influx following the depletion of SR Ca2+ stores, were markedly higher in denervated RANKmko EDL muscles. We speculated that extracellular Ca2+ influx mediated by STIM-1 may be important for the increase in [CaT] and the gain of force in denervated RANKmko EDL muscles. Overall, these findings showed for the first time that the RANKL/RANK interaction plays a role in denervation-induced muscle atrophy and dysfunction. PMID:27547781

  2. HGF mediated upregulation of lipocalin 2 regulates MMP9 through nuclear factor-κB activation.

    PubMed

    Koh, Sung Ae; Lee, Kyung Hee

    2015-10-01

    Lipocalin 2 (LCN2) is a member of lipocalin family that binds and transports a small lipophilic ligand, sharing a highly conserved tertiary structure and can be found as a monomer, homodimer, heterodimer with matrix metalloproteinase 9 (MMP9). The high molecule LCN2/MMP9 complex was found in several cancer types. Yet, the mechanisms of regulation between LCN2 with MMP9 in tumorigenesis is unclear. The aims of the present study were to identify the function of LCN2 associated with MMP9 in gastric cancer growth and metastasis. First, we confirmed that the expression level of LCN2 and MMP9 was upregulated by hepatocyte growth factor (HGF). To identify the association pathway of HGF-induced LCN2, the cells were treated with PI3-kinase inhibitor (LY294002), or MEK inhibitor (PD098059), or p38 inhibitor (SB203580) and then analyzed using western blotting. The HGF-mediated LCN2 protein level was decreased with LY294002. Also, the HGF-mediated MMP9 was decreased with LY294002. The role for LCN2 with HGF mediated MMP9 was determined by knockdown of LCN2. LCN2-sh RNA cells showed a decreased level of HGF-mediated MMP9. The HGF-mediated LCN2 protein level was decreased with treatment of the NFκB inhibitor. We confirmed the role of HGF-mediated LCN2. HGF-mediated cell proliferation and in vitro invasion was decreased in LCN2 knockdown cell. In conclusion, the present study showed that LCN2 upregulated MMP9 through PI3K/AKT/NFκB pathway in gastric cancer. LCN2 has a role in cell proliferation and cell invasion in gastric cancer, which may be a possible target for developing gastric cancer therapy.

  3. Modulation of mitochondrial dysfunction in neurodegenerative diseases via activation of nuclear factor erythroid-2-related factor 2 by food-derived compounds.

    PubMed

    Denzer, Isabel; Münch, Gerald; Friedland, Kristina

    2016-01-01

    Oxidative stress and mitochondrial dysfunction are early events in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). Mitochondria are important key players in cellular function based on mitochondrial energy production and their major role in cell physiology. Since neurons are highly depending on mitochondrial energy production due to their high energy demand and their reduced glycolytic capacity mitochondrial dysfunction has fatal consequences for neuronal function and survival. The transcription factor nuclear factor erythroid-2-related factor 2 (Nrf2) is the major regulator of cellular response to oxidative stress. Activation of Nrf2 induces the transcriptional regulation of antioxidant response element (ARE)-dependent expression of a battery of cytoprotective and antioxidant enzymes and proteins. Moreover, activation of Nrf2 protects mitochondria from dysfunction and promotes mitochondrial biogenesis. Therefore, the Nrf2/ARE pathway has become an attractive target for the prevention and treatment of oxidative stress-related neurodegenerative diseases. Small food-derived inducers of the Nrf2/ARE pathway including l-sulforaphane from broccoli and isoliquiritigenin from licorice displayed promising protection of mitochondrial function in models of oxidative stress and neurodegenerative diseases and represent a novel approach to prevent and treat aging-associated neurodegenerative diseases.

  4. The profile of immune modulation by cannabidiol (CBD) involves deregulation of nuclear factor of activated T cells (NFAT).

    PubMed

    Kaplan, Barbara L F; Springs, Alison E B; Kaminski, Norbert E

    2008-09-15

    Cannabidiol (CBD) is a cannabinoid compound derived from Cannabis Sativa that does not possess high affinity for either the CB1 or CB2 cannabinoid receptors. Similar to other cannabinoids, we demonstrated previously that CBD suppressed interleukin-2 (IL-2) production from phorbol ester plus calcium ionophore (PMA/Io)-activated murine splenocytes. Thus, the focus of the present studies was to further characterize the effect of CBD on immune function. CBD also suppressed IL-2 and interferon-gamma (IFN-gamma) mRNA expression, proliferation, and cell surface expression of the IL-2 receptor alpha chain, CD25. While all of these observations support the fact that CBD suppresses T cell function, we now demonstrate that CBD suppressed IL-2 and IFN-gamma production in purified splenic T cells. CBD also suppressed activator protein-1 (AP-1) and nuclear factor of activated T cells (NFAT) transcriptional activity, which are critical regulators of IL-2 and IFN-gamma. Furthermore, CBD suppressed the T cell-dependent anti-sheep red blood cell immunoglobulin M antibody forming cell (anti-sRBC IgM AFC) response. Finally, using splenocytes derived from CB1(-/-)/CB2(-/-) mice, it was determined that suppression of IL-2 and IFN-gamma and suppression of the in vitro anti-sRBC IgM AFC response occurred independently of both CB1 and CB2. However, the magnitude of the immune response to sRBC was significantly depressed in CB1(-/-)/CB2(-/-) mice. Taken together, these data suggest that CBD suppresses T cell function and that CB1 and/or CB2 play a critical role in the magnitude of the in vitro anti-sRBC IgM AFC response.

  5. Astragaloside IV ameliorates acute pancreatitis in rats by inhibiting the activation of nuclear factor-κB

    PubMed Central

    QIU, LEI; YIN, GUOJIAN; CHENG, LI; FAN, YUTING; XIAO, WENQIN; YU, GE; XING, MIAO; JIA, RONGRONG; SUN, RUIQING; MA, XIUYING; HU, GUOYONG; WANG, XINGPENG; TANG, MAOCHUN; ZHAO, YAN

    2015-01-01

    This study aimed to investigate the effects of astragaloside IV (AS-IV; 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol), which has been reported to have comprehensive pharmacological functions, on sodium taurocholate (NaTc)/L-arginine (L-Arg)-induced acute pancreatitis (AP) in rats in vivo and in rat pancreatic acinar cells in vitro. NaTc-induced experimental AP was induced in rats by injecting 4% NaTc (0.1 ml/100 g) in the retrograde direction of the biliopancreatic duct. L-Arg-induced experimental AP was induced in rats by 2 intraperitoneal injections of 20% L-arg (3 g/kg), with an interval of 1 h between the injections. The rats were pre-treated AS-IV (50 mg/kg) or the vehicle (DMSO) 2 h prior to the induction of AP. Enzyme-linked immunosorbent assay, H&E staining, myeloperoxidase (MPO) activity, reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry were used to evaluate the effects of AS-IV on AP. The results revealed that treatment with AS-IV significantly reduced serum amylase and lipase levels, pancreatic pathological alterations, the secretion of pro-inflammatory cytokines, MPO activity, and the protein expression of nuclear factor-κB (NF-κB) in vivo. Moreover, pre-treatment with AS-IV significantly increased the expression levels of manganese superoxide dismutase and cuprum/zinc superoxide dismutase. In the in vitro experiment, treatment of the cells with AS-IV aslo reduced rat pancreatic acinar cell necrosis and nuclear NF-κB activity, and enhanced the protein expression of superoxide dismutase. In conclusion, this study indicates that the protective effects of AS-IV on experimental AP in rats may be closely related to the inhibition of NF-κB. In addition, our results indicate that AS-IV may exert potential antioxidant effects on AP. Therefore, AS-IV may be an effective therapeutic agent for AP. PMID:25604657

  6. Pomegranate polyphenols and extract inhibit nuclear factor of activated T-cell activity and microglial activation in vitro and in a transgenic mouse model of Alzheimer disease.

    PubMed

    Rojanathammanee, Lalida; Puig, Kendra L; Combs, Colin K

    2013-05-01

    Alzheimer disease (AD) brain is characterized by extracellular plaques of amyloid β (Aβ) peptide with reactive microglia. This study aimed to determine whether a dietary intervention could attenuate microgliosis. Memory was assessed in 12-mo-old male amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice via Barnes maze testing followed by division into either a control-fed group provided free access to normal chow and water or a treatment group provided free access to normal chow and drinking water supplemented with pomegranate extract (6.25 mL/L) for 3 mo followed by repeat Barnes maze testing for both groups. Three months of pomegranate feeding decreased the path length to escape of mice compared with their initial 12-mo values (P < 0.05) and their control-fed counterparts (P < 0.05). Brains of the 3-mo study pomegranate-fed mice had lower tumor necrosis factor α (TNF-α) concentrations (P < 0.05) and lower nuclear factor of activated T-cell (NFAT) transcriptional activity (P < 0.05) compared with controls. Brains of the 3-mo pomegranate or control mice were also compared with an additional control group of 12-mo-old mice for histologic analysis. Immunocytochemistry showed that pomegranate- but not control-fed mice had attenuated microgliosis (P < 0.05) and Aβ plaque deposition (P < 0.05) compared with 12-mo-old mice. An additional behavioral study again used 12-mo-old male APP/PS1 mice tested by T-maze followed by division into a control group provided with free access to normal chow and sugar supplemented drinking water or a treatment group provided with normal chow and pomegranate extract-supplemented drinking water (6.25 mL/L) for 1 mo followed by repeat T-maze testing in both groups. One month of pomegranate feeding increased spontaneous alternations versus control-fed mice (P < 0.05). Cell culture experiments verified that 2 polyphenol components of pomegranate extract, punicalagin and ellagic acid, attenuated NFAT activity in a reporter cell

  7. Carotenoid derivatives inhibit nuclear factor kappa B activity in bone and cancer cells by targeting key thiol groups.

    PubMed

    Linnewiel-Hermoni, Karin; Motro, Yair; Miller, Yifat; Levy, Joseph; Sharoni, Yoav

    2014-10-01

    Aberrant activation of the nuclear factor kappa B (NFkB) transcription system contributes to cancer progression, and has a harmful effect on bone health. Several major components of the NFkB pathway such as IkB Kinase (IKK) and the NFkB subunits contain cysteine residues that are critical for their activity. The interaction of electrophiles with these cysteine residues results in NFkB inhibition. Carotenoids, hydrophobic plant pigments, are devoid of electrophilic groups, and we have previously demonstrated that carotenoid derivatives, but not the native compounds activate the Nrf2 transcription system. The aim of the current study was to examine whether carotenoid derivatives inhibit NFkB, and, if so, to determine the molecular mechanism underpinning the inhibitory action. We report in the present study that a mixture of oxidized derivatives, prepared by ethanol extraction from partially oxidized lycopene preparation, inhibited NFkB reporter gene activity. In contrast, the intact carotenoid was inactive. A series of synthetic dialdehyde carotenoid derivatives inhibited reporter activity as well as several stages of the NFkB pathway in both cancer and bone cells. The activity of the carotenoid derivatives depended on the reactivity of the electrophilic groups in reactions such as Michael addition to sulfhydryl groups of proteins. Specifically, carotenoid derivatives directly interacted with two key proteins of the NFkB pathway: the IKKβ and the p65 subunit. Direct interaction with IKKβ was found in an in vitro kinase assay with a recombinant enzyme. The inhibition by carotenoid derivatives of p65 transcriptional activity was observed in a reporter gene assay performed in the presence of excess p65. This inhibition action resulted, at least in part, from direct interaction of the carotenoid derivative with p65 leading to reduced binding of the protein to DNA as evidenced by electrophoretic mobility shift assay (EMSA) experiments. Importantly, we found by using

  8. Identifying panaxynol, a natural activator of nuclear factor erythroid-2 related factor 2 (Nrf2) from American ginseng as a suppressor of inflamed macrophage-induced cardiomyocyte hypertrophy

    PubMed Central

    Qu, Chen; Li, Bin; Lai, Yimu; Li, Hechu; Windust, Anthony; Hofseth, Lorne J.; Nagarkatti, Mitzi; Nagarkatti, Prakash; Wang, Xing Li; Tang, Dongqi; Janicki, Joseph S.; Tian, Xingsong; Cui, Taixing

    2015-01-01

    Ethnopharmacological relevance American ginseng is capable of ameliorating cardiac dysfunction and activating Nrf2, a master regulator of antioxidant defense, in the heart. This study was designed to isolate compounds from American ginseng and to determine those responsible for the Nrf2-mediated resolution of inflamed macrophage-induced cardiomyocyte hypertrophy. Materials and methods A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. A bioassay-based fractionization of American ginseng was performed to identify the putative substances which could activate Nrf2-mediated suppression of pro-inflammatory cytokine expression in macrophages and macrophage-mediated pro-hypertrophic growth in cardiomyocytes. Results A hexane fraction of an anti-inflammatory crude extract of American ginseng was found to be most effective in suppressing the inflammatory responses in macrophages. Preparative, reverse-phase HPLC and a comparative analysis by analytical scale LC–UV/MS revealed the hexane fraction contains predominantly C17 polyacetylenes and linolenic acid. Panaxynol, one of the major polyacetylenes, was found to be a potent Nrf2 activator. Panaxynol posttranscriptionally activated Nrf2 by inhibiting Kelch-like ECH-associated protein (Keap) 1-mediated degradation without affecting the binding of Keap1 and Nrf2. Moreover, panaxynol suppressed a selected set of cytokine expression via the activation of Nrf2 while minimally regulating nuclear factor-kappa B (NF-κB)-mediated cytokine expression in macrophages. It also dramatically inhibited the inflamed macrophage-mediated cardiomyocyte death and hypertrophy by activating Nrf2 in macrophages. Conclusions These results demonstrate that American ginseng-derived panaxynol is a specific Nrf2 activator and panaxynol-activated Nrf2 signaling is at least partly responsible for American ginseng-induced health benefit in the heart. PMID

  9. Transforming growth factor beta 1-responsive element: closely associated binding sites for USF and CCAAT-binding transcription factor-nuclear factor I in the type 1 plasminogen activator inhibitor gene.

    PubMed Central

    Riccio, A; Pedone, P V; Lund, L R; Olesen, T; Olsen, H S; Andreasen, P A

    1992-01-01

    Transforming growth factor beta (TGF-beta) is the name of a group of closely related polypeptides characterized by a multiplicity of effects, including regulation of extracellular proteolysis and turnover of the extracellular matrix. Its cellular mechanism of action is largely unknown. TGF-beta 1 is a strong and fast inducer of type 1 plasminogen activator inhibitor gene transcription. We have identified a TGF-beta 1-responsive element in the 5'-flanking region of the human type 1 plasminogen activator inhibitor gene and shown that it is functional both in its natural context and when fused to a heterologous nonresponsive promoter. Footprinting and gel retardation experiments showed that two different nuclear factors, present in extracts from both TGF-beta 1-treated and nontreated cells, bind to adjacent sequences contained in the responsive unit. A palindromic sequence binds a trans-acting factor(s) of the CCAAT-binding transcription factor-nuclear factor I family. A partially overlapping dyad symmetry interacts with a second protein that much evidence indicates to be USF. USF is a transactivator belonging to the basic helix-loop-helix family of transcription factors. Mutations which abolish the binding of either CCAAT-binding transcription factor-nuclear factor I or USF result in reduction of transcriptional activation upon exposure to TGF-beta 1, thus showing that both elements of the unit are necessary for the TGF-beta 1 response. We discuss the possible relationship of these findings to the complexity of the TGF-beta action. Images PMID:1549130

  10. Osteonecrosis of the jaw induced by receptor activator of nuclear factor-kappa B ligand (Denosumab) - Review

    PubMed Central

    Brizeno, Luiz-André-Cavalcante; de Sousa, Fabrício-Bitu; Mota, Mário-Rogério-Lima; Alves, Ana-Paula-Negreiros-Nunes

    2016-01-01

    Background Denosumab, an anti-resorptive agent, IgG2 monoclonal antibody for human Receptor activator of nuclear factor-kappa B ligand (RANKL), has been related to the occurrence of osteonecrosis of the jaws. Thus, the aim of this study was to review the literature from clinical case reports, regarding the type of patient and the therapeutic approach used for osteonecrosis of the jaws induced by chronic use of Denosumab. Material and Methods For this, a literature review was performed on PubMed, Medline and Cochrane databases, using the keywords “Denosumab” “anti-RANK ligand” and “Osteonecrosis of jaw”. To be included, articles should be a report or a serie of clinical cases, describing patients aged 18 years or over who used denosumab therapy and have received any therapy for ONJ. Results Thirteen complete articles were selected for this review, totaling 17 clinical cases. The majority of ONJ cases, patients receiving Denosumab as treatment for osteoporosis and prostate cancer therapy. In most cases, patients affected by ONJ were women aged 60 or over and posterior mandible area was the main site of involvement. Diabetes pre-treatment with bisphosphonates and exodontia were the most often risk factors related to the occurrence of this condition. It is concluded that the highest number of ONJ cases caused by the use of anti-RANKL agents occurred in female patients, aged 60 years or older, under treatment for osteoporosis and cancer metastasis, and the most affected region was the mandible posterior. Conclusions The results presented in this article are valid tool supporting the non-invasive mapping of facial vascularization. Key words:Denosumab, osteonecrosis, adverse effects, osteoporosis, antineoplastic protocols. PMID:26827069

  11. Anti-inflammatory and analgesic effect of plumbagin through inhibition of nuclear factor-κB activation.

    PubMed

    Luo, Pei; Wong, Yuen Fan; Ge, Lin; Zhang, Zhi Feng; Liu, Yuan; Liu, Liang; Zhou, Hua

    2010-12-01

    Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) (PL) is a naturally occurring yellow pigment found in the plants of the Plumbaginaceae, Droseraceae, Ancistrocladaceae, and Dioncophyllaceae families. It has been reported that PL exhibits anticarcinogenic, anti-inflammatory, and analgesic activities. However, the mechanism underlying its anti-inflammatory action remains unknown. In the current study, we investigated and characterized the anti-inflammatory and analgesic effects of PL orally administrated in a range of dosages from 5 to 20 mg/kg. We also examined the role of nuclear factor κB (NF-κB) and proinflammatory cytokines and mediators in this effect. The results showed that PL significantly and dose-dependently suppressed the paw edema of rats induced by carrageenan and various proinflammatory mediators, including histamine, serotonin, bradykinin, and prostaglandin E(2). PL reduced the number of writhing episodes of mice induced by the intraperitoneal injection of acetic acid, but it did not reduce the writhing episode numbers induced by MgSO(4) in mice or prolong the tail-flick reaction time of rats to noxious thermal pain. Mechanistic studies showed that PL effectively decreased the production of the proinflammatory cytokines interleukin 1β, interleukin 6, and tumor necrosis factor α. It also inhibited the expression of the proinflammatory mediators inducible nitric-oxide synthase and cyclooxygenase 2, whereas it did not inhibit the expression of cyclooxygenase 1. Further studies demonstrated that PL suppressed inhibitor of κBα phosphorylation and degradation, thus inhibiting the phosphorylation of the p65 subunit of NF-κB. This study suggests that PL has a potential to be developed into an anti-inflammatory agent for treating inflammatory diseases.

  12. Mutation of isocitrate dehydrogenase 1 induces glioma cell proliferation via nuclear factor-κB activation in a hypoxia-inducible factor 1-α dependent manner.

    PubMed

    Wang, Guoliang; Sai, Ke; Gong, Fanghe; Yang, Qunying; Chen, Furong; Lin, Jian

    2014-05-01

    Recently, mutations of the isocitrate dehydrogenase (IDH) 1 gene, which specifically occur in the majority of low-grade and secondary high-grade gliomas, have drawn particular attention of neuro-oncologists. Mutations of the IDH1 gene have been proposed to have significant roles in the tumorigenesis, progression and prognosis of gliomas. However, the molecular mechanism of the role of IDH1 mutants in gliomagenesis remains to be elucidated. The present study, showed that forced expression of an IDH1 mutant, of which the 132th amino acid residue arginine is substituted by histidine (IDH1R132H), promoted cell proliferation in cultured cells, while wild-type IDH1 overexpression had no effect on cell proliferation. Consistent with previous studies, it was also observed that expression of hypoxia-inducible factor 1-α (HIF1-α) was upregulated in IDH1R132H expressing cells with the induction of vascular endothelial growth factor (VEGF) expression. However, knockdown of VEGF via small RNA interference had no significant influence on the cell proliferation induced by overexpression of IDH1R132H, implying that another signaling pathway may be involved. Next, forced expression of IDH1R132H was found to activate nuclear factor-κB (NF-κB), since the inhibitory IκB protein (IκBα) was highly phosphorylated and the NF-κB p65 subunit was translocated into the nucleus. Notably, knockdown of HIF1-α significantly blocked NF-κB activation, which was induced by the overexpression of IDH1 mutants. In addition, expression of IDH1 mutants markedly induced the NF-κB target gene expression, including cyclin D1 and E and c-myc, which were involved in the regulation of cell proliferation. In conclusion, it was demonstrated that the IDH1 mutant activated NF-κB in a HIF1-α‑dependent manner and was involved in the regulation of cell proliferation.

  13. Plant Extracts of the Family Lauraceae: A Potential Resource for Chemopreventive Agents that Activate the Nuclear Factor-Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway

    PubMed Central

    Shen, Tao; Chen, Xue-Mei; Harder, Bryan; Long, Min; Wang, Xiao-Ning; Lou, Hong-Xiang; Wondrak, Georg T.; Ren, Dong-Mei; Zhang, Donna D.

    2015-01-01

    Cells and tissues counteract insults from exogenous or endogenous carcinogens through the expression of genes encoding antioxidants and phase II detoxifying enzymes regulated by antioxidant response element promoter regions. Nuclear factor-erythroid 2-related factor 2 plays a key role in regulating the antioxidant response elements-target gene expression. Hence, the Nrf2/ARE pathway represents a vital cellular defense mechanism against damage caused by oxidative stress and xenobiotics, and is recognized as a potential molecular target for discovering chemo-preventive agents. Using a stable antioxidant response element luciferase reporter cell line derived from human breast cancer MDA-MB-231 cells combined with a 96-well high-throughput screening system, we have identified a series of plant extracts from the family Lauraceae that harbor Nrf2-inducing effects. These extracts, including Litsea garrettii (ZK-08), Cinnamomum chartophyllum (ZK-02), C. mollifolium (ZK-04), C. camphora var. linaloolifera (ZK-05), and C. burmannii (ZK-10), promoted nuclear translocation of Nrf2, enhanced protein expression of Nrf2 and its target genes, and augmented intracellular glutathione levels. Cytoprotective activity of these extracts against two electrophilic toxicants, sodium arsenite and H2O2, was investigated. Treatment of human bronchial epithelial cells with extracts of ZK-02, ZK-05, and ZK-10 significantly improved cell survival in response to sodium arsenite and H2O2, while ZK-08 showed a protective effect against only H2O2. Importantly, their protective effects against insults from both sodium arsenite and H2O2 were Nrf2-dependent. Therefore, our data provide evidence that the selected plants from the family Lauraceae are potential sources for chemopreventive agents targeting the Nrf2/ARE pathway. PMID:24585092

  14. In vitro methylation of nuclear respiratory factor-2 binding sites suppresses the promoter activity of the human TOMM70 gene.

    PubMed

    Blesa, José R; Hegde, Anita A; Hernández-Yago, José

    2008-12-31

    TOMM70 is a subunit of the outer mitochondrial membrane translocase that plays a major role as a receptor of hydrophobic preproteins targeted to mitochondria. We have previously reported that two binding sites for transcription factor NRF-2 in the promoter region of the human TOMM70 gene are essential in activating transcription (Blesa et al., Mitochondrion 2004; 3:251-59. Blesa et al., Biochem Cell Biol 2006; 84:813-22). This region contains thirteen CpG methylation sites, three of which occur in the sequence 5'-CCGG-3' that is specifically recognized by HpaII methylase which modifies the internal cytosine residue. Interestingly, each NRF-2 site contains one CCGG sequence, allowing specific methylation of the NRF-2 sites and, therefore, providing an ideal model to study how methylation of these sites affects promoter activity. In this paper we report that site-specific methylation of the NRF-2 binding sites in the TOMM70 promoter down-regulated expression of a luciferase reporter in HeLa S3 cells. Electrophoretic mobility shift assays confirmed abrogation of NRF-2 binding at the methylated sites. These results suggest that methylation of the TOMM70 promoter in mammalian cells may silence TOMM70 expression. However, studies of methylation degree on DNAs from different sources found no methylation in the promoter regions of TOMM70 and other TOMM/TIMM family genes. Thus, although in vitro methylation inactivates the expression of TOMM70, our results suggest that this is not the mechanism modulating its expression in vivo. Since a number of nuclear genes encoding mitochondrial translocases have NRF-2 binding sequences containing CpG methylation sites, a possible role of methylation as a regulatory mechanism of mitochondrial biogenesis can be ruled out.

  15. [Role of nuclear factor of activated T-cells cytoplasmic 1 on vascular calcification in rats with chronic renal failure].

    PubMed

    Zhang, J X; Xu, J S; Han, Y Y; Bai, Y L; Cui, L W; Zhang, H R; Zhang, S L

    2017-02-14

    Objective: To explore the role of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) on vascular calcification in chronic renal failure rats. Methods: Nineteen male Sprague-Dawley (SD) rats were randomly divided into three groups: sham-operated group (n=6), 5/6 Nephrectomy (Nx) group (n=6), 5/6 Nx+ calcitriol group (n=7). Vascular calcification was determined by von Kossa staining and orthocresolphthalein complexone (OCPC) method. Protein expressions of NFATc1 and runt-related transcription factor 2 (Runx2) in aortas were measured by immunohistochemistry.In vitro, vascular smooth muscle cells (VSMCs) were primarily cultured and calcification was induced by β-glycerophosphate (β-GP). These cells were then randomly divided into control group, calcification group (10 mmol/L β-GP) and cyclosporin A (CsA) intervention group (10 mmol/L β-GP+ 1 μg/ml CsA). Calcium deposition was measured by Alizarin red staining and OCPC method; alkaline phosphatase (ALP) activity was measured by enzyme-linked immunosorbent assay. RT-PCR and Western blotting were used to observe the mRNA and protein expression of VSMCs NFATc1 and Runx2 respectively. Results: Compared to that in sham-operated and 5/6 Nx group, the expression of NFATc1 was obviously up-regulated in 5/6 Nx+ calcitriol group (7.20±0.46 vs 1.52±0.77, 2.04±1.31, P<0.05). In vitro, VSMCs calcification was successfully induced by high phosphorus environment, and RT-PCR and Western blotting showed that the expressions of NFATc1 and Runx2 were up-regulated (P<0.05). The calcification level in CsA intervention group was lower than that in calcification group [(60.86±7.95) vs (107.20±11.07) mg/g, P<0.05], and expression of Runx2 (mRNA and protein level) and ALP activity [(48.63±3.02) vs (98.75±3.46) U/g, P<0.05] decreased as well. Conclusion: NFATc1 contributes to accelerating vascular calcification in rat with chronic renal failure, the possible mechanism of which is that NFATc1 promotes VSMCs transformation to

  16. Activation of nicotinamide N-methyltransferase gene promoter by hepatocyte nuclear factor-1beta in human papillary thyroid cancer cells.

    PubMed

    Xu, Jimin; Capezzone, Marco; Xu, Xiao; Hershman, Jerome M

    2005-02-01

    We previously demonstrated that the human nicotinamide N-methytransferase (NNMT) gene was highly expressed in many papillary thyroid cancers and cell lines. The expression in other papillary and follicular cancers or cell lines and normal thyroid cells was low or undetectable. To gain an understanding of the molecular mechanism of this cell-specific expression, the NNMT promoter was cloned and studied by luciferase reporter gene assay. The promoter construct was expressed highly in papillary cancer cell lines, including those with higher (e.g. BHP 2-7) and lower (e.g. BHP 14-9) NNMT gene expression, and expressed weakly in follicular thyroid cancer cell lines. Further study with 5'-deletion promoter construct suggested that the NNMT promoter was regulated differently in BHP 2-7 and BHP 14-9 cells. In BHP 2-7 cells, promoter activity was dependent on an upstream sequence. In BHP 14-9 cells, sequence in the basal promoter region contributed notably to the overall promoter activity. RT-PCR or Western blot analysis indicated that hepatocyte nuclear factor-1beta (HNF-1beta) was expressed in only papillary cancer cell lines with high NNMT gene expression. HNF-1beta was not expressed or expressed very weakly in other papillary, follicular, and Hurthle cancer cell lines and primary cultures of normal thyroid cells and benign thyroid conditions. A HNF-1 binding site was identified in the NNMT basal promoter region. Mutations in this site decreased NNMT promoter activity in the HNF-1beta-positive BHP 2-7 cells, but not in the HNF-1beta-negative BHP 14-9 cells. HNF-1beta bound to the HNF-1 site specifically as a homodimer as determined by gel retardation assays with HNF-1beta-specific antibody. Cotransfection of a HNF-1beta expression plasmid increased NNMT promoter activity significantly in both HNF-1beta-positive and -negative thyroid cancer cell lines and Hep G2 liver cancer cells. Furthermore, transient expression of HNF-1beta in BHP 14-9 cells increased endogenous NNMT

  17. Src subfamily kinases regulate nuclear export and degradation of transcription factor Nrf2 to switch off Nrf2-mediated antioxidant activation of cytoprotective gene expression.

    PubMed

    Niture, Suryakant K; Jain, Abhinav K; Shelton, Phillip M; Jaiswal, Anil K

    2011-08-19

    Nrf2 (NF-E2-related factor 2) is a nuclear transcription factor that in response to chemical and radiation stress regulates coordinated induction of a battery of cytoprotective gene expressions leading to cellular protection. In this study, we investigated the role of Src kinases in the regulation of Nrf2 and downstream signaling. siRNA-mediated inhibition of Fyn, Src, Yes, and Fgr, but not Lyn, in mouse hepatoma Hepa-1 cells, led to nuclear accumulation of Nrf2 and up-regulation of Nrf2 downstream gene expression. Mouse embryonic fibroblasts with combined deficiency of Fyn/Src/Yes/Fgr supported results from siRNA. In addition, steady-state overexpression of Fyn, Src, and Yes phosphorylated Nrf2Tyr568 that triggered nuclear export and degradation of Nrf2 and down-regulation of Nrf2 downstream gene expression. Exposure of cells to antioxidant, oxidant, or UV radiation increased nuclear import of Fyn, Src, and Yes kinases, which phosphorylated Nrf2Tyr568 resulting in nuclear export and degradation of Nrf2. Further analysis revealed that stress-activated GSK3β acted upstream to the Src kinases and phosphorylated the Src kinases, leading to their nuclear localization and Nrf2 phosphorylation. The overexpression of Src kinases in Hepa-1 cells led to decreased Nrf2, increased apoptosis, and decreased cell survival. Mouse embryonic fibroblasts deficient in Src kinases showed nuclear accumulation of Nrf2, induction of Nrf2 and downstream gene expression, reduced apoptosis, and increased cell survival. The studies together demonstrate that Src kinases play a critical role in nuclear export and degradation of Nrf2, thereby providing a negative feedback mechanism to switch off Nrf2 activation and restore normal cellular homeostasis.

  18. Calcineurin/nuclear factor of activated T cells-coupled vanilliod transient receptor potential channel 4 ca2+ sparklets stimulate airway smooth muscle cell proliferation.

    PubMed

    Zhao, Limin; Sullivan, Michelle N; Chase, Marlee; Gonzales, Albert L; Earley, Scott

    2014-06-01

    Proliferation of airway smooth muscle cells (ASMCs) contributes to the remodeling and irreversible obstruction of airways during severe asthma, but the mechanisms underlying this disease process are poorly understood. Here we tested the hypothesis that Ca(2+) influx through the vanilliod transient receptor potential channel (TRPV) 4 stimulates ASMC proliferation. We found that synthetic and endogenous TRPV4 agonists increase proliferation of primary ASMCs. Furthermore, we demonstrate that Ca(2+) influx through individual TRPV4 channels produces Ca(2+) microdomains in ASMCs, called "TRPV4 Ca(2+) sparklets." We also show that TRPV4 channels colocalize with the Ca(2+)/calmodulin-dependent protein phosphatase calcineurin in ASMCs. Activated calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors cytosolic (c) to allow nuclear translocation and activation of synthetic transcriptional pathways. We show that ASMC proliferation in response to TRPV4 activity is associated with calcineurin-dependent nuclear translocation of the NFATc3 isoform tagged with green florescent protein. Our findings suggest that Ca(2+) microdomains created by TRPV4 Ca(2+) sparklets activate calcineurin to stimulate nuclear translocation of NFAT and ASMC proliferation. These findings further suggest that inhibition of TRPV4 could diminish asthma-induced airway remodeling.

  19. Basiliolides, a class of tetracyclic C19 dilactones from Thapsia garganica, release Ca(2+) from the endoplasmic reticulum and regulate the activity of the transcription factors nuclear factor of activated T cells, nuclear factor-kappaB, and activator protein 1 in T lymphocytes.

    PubMed

    Navarrete, Carmen; Sancho, Rocío; Caballero, Francisco J; Pollastro, Federica; Fiebich, Bernd L; Sterner, Olov; Appendino, Giovanni; Muñoz, Eduardo

    2006-10-01

    Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiology. We have investigated the effects of basiliolides, a novel class of C19 dilactones isolated from Thapsia garganica, on Ca(2+) mobilization in T cells. Basiliolide A1 induced a rapid mobilization of intracellular Ca(2+) in the leukemia T-cell line Jurkat. First, a rapid calcium peak was observed and inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through store-operated calcium release-activated Ca(2+) (CRAC) channels and sensitive to inhibition by EGTA, and by the CRAC channel inhibitor N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Basiliolide A1 mobilized Ca(2+) from ER stores, but in contrast to thapsigargin, it did not induce apoptosis. Basiliolide A1 induced nuclear factor of activated T cells 1 dephosphorylation and activation that was inhibited by BTP-2 and cyclosporine A. In addition, we found that basiliolide A1 alone did not mediate IkappaBalpha degradation or RelA phosphorylation (ser536), but it synergized with phorbol 12-myristate 13-acetate to induce a complete degradation of the nuclear factor-kappaB inhibitory protein and to activate the c-Jun NH(2)-terminal kinase. Moreover, basiliolide A1 regulated both interleukin-2 and tumor necrosis factor-alpha gene expression at the transcriptional level. In basiliolide B, oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1. In contrast, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. These findings qualify these compounds as new probes to investigate intracellular calcium homeostasis.

  20. Nuclear factor-kappaB activation in axons and Schwann cells in experimental sciatic nerve injury and its role in modulating axon regeneration: studies with etanercept.

    PubMed

    Smith, Darrell; Tweed, Christopher; Fernyhough, Paul; Glazner, Gordon W

    2009-06-01

    Early inflammatory events may inhibit functional recovery after injury in both the peripheral and central nervous systems. We investigated the role of the inflammatory tumor necrosis factor/nuclear factor-kappaB (NF-kappaB) axis on events subsequent to sciatic nerve crush injury in adult rats. Electrophoretic mobility shift assays revealed that within 6 hours after crush, NF-kappaB DNA-binding activity increased significantly in a 1-cm section around the crush site. By immunofluorescence staining, there was increased nuclear localization of the NF-kappaB subunits p50 but not p65 or c-Rel in Schwann cells but no obvious inflammatory cell infiltration. In rats injected subcutaneously with etanercept, a tumor necrosis factor receptor chimera that binds free cytokine, the injury-induced rise in NF-kappaB DNA-binding activity was inhibited, and nuclear localization of p50 in Schwann cells was lowered after the injury. Axonal growth 3 days after nerve crush assessed with immunofluorescence for GAP43 demonstrated that the regeneration distance of leading axons from the site of nerve crush was greater in etanercept-treated animals than in saline-treated controls. These data indicate that tumor necrosis factor mediates rapid activation of injury-induced NF-kappaB DNA binding in Schwann cells and that these events are associated with inhibition of postinjury axonal sprouting.

  1. Death-domain associated protein-6 (DAXX) mediated apoptosis in hantavirus infection is counter-balanced by activation of interferon-stimulated nuclear transcription factors

    SciTech Connect

    Khaiboullina, Svetlana F.; Morzunov, Sergey P.; Boichuk, Sergei V.; Palotás, András; Jeor, Stephen St.; Lombardi, Vincent C.; Rizvanov, Albert A.

    2013-09-01

    Hantaviruses are negative strand RNA species that replicate predominantly in the cytoplasm. They also activate numerous cellular responses, but their involvement in nuclear processes is yet to be established. Using human umbilical vein endothelial cells (HUVECs), this study investigates the molecular finger-print of nuclear transcription factors during hantavirus infection. The viral-replication-dependent activation of pro-myelocytic leukemia protein (PML) was followed by subsequent localization in nuclear bodies (NBs). PML was also found in close proximity to activated Sp100 nuclear antigen and interferon-stimulated gene 20 kDa protein (ISG-20), but co-localization with death-domain associated protein-6 (DAXX) was not observed. These data demonstrate that hantavirus triggers PML activation and localization in NBs in the absence of DAXX-PLM-NB co-localization. The results suggest that viral infection interferes with DAXX-mediated apoptosis, and expression of interferon-activated Sp100 and ISG-20 proteins may indicate intracellular intrinsic antiviral attempts.

  2. Glycogen synthase kinase 3{beta} regulation of nuclear factor of activated T-cells isoform c1 in the vascular smooth muscle cell response to injury

    SciTech Connect

    Chow Winsion; Hou Guangpei; Bendeck, Michelle P.

    2008-10-01

    The migration and proliferation of vascular smooth muscle cells (vSMCs) are critical events in neointima formation during atherosclerosis and restenosis. The transcription factor nuclear factor of activated T-cells-isoform c1 (NFATc1) is regulated by atherogenic cytokines, and has been implicated in the migratory and proliferative responses of vSMCs through the regulation of gene expression. In T-cells, calcineurin de-phosphorylates NFATc1, leading to its nuclear import, while glycogen synthase kinase 3 {beta} (GSK3{beta}) phosphorylates NFATc1 and promotes its nuclear export. However, the relationship between NFATc1 and GSK3{beta} has not been studied during SMC migration and proliferation. We investigated this by scrape wounding vSMCs in vitro, and studying wound repair. NFATc1 protein was transiently increased, reaching a peak at 8 h after wounding. Cell fractionation and immunocytochemistry revealed that NFATc1 accumulation in the nucleus was maximal at 4 h after injury, and this was coincident with a significant 9 fold increase in transcriptional activity. Silencing NFATc1 expression with siRNA or inhibition of NFAT with cyclosporin A (CsA) attenuated wound closure by vSMCs. Phospho-GSK3{beta} (inactive) increased to a peak at 30 min after injury, preceding the nuclear accumulation of NFATc1. Overexpression of a constitutively active mutant of GSK3{beta} delayed the nuclear accumulation of NFATc1, caused a 50% decrease in NFAT transcriptional activity, and attenuated vSMC wound repair. We conclude that NFATc1 promotes the vSMC response to injury, and that inhibition of GSK3{beta} is required for the activation of NFAT during wound repair.

  3. Human Factors Research and Nuclear Safety.

    ERIC Educational Resources Information Center

    Moray, Neville P., Ed.; Huey, Beverly M., Ed.

    The Panel on Human Factors Research Needs in Nuclear Regulatory Research was formed by the National Research Council in response to a request from the Nuclear Regulatory Commission (NRC). The NRC asked the research council to conduct an 18-month study of human factors research needs for the safe operation of nuclear power plants. This report…

  4. Glycemia-dependent Nuclear Factor κB Activation Contributes to Mechanical Allodynia in Rats with Chronic Postischemia Pain

    PubMed Central

    Ross-Huot, Marie-Christine; Laferrière, André; Khorashadi, Mina; Coderre, Terence J.

    2015-01-01

    Background Ischemia-reperfusion injury causes chronic postischemia pain (CPIP), and rats with higher glycemia during ischemia-reperfusion injury exhibit increased allodynia. Glycemia-induced elevation of nuclear factor kappa-B (NFκB) may contribute to increased allodynia. Methods Glycemia during a 3 h ischemia-reperfusion injury was manipulated by: normal feeding; or normal feeding with administration of insulin; dextrose; or insulin/dextrose. In these groups, NFκB was measured in ipsilateral hind paw muscle and spinal dorsal horn by ELISA, and SN50, an NFκB inhibitor, was administered to determine its differential anti-allodynic effects depending on glycemia. Results CPIP fed/insulin rats (12.03 ± 4.9, N = 6) had less allodynia than fed, fed/insulin/dextrose and fed/dextrose rats (6.29 ± 3.37 N = 7, 4.57 ± 3.03 g, N = 6, 2.95 ± 1.10, N = 9), respectively. Compared to fed rats (0.209 ± 0.022, N = 7), NFκB in ipsilateral plantar muscles was significantly lower for fed/insulin rats and significantly higher for fed/dextrose rats (0.152 ± 0.053, N = 6; 0.240 ± 0.057, N = 7, respectively). Furthermore, NFκB in the dorsal horn of fed, fed/insulin/dextrose and fed/dextrose rats (0.293 ± 0.049, N = 6) was significantly higher than in fed/insulin animals (0.267 ± 0.037, N = 6). The anti-allodynic SN50 dose-response curves of CPIP rats in the fed/insulin/dextrose, fed/dextrose and fed conditions exhibited a rightward shift compared to the fed/insulin group. The threshold SN50 dose of CPIP fed/dextrose, fed/insulin/dextrose and fed rats (328.94 ± 92.4, 77.80 ± 44.50 and 24.89 ± 17.20, respectively) was higher than that for fed/insulin rats (4.06 ± 7.04). Conclusions NFκB was activated in a glycemia-dependent manner in CPIP rats. Hypoglycemic rats were more sensitive to SN50 than rats with higher glycemia. The finding that SN50 reduces mechanical allodynia suggests that NFκB inhibitors might be useful for treating postischemia pain. PMID:23695173

  5. Statins suppress glucose-induced plasminogen activator inhibitor-1 expression by regulating RhoA and nuclear factor-κB activities in cardiac microvascular endothelial cells.

    PubMed

    Ni, Xiao-Qing; Zhu, Jian-Hua; Yao, Ning-Hua; Qian, Juan; Yang, Xiang-Jun

    2013-01-01

    The aim of this study was to investigate the possible proinflammatory signaling pathways involved in statin inhibition of glucose-induced plasminogen activator inhibitor-1 (PAI-1) expression in cardiac microvascular endothelial cells (CMECs). Primary rat CMECs were grown in the presence of 5.7 or 23 mmol/L glucose. PAI-1 mRNA and protein expression levels were measured by realtime polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay, respectively. A pull-down assay was performed to determine RhoA activity. IκBα protein expression was measured by Western blotting, nuclear factor (NF)-κB activation was detected by electrophoretic mobility shift assay and its transcription activity was determined by a dual luciferase reporter gene assay. PAI-1 mRNA and protein expression levels were both increased with high glucose concentrations, but they were significantly suppressed by simvastatin and atorvastatin treatment (P < 0.01) and the effects were reversed by mevalonate (100 μmol/L) and geranylgeranyl pyrophosphate (10 μmol/L) but not farnesyl pyrophosphate (10 μmol/L). Such effects were similar to those of a RhoA inhibitor, C3 exoenzyme (5 μg/mL), inhibitors of RhoA kinase (ROCK), Y-27632 (10 μmol/L) and hydroxyfasudil (10 μmol/L) and an NF-κB inhibitor, BAY 11-7082 (5 μmol/L). High glucose-induced RhoA and NF-κB activations in CMECs were both significantly inhibited by statins (P < 0.01). Simvastatin and atorvastatin equally suppress high glucose-induced PAI-1 expression. These effects of statins may occur partly by regulating the RhoA/ROCK-NF-κB pathway. The multifunctional roles of statins may be particularly beneficial for patients with metabolic syndrome.

  6. Effect of Hyperketonemia (Acetoacetate) on Nuclear Factor-κB and p38 Mitogen-Activated Protein Kinase Activation Mediated Intercellular Adhesion Molecule 1 Upregulation in Endothelial Cells

    PubMed Central

    Rains, Justin L.

    2015-01-01

    Abstract Background: Hyperketonemia is a pathological condition observed in patients with type 1 diabetes and ketosis-prone diabetes (KPD), which results in increased blood levels of acetoacetate (AA) and β-hydroxybutyrate (BHB). Frequent episodes of hyperketonemia are associated with a higher incidence of vascular disease. We examined the hypothesis that hyperketonemia activates the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways that regulate intercellular adhesion molecule 1 (ICAM-1) expression in endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured with AA (0–8 mM) or BHB (0–10 mM) for 0–24 hr. Western blotting was used to determine NF-κB activation in whole-cell lysates. ICAM-1 expression was measured using flow cytometry. Results: Results show a 2.4-fold increase in NF-κB activation in cells treated with 8 mM AA compared to the control. BHB had little or no effect on NF-κB activation. Pretreatment with a reactive oxygen species (ROS) inhibitor [N-acetyl-l-cysteine (NAC)] reduced NF-κB to near-control levels. The expression of AA-induced ICAM-1 was significantly reduced when cells were pretreated with either NAC or p38 MAPK inhibitor. Conclusions: These results suggest that NF-κB and p38 MAPK mediate upregulation of ICAM-1 expression in endothelial cells exposed to elevated levels of AA, which may contribute to the development of vascular disease in diabetes. PMID:25489974

  7. PROX1 promotes human glioblastoma cell proliferation and invasion via activation of the nuclear factor-κB signaling pathway.

    PubMed

    Xu, Xuchang; Wan, Xuefeng; Wei, Xinting

    2017-02-01

    Prospero homeobox protein 1 (PROX1) is highly expressed in high-grade malignant astrocytic gliomas. However, the role of PROX1 in the pathogenesis of glioblastoma multiforme (GBM) remains unclear. The present study overexpressed PROX1 in human GBM cell lines and examined its effects on cell growth, tumorigenesis, and invasiveness. In addition, the involvement of the nuclear factor‑κB (NF‑κB) signaling pathway in the action of PROX1 was examined. It was identified that overexpression of PROX1 significantly increased the proliferation and colony formation of glioblastoma cells, compared with empty vector‑transfected controls. Furthermore, ectopic expression of PROX1 promoted the growth of GBM xenograft tumors. Western blot analysis revealed that PROX1 overexpression induced nuclear accumulation of NF‑κB p65 and upregulated the expression levels of the NF‑κB responsive genes cyclin D1 and matrix metallopeptidase 9. An NF‑κB reporter assay demonstrated that PROX1‑overexpressing glioblastoma cells had significantly greater NF‑κB‑dependent reporter activities compared with empty vector‑transfected controls. Transfection of a dominant inhibitor of κBα mutant into PROX1‑overexpressing cells significantly impaired their proliferation and invasion capacities, which was accompanied by reduced levels of nuclear NF‑κB p65. Collectively, these data indicated that PROX1 serves an oncogenic role in GBM and promotes cell proliferation and invasiveness potentially via activation of the NF‑κB signaling pathway. Therefore, PROX1 may represent a potential target for the treatment of GBM.

  8. Baicalin attenuates focal cerebral ischemic reperfusion injury through inhibition of nuclear factor {kappa}B p65 activation

    SciTech Connect

    Xue, Xia; Qu, Xian-Jun; Yang, Ying; Sheng, Xie-Huang; Cheng, Fang; Jiang, E-Nang; Wang, Jian-hua; Bu, Wen; Liu, Zhao-Ping

    2010-12-17

    Research highlights: {yields} Permanent NF-{kappa}B p65 activation contributes to the infarction after ischemia-reperfusion injury in rats. {yields} Baicalin can markedly inhibit the nuclear NF-{kappa}B p65 expression and m RNA levels after ischemia-reperfusion injury in rats. {yields} Baicalin decreased the cerebral infarction area via inhibiting the activation of nuclear NF-{kappa}B p65. -- Abstract: Baicalin is a flavonoid compound purified from plant Scutellaria baicalensis Georgi. We aimed to evaluate the neuroprotective effects of baicalin against cerebral ischemic reperfusion injury. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 24 h. Baicalin at doses of 50, 100 and 200 mg/kg was intravenously injected after ischemia onset. Twenty-four hours after reperfusion, the neurological deficit was scored and infarct volume was measured. Hematoxylin and eosin (HE) staining was performed to analyze the histopathological changes of cortex and hippocampus neurons. We examined the levels of NF-{kappa}B p65 in ischemic cortexes by Western blot analysis and RT-PCR assay. The results showed that the neurological deficit scores were significantly decreased from 2.0 {+-} 0.7 to 1.2 {+-} 0.4 and the volume of infarction was reduced by 25% after baicalin injection. Histopathological examination showed that the increase of neurons with pycnotic shape and condensed nuclear in cortex and hippocampus were not observed in baicalin treated animals. Further examination showed that NF-{kappa}B p65 in cortex was increased after ischemia reperfusion injury, indicating the molecular mechanism of ischemia reperfusion injury. The level of NF-{kappa}B p65 was decreased by 73% after baicalin treatment. These results suggest that baicalin might be useful as a potential neuroprotective agent in stroke therapy. The neuroprotective effects of baicalin may relate to inhibition of NF-{kappa}B p65.

  9. NRC-interacting factor 1 is a novel cotransducer that interacts with and regulates the activity of the nuclear hormone receptor coactivator NRC.

    PubMed

    Mahajan, Muktar A; Murray, Audrey; Samuels, Herbert H

    2002-10-01

    We previously reported the cloning and characterization of a novel nuclear hormone receptor transcriptional coactivator, which we refer to as NRC. NRC is a 2,063-amino-acid nuclear protein which contains a potent N-terminal activation domain and several C-terminal modules which interact with CBP and ligand-bound nuclear hormone receptors as well as c-Fos and c-Jun. In this study we sought to clone and identify novel factors that interact with NRC to modulate its transcriptional activity. Here we describe the cloning and characterization of a novel protein we refer to as NIF-1 (NRC-interacting factor 1). NIF-1 was cloned from rat pituitary and human cell lines and was found to interact in vivo and in vitro with NRC. NIF-1 is a 1,342-amino-acid nuclear protein containing a number of conserved domains, including six Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif. Zinc fingers 1 to 3 are potential DNA-binding BED finger domains recently proposed to play a role in altering local chromatin architecture. We mapped the interaction domains of NRC and NIF-1. Although NIF-1 does not directly interact with nuclear receptors, it markedly enhances ligand-dependent transcriptional activation by nuclear hormone receptors in vivo as well as activation by c-Fos and c-Jun. These results, and the finding that NIF-1 interacts with NRC in vivo, suggest that NIF-1 functions to regulate transcriptional activation through NRC. We suggest that NIF-1, and factors which associate with coactivators but not receptors, be referred to as cotransducers, which act in vivo either as part of a coactivator complex or downstream of a coactivator complex to modulate transcriptional activity. Our findings suggest that NIF-1 may be a functional component of an NRC complex and acts as a regulator or cotransducer of NRC function.

  10. The role of serum osteoprotegerin and receptor-activator of nuclear factor-κB ligand in metabolic bone disease of women after obesity surgery.

    PubMed

    Balsa, José A; Lafuente, Christian; Gómez-Martín, Jesús M; Galindo, Julio; Peromingo, Roberto; García-Moreno, Francisca; Rodriguez-Velasco, Gloria; Martínez-Botas, Javier; Gómez-Coronado, Diego; Escobar-Morreale, Héctor F; Botella-Carretero, José I

    2016-11-01

    Metabolic bone disease may appear as a complication of obesity surgery. Because an imbalance in the osteoprotegerin and receptor-activator of nuclear factor-κB ligand system may underlie osteoporosis, we aimed to study this system in humans in the metabolic bone disease occurring after obesity surgery. In this study we included sixty women with a mean age of 47 ± 10 years studied 7 ± 2 years after bariatric surgery. The variables studied were bone mineral density, β-isomer of C-terminal telopeptide of type I collagen cross-links (a bone resorption marker), the bone formation markers osteocalcin and N-terminal propeptide of procollagen 1, serum osteoprotegerin and receptor-activator of nuclear factor-κB ligand. Serum osteoprotegerin inversely correlated with the bone remodeling markers osteocalcin, β-isomer of C-terminal telopeptide of type I collagen cross-links and N-terminal propeptide of procollagen 1. The osteoprotegerin and receptor-activator of nuclear factor-κB ligand ratio also correlated inversely with serum parathormone and osteocalcin. Bone mineral density at the lumbar spine was associated with age (β = -0.235, P = 0.046), percentage of weight loss (β = 0.421, P = 0.001) and osteoprotegerin and receptor-activator of nuclear factor-κB ligand ratio (β = 0.259, P = 0.029) in stepwise multivariate analysis (R (2) = 0.29, F = 7.49, P < 0.001). Bone mineral density at the hip site was associated only with percentage of weight loss (β = 0.464, P < 0.001) in stepwise multivariate regression (R (2) = 0.21, F = 15.1, P < 0.001). These data show that the osteoprotegerin and receptor-activator of nuclear factor-κB ligand system is associated with bone markers and bone mineral density at the lumbar spine after obesity surgery.

  11. The effect of ex vivo CDDO-Me activation on nuclear factor erythroid 2-related factor 2 pathway in white blood cells from patients with septic shock.

    PubMed

    Noel, Sanjeev; Zheng, Laura; Navas-Acien, Ana; Fuchs, Ralph J

    2014-11-01

    Nuclear factor erythroid 2-related factor 2 (NRF2) has been shown to protect against experimental sepsis in mice and lipopolysaccharide (LPS)-induced inflammation in ex vivo white blood cells from healthy subjects by upregulating cellular antioxidant genes. The objective of this study was to test the hypothesis that ex vivo methyl 2-cyano-3,12-dioxoolean-1,9-dien-28-oate (CDDO-Me) activates NRF2-regulated antioxidant genes in white blood cells from patients with septic shock and protects against LPS-induced inflammation and reactive oxidative species production. Peripheral blood was collected from 18 patients with septic shock who were being treated in medical and surgical intensive care units. Real-time polymerase chain reaction was used to quantify the expression of NRF2 target genes (NQO1, HO-1, GCLM, and FTL) and IL-6 in peripheral blood mononuclear cells (PBMCs), monocytes, and neutrophils after CDDO-Me treatment alone or after subsequent LPS exposure. Superoxide anion (O2) was measured to assess the effect of CDDO-Me pretreatment on subsequent LPS exposure. Treatment with CDDO-Me increased the gene expression of NQO1 (P = 0.04) and decreased the expression of HO-1 (P = 0.03) in PBMCs from patients with septic shock. Purified monocytes exhibited significant increases in the expression of NQO1 (P = 0.01) and GCLM (P = 0.003) after CDDO-Me treatment. Levels of other NRF2 target genes (HO-1 and FTL) remained similar to those of vehicle-treated cells. Peripheral blood mononuclear cells showed a trend toward increased IL-6 gene expression after CDDO-Me treatment, whereas purified monocytes showed a trend toward decreased IL-6. There was no discernible trend in the IL-6 expression subsequent to LPS treatment in either vehicle-treated or CDDO-Me-treated PBMCs and monocytes. Treatment with CDDO-Me significantly increased O2 production in PBMCs (P = 0.04). Although CDDO-Me pretreatment significantly attenuated O2 production to subsequent LPS exposure (P = 0.03), the

  12. Analysis and Quantitation of NF-[kappa]B Nuclear Translocation in Tumor Necrosis Factor Alpha (TNF-[alpha]) Activated Vascular Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Fuseler, John W.; Merrill, Dana M.; Rogers, Jennifer A.; Grisham, Matthew B.; Wolf, Robert E.

    2006-07-01

    Nuclear factor kappa B (NF-[kappa]B) is a heterodimeric transcription factor typically composed of p50 and p65 subunits and is a pleiotropic regulator of various inflammatory and immune responses. In quiescent cells, p50/p65 dimers are sequestered in the cytoplasm bound to its inhibitors, the I-[kappa]Bs, which prevent entry into the nucleus. Following cellular stimulation, the I-[kappa]Bs are rapidly degraded, activating NF-[kappa]B. The active form of NF-[kappa]B rapidly translocates into the nucleus, binding to consensus sequences in the promoter/enhancer region of various genes, promoting their transcription. In human vascular endothelial cells activated with tumor necrosis factor-alpha, the activation and translocation of NF-[kappa]B is rapid, reaching maximal nuclear localization by 30 min. In this study, the appearance of NF-[kappa]B (p65 subunit, p65-NF-[kappa]B) in the nucleus visualized by immunofluorescence and quantified by morphometric image analysis (integrated optical density, IOD) is compared to the appearance of activated p65-NF-[kappa]B protein in the nucleus determined biochemically. The appearance of p65-NF-[kappa]B in the nucleus measured by fluorescence image analysis and biochemically express a linear correlation (R2 = 0.9477). These data suggest that localization and relative protein concentrations of NF-[kappa]B can be reliably determined from IOD measurements of the immunofluorescent labeled protein.

  13. Effects of anti-IgM suppression on polyclonally activated murine B cells: analysis of immunoglobulin mRNA, gene specific nuclear factors and cell cycle distribution.

    PubMed Central

    Marcuzzi, A; Van Ness, B; Rouse, T; Lafrenz, D

    1989-01-01

    Polyclonal activation of murine B cells with bacterial lipopolysaccharide (LPS) and dextran sulfate (DxS) results in cell proliferation as well as increased immunoglobulin gene transcription and antibody secretion. When added to B cell cultures during mitogen activation, anti-mu antibody suppresses the rate of proliferation and immunoglobulin gene expression. Using this model system we show that co-cultures of B cells with LPS/DxS and anti-mu resulted in a decrease of both mu and kappa chain mRNA. Suppression did not prevent B cell entry into cycle nor a significant alteration in the distribution of cells in phases of cell cycle, although it did prolong the cycle transit time in a dose dependent fashion as determined by bromodeoxyuridine pulse labelling. Analysis of B cell specific nuclear binding factors, which previously have been shown to be important in regulating immunoglobulin gene transcription were examined. Results show that the kappa-specific enhancer binding activity of NF-kappa B was induced in activated as well as suppressed cultures. The lymphoid specific factor NF-A2, which recognizes the octamer sequence motif in the promoters of immunoglobulin genes, was induced by the polyclonal activation but was selectively lost in extracts from suppressed cells. Thus, specific regulation of the nuclear factor which plays a critical role in both heavy and light chain immunoglobulin gene expression may contribute to the transcriptional suppression observed in anti-mu treated B cells. Images PMID:2481271

  14. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

    PubMed Central

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais ML; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-01-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells. PMID:19183502

  15. Cocaine induces cell death and activates the transcription nuclear factor kappa-B in PC12 cells.

    PubMed

    Lepsch, Lucilia B; Munhoz, Carolina D; Kawamoto, Elisa M; Yshii, Lidia M; Lima, Larissa S; Curi-Boaventura, Maria F; Salgado, Thais M L; Curi, Rui; Planeta, Cleopatra S; Scavone, Cristoforo

    2009-02-01

    Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-kappaB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-kappaB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-kappaB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-kappaB activation. Inhibition of NF-kappaB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-kappaB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

  16. AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

    SciTech Connect

    Adam, Tasneem; Opie, Lionel H.; Essop, M. Faadiel

    2010-07-30

    Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

  17. Ca2+/calmodulin-dependent kinase II contributes to inhibitor of nuclear factor-kappa B kinase complex activation in Helicobacter pylori infection.

    PubMed

    Maubach, Gunter; Sokolova, Olga; Wolfien, Markus; Rothkötter, Hermann-Josef; Naumann, Michael

    2013-09-15

    Helicobacter pylori, a class I carcinogen, induces a proinflammatory response by activating the transcription factor nuclear factor-kappa B (NF-κB) in gastric epithelial cells. This inflammatory condition could lead to chronic gastritis, which is epidemiologically and biologically linked to the development of gastric cancer. So far, there exists no clear knowledge on how H. pylori induces the NF-κB-mediated inflammatory response. In our study, we investigated the role of Ca(2+) /calmodulin-dependent kinase II (CAMKII), calmodulin, protein kinases C (PKCs) and the CARMA3-Bcl10-MALT1 (CBM) complex in conjunction with H. pylori-induced activation of NF-κB via the inhibitor of nuclear factor-kappa B kinase (IKK) complex. We use specific inhibitors and/or RNA interference to assess the contribution of these components. Our results show that CAMKII and calmodulin contribute to IKK complex activation and thus to the induction of NF-κB in response to H. pylori infection, but not in response to TNF-α. Thus, our findings are specific for H. pylori infected cells. Neither the PKCs α, δ, θ, nor the CBM complex itself is involved in the activation of NF-κB by H. pylori. The contribution of CAMKII and calmodulin, but not PKCs/CBM to the induction of an inflammatory response by H. pylori infection augment the understanding of the molecular mechanism involved and provide potential new disease markers for the diagnosis of gastric inflammatory diseases including gastric cancer.

  18. Activation of nuclear factor-kappaB during doxorubicin-induced apoptosis in endothelial cells and myocytes is pro-apoptotic: the role of hydrogen peroxide.

    PubMed Central

    Wang, Suwei; Kotamraju, Srigiridhar; Konorev, Eugene; Kalivendi, Shasi; Joseph, Joy; Kalyanaraman, Balaraman

    2002-01-01

    Doxorubicin (DOX) is a widely used anti-tumour drug. Cardiotoxicity is a major toxic side effect of DOX therapy. Although recent studies implicated an apoptotic pathway in DOX-induced cardiotoxicity, the mechanism of DOX-induced apoptosis remains unclear. In the present study, we investigated the role of reactive oxygen species and the nuclear transcription factor nuclear factor kappaB (NF-kappaB) during apoptosis induced by DOX in bovine aortic endothelial cells (BAECs) and adult rat cardiomyocytes. DOX-induced NF-kappaB activation is both dose- and time-dependent, as demonstrated using electrophoretic mobility-shift assay and luciferase and p65 (Rel A) nuclear-translocation assays. Addition of a cell-permeant iron metalloporphyrin significantly suppressed NF-kappaB activation and apoptosis induced by DOX. Overexpression of glutathione peroxidase, which detoxifies cellular H(2)O(2), significantly decreased DOX-induced NF-kappaB activation and apoptosis. Inhibition of DOX-induced NF-kappaB activation by a cell-permeant peptide SN50 that blocks translocation of the NF-kappaB complex into the nucleus greatly diminished DOX-induced apoptosis. Apoptosis was inhibited when IkappaB mutant vector, another NF-kappaB inhibitor, was added to DOX-treated BAECs. These results suggest that NF-kappaB activation in DOX-treated endothelial cells and myocytes is pro-apoptotic, in contrast with DOX-treated cancer cells, where NF-kappaB activation is anti-apoptotic. Removal of intracellular H(2)O(2) protects endothelial cells and myocytes from DOX-induced apoptosis, possibly by inhibiting NF-kappaB activation. These findings suggest a novel mechanism for enhancing the therapeutic efficacy of DOX. PMID:12139490

  19. Nuclear Factor of Activated T Cells and Cytokines Gene Expression of the T Cells in AIDS Patients with Immune Reconstitution Inflammatory Syndrome during Highly Active Antiretroviral Therapy

    PubMed Central

    Chen, Heling; Xie, Yirui; Su, Junwei; Huang, Ying; Xu, Lijun; Yin, Michael; Zhou, Qihui

    2017-01-01

    Background. The etiology of immune reconstitution inflammatory syndrome (IRIS) in AIDS patients after the initiation of HAART remains unknown. Several researches indicated that the development of IRIS is associated with the production and variation of cytokines, whose gene expression are closely related to the Ca2+/CN-nuclear factor of activated T cells (NFAT) pathway. Methods. We studied the expression of NFAT isoforms and their major target cytokines genes in peripheral blood CD3+ T cells of subjects through fluorescence quantitative PCR and explored the expression changes of these genes before and after HAART. Results. After the initiation of HARRT, NFAT1, IL-6, and IL-8 gene expression showed a reversal trend in the CD3+ T cells of the IRIS group and changed from low expression before HARRT to high expression after HARRT. In particular, the relative gene expression of NFAT1 was markedly higher compared with the other three isoforms. The IRIS group also showed higher NFAT4, NFAT2, NFAT1, IL-1β, IL-10, IL-2, IL-18, and TNF-α gene expression than the non-IRIS group. Conclusion. This study suggested that high expression levels of IL-2, IL-6, IL-8, TNF-α, IL-1β, IL-10, IL-12, and IL-18 can predict the risk of IRIS. The increased expression of NFAT1 and NFAT4 may promote the expression of cytokines, such as IL-6, IL-8, and TNF-α, which may promote the occurrence of IRIS. PMID:28316373

  20. Teaching Activities on Horizontal Nuclear Proliferation.

    ERIC Educational Resources Information Center

    Zola, John

    1990-01-01

    Provides learning activities concerning the horizontal proliferation of nuclear weapons. Includes step-by-step directions for four activities: (1) the life cycle of nuclear weapons; (2) nuclear nonproliferation: pros and cons; (3) the nuclear power/nuclear weapons connection; and (4) managing nuclear proliferation. (NL)

  1. Ethanol induces rapid lipid peroxidation and activation of nuclear factor-kappa B in cerebral vascular smooth muscle: relation to alcohol-induced brain injury in rats.

    PubMed

    Altura, Burton M; Gebrewold, Asefa; Zhang, Aimin; Altura, Bella T

    2002-06-07

    The present study was designed to test the hypothesis that acute administration of alcohol (ethanol) to primary cultured cerebral vascular smooth muscle cells will cause lipid peroxidation, inhibition of IkappaB phosphorylation, and inhibition of nuclear transcription factor-kappa B (NF-kappaB). Ethanol (10, 25, 100 mM) resulted in concentration-dependent rises in malondialdehyde in as little as 30-45 min after exposure to the alcohol, rising to levels 2.5-10x normal after 18-24 h. Using EMSA assays and specific antibodies, ethanol caused three DNA-binding proteins (p50, p65, c-Rel) to rise in nuclear extracts in a concentration-dependent manner. Using a rabbit antibody, IkappaB phosphorylation (and degradation) was stimulated by ethanol (in a concentration-dependent manner) and inhibited by a low concentration of the NF-kappaB inhibitor, pyrrolidine dithiocarbamate. These new biochemical and molecular data indicate that ethanol, even in physiologic concentrations, can elicit rapid lipid peroxidation and activation of NF-kappaB in cerebral vascular muscle cells. The present results when viewed in light of other recently published data suggest that ethanol-induced lipid peroxidation and activation of nuclear transcription factors probably play important roles in alcohol-induced brain-vascular damage, neurobehavioral actions and stroke.

  2. Role of mitogen-activated protein kinases and nuclear factor-kappa B in 1,3-dichloro-2-propanol-induced hepatic injury

    PubMed Central

    Lee, In-Chul; Lee, Sang-Min; Ko, Je-Won; Park, Sung-Hyeuk; Shin, In-Sik; Moon, Changjong; Kim, Sung-Ho

    2016-01-01

    In this study, the potential hepatotoxicity of 1,3-dichloro-2-propanol and its hepatotoxic mechanisms in rats was investigated. The test chemical was administered orally to male rats at 0, 27.5, 55, and 110 mg/kg body weight. 1,3-Dichloro-2-propanol administration caused acute hepatotoxicity, as evidenced by an increase in serum aminotransferases, total cholesterol, and total bilirubin levels and a decrease in serum glucose concentration in a dose-dependent manner with corresponding histopathological changes in the hepatic tissues. The significant increase in malondialdehyde content and the significant decrease in glutathione content and antioxidant enzyme activities indicated that 1,3-dichloro-2-propanol-induced hepatic damage was mediated through oxidative stress, which caused a dose-dependent increase of hepatocellular apoptotic changes in the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and immunohistochemical analysis for caspase-3. The phosphorylation of mitogen-activated protein kinases caused by 1,3-dichloro-2-propanol possibly involved in hepatocellular apoptotic changes in rat liver. Furthermore, 1,3-dichloro-2-propanol induced an inflammatory response through activation of nuclear factor-kappa B signaling that coincided with the induction of pro-inflammatory mediators or cytokines in a dose-dependent manner. Taken together, these results demonstrate that hepatotoxicity may be related to oxidative stress-mediated activation of mitogen-activated protein kinases and nuclear factor-kappa B-mediated inflammatory response. PMID:27051440

  3. MicroRNA-19a mediates gastric carcinoma cell proliferation through the activation of nuclear factor-κB.

    PubMed

    Yang, Fan; Wang, Hongjian; Jiang, Zhenyu; Hu, Anxiang; Chu, Lisha; Sun, Yiling; Han, Junqing

    2015-10-01

    In gastric carcinoma, the nuclear factor‑κB (NF‑κB) signaling pathway is highly active, and the constitutive activation of NF‑κB prompts malignant cell proliferation. MicroRNAs are considered to be important mediators in the regulation of the NF‑κB signaling pathway. The present study predominantly focussed on the effects of microRNA (miR)‑19a on NF‑κB activation. Reverse transcription‑quantitative polymerase chain reaction was used to quantify the relative levels of miR‑19a in gastric carcinoma cells. MTT assays were used to determine the effect of miR‑19a on cellular proliferation. To detect the activation of NF‑κB, western blotting was performed to measure the protein levels of NF‑κB and the products of its downstream target genes. To define the target genes, luciferase reporter assays were used. miR‑19a was found to be markedly upregulated in gastric carcinoma cells. The overexpression of miR‑19a resulted in proliferation and enhanced migratory capabilities of the MGC‑803 gastric carcinoma cell line. The results of the western blot analysis demonstrated that the protein levels of p65 increased when the MGC‑803 cells were transfected with miR‑19a mimics. In addition, the downstream target genes of miR‑19a, including intercellular adhesion molecule, vascular cell adhesion molecule and monocyte chemoattractant protein‑1, were upregulated. The results of the luciferase assay indicated that IκB‑α was the target gene of miR‑19a. Therefore, the results of the present study suggested that miR‑19a enhances malignant gastric cell proliferation by constitutively activating the NF‑κB signaling pathway.

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

    PubMed Central

    1992-01-01

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

  5. Extracellular matrix metalloproteinase inducer (CD147) is a novel receptor on platelets, activates platelets, and augments nuclear factor kappaB-dependent inflammation in monocytes.

    PubMed

    Schmidt, Roland; Bültmann, Andreas; Fischel, Sina; Gillitzer, Angelika; Cullen, Paul; Walch, Axel; Jost, Philipp; Ungerer, Martin; Tolley, Neal D; Lindemann, Stephan; Gawaz, Meinrad; Schömig, Albert; May, Andreas E

    2008-02-15

    In atherosclerosis, circulating platelets interact with endothelial cells and monocytes, leading to cell activation and enhanced recruitment of leukocytes into the vascular wall. The invasion of monocytes is accompanied by overexpression of matrix metalloproteinases (MMPs), which are thought to promote atherosclerosis and trigger plaque rupture. Following interaction with itself, the extracellular matrix metalloproteinase inducer (EMMPRIN) induces MMP synthesis via a little-known intracellular pathway. Recently, we showed upregulation of EMMPRIN on monocytes during acute myocardial infarction. EMMPRIN also stimulates secretion of MMP-9 by monocytes and of MMP-2 by smooth muscle cells, indicating that it may be an important regulator of MMP activity. Expression of EMMPRIN on platelets has not been described until now. Here, we demonstrate that resting platelets show low surface expression of EMMPRIN, which is upregulated by various platelet stimulators (flow cytometry). EMMPRIN is located in the open canalicular system and in alpha granules of platelets (according to electron microscopy and sucrose gradient ultracentrifugation). Platelet stimulation with recombinant EMMPRIN-Fc induced surface expression of CD40L and P-selectin (according to flow cytometry), suggesting that EMMPRIN-EMMPRIN interaction activates platelets. Coincubation of platelets with monocytes induced EMMPRIN-mediated nuclear factor kappaB activation (according to Western blot) in monocytes with increased MMP-9 (zymography), interleukin-6, and tumor necrosis factor-alpha secretion (according to ELISA) by monocytes. In conclusion, EMMPRIN displays a new platelet receptor that is upregulated on activated platelets. Binding of EMMPRIN to platelets fosters platelet degranulation. Platelet-monocyte interactions via EMMPRIN stimulate nuclear factor kappaB-driven inflammatory pathways in monocytes, such as MMP and cytokine induction. Thus, EMMPRIN may represent a novel target to diminish the burden of

  6. Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4α dimerisation and transcriptional activity

    PubMed Central

    Eeckhoute, Jérôme; Oxombre, Bénédicte; Formstecher, Pierre; Lefebvre, Philippe; Laine, Bernard

    2003-01-01

    Hepatocyte Nuclear Factor 4α (HNF4α, NR2A1) is central to hepatocyte and pancreatic β-cell functions. Along with retinoid X receptor α (RXRα), HNF4α belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRα H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4α H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4α LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4α transcriptional activity by a dominant-negative HNF4α mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4α function since its deletion abolished HNF4α transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors. PMID:14602925

  7. Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4alpha dimerisation and transcriptional activity.

    PubMed

    Eeckhoute, Jérôme; Oxombre, Bénédicte; Formstecher, Pierre; Lefebvre, Philippe; Laine, Bernard

    2003-11-15

    Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.

  8. HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

    PubMed Central

    Mastropietro, Giuliana; Tiscornia, Inés; Perelmuter, Karen; Astrada, Soledad; Bollati-Fogolín, Mariela

    2015-01-01

    The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest. PMID:25861164

  9. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    EPA Science Inventory

    Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic an...

  10. Involvement of PPAR gamma co-activator-1, nuclear respiratory factors 1 and 2, and PPAR alpha in the adaptive response to endurance exercise.

    PubMed

    Baar, Keith

    2004-05-01

    Endurance exercise training induces an increase in the respiratory capacity of muscle, resulting in an increased capacity to generate ATP as well as improved efficiency of muscle contraction. Such adaptations are largely the result of a coordinated genetic response that increases mitochondrial proteins, fatty acid oxidation enzymes and the exercise- and insulin-stimulated glucose transporter GLUT4, and shifts the contractile and regulatory proteins to their more efficient isoforms. In recent years a number of the transcriptional regulators involved in this genetic response have been identified and these factors can be classified into two different groups. The first group comprises transcription factors such as nuclear respiratory factors (NRF) 1 and 2 and PPAR alpha that bind DNA in a sequence-specific manner. The second group, referred to as transcriptional co-activators, alter transcription without directly binding to DNA. The PPAR gamma co-activator (PGC) family of proteins have been identified as the central family of transcriptional co-activators for induction of mitochondrial biogenesis. PGC-1 alpha is activated by exercise, and is sufficient to produce the endurance phenotype through direct interactions with NRF-1 and PPAR alpha, and potentially NRF-2. Furthering the understanding of the activation of PGC proteins following exercise has implications beyond improving athletic performance, including the possibility of providing targets for the treatment of frailty in the elderly, obesity and diseases such as mitochondrial myopathies and diabetes.

  11. BaeR protein acts as an activator of nuclear factor-kappa B and Janus kinase 2 to induce inflammation in murine cell lines.

    PubMed

    Lee, Seung-Jin; Birhanu, Biruk Tesfaye; Awji, Elias Gebru; Kim, Myung Hee; Park, Ji-Yong; Suh, Joo-Won; Park, Seung-Chun

    2016-09-01

    BaeR, a response regulator protein, takes part in multidrug efflux, bacterial virulence activity, and other biological functions. Recently, BaeR was shown to induce inflammatory responses by activating the mitogen-activated protein kinases (MAPKs). In this study, we investigated additional pathways used by BaeR to induce an inflammatory response. BaeR protein was purified from Salmonella enterica Paratyphi A and subcloned into a pPosKJ expression vector. RAW 264.7 cells were treated with BaeR, and RNA was extracted by TRIzol reagent for RT-PCR. Cytokine gene expression was analyzed by using the comparative cycle threshold method, while western blotting and ELISA were used to assess protein expression. We confirmed that BaeR activates nuclear factor-kappa B (NF-κB), thereby inducing an inflammatory response and increases the production of interleukins (IL-)1β and IL-6. During this process, the Janus kinase 2 (JAK2)-STAT1 signaling pathway was activated, resulting in an increase in the release of interferons I and II. Additionally, COX-2 was activated and its expression increased with time. In conclusion, BaeR induced an inflammatory response through activation of NF-κB in addition to the MAPKs. Furthermore, activation of the JAK2-STAT1 pathway and COX-2 facilitated the cytokine binding activity, suggesting an additional role for BaeR in the modulation of the immune system of the host and the virulence activity of the pathogen.

  12. The nuclear splicing factor RNA binding motif 5 promotes caspase activation in human neuronal cells, and increases after traumatic brain injury in mice

    PubMed Central

    Jackson, Travis C; Du, Lina; Janesko-Feldman, Keri; Vagni, Vincent A; Dezfulian, Cameron; Poloyac, Samuel M; Jackson, Edwin K; Clark, Robert SB; Kochanek, Patrick M

    2015-01-01

    Splicing factors (SFs) coordinate nuclear intron/exon splicing of RNA. Splicing factor disturbances can cause cell death. RNA binding motif 5 (RBM5) and 10 (RBM10) promote apoptosis in cancer cells by activating detrimental alternative splicing of key death/survival genes. The role(s) of RBM5/10 in neurons has not been established. Here, we report that RBM5 knockdown in human neuronal cells decreases caspase activation by staurosporine. In contrast, RBM10 knockdown augments caspase activation. To determine whether brain injury alters RBM signaling, we measured RBM5/10 protein in mouse cortical/hippocampus homogenates after controlled cortical impact (CCI) traumatic brain injury (TBI) plus hemorrhagic shock (CCI+HS). The RBM5/10 staining was higher 48  to 72 hours after injury and appeared to be increased in neuronal nuclei of the hippocampus. We also measured levels of other nuclear SFs known to be essential for cellular viability and report that splicing factor 1 (SF1) but not splicing factor 3A (SF3A) decreased 4  to 72 hours after injury. Finally, we confirm that RBM5/10 regulate protein expression of several target genes including caspase-2, cellular FLICE-like inhibitory protein (c-FLIP), LETM1 Domain-Containing Protein 1 (LETMD1), and amyloid precursor-like protein 2 (APLP2) in neuronal cells. Knockdown of RBM5 appeared to increase expression of c-FLIP(s), LETMD1, and APLP2 but decrease caspase-2. PMID:25586139

  13. Citalopram protects against cold-restraint stress-induced activation of brain-derived neurotrophic factor and expression of nuclear factor kappa-light-chain-enhancer of activated B cells in rats.

    PubMed

    Garabadu, Debapriya; Reddy, B C M Harshavardhan; Krishnamurthy, Sairam

    2015-02-01

    The present study evaluates the protective effect of citalopram against cold-restraint stress (CRS) paradigm. Rats were pretreated with citalopram (0.1, 1.0, and 10.0 mg/kg) acutely and repeatedly for 21 days before exposure to the CRS procedure. None of the doses of citalopram attenuated CRS-induced gastric ulcers in the acute study. In contrast, repeated pretreatment of citalopram at a dose level of 0.1 mg/kg attenuated the CRS-induced gastric ulcers. Citalopram (0.1 mg/kg) diminished CRS-induced increase in plasma corticosterone, but not plasma norepinephrine level in the chronic study indicating its effect on hypothalamic-pituitary-adrenal axis function. Repeated citalopram (0.1 mg/kg) pretreatment attenuated CRS-induced changes in serotonin turnover in the hippocampus and amygdala. Moreover, repeated pretreatment with citalopram (0.1 mg/kg) mitigated the CRS-induced increase in the expression of brain-derived neurotrophic factor (BDNF) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in the hippocampus and amygdala. These results suggest that there is a region- and a dose-specific effect of citalopram on CRS-induced BDNF-NFκB activation. Therefore, citalopram showed antistress activity in the CRS model through changes in the stress-responsive pathways such as hypothalamic-pituitary-adrenal-axis and brain serotonergic system apart from decreasing the expression of BDNF and NFκB.

  14. TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

    PubMed

    Pype, S; Declercq, W; Ibrahimi, A; Michiels, C; Van Rietschoten, J G; Dewulf, N; de Boer, M; Vandenabeele, P; Huylebroeck, D; Remacle, J E

    2000-06-16

    CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

  15. Changes in chromatin accessibility across the GM-CSF promoter upon T cell activation are dependent on nuclear factor kappaB proteins.

    PubMed

    Holloway, Adele F; Rao, Sudha; Chen, Xinxin; Shannon, M Frances

    2003-02-17

    Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a key cytokine in myelopoiesis and aberrant expression is associated with chronic inflammatory disease and myeloid leukemias. This aberrant expression is often associated with constitutive nuclear factor (NF)-kappaB activation. To investigate the relationship between NF-kappaB and GM-CSF transcription in a chromatin context, we analyzed the chromatin structure of the GM-CSF gene in T cells and the role of NF-kappaB proteins in chromatin remodeling. We show here that chromatin remodeling occurs across a region of the GM-CSF gene between -174 and +24 upon T cell activation, suggesting that remodeling is limited to a single nucleosome encompassing the proximal promoter. Nuclear NF-kappaB levels appear to play a critical role in this process. In addition, using an immobilized template assay we found that the ATPase component of the SWI/SNF chromatin remodeling complex, brg1, is recruited to the GM-CSF proximal promoter in an NF-kappaB-dependent manner in vitro. These results suggest that chromatin remodeling across the GM-CSF promoter in T cells is a result of recruitment of SWI/SNF type remodeling complexes by NF-kappaB proteins binding to the CD28 response region of the promoter.

  16. Human labour is associated with nuclear factor-kappaB activity which mediates cyclo-oxygenase-2 expression and is involved with the 'functional progesterone withdrawal'.

    PubMed

    Allport, V C; Pieber, D; Slater, D M; Newton, R; White, J O; Bennett, P R

    2001-06-01

    Human labour is associated with the up-regulation of prostaglandins within the uterus, synthesized via the type-2 cyclo-oxygenase enzyme (COX-2). These lead to remodelling of the fetal membranes and cervix and to stimulation of myometrial contractions. In the human, the principal source of prostaglandins is the amnion. Progesterone acts to promote myometrial quiescence, and in many species the onset of labour is preceded by withdrawal of progesterone. Humans show no systemic progesterone withdrawal, although biochemical changes within the uterus are similar to those in other species. A mutual negative interaction between the transcription factor nuclear factor (NF)-kappaB and the progesterone receptor (PR) has been reported. Using transient transfections and assays for transcriptional activation and promoter binding, we have shown that there is constitutive activity of NF-kappaB in amnion cells at the time of labour, and that COX-2 expression depends upon NF-kappaB. In cells obtained before labour, in which NF-kappaB activity is low, increasing the concentration of PR represses NF-kappaB dependent transcription, while stimulation with IL-1beta both increases NF-kappaB activity and represses PR activity. Our data suggest that human labour is associated with constitutive NF-kappaB activity within the amnion, which functions to increase the expression of COX-2 and appears to contribute to the 'functional progesterone withdrawal'.

  17. Addition and correction: the NF-kappa B-like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor.

    PubMed

    Traincard, F; Ponte, E; Pun, J; Coukell, B; Veron, M

    2001-10-01

    We have previously reported that a NF-kappa B transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide kappa B sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-kappa B-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kappa B pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kappa B signal transduction pathway in Dictyostelium.

  18. The Chromatin Regulator DMAP1 Modulates Activity of the Nuclear Factor κB (NF-κB) Transcription Factor Relish in the Drosophila Innate Immune Response*

    PubMed Central

    Goto, Akira; Fukuyama, Hidehiro; Imler, Jean-Luc; Hoffmann, Jules A.

    2014-01-01

    The host defense of the model organism Drosophila is under the control of two major signaling cascades controlling transcription factors of the NF-κB family, the Toll and the immune deficiency (IMD) pathways. The latter shares extensive similarities with the mammalian TNF-R pathway and was initially discovered for its role in anti-Gram-negative bacterial reactions. A previous interactome study from this laboratory reported that an unexpectedly large number of proteins are binding to the canonical components of the IMD pathway. Here, we focus on DNA methyltransferase-associated protein 1 (DMAP1), which this study identified as an interactant of Relish, a Drosophila transcription factor reminiscent of the mammalian p105 NF-κB protein. We show that silencing of DMAP1 expression both in S2 cells and in flies results in a significant reduction of Escherichia coli-induced expression of antimicrobial peptides. Epistatic analysis indicates that DMAP1 acts in parallel or downstream of Relish. Co-immunoprecipitation experiments further reveal that, in addition to Relish, DMAP1 also interacts with Akirin and the Brahma-associated protein 55 kDa (BAP55). Taken together, these results reveal that DMAP1 is a novel nuclear modulator of the IMD pathway, possibly acting at the level of chromatin remodeling. PMID:24947515

  19. Receptor Activator for Nuclear Factor kappa B Ligand (RANKL) as an osteoimmune key regulator in bone physiology and pathology.

    PubMed

    Narducci, Paola; Bareggi, Renato; Nicolin, Vanessa

    2011-02-01

    The strength and integrity of the human skeleton depends on a delicate equilibrium between bone resorption and bone formation. Bone resorption is an elementary cellular activity in the modelling of the skeleton during growth and development. Later in life a most important physiological process in the skeleton is bone remodelling, which is locally initiated by resorption. During remodelling bone resorption is coupled to new bone formation that ensures renewal of bone with only minor local and temporary bone loss. Cells responsible for bone resorption and subsequent bone formation are the osteoclasts and osteoblasts, respectively. The osteoclast is derived from the pluripotent hematopoietic stem cell, which gives rise to a myeloid stem cell that can further differentiate into megakaryocytes, granulocytes, monocytes/macrophages and osteoclasts. The respective bone resorbing and forming actions of osteoclasts and osteoblasts are finely coupled, so that bone mass remains remarkably stable in a healthy adult. Imbalance between osteoclast and osteoblast activities can arise from a wide variety of hormonal changes or perturbations of inflammatory and growth factors resulting in postmenopausal osteoporosis, Paget's disease, lytic bone metastases, or rheumatoid arthritis, leading to increased bone resorption and crippling bone damage. In view of the critical role of osteoclasts in diverse pathology, there has been immense effort aimed at understanding the biology of this unique cell. The present review is focused on the current knowledge of the mechanisms that regulate the functional links between bone turnover and the immune system helping us to understand the main factors that lead to bone loss observed in osteoporosis, cancer and in rheumatoid arthritis. The aim of this review paper is to consider the key molecular interactions involved in the formation of osteoclast cells in normal and pathological conditions.

  20. Cystic Fibrosis Transmembrane Conductance Regulator Controls Lung Proteasomal Degradation and Nuclear Factor-κB Activity in Conditions of Oxidative Stress

    PubMed Central

    Boncoeur, Emilie; Roque, Telma; Bonvin, Elise; Saint-Criq, Vinciane; Bonora, Monique; Clement, Annick; Tabary, Olivier; Henrion-Caude, Alexandra; Jacquot, Jacky

    2008-01-01

    Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-κB/IκB-α signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr−/−) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr+/+) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-κB inhibitor IκB-α. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-κB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl− channel by CFTRinh-172 in the normal bronchial immortalized cell line 16HBE14o− increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-κB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl− channel activity is crucial for regulation of lung proteasomal degradation and NF-κB activity in conditions of oxidative stress. PMID:18372427

  1. Interference of fisetin with targets of the nuclear factor-κB signal transduction pathway activated by Epstein-Barr virus encoded latent membrane protein 1.

    PubMed

    Li, Rong; Liang, Hong-Ying; Li, Ming-Yong; Lin, Chun-Yan; Shi, Meng-Jie; Zhang, Xiu-Juan

    2014-01-01

    Fisetin is an effective compound extracted from lacquer which has been used in the treatment of various diseases. Preliminary data indicate that it also exerts specific anti-cancer effects. However, the manner in which fisetin regulates cancer growth remains unknown. In this study, we elucidated interference of fisetin with targets of the nuclear factorκB signal transduction pathway activated by Epstein-Barr virus encoding latent membrane protein 1 (LMP1)in nasopharyngeal carcinoma (NPC) cells, Results showed that fisetin inhibited the survival rate of CNE-LMP1 cells and NF-κB activation caused by LMP1. Fisetin also suppressed nuclear translocation of NF-κB (p65) and IκBα phosphorylation, while inhibiting CyclinD1, all key targets of the NF-κB signal transduction pathway. It was suggested that interference effects of fisetin with signal transduction activated by LMP1 encoded by the Epstein-Barr virus may play an important role in its anticancer potential.

  2. Antioxidant activity in lingonberries (Vaccinium vitis-idaea L.) and its inhibitory effect on activator protein-1, nuclear factor-kappaB, and mitogen-activated protein kinases activation.

    PubMed

    Wang, Shiow Y; Feng, Rentian; Bowman, Linda; Penhallegon, Ross; Ding, Min; Lu, Y

    2005-04-20

    Lingonberry has been shown to contain high antioxidant activity. Fruits from different cultivars of lingonberry (Vaccinium vitis-idaea L.) were evaluated for fruit quality, antioxidant activity, and anthocyanin and phenolic contents. The fruit soluble solids, titratable acids, antioxidant capacity, and anthocyanin and phenolic contents varied with cultivars. Lingonberries contain potent free radical scavenging activities for DPPH*, ROO*, *OH, and O2*- radicals. Pretreatment of JB6 P+ mouse epidermal cells with lingonberry extracts produced a dose-dependent inhibition on the activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) induced by either 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B (UVB). Lingonberry extract blocked UVB-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling members ERK1, ERK2, p38, and MEK1/2 but not JNK. Lingonberry extract also prevented TPA-induced phosphorylation of ERK1, ERK2, and MEK1/2. Results of soft agar assays indicated that lingonberry extract suppressed TPA-induced neoplastic transformation of JB6 P(+) cells in a dose-dependent manner. Lingonberry extract also induced the apoptosis of human leukemia HL-60 cells in a dose-independent manner. These results suggest that ERK1, ERK2, and MEK1/2 may be the primary targets of lingonberry that result in suppression of AP-1, NF-kappaB, and neoplastic transformation in JB6 P(+) cells and causes cancer cell death by an apoptotic mechanism in human leukemia HL-60 cells.

  3. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells.

    PubMed

    Zhu, Fei; Yue, Wanfu; Wang, Yongxia

    2014-10-01

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages.

  4. A new phenolic derivative with soluble epoxide hydrolase and nuclear factor-kappaB inhibitory activity from the aqueous extract of Acacia catechu.

    PubMed

    Sun, Ya Nan; Li, Wei; Song, Seok Bean; Yan, Xi Tao; Zhao, Yan; Jo, A Reum; Kang, Jong Seong; Young Ho, Kim

    2016-09-01

    One novel phenolic compound, (4S,5R)-4-(3,4-dihydroxyphenyl)-5-(3-oxobutyl)dihydrofuran-2(3H)-one (1), as well as 12 known compounds (2-13) was obtained from the aqueous extract of Acacia catechu and their chemical structures were determined by spectroscopic analysis. Compounds 8 and 9 exhibited significant soluble epoxide hydrolase (sEH) inhibitory activities with IC50 values of 26.6 ± 0.5 and 24.4 ± 5.6 μM, respectively. Compounds 7-10 showed significant inhibitory effects on TNFα-induced nuclear factor kappa B (NF-κB) transcriptional activity in a dose-dependent manner, with IC50 values ranging from 11.15 to 19.45 μM.

  5. C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

    PubMed Central

    Oller, Jorge; Alfranca, Arántzazu; Méndez-Barbero, Nerea; Villahoz, Silvia; Lozano-Vidal, Noelia; Martín-Alonso, Mara; Arroyo, Alicia G.; Escolano, Amelia; Armesilla, Angel Luis

    2015-01-01

    Emerging evidence indicates that the metalloproteinase Adamts-1 plays a significant role in the pathophysiology of vessel remodeling, but little is known about the signaling pathways that control Adamts-1 expression. We show that vascular endothelial growth factor (VEGF), angiotensin-II, interleukin-1β, and tumor necrosis factor α, stimuli implicated in pathological vascular remodeling, increase Adamts-1 expression in endothelial and vascular smooth muscle cells. Analysis of the intracellular signaling pathways implicated in this process revealed that VEGF and angiotensin-II upregulate Adamts-1 expression via activation of differential signaling pathways that ultimately promote functional binding of the NFAT or C/EBPβ transcription factors, respectively, to the Adamts-1 promoter. Infusion of mice with angiotensin-II triggered phosphorylation and nuclear translocation of C/EBPβ proteins in aortic cells concomitantly with an increase in the expression of Adamts-1, further underscoring the importance of C/EBPβ signaling in angiotensin-II-induced upregulation of Adamts-1. Similarly, VEGF promoted NFAT activation and subsequent Adamts-1 induction in aortic wall in a calcineurin-dependent manner. Our results demonstrate that Adamts-1 upregulation by inducers of pathological vascular remodeling is mediated by specific signal transduction pathways involving NFAT or C/EBPβ transcription factors. Targeting of these pathways may prove useful in the treatment of vascular disease. PMID:26217013

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

    PubMed Central

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

    1994-01-01

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

  7. Anti-cancer effect of bee venom on colon cancer cell growth by activation of death receptors and inhibition of nuclear factor kappa B

    PubMed Central

    Zheng, Jie; Lee, Hye Lim; Ham, Young Wan; Song, Ho Sueb; Song, Min Jong; Hong, Jin Tae

    2015-01-01

    Bee venom (BV) has been used as a traditional medicine to treat arthritis, rheumatism, back pain, cancerous tumors, and skin diseases. However, the effects of BV on the colon cancer and their action mechanisms have not been reported yet. We used cell viability assay and soft agar colony formation assay for testing cell viability, electro mobility shift assay for detecting DNA binding activity of nuclear factor kappa B (NF-κB) and Western blotting assay for detection of apoptosis regulatory proteins. We found that BV inhibited growth of colon cancer cells through induction of apoptosis. We also found that the expression of death receptor (DR) 4, DR5, p53, p21, Bax, cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 was increased by BV treatment in a dose dependent manner (0–5 μg/ml). Consistent with cancer cell growth inhibition, the DNA binding activity of nuclear factor kappa B (NF-κB) was also inhibited by BV treatment. Besides, we found that BV blocked NF-κB activation by directly binding to NF-κB p50 subunit. Moreover, combination treatment with BV and p50 siRNA or NF-κB inhibitor augmented BV-induced cell growth inhibition. However, p50 mutant plasmid (C62S) transfection partially abolished BV-induced cell growth inhibiton. In addition, BV significantly suppressed tumor growth in vivo. Therefore, these results suggested that BV could inhibit colon cancer cell growth, and these anti-proliferative effects may be related to the induction of apoptosis by activation of DR4 and DR5 and inhibition of NF-κB. PMID:26561202

  8. Silica nanoparticles induce oxidative stress, inflammation, and endothelial dysfunction in vitro via activation of the MAPK/Nrf2 pathway and nuclear factor-κB signaling

    PubMed Central

    Guo, Caixia; Xia, Yinye; Niu, Piye; Jiang, Lizhen; Duan, Junchao; Yu, Yang; Zhou, Xianqing; Li, Yanbo; Sun, Zhiwei

    2015-01-01

    Despite the widespread application of silica nanoparticles (SiNPs) in industrial, commercial, and biomedical fields, their response to human cells has not been fully elucidated. Overall, little is known about the toxicological effects of SiNPs on the cardiovascular system. In this study, SiNPs with a 58 nm diameter were used to study their interaction with human umbilical vein endothelial cells (HUVECs). Dose- and time-dependent decrease in cell viability and damage on cell plasma-membrane integrity showed the cytotoxic potential of the SiNPs. SiNPs were found to induce oxidative stress, as evidenced by the significant elevation of reactive oxygen species generation and malondialdehyde production and downregulated activity in glutathione peroxidase. SiNPs also stimulated release of cytoprotective nitric oxide (NO) and upregulated inducible nitric oxide synthase (NOS) messenger ribonucleic acid, while downregulating endothelial NOS and ET-1 messenger ribonucleic acid, suggesting that SiNPs disturbed the NO/NOS system. SiNP-induced oxidative stress and NO/NOS imbalance resulted in endothelial dysfunction. SiNPs induced inflammation characterized by the upregulation of key inflammatory mediators, including IL-1β, IL-6, IL-8, TNFα, ICAM-1, VCAM-1, and MCP-1. In addition, SiNPs triggered the activation of the Nrf2-mediated antioxidant system, as evidenced by the induction of nuclear factor-κB and MAPK pathway activation. Our findings demonstrated that SiNPs could induce oxidative stress, inflammation, and NO/NOS system imbalance, and eventually lead to endothelial dysfunction via activation of the MAPK/Nrf2 pathway and nuclear factor-κB signaling. This study indicated a potential deleterious effect of SiNPs on the vascular endothelium, which warrants more careful assessment of SiNPs before their application. PMID:25759575

  9. Peroxisome Proliferator-Activated Receptor α Reduces Endothelin-1-Caused Cardiomyocyte Hypertrophy by Inhibiting Nuclear Factor-κB and Adiponectin

    PubMed Central

    Jen, Hsu-Lung; Liu, Po-Len; Chen, Jaw-Wen; Lin, Shing-Jong

    2016-01-01

    Peroxisome proliferator-activated receptor α (PPARα) plays a role in the pathogenesis of cardiac hypertrophy, although its underlying mechanism remains unclear. The purpose of this study was to evaluate the effect of PPARα activation on endothelin-1- (ET-1-) caused cardiomyocyte hypertrophy and explore its underlying mechanisms. Human cardiomyocytes (HCMs) were cultured with or without ET-1, whereafter the inhibitory effects of fenofibrate, a PPARα activator, on cell size and adiponectin protein were tested. We examined the activation of extracellular signal-regulated kinase (ERK) and p38 proteins caused by ET-1 and the inhibition of the ERK and p38 pathways on ET-1-induced cell size and adiponectin expression. Moreover, we investigated the interaction of PPARα with adiponectin and nuclear factor-κB (NF-κB) by electrophoretic mobility shift assays and coimmunoprecipitation. ET-1 treatment significantly increased cell size, suppressed PPARα expression, and enhanced the expression of adiponectin. Pretreatment with fenofibrate inhibited the increase in cell size and enhancement of adiponectin expression. ET-1 significantly activated the ERK and p38 pathways, whereas PD98059 and SB205380, respectively, inhibited them. Our results suggest that activated PPARα can decrease activation of adiponectin and NF-κB and inhibit ET-1-induced cardiomyocyte hypertrophy. PMID:27807394

  10. Activation Experiments for Nuclear Astrophysics

    SciTech Connect

    Sonnabend, K.; Mueller, S.; Pietralla, N.; Savran, D.; Schnorrenberger, L.; Hasper, J.; Zilges, A.

    2009-01-28

    The study of ({gamma},n) reactions can be used to constrain the theoretical predictions of the neutron capture cross sections of short-lived branching points in the s process. The usability of the activation technique to study these ({gamma},n) reactions is discussed as one example of an activation experiment in nuclear astrophysics. Two photon sources using bremsstrahlung and laser-Compton backscattered photons where such experiments were carried out are compared.

  11. PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

    PubMed

    Tanaka, Hiroshi; Hoshikawa, Yutaka; Oh-hara, Tomoko; Koike, Sumie; Naito, Mikihiko; Noda, Tetsuo; Arai, Hiroyuki; Tsuruo, Takashi; Fujita, Naoya

    2009-04-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments

  12. Increase in antioxidant activity by sheep/goat whey protein through nuclear factor-like 2 (Nrf2) is cell type dependent.

    PubMed

    Kerasioti, Efthalia; Stagos, Dimitrios; Tzimi, Aggeliki; Kouretas, Dimitrios

    2016-11-01

    The aim of the present study was to investigate the molecular mechanisms through which sheep/goat whey protein exerts its antioxidant activity. Thus, it was examined whey protein's effects on the expression of transcription factor, nuclear factor-like 2 (Nrf2) and on the expression and activity of a number of antioxidant and phase II enzymes, superoxide dismutase (SOD), catalase (CAT), heme oxygenase 1 (HO-1), synthase glutamyl cysteine (GCS) and glutathione-s-transferase (GST), in muscle C2C12 and EA.hy926 endothelial cells. C2C12 and EA.hy926 cells were treated with sheep/goat whey protein (0.78 and 3.12 mg/ml) and incubated for 3, 6, 12, 18 and 24 h. Whey protein increased significantly the expression of Nrf2 only in EA.hy926 cells. Also, the expression of SOD, HO-1, CAT and the activity of SOD, CAT and GST were increased significantly in both cells types. The expression of GCS was increased significantly only in C2C12 cells. Sheep/goat whey protein was shown for the first time to exert its antioxidant activity through Nrf2-dependent mechanism in endothelial cells and Nrf2-independent mechanism in muscle cells. Thus, Nrf2 could be a target for food supplements containing whey protein in order to prevent oxidative stress damages and diseases related to endothelium.

  13. Berberine inhibits tumor necrosis factor-α-induced expression of inflammatory molecules and activation of nuclear factor-κB via the activation of AMPK in vascular endothelial cells.

    PubMed

    Liu, Su-Jian; Yin, Cai-Xia; Ding, Ming-Chao; Wang, Yi-Zhong; Wang, Hong

    2015-10-01

    Berberine, which is a well‑known drug used in traditional medicine, has been demonstrated to exert diverse pharmacological effects, including anti‑inflammatory effects. However, whether berberine can affect the production of inflammatory molecules in vascular endothelial cells remains to be elucidated. Therefore, the present study aimed to determine the effects of berberine, and the underlying molecular mechanisms of these effects. The effect of berberine on tumor necrosis factor (TNF)‑α‑induced inflammatory molecule expression was examined in cultured human aortic endothelial cells (HAECs). The HAECs were stimulated with TNF‑α and incubated with or without berberine. The activation of nuclear factor (NF)‑κB and adenosine monophosphate‑activated protein kinase (AMPK) were analyzed using western blotting, and the protein secretion of intercellular adhesion molecule (ICAM)‑1 and monocyte chemoattractant protein (MCP)‑1 was measured using ELISA kits. The mRNA expression levels of ICAM‑1 and MCP‑1 were analyzed using reverse transcription‑quantitative polymerase chain reaction. The results of the present study demonstrated that berberine significantly inhibited the TNF‑α‑induced expression of ICAM‑1 and MCP‑1, as well as the activation of NF‑κB in the HAECs. These effects were attenuated following co‑treatment with AMPK inhibitor compound C, or specific small interfering RNAs. In conclusion, the results of the present study indicated that berberine inhibits the TNF‑α‑induced expression of ICAM‑1 and MCP‑1, and the activation of NF‑κB in HAECs in vitro, possibly through the AMPK‑dependent pathway.

  14. The variant hepatocyte nuclear factor 1 activates the P1 promoter of the human alpha-folate receptor gene in ovarian carcinoma.

    PubMed

    Tomassetti, Antonella; Mangiarotti, Fabio; Mazzi, Mimma; Sforzini, Sabrina; Miotti, Silvia; Galmozzi, Enrico; Elwood, Patrick C; Canevari, Silvana

    2003-02-01

    The alpha folate receptor (alpha FR) is a membrane glycoprotein that binds folates, and mediates their uptake and that of antifolate drugs. alpha FR is absent on ovarian surface epithelium (OSE) but is detectable during early transforming events in this epithelium, with increasing expression levels in association with tumor progression. Analysis of transcriptional regulation of the alpha FR gene have revealed two promoter regions, P1 and P4, flanking exons 1 and 4, respectively, and a requirement for three SP1 sites and an INR element for optimal P4 activity. Here, we focused on the P1 transcription regulation in ovarian carcinoma cells. RNase protection assay indicated that the 5'-untranslated region is heterogeneous because of different start sites and alternative splicing of exon 3. A core region of the P1 promoter was sufficient for maximal promoter activity in ovarian carcinoma cell lines but not in OSE cells or in alpha FR-nonexpressing cell lines. Deletion and mutation analysis of this core promoter identified a cis-regulatory element at position +27 to +33 of the untranslated exon 1, which is responsible for maximum P1 activity. This element formed an abundant DNA-protein complex with nuclear proteins from ovarian cancer cells but not from other cell lines or OSE cells. Competition experiments and supershift assays demonstrated binding of the P1 cis-regulatory element by a transcription factor involved in embryonic development, the variant hepatocyte nuclear factor-1 (vHNF1). Analysis of RNA from various cell lines and surgical specimens confirmed that vHNF1 is expressed in ovarian carcinomas. Thus, vHNF1 regulates tissue-specific transcription in ovarian carcinoma.

  15. Interleukin-4 and interleukin-10 modulate nuclear factor kappaB activity and nitric oxide synthase-2 expression in Theiler's virus-infected brain astrocytes.

    PubMed

    Molina-Holgado, Eduardo; Arévalo-Martín, Angel; Castrillo, Antonio; Boscá, Lisardo; Vela, José M; Guaza, Carmen

    2002-06-01

    In brain astrocytes, nuclear factor kappaB (NF-kappaB) is activated by stimuli that produce cellular stress causing the expression of genes involved in defence, including the inducible nitric oxide synthase (NOS-2). Theiler's murine encephalomyelitis virus (TMEV) induces a persistent CNS infection and chronic immune-mediated demyelination, similar to human multiple sclerosis. The cytokines interleukin (IL)-4 and IL-10 inhibit the expression of proinflammatory cytokines, counteracting the inflammatory process. Our study reports that infection of cultured astrocytes with TMEV resulted in a time-dependent phosphorylation of IkappaBalpha, degradation of IkappaBalpha and IkappaBbeta, activation of NF-kappaB and expression of NOS-2. The proteasome inhibitor MG-132 blocked TMEV-induced nitrite accumulation, NOS-2 mRNA expression and phospho-IkappaBalpha degradation, suggesting NF-kappaB-dependent NOS-2 expression. Pretreatment of astrocytes with IL-4 or IL-10 decreased p65 nuclear translocation, NF-kappaB binding activity and NOS-2 transcription. IL-4 and IL-10 caused an accumulation of IkappaBalpha in TMEV-infected astrocytes without affecting IkappaBbeta levels. The IkappaB kinase activity and the degradation rate of both IkappaBs were not modified by either cytokine, suggesting de novo synthesis of IkappaBalpha. Indeed, IL-4 or IL-10 up-regulated IkappaBalpha mRNA levels after TMEV infection. Therefore, the accumulation of IkappaBalpha might impair the translocation of the NF-kappaB to the nucleus, mediating the inhibition of NF-kappaB activity. Overall, these data suggest a novel mechanism of action of IL-4 and IL-10, which abrogates NOS-2 expression in viral-infected glial cells.

  16. Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.

    PubMed

    Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi

    2002-11-01

    We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

  17. Effect of The Receptor Activator of Nuclear Factor кB and RANK Ligand on In Vitro Differentiation of Cord Blood CD133+ Hematopoietic Stem Cells to Osteoclasts

    PubMed Central

    Kalantari, Nasim; Abroun, Saeid; Soleimani, Masoud; Kaviani, Saeid; Azad, Mehdi; Eskandari, Fatemeh; Habibi, Hossein

    2016-01-01

    Objective Receptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface. Materials and Methods In this experimental study, CD133+ hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes. Results Hematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells. Conclusion Presence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast. PMID:27602313

  18. Arctigenin suppresses receptor activator of nuclear factor κB ligand (RANKL)-mediated osteoclast differentiation in bone marrow-derived macrophages.

    PubMed

    Kim, A-Ram; Kim, Hyuk Soon; Lee, Jeong Min; Choi, Jung Ho; Kim, Se Na; Kim, Do Kyun; Kim, Ji Hyung; Mun, Se Hwan; Kim, Jie Wan; Jeon, Hyun Soo; Kim, Young Mi; Choi, Wahn Soo

    2012-05-05

    Osteoclasts, multinucleated bone-resorbing cells, are closely associated with bone diseases such as rheumatoid arthritis and osteoporosis. Osteoclasts are derived from hematopoietic precursor cells, and their differentiation is mediated by two cytokines, including macrophage colony stimulating factor and receptor activator of nuclear factor κB ligand (RANKL). Previous studies have shown that arctigenin exhibits an anti-inflammatory effect. However, the effect of arctigenin on osteoclast differentiation is yet to be elucidated. In this study, we found that arctigenin inhibited RANKL-mediated osteoclast differentiation in bone marrow macrophages in a dose-dependent manner and suppressed RANKL-mediated bone resorption. Additionally, the expression of typical marker proteins, such as NFATc1, c-Fos, TRAF6, c-Src, and cathepsin K, were significantly inhibited. Arctigenin inhibited the phosphorylation of Erk1/2, but not p38 and JNK, in a dose-dependent manner. Arctigenin also dramatically suppressed immunoreceptor tyrosine-based activation motif-mediated costimulatory signaling molecules, including Syk and PLCγ2, and Gab2. Notably, arctigenin inhibited the activation of Syk through RANKL stimulation. Furthermore, arctigenin prevented osteoclast differentiation in the calvarial bone of mice following stimulation with lipopolysaccharide. Our results show that arctigenin inhibits osteoclast differentiation in vitro and in vivo. Therefore, arctigenin may be useful for treating rheumatoid arthritis and osteoporosis.

  19. Diosmetin ameliorates the severity of cerulein-induced acute pancreatitis in mice by inhibiting the activation of the nuclear factor-κB.

    PubMed

    Yu, Ge; Wan, Rong; Yin, Guojian; Xiong, Jie; Hu, Yanling; Xing, Miao; Cang, Xiaofeng; Fan, Yuting; Xiao, Wenqin; Qiu, Lei; Wang, Xingpeng; Hu, Guoyong

    2014-01-01

    Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), the aglycone part of the flavonoid glycosides diosmin occurs naturally in citrus fruit, was considered to exhibit anti-inflammatory and antioxidant properties. Our study aimed to investigate the effect of diosmetin in a murine model of cerulein-induced acute pancreatitis (AP). Experimental AP was induced in mice by seven intraperitoneal injection of cerulein (50 ug/kg) at hourly intervals. Diosmetin (100 mg/kg) or vehicle was pretreated 2 h before the first cerulein injection. After 6 h, 9 h, 12 h of the first cerulein injection, the severity of acute pancreatitis was evaluated biochemically and morphologically. Pretreatment with diosmetin significantly reduced serum levels of amylase and lipase; the histological injury; the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6; myeloperoxidase (MPO) activity, trypsinogen activation peptide (TAP) level, the expression of inducible nitric oxide synthase (iNOS); and the nuclear factor (NF)-κB activation in cerulein-induced AP. This study showed that administration of diosmetin demonstrated a beneficial effect on the course of cerulein-induced AP in mice. Therefore, diosmetin may become a new therapeutic agent in future clinical trials for treatment of AP.

  20. Asiatic acid inhibits cardiac hypertrophy by blocking interleukin-1β-activated nuclear factor-κB signaling in vitro and in vivo

    PubMed Central

    Xu, Xiaohan; Si, Linjie; Xu, Jing; Yi, Chenlong; Wang, Fang; Gu, Weijuan

    2015-01-01

    Background Activated interleukin (IL)-1β signaling pathway is closely associated with pathological cardiac hypertrophy. This study investigated whether asiatic acid (AA) could inhibit IL-1β-related hypertrophic signaling, and thus suppressing the development of cardiac hypertrophy. Methods Transverse aortic constriction (TAC) induced cardiac hypertrophy in C57BL/6 mice and cultured neonatal cardiac myocytes stimulated with IL-1β were used to evaluate the role of AA in cardiac hypertrophy. The expression of atrial natriuretic peptide (ANP) was evaluated by quantitative polymerase chain reaction (qPCR) and the nuclear factor (NF)-κB binding activity was measured by electrophoretic mobility shift assays (EMSA). Results AA pretreatment significantly attenuated the IL-1β-induced hypertrophic response of cardiomyocytes as reflected by reduction in the cardiomyocyte surface area and the inhibition of ANP mRNA expression. The protective effect of AA on IL-1β-stimulated cardiomyocytes was associated with the reduction of NF-κB binding activity. In addition, AA prevented TAC-induced cardiac hypertrophy in vivo. It was found that AA markedly reduced the excessive expression of IL-1β and ANP, and inhibited the activation of NF-κB in the hypertrophic myocardium. Conclusions Our data suggest that AA may be a novel therapeutic agent for cardiac hypertrophy. The inhibition of IL-1β-activated NF-κB signaling may be the mechanism through which AA prevents cardiac hypertrophy. PMID:26623102

  1. Positive Regulation by γ-Aminobutyric Acid B Receptor Subunit-1 of Chondrogenesis through Acceleration of Nuclear Translocation of Activating Transcription Factor-4*

    PubMed Central

    Takahata, Yoshifumi; Hinoi, Eiichi; Takarada, Takeshi; Nakamura, Yukari; Ogawa, Shinya; Yoneda, Yukio

    2012-01-01

    A view that signaling machineries for the neurotransmitter γ-aminobutyric acid (GABA) are functionally expressed by cells outside the central nervous system is now prevailing. In this study, we attempted to demonstrate functional expression of GABAergic signaling molecules by chondrocytes. In cultured murine costal chondrocytes, mRNA was constitutively expressed for metabotropic GABAB receptor subunit-1 (GABABR1), but not for GABABR2. Immunohistochemical analysis revealed the predominant expression of GABABR1 by prehypertrophic to hypertrophic chondrocytes in tibial sections of newborn mice. The GABABR agonist baclofen failed to significantly affect chondrocytic differentiation determined by Alcian blue staining and alkaline phosphatase activity in cultured chondrocytes, whereas newborn mice knocked out of GABABR1 (KO) showed a decreased body size and delayed calcification in hyoid bone and forelimb and hindlimb digits. Delayed calcification was also seen in cultured metatarsals from KO mice with a marked reduction of Indian hedgehog gene (Ihh) expression. Introduction of GABABR1 led to synergistic promotion of the transcriptional activity of activating transcription factor-4 (ATF4) essential for normal chondrogenesis, in addition to facilitating ATF4-dependent Ihh promoter activation. Although immunoreactive ATF4 was negligibly detected in the nucleus of chondrocytes from KO mice, ATF4 expression was again seen in the nucleus and cytoplasm after the retroviral introduction of GABABR1 into cultured chondrocytes from KO mice. In nuclear extracts of KO chondrocytes, a marked decrease was seen in ATF4 DNA binding. These results suggest that GABABR1 positively regulates chondrogenesis through a mechanism relevant to the acceleration of nuclear translocation of ATF4 for Ihh expression in chondrocytes. PMID:22879594

  2. Pancreatic β-cell dysfunction in polycystic ovary syndrome: role of hyperglycemia-induced nuclear factor-κB activation and systemic inflammation

    PubMed Central

    Malin, Steven K.; Kirwan, John P.; Sia, Chang Ling

    2015-01-01

    In polycystic ovary syndrome (PCOS), oxidative stress is implicated in the development of β-cell dysfunction. However, the role of mononuclear cell (MNC)-derived inflammation in this process is unclear. We determined the relationship between β-cell function and MNC-derived nuclear factor-κB (NF-κB) activation and tumor necrosis factor-α (TNF-α) secretion in response to a 2-h 75-g oral glucose tolerance test (OGTT) in normoglycemic women with PCOS (15 lean, 15 obese) and controls (16 lean, 14 obese). First- and second-phase β-cell function was calculated as glucose-stimulated insulin secretion (insulin/glucose area under the curve for 0–30 and 60–120 min, respectively) × insulin sensitivity (Matsuda Index derived from the OGTT). Glucose-stimulated NF-κB activation and TNF-α secretion from MNC, and fasting plasma thiobarbituric acid-reactive substances (TBARS) and high-sensitivity C-reactive protein (hs-CRP) were also assessed. In obese women with PCOS, first- and second-phase β-cell function was lower compared with lean and obese controls. Compared with lean controls, women with PCOS had greater change from baseline in NF-κB activation and TNF-α secretion, and higher plasma TBARS. β-Cell function was inversely related to NF-κB activation (1st and 2nd) and TNF-α secretion (1st), and plasma TBARS and hs-CRP (1st and 2nd). First- and second-phase β-cell function also remained independently linked to NF-κB activation after adjustment for body fat percentage and TBARS. In conclusion, β-cell dysfunction in PCOS is linked to hyperglycemia-induced NF-κB activation from MNC and systemic inflammation. These data suggest that in PCOS, inflammation may play a role in impairing insulin secretion before the development of overt hyperglycemia. PMID:25714674

  3. The nuclear factor kappa B (NF-κB) activation is required for phagocytosis of staphylococcus aureus by RAW 264.7 cells

    SciTech Connect

    Zhu, Fei Yue, Wanfu; Wang, Yongxia

    2014-10-01

    Nuclear factor kappa B (NF-κB) is a ubiquitous transcription factor which controls the expression of various genes involved in immune responses. However, it is not clear whether NF-κB activation is critical for phagocytosis when Staphylococcus aureus is the pathogen. Using oligonucleotide microarrays, we investigated whether NF-κB cascade genes are altered in a mouse leukemic monocyte macrophage cell line (RAW 264.7) when the cells were stimulated to activate a host innate immune response against live S. aureus or heat-inactivated S. aureus (HISA). NF-κB cascade genes such as Nfκb1, Nfκbiz, Nfκbie, Rel, Traf1 and Tnfaip3 were up-regulated by all treatments at one hour after incubation. NF-κB play an important role in activating phagocytosis in RAW 264.7 cells infected with S. aureus. Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus and decreased the expression of NFκB1, IL1α, IL1β and TLR2 in this cell line. Our results demonstrate that S. aureus may activate the NF-κB pathway and that NF-κB activation is required for phagocytosis of S. aureus by macrophages. - Highlights: • NF-κB cascade genes such as Nfκb1 and Traf1 were up-regulated by heat-inactivated S. aureus. • Inhibition of NF-κB significantly blocked phagocytosis of fluorescently labeled S. aureus. • NF-κB activation is required for phagocytosis of S. aureus by macrophages.

  4. Keap1 silencing boosts lipopolysaccharide-induced transcription of interleukin 6 via activation of nuclear factor κB in macrophages

    SciTech Connect

    Lv, Peng; Xue, Peng; Dong, Jian; Peng, Hui; Clewell, Rebecca; Wang, Aiping; Wang, Yue; Peng, Shuangqing; Qu, Weidong; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-11-01

    Interleukin-6 (IL6) is a multifunctional cytokine that regulates immune and inflammatory responses. Multiple transcription factors, including nuclear factor κB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2), regulate IL6 transcription. Kelch-like ECH-associated protein 1 (Keap1) is a substrate adaptor protein for the Cullin 3-dependent E3 ubiquitin ligase complex, which regulates the degradation of many proteins, including Nrf2 and IκB kinase β (IKKβ). Here, we found that stable knockdown of Keap1 (Keap1-KD) in RAW 264.7 (RAW) mouse macrophages and human monocyte THP-1 cells significantly increased expression of Il6, and Nrf2-target genes, under basal and lipopolysaccharide (LPS, 0.001–0.1 μg/ml)-challenged conditions. However, Nrf2 activation alone, by tert-butylhydroquinone treatment of RAW cells, did not increase expression of Il6. Compared to cells transduced with scrambled non-target negative control shRNA, Keap1-KD RAW cells showed enhanced protein levels of IKKβ and increased expression and phosphorylation of NF-κB p65 under non-stressed and LPS-treated conditions. Because the expression of Il6 in Keap1-KD RAW cells was significantly attenuated by silencing of Ikkβ, but not Nrf2, it appears that stabilized IKKβ is responsible for the enhanced transactivation of Il6 in Keap1-KD cells. This study demonstrated that silencing of Keap1 in macrophages boosts LPS-induced transcription of Il6 via NF-κB activation. Given the importance of IL6 in the inflammatory response, the Keap1–IKKβ–NF-κB pathway may be a novel target for treatment and prevention of inflammation and associated disorders. - Highlights: • Knockdown of Keap1 increases expression of Il6 in macrophages. • Silencing of Keap1 results in protein accumulation of IKKβ and NF-κB p65. • Induction of Il6 resulting from Keap1 silencing is attributed to NF-κB activation.

  5. Involvement of mitogen-activated protein kinases (MAPKs) during testicular ischemia-reperfusion injury in nuclear factor-kappaB knock-out mice.

    PubMed

    Minutoli, Letteria; Antonuccio, Pietro; Polito, Francesca; Bitto, Alessandra; Fiumara, Tiziana; Squadrito, Francesco; Nicotina, Piero Antonio; Arena, Salvatore; Marini, Herbert; Romeo, Carmelo; Altavilla, Domenica

    2007-07-12

    Nuclear factor kappa-B (NF-kappaB), extracellular regulated kinase (ERK 1/2) and c-jun-N terminal kinase (JNK) play an important role in testicular ischemia. We investigated the patterns of ERK1/2, JNK and p38 activation in NF-kappaB knockout (KO) mice subjected to testicular torsion. KO and normal littermate wild-type (WT) animals underwent at 1 h testicular ischemia followed by 24 h reperfusion (TI/R). Sham testicular ischemia-reperfusion mice served as controls. ERK 1/2, JNK and p38 expression by western blot analysis, tumor necrosis factor-alpha (TNF-alpha) expression (RT-PCR and western blot analysis) and a complete histological examination were carried out. TI/R caused a greater increase in phosphorylated form of ERK 1/2 in KO mice than in WT animals in either the ischemic testis and the contralateral one. By contrary, active form of JNK and p38 were completely abrogated in both testes of KO mice, while WT animals showed a significant activation of those kinases in both testes. TNF-alpha expression was markedly reduced in KO mice when compared to WT mice either at the mRNA and the protein level. Finally TI/R-induced histological damage was markedly reduced in KO mice. Our data indicate that NF-kappaB plays a pivotal role in the development of testicular ischemia-reperfusion injury and suggest that, in the absence of the transcriptional factor, the up-stream signal JNK and p38 may be abrogated while ERK 1/2 activity is enhanced.

  6. Nuclear factor of activated T cells (NFATc4) is required for BDNF-dependent survival of adult-born neurons and spatial memory formation in the hippocampus.

    PubMed

    Quadrato, Giorgia; Benevento, Marco; Alber, Stefanie; Jacob, Carolin; Floriddia, Elisa M; Nguyen, Tuan; Elnaggar, Mohamed Y; Pedroarena, Christine M; Molkentin, Jeffrey D; Di Giovanni, Simone

    2012-06-05

    New neurons generated in the adult dentate gyrus are constantly integrated into the hippocampal circuitry and activated during encoding and recall of new memories. Despite identification of extracellular signals that regulate survival and integration of adult-born neurons such as neurotrophins and neurotransmitters, the nature of the intracellular modulators required to transduce those signals remains elusive. Here, we provide evidence of the expression and transcriptional activity of nuclear factor of activated T cell c4 (NFATc4) in hippocampal progenitor cells. We show that NFATc4 calcineurin-dependent activity is required selectively for survival of adult-born neurons in response to BDNF signaling. Indeed, cyclosporin A injection and stereotaxic delivery of the BDNF scavenger TrkB-Fc in the mouse dentate gyrus reduce the survival of hippocampal adult-born neurons in wild-type but not in NFATc4(-/-) mice and do not affect the net rate of neural precursor proliferation and their fate commitment. Furthermore, associated with the reduced survival of adult-born neurons, the absence of NFATc4 leads to selective defects in LTP and in the encoding of hippocampal-dependent spatial memories. Thus, our data demonstrate that NFATc4 is essential in the regulation of adult hippocampal neurogenesis and identify NFATc4 as a central player of BDNF-driven prosurvival signaling in hippocampal adult-born neurons.

  7. Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways.

    PubMed

    Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

    2015-08-01

    The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity.

  8. Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways

    PubMed Central

    Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

    2015-01-01

    Abstract The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity. PMID:26061361

  9. Enhancement of CYP3A4 activity in Hep G2 cells by lentiviral transfection of hepatocyte nuclear factor-1 alpha.

    PubMed

    Chiang, Tsai-Shin; Yang, Kai-Chiang; Chiou, Ling-Ling; Huang, Guan-Tarn; Lee, Hsuan-Shu

    2014-01-01

    Human hepatoma cell lines are commonly used as alternatives to primary hepatocytes for the study of drug metabolism in vitro. However, the phase I cytochrome P450 (CYP) enzyme activities in these cell lines occur at a much lower level than their corresponding activities in primary hepatocytes, and thus these cell lines may not accurately predict drug metabolism. In the present study, we selected hepatocyte nuclear factor-1 alpha (HNF1α) from six transcriptional regulators for lentiviral transfection into Hep G2 cells to optimally increase their expression of the CYP3A4 enzyme, which is the major CYP enzyme in the human body. We subsequently found that HNF1α-transfected Hep G2 enhanced the CYP3A4 expression in a time- and dose-dependent manner and the activity was noted to increase with time and peaked 7 days. With a multiplicity of infection (MOI) of 100, CYP3A4 expression increased 19-fold and enzyme activity more than doubled at day 7. With higher MOI (1,000 to 3,000), the activity increased 8- to 10-fold; however, it was noted the higher MOI, the higher cell death rate and lower cell survival. Furthermore, the CYP3A4 activity in the HNF1α-transfected cells could be induced by CYP3A4-specific inducer, rifampicin, and metabolized nifedipine in a dose-dependent manner. With an MOI of 3,000, nifedipine-metabolizing activity was 6-fold of control and as high as 66% of primary hepatocytes. In conclusion, forceful delivery of selected transcriptional regulators into human hepatoma cells might be a valuable method to enhance the CYP activity for a more accurate determination of drug metabolism in vitro.

  10. Fisetin Ameliorated Photodamage by Suppressing the Mitogen-Activated Protein Kinase/Matrix Metalloproteinase Pathway and Nuclear Factor-κB Pathways.

    PubMed

    Chiang, Hsiu-Mei; Chan, Shih-Yun; Chu, Yin; Wen, Kuo-Ching

    2015-05-13

    Ultraviolet (UV) irradiation is one of the most important extrinsic factors contributing to skin photodamage. After UV irradiation, a series of signal transductions in the skin will be activated, leading to inflammatory response and photoaged skin. In this study, fisetin, a flavonol that exists in fruits and vegetables, was investigated for its photoprotective effects. The results revealed that 5-25 μM fisetin inhibits cyclooxygenase-2 (COX-2) and matrix metalloproteinase (MMP)-1, MMP-3, MMP-9 expression induced by ultraviolet B (UVB) irradiation in human skin fibroblasts. In addition, fisetin suppressed UVB-induced collagen degradation. With regard to its effect on upper-stream signal transduction, we found that fisetin reduced the expression of ultraviolet (UV)-induced ERK, JNK, and p38 phosphorylation in the mitogen-activated protein kinase (MAP kinase) pathway. Furthermore, fisetin reduced inhibitor κB (IκB) degradation and increased the amount of p65, which is a major subunit of nuclear factor-κB (NF-κB), in cytoplasm. It also suppressed NF-κB translocated to the nucleus and inhibited cAMP response element-binding protein (CREB) Ser-133 phosphorylation level in the phosphoinositide 3-kinase/protein kinase B/CREB (PI3K/AKT/CREB) pathway. Finally, fisetin inhibited UV-induced intracellular reactive oxygen species (ROS), prostaglandin E2 (PGE2), and nitric oxide (NO) generation. The mentioned effects and mechanisms suggest that fisetin can be used in the development of photoprotective agents.

  11. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB*

    PubMed Central

    Tomasello, Danielle L.; Gancarz-Kausch, Amy M.; Dietz, David M.; Bhattacharjee, Arin

    2015-01-01

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene. PMID:26100633

  12. Transcriptional Regulation of the Sodium-activated Potassium Channel SLICK (KCNT2) Promoter by Nuclear Factor-κB.

    PubMed

    Tomasello, Danielle L; Gancarz-Kausch, Amy M; Dietz, David M; Bhattacharjee, Arin

    2015-07-24

    Although recent studies have shown the sodium-activated potassium channel SLACK (KCNT1) can contribute to neuronal excitability, there remains little information on the physiological role of the closely related SLICK (KCNT2) channel. Activation of SLICK channels may be important during pathological states such as ischemia, in which an increase in intracellular sodium and chloride can perturb membrane potential and ion homeostasis. We have identified two NFκB-binding sites within the promoter region of the human SLICK (KCNT2) and orthologous rat Slick (Kcnt2) genes, suggesting that conditions in which NFκB transcriptional activity is elevated promote expression of this channel. NFκB binding to the rat Slick promoter was confirmed in vivo by ChIP analyses, and NFκB was found differentially bound to the two sites. We verified NFκB transcriptional regulation of SLICK/Slick by mutational analyses and studying gene expression by luciferase assay in P19 cells, where NFκB is constitutively active. For the rat gene, activation of the Slick promoter was found to be additive in single NFκB mutations and synergistic in double mutations. Unexpectedly, for the human gene, NFκB exhibited cooperativity in activating the SLICK promoter. The human SLICK promoter constructs were then tested under hypoxic conditions in PC-12 cells, where NFκB is not active. Only under hypoxic conditions could luciferase activity be detected; the double NFκB mutant construct failed to exhibit activity. Transcriptional regulation of Slick by NFκB was verified in primary neurons. The Slick transcript decreased 24 h after NFκB inhibition. Our data show SLICK expression is predominantly under the control of NFκB. Because neuronal NFκB activation occurs during stressful stimuli such as hypoxia and injury, our findings suggest that SLICK is a neuroprotective gene.

  13. Hydrogen inhalation reduced epithelial apoptosis in ventilator-induced lung injury via a mechanism involving nuclear factor-kappa B activation

    SciTech Connect

    Huang, Chien-Sheng; Kawamura, Tomohiro; Peng, Ximei; Tochigi, Naobumi; Shigemura, Norihisa; Billiar, Timothy R.; Nakao, Atsunori; Toyoda, Yoshiya

    2011-05-06

    Highlights: {yields} Hydrogen is a regulatory molecule with antiinflammatory and antiapoptotic protective effects. {yields} There is very limited information on the pathways regulated in vivo by the hydrogen. {yields} Antiapoptotic abilities of hydrogen were explained by upregulation of the antiapoptotic gene. {yields} NF{kappa}B activation during hydrogen treatment was correlated with elevated antiapoptotic protein. {yields} NF{kappa}B activation associated with increase Bcl-2 may contribute to cytoprotection of hydrogen. -- Abstract: We recently demonstrated the inhalation of hydrogen gas, a novel medical therapeutic gas, ameliorates ventilator-induced lung injury (VILI); however, the molecular mechanisms by which hydrogen ameliorates VILI remain unclear. Therefore, we investigated whether inhaled hydrogen gas modulates the nuclear factor-kappa B (NF{kappa}B) signaling pathway. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg or 10 ml/kg without positive end-expiratory pressure). The ventilator delivered either 2% nitrogen or 2% hydrogen in balanced air. NF{kappa}B activation, as indicated by NF{kappa}B DNA binding, was detected by electrophoretic mobility shift assays and enzyme-linked immunosorbent assay. Hydrogen gas inhalation increased NF{kappa}B DNA binding after 1 h of ventilation and decreased NF{kappa}B DNA binding after 2 h of ventilation, as compared with controls. The early activation of NF{kappa}B during hydrogen treatment was correlated with elevated levels of the antiapoptotic protein Bcl-2 and decreased levels of Bax. Hydrogen inhalation increased oxygen tension, decreased lung edema, and decreased the expression of proinflammatory mediators. Chemical inhibition of early NF{kappa}B activation using SN50 reversed these protective effects. NF{kappa}B activation and an associated increase in the expression of Bcl-2 may contribute, in part, to the

  14. The transcription factor nuclear factor-kappa B and cancer.

    PubMed

    Escárcega, R O; Fuentes-Alexandro, S; García-Carrasco, M; Gatica, A; Zamora, A

    2007-03-01

    Since the discovery of nuclear factor-kappa B (NF-kappaB) in 1986, many studies have been conducted showing the link between the NF-kappaB signalling pathway and control of the inflammatory response. Today it is well known that control of the inflammatory response and apoptosis is closely related to the activation of NF-kappaB. Three NF-kappaB activation pathways exist. The first (the classical pathway) is normally triggered in response to microbial and viral infections or exposure to pro-inflammatory cytokines that activate the tripartite IKK complex, leading to phosphorylation-induced IkappaB degradation and depends mainly on IKKbeta activity. The second (the alternative pathway), leads to selective activation of p52:RelB dimers by inducing the processing of the NF-kappaB2/p100 precursor protein, which mostly occurs as a heterodimer with RelB in the cytoplasm. This pathway is triggered by certain members of the tumour necrosis factor cytokine family, through selective activation of IKKalpha homodimers by the upstream kinase NIK. The third pathway is named CK2 and is IKK independent. NF-kappaB acts through the transcription of anti-apoptotic proteins, leading to increased proliferation of cells and tumour growth. It is also known that some drugs act directly in the inhibition of NF-kappaB, thus producing regulation of apoptosis; some examples are aspirin and corticosteroids. Here we review the role of NF-kappaB in the control of apoptosis, its link to oncogenesis, the evidence of several studies that show that NF-kappaB activation is closely related to different cancers, and finally the potential target of NF-kappaB as cancer therapy.

  15. Fat-specific protein 27 modulates nuclear factor of activated T cells 5 and the cellular response to stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fat-specific protein 27 (FSP27), a member of the cell death-inducing DNA fragmentation factor a-like effector (Cide) family, is highly expressed in adipose tissues and is a lipid droplet (LD)-associated protein that induces the accumulation of LDs. Using a yeast two-hybrid system to examine potentia...

  16. Factorized molecular wave functions: Analysis of the nuclear factor

    SciTech Connect

    Lefebvre, R.

    2015-06-07

    The exact factorization of molecular wave functions leads to nuclear factors which should be nodeless functions. We reconsider the case of vibrational perturbations in a diatomic species, a situation usually treated by combining Born-Oppenheimer products. It was shown [R. Lefebvre, J. Chem. Phys. 142, 074106 (2015)] that it is possible to derive, from the solutions of coupled equations, the form of the factorized function. By increasing artificially the interstate coupling in the usual approach, the adiabatic regime can be reached, whereby the wave function can be reduced to a single product. The nuclear factor of this product is determined by the lowest of the two potentials obtained by diagonalization of the potential matrix. By comparison with the nuclear wave function of the factorized scheme, it is shown that by a simple rectification, an agreement is obtained between the modified nodeless function and that of the adiabatic scheme.

  17. Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta.

    PubMed

    Tian, Qingping; Miyazaki, Ryohei; Ichiki, Toshihiro; Imayama, Ikuyo; Inanaga, Keita; Ohtsubo, Hideki; Yano, Kotaro; Takeda, Kotaro; Sunagawa, Kenji

    2009-05-01

    Telmisartan, an angiotensin II type 1 receptor antagonist, was reported to be a partial agonist of peroxisome proliferator-activated receptor-gamma. Although peroxisome proliferator-activated receptor-gamma activators have been shown to have an anti-inflammatory effect, such as inhibition of cytokine production, it has not been determined whether telmisartan has such effects. We examined whether telmisartan inhibits expression of interleukin-6 (IL-6), a proinflammatory cytokine, in vascular smooth muscle cells. Telmisartan, but not valsartan, attenuated IL-6 mRNA expression induced by tumor necrosis factor-alpha (TNF-alpha). Telmisartan decreased TNF-alpha-induced IL-6 mRNA and protein expression in a dose-dependent manner. Because suppression of IL-6 mRNA expression was prevented by pretreatment with GW9662, a specific peroxisome proliferator-activated receptor-gamma antagonist, peroxisome proliferator-activated receptor-gamma may be involved in the process. Telmisartan suppressed IL-6 gene promoter activity induced by TNF-alpha. Deletion analysis suggested that the DNA segment between -150 bp and -27 bp of the IL-6 gene promoter that contains nuclear factor kappaB and CCAAT/enhancer-binding protein-beta sites was responsible for telmisartan suppression. Telmisartan attenuated TNF-alpha-induced nuclear factor kappaB- and CCAAT/enhancer-binding protein-beta-dependent gene transcription and DNA binding. Telmisartan also attenuated serum IL-6 level in TNF-alpha-infused mice and IL-6 production from rat aorta stimulated with TNF-alpha ex vivo. These data suggest that telmisartan may attenuate inflammatory process induced by TNF-alpha in addition to the blockade of angiotensin II type 1 receptor. Because both TNF-alpha and angiotensin II play important roles in atherogenesis through enhancement of vascular inflammation, telmisartan may be beneficial for treatment of not only hypertension but also vascular inflammatory change.

  18. Inhibition of the nuclear factor kappa B (NF-kappa B) pathway by tetracyclic kaurene diterpenes in macrophages. Specific effects on NF-kappa B-inducing kinase activity and on the coordinate activation of ERK and p38 MAPK.

    PubMed

    Castrillo, A; de Las Heras, B; Hortelano, S; Rodriguez, B; Villar, A; Bosca, L

    2001-05-11

    The anti-inflammatory action of most terpenes has been explained in terms of the inhibition of nuclear factor kappaB (NF-kappaB) activity. Ent-kaurene diterpenes are intermediates of the synthesis of gibberellins and inhibit the expression of NO synthase-2 and the release of tumor necrosis factor-alpha in J774 macrophages challenged with lipopolysaccharide. These diterpenes inhibit NF-kappaB and IkappaB kinase (IKK) activation in vivo but failed to affect in vitro the function of NF-kappaB, the phosphorylation and targeting of IkappaBalpha, and the activity of IKK-2. Transient expression of NF-kappaB-inducing kinase (NIK) activated the IKK complex and NF-kappaB, a process that was inhibited by kaurenes, indicating that the inhibition of NIK was one of the targets of these diterpenes. These results show that kaurenes impair the inflammatory signaling by inhibiting NIK, a member of the MAPK kinase superfamily that interacts with tumor necrosis factor receptor-associated factors, and mediate the activation of NF-kappaB by these receptors. Moreover, kaurenes delayed the phosphorylation of p38, ERK1, and ERK2 MAPKs, but not that of JNK, in response to lipopolysaccharide treatment of J774 cells. The absence of a coordinate activation of MAPK and IKK might contribute to a deficient activation of NF-kappaB that is involved in the anti-inflammatory activity of these molecules.

  19. Isoliquiritigenin Inhibits Metastatic Breast Cancer Cell-induced Receptor Activator of Nuclear Factor Kappa-B Ligand/Osteoprotegerin Ratio in Human Osteoblastic Cells

    PubMed Central

    Lee, Sun Kyoung; Park, Kwang-Kyun; Kim, Ki Rim; Kim, Hyun-Jeong; Chung, Won-Yoon

    2015-01-01

    Bone destruction induced by the metastasis of breast cancer cells is a frequent complication that is caused by the interaction between cancer cells and bone cells. Receptor activator of nuclear factor kappa-B ligand (RANKL) and the endogenous soluble RANKL inhibitor, osteoprotegerin (OPG), directly play critical roles in the differentiation, activity, and survival of osteoclasts. In patients with bone metastases, osteoclastic bone resorption promotes the majority of skeletal-related events and propagates bone metastases. Therefore, blocking osteoclast activity and differentiation via RANKL inhibition can be a promising therapeutic approach for cancer-associated bone diseases. We investigated the potential of isoliquiritigenin (ISL), which has anti-proliferative, anti-angiogenic, and anti-invasive effects, as a preventive and therapeutic agent for breast cancer cell-induced bone destruction. ISL at non-toxicity concentrations significantly inhibited the RANKL/OPG ratio by reducing the production of RANKL and restoring OPG production to control levels in hFOB1.19 cells stimulated with conditioned medium (CM) of MDA-MB-231 cells. In addition, ISL reduced the expression of cyclooxygenase-2 in hFOB1.19 cells stimulated by CM of MDA-MB-231 cells. Therefore, ISL may have inhibitory potential on breast cancer-induced bone destruction. PMID:26734591

  20. Expression of the receptor activator of nuclear factor-kB ligand in peripheral blood mononuclear cells in patients with acute Charcot neuroarthropathy

    PubMed Central

    Bergamini, Alberto; Bolacchi, Francesca; Pesce, Caterina Delfina; Veneziano, Giada; Uccioli, Luigi; Girardi, Valentina; De Corato, Laura; Mondillo, Maria Teresa; Squillaci, Ettore

    2016-01-01

    Introduction. The receptor activator of nuclear factor-kB (RANK), ligand (RANK-L) and osteoprotegerin (OPG) are implicated in the pathogenesis of acute Charcot neuroarthropathy (CN). Materials and Methods. This study aimed to investigate the expression of RANK-L and OPG in peripheral blood mononuclear cells (PBMC) from patients with acute CN. Results. We found that the expression of RANK-L was lower in patients with acute CN as compared with diabetic control subjects and healthy control participants; whereas OPG expression was not detected in patients and in both control groups. RANK-L expression at the onset of disease was inversely correlated with the index of polyunsaturation (PUI), a bone marrow MRS-derived measurable index that allows evaluation of disease activity in acute CN, and recovery time. Finally, the expression of RANK-L increased at the time of healing compared with the values found during the acute phase. Conclusions. In conclusion, our preliminary data provide a first step in applying analysis of RANK-L expression in peripheral blood cells to the diagnosis of acute CN. Based on our data we also suggest that analysis of RANK-L expression could be a complementary tool that can be employed to obtain quantitative parameters that may help clinicians to monitor disease activity in patients with acute CN. PMID:28090190

  1. Protective mechanism of Korean Red Ginseng in cisplatin-induced ototoxicity through attenuation of nuclear factor-κB and caspase-1 activation.

    PubMed

    Kim, Su-Jin; Kwak, Hyun Jeong; Kim, Dae-Seung; Choi, Hyun-Myung; Sim, Jung-Eun; Kim, Sung-Hoon; Um, Jae-Young; Hong, Seung-Heon

    2015-07-01

    Cisplatin is an effective anti-cancer drug; however, one of its side effects is irreversible sensorineural hearing damage. Korean Red Ginseng (KRG) has been used clinically for the treatment of various diseases; however, the underlying mechanism of KRG treatment of ototoxicity has not been studied extensively. The present study aimed to further investigate the mechanism of KRG on cisplatin-induced toxicity in auditory HEI-OC1 cells in vitro, as well as in vivo. The pharmacological effects of KRG on cisplatin-induced changes in the hearing threshold of mice were determined, as well as the effect on the impairment of hair cell arrays. In addition, in order to elucidate the protective mechanisms of KRG, the regulatory effects of KRG on cisplatin-induced apoptosis-associated gene levels and nuclear factor-κB (NF-κB) activation were investigated in auditory cells. The results revealed that KRG prevented cisplatin-induced alterations in the hearing threshold of mice as well as the destruction of hair cell arrays in rat organ of Corti primary explants. In addition, KRG inhibited cisplatin-mediated cell toxicity, reactive oxygen species generation, interleukin-6 production, cytochrome c release and activation of caspases-3 in the HEI-OC1 auditory cell line. Furthermore, the results demonstrated that KRG inhibited the activation of NF-κB and caspase-1. In conclusion, these results provided a model for the pharmacological mechanism of KRG and provided evidence for potential therapies against ototoxicity.

  2. Aloperine attenuates hydrogen peroxide-induced injury via anti-apoptotic activity and suppression of the nuclear factor-κB signaling pathway

    PubMed Central

    Ren, Dongliang; Ma, Weisong; Guo, Baozhen; Wang, Shunyi

    2017-01-01

    Aloperine is an alkaloid that exerts significant inhibitive effects on acute inflammation and Type III and IV hypersensitivity caused by a variety of inflammatory agents. The aims of the present study were to investigate whether the protective effect of aloperine attenuates hydrogen peroxide (H2O2)-induced injury, and to identify the underlying mechanisms involved. Nucleus pulposus cells were extracted from adult male Sprague-Dawley rats, and incubated with fresh medium containing 200 µM H2O2 for 24 h. In the study, treatment with aloperine significantly increased cell viability and suppressed apoptosis in H2O2-treated nucleus pulposus cells in a dose-dependent manner. In addition, 10 and 100 nM aloperine significantly inhibited H2O2-induced tumor necrosis factor-α and interleukin-6 activities, and significantly increased the H2O2-reduced superoxide dismutase and glutathione peroxidase activities in nucleus pulposus cells (all P<0.01). However, aloperine treatment (10 and 100 nM) significantly reduced the H2O2-induced caspase-9 activity in nucleus pulposus cells. Furthermore, addition of 10 and 100 nM aloperine significantly suppressed nuclear factor-κB (NF-κB) and phosphorylated-protein kinase B expression levels in H2O2-treated nucleus pulposus cells. In conclusion, the protective effect of aloperine attenuated H2O2-induced injury via hyperproliferation, its anti-apoptotic activity and suppression of the NF-κB signaling pathway. PMID:28123508

  3. Estrogen controls embryonic stem cell proliferation via store-operated calcium entry and the nuclear factor of activated T-cells (NFAT).

    PubMed

    Wong, Chun-Kit; So, Wing-Yan; Law, Sau-Kwan; Leung, Fung-Ping; Yau, Ka-Long; Yao, Xiaoqiang; Huang, Yu; Li, Xiangdong; Tsang, Suk-Ying

    2012-06-01

    Embryonic stem cells (ESCs) can self-renew indefinitely and differentiate into all cell lineages. Calcium is a universal second messenger which regulates a number of cellular pathways. Previous studies showed that store-operated calcium channels (SOCCs) but not voltage-operated calcium channels are present in mouse ESCs (mESCs). In this study, store-operated calcium entry (SOCE) was found to exist in mESCs using confocal microscopy. SOCC blockers lanthanum, 2-aminoethoxydiphenyl borate (2-APB) and SKF-96365 reduced mESC proliferation in a concentration-dependent manner, suggesting that SOCE is important for ESC proliferation. Pluripotent markers, Sox-2, Klf-4, and Nanog, were down-regulated by 2-APB, suggesting that self-renewal property of mESCs relies on SOCE. 17β-estradiol (E2) enhanced mESC proliferation. This enhanced proliferation was associated with an increment of SOCE. Both stimulated proliferation and increased SOCE could be reversed by SOCC blockers suggesting that E2 mediates its stimulatory effect on proliferation via enhancing SOCE. Also, cyclosporin A and INCA-6, inhibitors of calcineurin [phosphatase that de-phosphorylates and activates nuclear factor of activated T-cells (NFAT)], reversed the proliferative effect of E2, indicating that NFAT is involved in E2-stimulated proliferation. Interestingly, E2 caused the nuclear translocation of NFATc4, and this could be reversed by 2-APB. These results suggested that NFATc4 is the downstream target of E2-induced SOCE. The present investigation provides the first line of evidence that SOCE and NFAT are crucial for ESCs to maintain their unique characteristics. In addition, the present investigation also provides novel information on the mechanisms of how E2, an important female sex hormone, affects ESC proliferation.

  4. Andrographolide Inhibits Nuclear Factor-κB Activation through JNK-Akt-p65 Signaling Cascade in Tumor Necrosis Factor-α-Stimulated Vascular Smooth Muscle Cells

    PubMed Central

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Lee, Lin-Wen; Sheu, Joen-Rong

    2014-01-01

    Critical vascular inflammation leads to vascular dysfunction and cardiovascular diseases, including abdominal aortic aneurysms, hypertension, and atherosclerosis. Andrographolide is the most active and critical constituent isolated from the leaves of Andrographis paniculata, a herbal medicine widely used for treating anti-inflammation in Asia. In this study, we investigated the mechanisms of the inhibitory effects of andrographolide in vascular smooth muscle cells (VSMCs) exposed to a proinflammatory stimulus, tumor necrosis factor-α (TNF-α). Treating TNF-α-stimulated VSMCs with andrographolide suppressed the expression of inducible nitric oxide synthase in a concentration-dependent manner. A reduction in TNF-α-induced c-Jun N-terminal kinase (JNK), Akt, and p65 phosphorylation was observed in andrographolide-treated VSMCs. However, andrographolide affected neither IκBα degradation nor p38 mitogen-activated protein kinase or extracellular signal-regulated kinase 1/2 phosphorylation under these conditions. Both treatment with LY294002, a phosphatidylinositol 3-kinase/Akt inhibitor, and treatment with SP600125, a JNK inhibitor, markedly reversed the andrographolide-mediated inhibition of p65 phosphorylation. In addition, LY294002 and SP600125 both diminished Akt phosphorylation, whereas LY294002 had no effects on JNK phosphorylation. These results collectively suggest that therapeutic interventions using andrographolide can benefit the treatment of vascular inflammatory diseases, and andrographolide-mediated inhibition of NF-κB activity in TNF-α-stimulated VSMCs occurs through the JNK-Akt-p65 signaling cascade, an IκBα-independent mechanism. PMID:25114952

  5. Deletion Of XIAP reduces the severity of acute pancreatitis via regulation of cell death and nuclear factor-κB activity.

    PubMed

    Liu, Yong; Chen, Xiao-Dong; Yu, Jiang; Chi, Jun-Lin; Long, Fei-Wu; Yang, Hong-Wei; Chen, Ke-Ling; Lv, Zhao-Ying; Zhou, Bin; Peng, Zhi-Hai; Sun, Xiao-Feng; Li, Yuan; Zhou, Zong-Guang

    2017-03-16

    Severe acute pancreatitis (SAP) still remains a clinical challenge, not only for its high mortality but the uncontrolled inflammatory progression from acute pancreatitis (AP) to SAP. Cell death, including apoptosis and necrosis are critical pathology of AP, since the severity of pancreatitis correlates directly with necrosis and inversely with apoptosis Therefore, regulation of cell death from necrosis to apoptosis may have practicably therapeutic value. X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the inhibitor of apoptosis proteins (IAP) family, but its function in AP remains unclear. In the present study, we investigated the potential role of XIAP in regulation of cell death and inflammation during acute pancreatitis. The in vivo pancreatitis model was induced by the administration of cerulein with or without lipopolysaccharide (LPS) or by the administration of l-arginine in wild-type or XIAP-deficient mice, and ex vivo model was induced by the administration of cerulein+LPS in AR42J cell line following XIAP inhibition. The severity of acute pancreatitis was determined by serum amylase activity and histological grading. XIAP deletion on cell apoptosis, necrosis and inflammatory response were examined. Caspases activities, nuclear factor-κB (NF-κB) activation and receptor-interacting protein kinase1 (RIP1) degradation were assessed by western blot. Deletion of XIAP resulted in the reduction of amylase activity, decrease of NF-κB activation and less release of TNF-α and IL-6, together with increased caspases activities and RIP1 degradation, leading to enhanced apoptosis and reduced necrosis in pancreatic acinar cells and ameliorated the severity of acute pancreatitis. Our results indicate that deletion of XIAP switches cell death away from necrosis to apoptosis and decreases the inflammatory response, effectively attenuating the severity of AP/SAP. The critical role of XIAP in cell death and inflammation suggests that

  6. Release of positive transcription elongation factor b (P-TEFb) from 7SK small nuclear ribonucleoprotein (snRNP) activates hexamethylene bisacetamide-inducible protein (HEXIM1) transcription.

    PubMed

    Liu, Pingyang; Xiang, Yanhui; Fujinaga, Koh; Bartholomeeusen, Koen; Nilson, Kyle A; Price, David H; Peterlin, B Matija

    2014-04-04

    By phosphorylating negative elongation factors and the C-terminal domain of RNA polymerase II (RNAPII), positive transcription elongation factor b (P-TEFb), which is composed of CycT1 or CycT2 and CDK9, activates eukaryotic transcription elongation. In growing cells, it is found in active and inactive forms. In the former, free P-TEFb is a potent transcriptional coactivator. In the latter, it is inhibited by HEXIM1 or HEXIM2 in the 7SK small nuclear ribonucleoprotein (snRNP), which contains, additionally, 7SK snRNA, methyl phosphate-capping enzyme (MePCE), and La-related protein 7 (LARP7). This P-TEFb equilibrium determines the state of growth and proliferation of the cell. In this study, the release of P-TEFb from the 7SK snRNP led to increased synthesis of HEXIM1 but not HEXIM2 in HeLa cells, and this occurred only from an unannotated, proximal promoter. ChIP with sequencing revealed P-TEFb-sensitive poised RNA polymerase II at this proximal but not the previously annotated distal HEXIM1 promoter. Its immediate upstream sequences were fused to luciferase reporters and were found to be responsive to many P-TEFb-releasing compounds. The superelongation complex subunits AF4/FMR2 family member 4 (AFF4) and elongation factor RNA polymerase II 2 (ELL2) were recruited to this proximal promoter after P-TEFb release and were required for its transcriptional effects. Thus, P-TEFb regulates its own equilibrium in cells, most likely to maintain optimal cellular homeostasis.

  7. Activated nuclear transcription factor {kappa}B in patients with myocarditis and dilated cardiomyopathy-relation to inflammation and cardiac function

    SciTech Connect

    Alter, Peter . E-mail: palter@med.uni-marburg.de; Rupp, Heinz; Maisch, Bernhard

    2006-01-06

    Objectives and background: Myocarditis is caused by various agents and autoimmune processes. It is unknown whether viral genome persistence represents inactive remnants of previous infections or whether it is attributed to ongoing adverse processes. The latter also applies to the course of autoimmune myocarditis. One principal candidate for an adverse remodeling is nuclear factor-{kappa}B (NF{kappa}B). Methods: A total of 93 patients with suspected myocarditis/cardiomyopathy was examined. Hemodynamics were assessed by echocardiography as well as right and left heart catheterization. Endomyocardial biopsies were taken from the left ventricle. Biopsies were examined by immunohistochemistry and PCR for viral genomes. Selective immunostaining of activated NF{kappa}B was performed. Results: NF{kappa}B was increased in patients with myocarditis when compared with controls (11.1 {+-} 7.1% vs. 5.0 {+-} 5.3%, P < 0.005) whereas dilated cardiomyopathy showed no significant increase. Patients with myocarditis and preserved left ventricular function exhibited increased activated NF{kappa}B when compared with reduced function (r {sup 2} = 0.72, P < 0.001). In parallel, inverse correlation of NF{kappa}B and left ventricular enddiasstolic volume was found (r {sup 2} = 0.43, P < 0.02). Increased activated NF{kappa}B was found in adenovirus persistence when compared with controls (P = 0.001). Only a trend of increased NF{kappa}B activation was seen in cytomegalovirus persistence. Parvovirus B19 persistence did not affect NF{kappa}B activation. Conclusions: Increased activation of NF{kappa}B is related to inflammatory processes in myocarditis. Since activated NF{kappa}B correlates with left ventricular function, it could be assumed that NF{kappa}B activation occurs at early stages of inflammation. Potentially, NF{kappa}B could inhibit loss of cardiomyocytes by apoptosis and protect from cardiac dilation. Since NF{kappa}B is a crucial key transcription factor of inflammation, its

  8. Cyclosporin A promotes proliferating cell nuclear antigen expression and migration of human cytotrophoblast cells via the mitgen-activated protein kinase-3/1-mediated nuclear factor-κB signaling pathways.

    PubMed

    Wang, Song-Cun; Yu, Min; Li, Yan-Hong; Piao, Hai-Lan; Tang, Chuan-Lin; Sun, Chan; Zhu, Rui; Li, Ming Qing; Jin, Li-Ping; Li, Da-Jin; Du, Mei-Rong

    2013-01-01

    Our previous studies have demonstrated that cyclosporin A (CsA) promotes the proliferation and migration of human trophoblasts via the mitgen-activated protein kinase-3/1 (MAPK3/1) pathway. In the present study, we further investigated the role of nuclear factor (NF)-κB in the CsA-induced trophoblast proliferating cell nuclear antigen (PCNA) expression and migration, and its relationship to MAPK3/1 signal. Flow cytometry was used to analyze the expression of PCNA in trophoblasts. The migration of human primary trophoblasts was determined by wound-healing assay and transwell migration assay. Western blot analysis was performed to evaluate the activation of NF-κB p65 and NF-κB inhibitory protein I-κB in human trophoblasts. We found that treatment with CsA promotes PCNA expression and migration of human trophoblast in a dose-associated manner. Blocking of the MAPK3/1 signal abrogated the enhanced PCNA expression and migration in trophoblasts by CsA. In addition, CsA increased the phosphorylation of NF-κB p65 and the inhibitor I-κB in human trophoblasts in a time-related manner. Pretreatment with MAPK3/1 inhibitor U0126 abrogated the phosphorylation of NF-κB p65 and I-κB. Accordingly, the CsA-induced enhancement of PCNA expression and migration in trophoblasts was also decreased. This CsA-induced enhancement in the expression and migration of trophoblasts was abolished by pretreatment with pyrrolidine dithiocarbamate, a specific NF-κB inhibitor. Thus, our results suggest that CsA promotes PCNA expression and migration of human trophoblasts via MAPK-mediated NF-κB activation.

  9. Electrophilic nitro-fatty acids prevent astrocyte-mediated toxicity to motor neurons in a cell model of familial amyotrophic lateral sclerosis via nuclear factor erythroid 2-related factor activation.

    PubMed

    Diaz-Amarilla, Pablo; Miquel, Ernesto; Trostchansky, Andrés; Trias, Emiliano; Ferreira, Ana M; Freeman, Bruce A; Cassina, Patricia; Barbeito, Luis; Vargas, Marcelo R; Rubbo, Homero

    2016-06-01

    Nitro-fatty acids (NO2-FA) are electrophilic signaling mediators formed in tissues during inflammation, which are able to induce pleiotropic cytoprotective and antioxidant pathways including up regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2) responsive genes. Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motor neurons associated to an inflammatory process that usually aggravates the disease progression. In ALS animal models, the activation of the transcription factor Nrf2 in astrocytes confers protection to neighboring neurons. It is currently unknown whether NO2-FA can exert protective activity in ALS through Nrf2 activation. Herein we demonstrate that nitro-arachidonic acid (NO2-AA) or nitro-oleic acid (NO2-OA) administrated to astrocytes expressing the ALS-linked hSOD1(G93A) induce antioxidant phase II enzyme expression through Nrf2 activation concomitant with increasing intracellular glutathione levels. Furthermore, treatment of hSOD1(G93A)-expressing astrocytes with NO2-FA prevented their toxicity to motor neurons. Transfection of siRNA targeted to Nrf2 mRNA supported the involvement of Nrf2 activation in NO2-FA-mediated protective effects. Our results show for the first time that NO2-FA induce a potent Nrf2-dependent antioxidant response in astrocytes capable of preventing motor neurons death in a culture model of ALS.

  10. Expression of nuclear factor of activated T cells (NFAT) and downstream muscle-specific proteins in ground squirrel skeletal and heart muscle during hibernation.

    PubMed

    Zhang, Yichi; Storey, Kenneth B

    2016-01-01

    The thirteen-lined ground squirrel (Ictidomys tridecemlineatus) undergoes remarkable adaptive changes during hibernation. Interestingly, skeletal muscle remodelling occurs during the torpor-arousal cycle of hibernation to prevent net muscle loss despite inactivity. Reversible cardiomyocyte hypertrophy occurs in cardiac muscle, allowing the heart to preserve cardiac output during hibernation, while avoiding chronic maladaptive hypertrophy post-hibernation. We propose that calcium signalling proteins [calcineurin (Cn), calmodulin (CaM), and calpain], the nuclear factor of activated T cell (NFAT) family of transcription factors, and the NFAT targets myoferlin and myomaker contribute significantly to adaptations taking place in skeletal and cardiac muscle during hibernation. Protein-level analyses were performed over several conditions: euthermic room temperature (ER), euthermic cold room (EC), entrance into (EN), early (ET), and late torpor (LT) time points, in addition to early (EA), interbout (IA), and late arousal (LA) time points using immunoblotting and DNA-protein interaction (DPI) enzyme-linked immunosorbent assay (ELISAs). In skeletal and cardiac muscle, NFATc2 protein levels were elevated during torpor. NFATc4 increased throughout the torpor-arousal cycle in both tissues, and NFATc1 showed this trend in cardiac muscle only. NFATc3 showed an elevation in DNA-binding activity but not expression during torpor. Myoferlin protein levels dramatically increased during torpor in both skeletal and cardiac muscle. Myomaker levels also increased significantly in cardiac muscle during torpor. Cardiac Cn levels remained stable, whereas CaM and calpain decreased throughout the torpor-arousal cycle. Activation and/or upregulation of NFATc2, c3, myoferlin, and myomaker at torpor could be part of a stress-response mechanism to preserve skeletal muscle mass, whereas CaM and calpain appear to initiate the rapid reversal of cardiac hypertrophy during arousal through

  11. The Emerging Roles of the Calcineurin-Nuclear Factor of Activated T-Lymphocytes Pathway in Nervous System Functions and Diseases

    PubMed Central

    Kimaro, Wahabu Hamisi; Etet, Paul F. Seke

    2016-01-01

    The ongoing epidemics of metabolic diseases and increase in the older population have increased the incidences of neurodegenerative diseases. Evidence from murine and cell line models has implicated calcineurin-nuclear factor of activated T-lymphocytes (NFAT) signaling pathway, a Ca2+/calmodulin-dependent major proinflammatory pathway, in the pathogenesis of these diseases. Neurotoxins such as amyloid-β, tau protein, and α-synuclein trigger abnormal calcineurin/NFAT signaling activities. Additionally increased activities of endogenous regulators of calcineurin like plasma membrane Ca2+-ATPase (PMCA) and regulator of calcineurin 1 (RCAN1) also cause neuronal and glial loss and related functional alterations, in neurodegenerative diseases, psychotic disorders, epilepsy, and traumatic brain and spinal cord injuries. Treatment with calcineurin/NFAT inhibitors induces some degree of neuroprotection and decreased reactive gliosis in the central and peripheral nervous system. In this paper, we summarize and discuss the current understanding of the roles of calcineurin/NFAT signaling in physiology and pathologies of the adult and developing nervous system, with an emphasis on recent reports and cutting-edge findings. Calcineurin/NFAT signaling is known for its critical roles in the developing and adult nervous system. Its role in physiological and pathological processes is still controversial. However, available data suggest that its beneficial and detrimental effects are context-dependent. In view of recent reports calcineurin/NFAT signaling is likely to serve as a potential therapeutic target for neurodegenerative diseases and conditions. This review further highlights the need to characterize better all factors determining the outcome of calcineurin/NFAT signaling in diseases and the downstream targets mediating the beneficial and detrimental effects. PMID:27597899

  12. Transcriptional activation of LON Gene by a new form of mitochondrial stress: A role for the nuclear respiratory factor 2 in StAR overload response (SOR).

    PubMed

    Bahat, Assaf; Perlberg, Shira; Melamed-Book, Naomi; Isaac, Sara; Eden, Amir; Lauria, Ines; Langer, Thomas; Orly, Joseph

    2015-06-15

    High output of steroid hormone synthesis in steroidogenic cells of the adrenal cortex and the gonads requires the expression of the steroidogenic acute regulatory protein (StAR) that facilitates cholesterol mobilization to the mitochondrial inner membrane where the CYP11A1/P450scc enzyme complex converts the sterol to the first steroid. Earlier studies have shown that StAR is active while pausing on the cytosolic face of the outer mitochondrial membrane while subsequent import of the protein into the matrix terminates the cholesterol mobilization activity. Consequently, during repeated activity cycles, high level of post-active StAR accumulates in the mitochondrial matrix. To prevent functional damage due to such protein overload effect, StAR is degraded by a sequence of three to four ATP-dependent proteases of the mitochondria protein quality control system, including LON and the m-AAA membranous proteases AFG3L2 and SPG7/paraplegin. Furthermore, StAR expression in both peri-ovulatory ovarian cells, or under ectopic expression in cell line models, results in up to 3-fold enrichment of the mitochondrial proteases and their transcripts. We named this novel form of mitochondrial stress as StAR overload response (SOR). To better understand the SOR mechanism at the transcriptional level we analyzed first the unexplored properties of the proximal promoter of the LON gene. Our findings suggest that the human nuclear respiratory factor 2 (NRF-2), also known as GA binding protein (GABP), is responsible for 88% of the proximal promoter activity, including the observed increase of transcription in the presence of StAR. Further studies are expected to reveal if common transcriptional determinants coordinate the SOR induced transcription of all the genes encoding the SOR proteases.

  13. The Inhibitory Effect of Angelica tenuissima Water Extract on Receptor Activator of Nuclear Factor-Kappa-B Ligand-Induced Osteoclast Differentiation and Bone Resorbing Activity of Mature Osteoclasts.

    PubMed

    Ahn, Sung-Jun; Baek, Jong Min; Cheon, Yoon-Hee; Park, Sun-Hyang; Lee, Myeung Su; Oh, Jaemin; Kim, Ju-Young

    2015-01-01

    Angelica tenuissima has been traditionally used in oriental medicine for its therapeutic effects in headache, toothache, and flu symptoms. It also exerts anti-inflammatory activity via the inhibition of the expression of cyclooxygenase-2 (COX-2). However, the effect of Angelica tenuissima on osteoclast differentiation has not been identified until recently. In this study, we first confirmed that Angelica tenuissima water extract (ATWE) significantly interrupted the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) in a dose-dependent manner without any cytotoxicity. Next, we clarified the underlying mechanisms linking the suppression effects of ATWE on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. At the molecular level, ATWE induced the dephosphorylation of c-Jun N-terminal kinase (JNK) and Akt and decreased the degradation of IκB in RANKL-dependent early signaling pathways. Subsequently, ATWE caused impaired activation of the protein and mRNA levels of c-Fos and nuclear factor of activated T cell c1 (NFATc1). Moreover, the disassembly of filamentous actin (F-actin) ring and anti-resorptive activity of mature osteoclasts were triggered by ATWE treatment. Although ATWE did not enhance osteogenesis in primary osteoblasts, our results showed that ATWE is a potential candidate for anti-resorptive agent in osteoporosis, a common metabolic bone disorder.

  14. Autophagy Mediates HBx-Induced Nuclear Factor-κB Activation and Release of IL-6, IL-8, and CXCL2 in Hepatocytes.

    PubMed

    Luo, Millore X M; Wong, Sunny H; Chan, Matthew T V; Yu, Le; Yu, Sidney S B; Wu, Feng; Xiao, Zhangang; Wang, Xiaojuan; Zhang, Lin; Cheng, Alfred S L; Ng, Simon S M; Chan, Francis K L; Cho, Chi H; Yu, Jun; Sung, Joseph J Y; Wu, William K K

    2015-10-01

    Hepatitis B virus (HBV) and one of its encoded proteins, HBV X protein (HBx), have been shown to induce autophagy in hepatoma cells. Substantial evidence indicates that autophagy is a potent suppressor of inflammation. However, sporadic reports suggest that autophagy could promote pro-inflammatory cytokine expression and inflammation in some biological contexts. Here, we show that overexpression of HBx induces LC3B-positive autophagosome formation, increases autophagic flux and enhances the expression of ATG5, ATG7, and LC3B-II in normal hepatocytes. Abrogation of autophagy by small interfering RNA against ATG5 and ATG7 prevents HBx-induced formation of autophagosomes. Autophagy inhibition also abrogates HBx-induced activation of nuclear factor-κB (NF-κB) and production of interleukin-6 (IL-6), IL-8, and CXCL2. These findings suggest that autophagy is required for HBx-induced NF-κB activation and pro-inflammatory cytokine production and could shed new light on the complex role of autophagy in the modulation of inflammation.

  15. Gypenoside Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of Mitogen-Activated Protein Kinase Mediated Nuclear Factor Kappa B Pathway In Vitro and In Vivo

    PubMed Central

    Yu, Haijie; Shi, Liye; Qi, Guoxian; Zhao, Shijie; Gao, Yuan; Li, Yuzhe

    2016-01-01

    Gypenoside (GP) is the major effective component of Gynostemma pentaphyllum and has been shown to encompass a variety of pharmacological activities. In this study, we investigated whether GP is able to protect cardiomyocytes against injury myocardial ischemia–reperfusion (I/R) injury by using in vitro oxygen-glucose deprivation–reoxygenation (OGD/R) H9c2 cell model and in vivo myocardial I/R rat model. We found that GP pre-treatment alleviated the impairments on the cardiac structure and function in I/R injured rats. Moreover, pre-treatment with GP significantly inhibited IκB-α phosphorylation and nuclear factor (NF)-κB p65 subunit translocation into nuclei. GP and the MAPK pathway inhibitors also reduced the phosphorylation of ERK, JNK, and p38 in vitro. Specific inhibition of ERK, JNK, and p38 increased the cell viability of OGD/R injured cells. Taken together, our data demonstrated that GP protects cardiomyocytes against I/R injury by inhibiting NF-κB p65 activation via the MAPK signaling pathway both in vitro and in vivo. These findings suggest that GP may be a promising agent for the prevention or treatment of myocardial I/R injury. PMID:27313532

  16. Upregulation of Scavenger Receptor BI by Hepatic Nuclear Factor 4α through a Peroxisome Proliferator-Activated Receptor γ-Dependent Mechanism in Liver

    PubMed Central

    Zhang, Yi; Shen, Chen; Ai, Ding; Xie, Xuefen; Zhu, Yi

    2011-01-01

    Hepatic nuclear factor 4α (HNF4α) modulates the transcriptional activation of numerous metabolic genes in liver. In this study, gene-array analysis revealed that HNF4α overexpression increased peroxisome proliferator-activated receptorγ (PPARγ) greatly in cultured rat primary hepatocytes. PPAR-response-element-driven reporter gene expression could be elevated by HNF4α. Bioinformatics analysis revealed a high-affinity HNF4α binding site in the human PPARγ2 promoter and in vitro experiments showed that this promoter could be transactivated by HNF4α. The presence of HNF4α on the promoter was then confirmed by ChIP assay. In vivo, hepatic overexpression of HNF4α decreased cholesterol levels both in plasma and liver and several hepatic genes related to cholesterol metabolism, including scavenger receptor BI (SR-BI), were upregulated. The upregulation of SR-BI by HNF4α could be inhibited by a PPARγ antagonist in vitro. In conclusion, HNF4α regulates cholesterol metabolism in rat by modulating the expression of SR-BI in the liver, in which the upregulation of PPARγ was involved. PMID:22190905

  17. Activating the AKT2-nuclear factor-κB-lipocalin-2 axis elicits an inflammatory response in age-related macular degeneration.

    PubMed

    Ghosh, Sayan; Shang, Peng; Yazdankhah, Meysam; Bhutto, Imran; Hose, Stacey; Montezuma, Sandra R; Luo, Tianqi; Chattopadhyay, Sreya; Qian, Jiang; Lutty, Gerard A; Ferrington, Deborah A; Zigler, J Samuel; Sinha, Debasish

    2017-04-01

    Age-related macular degeneration (AMD) is a complex and progressive degenerative eye disease resulting in severe loss of central vision. Recent evidence indicates that immune system dysregulation could contribute to the development of AMD. We hypothesize that defective lysosome-mediated clearance causes accumulation of waste products in the retinal pigmented epithelium (RPE), activating the immune system and leading to retinal tissue injury and AMD. We have generated unique genetically engineered mice in which lysosome-mediated clearance (both by phagocytosis and autophagy) in RPE cells is compromised, causing the development of features of early AMD. Our recent data indicate a link between lipocalin-2 (LCN-2) and the inflammatory responses induced in this mouse model. We show that nuclear factor-κB (NF-κB) and STAT-1 may function as a complex in our animal model system, together controlling the upregulation of LCN-2 expression in the retina and stimulating an inflammatory response. This study revealed increased infiltration of LCN-2-positive neutrophils in the choroid and retina of early AMD patients as compared with age-matched controls. Our results demonstrate that, both in our animal model and in human AMD, the AKT2-NF-κB-LCN-2 signalling axis is involved in activating the inflammatory response, making this pathway a potential target for AMD treatment. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  18. Cyclic stretch induces cyclooxygenase-2 gene expression in vascular endothelial cells via activation of nuclear factor kappa-{beta}

    SciTech Connect

    Zhao, Haige; Hiroi, Toyoko; Hansen, Baranda S.; Rade, Jeffrey J.

    2009-11-27

    Vascular endothelial cells respond to biomechanical forces, such as cyclic stretch and shear stress, by altering gene expression. Since endothelial-derived prostanoids, such as prostacyclin and thromboxane A{sub 2}, are key mediators of endothelial function, we investigated the effects of cyclic stretch on the expression of genes in human umbilical vein endothelial cells controlling prostanoid synthesis: cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS) and thromboxane A{sub 2} synthase (TXAS). COX-2 and TXAS mRNAs were upregulated by cyclic stretch for 24 h. In contrast, PGIS mRNA was decreased and stretch had no effect on COX-1 mRNA expression. We further show that stretch-induced upregulation of COX-2 is mediated by activation of the NF-{kappa}{beta} signaling pathway.

  19. Amadori adducts activate nuclear factor-κB-related proinflammatory genes in cultured human peritoneal mesothelial cells

    PubMed Central

    Nevado, Julián; Peiró, Concepción; Vallejo, Susana; El-Assar, Mariam; Lafuente, Nuria; Matesanz, Nuria; Azcutia, Veronica; Cercas, Elena; Sánchez-Ferrer, Carlos F; Rodríguez-Mañas, Leocadio

    2005-01-01

    Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean age 38.7±16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml−1), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF-κB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF-κB-related proinflammatory molecules, including cytokines, such as TNF-α, IL-1β or IL-6, and enzymes, such as cyclooxygenase-2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF-κB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF-κB-related proinflammatory signals in human mesothelial cells. PMID:15997235

  20. Nuclear Factor κ-B Is Activated in the Pulmonary Vessels of Patients with End-Stage Idiopathic Pulmonary Arterial Hypertension

    PubMed Central

    Price, Laura C.; Caramori, Gaetano; Perros, Frederic; Meng, Chao; Gambaryan, Natalia; Dorfmuller, Peter; Montani, David; Casolari, Paolo; Zhu, Jie; Dimopoulos, Konstantinos; Shao, Dongmin; Girerd, Barbara; Mumby, Sharon; Proudfoot, Alastair; Griffiths, Mark; Papi, Alberto; Humbert, Marc; Adcock, Ian M.; Wort, S. John

    2013-01-01

    Objectives To assess activation of the inflammatory transcription factor NF-kappa B (NF-κB) in human idiopathic pulmonary arterial hypertension (PAH). Background Idiopathic PAH is a severe progressive disease characterized by pulmonary vascular remodeling and excessive proliferation of vascular cells. Increasing evidence indicates that inflammation is important in disease pathophysiology. Methods NF-κB-p65 and CD68, CD20 and CD45 were measured by immunohistochemistry and confocal microscopy on lung specimens from patients with idiopathic PAH (n = 12) and controls undergoing lung surgery (n = 14). Clinical data were recorded for all patients including invasive pulmonary hemodynamics for the PAH patients. Immunohistochemical images were analyzed by blinded observers to include standard pulmonary vascular morphometry; absolute macrophage counts/mm2 and p65-positivity (p65+) using composite images and image-analysis software; and cytoplasmic:nuclear p65+ of individual pulmonary arterial endothelial and smooth muscle cells (PASMC) in 10–20 pulmonary arteries or arterioles per subject. The expression of ET-1 and CCL5 (RANTES) in whole lung was determined by RT-qPCR. Results Macrophage numbers were increased in idiopathic PAH versus controls (49.0±4.5 vs. 7.95±1.9 macrophages/100 mm2, p<0.0001): these macrophages demonstrated more nuclear p65+ than in macrophages from controls (16.9±2.49 vs. 3.5±1.25%, p<0.001). An increase in p65+ was also seen in perivascular lymphocytes in patients with PAH. Furthermore, NF-κB activation was increased in pulmonary arterial endothelial cells (62.3±2.9 vs. 14.4±3.8, p<0.0001) and PASMC (22.6±2.3 vs. 11.2±2.0, p<0.001) in patients with PAH versus controls, with similar findings in arterioles. Gene expression of both ET-1 mRNA ((0.213±0.069 vs. 1.06±0.23, p<0.01) and CCL5 (RANTES) (0.16±0.045 vs. 0.26±0.039, p<0.05) was increased in whole lung homogenates from patients with PAH. Conclusions NF-κB is activated in

  1. Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

    PubMed

    Tsang, Daisy P F; Wu, William K K; Kang, Wei; Lee, Ying-Ying; Wu, Feng; Yu, Zhuo; Xiong, Lei; Chan, Anthony W; Tong, Joanna H; Yang, Weiqin; Li, May S M; Lau, Suki S; Li, Xiangchun; Lee, Sau-Dan; Yang, Yihua; Lai, Paul B S; Yu, Dae-Yeul; Xu, Gang; Lo, Kwok-Wai; Chan, Matthew T V; Wang, Huating; Lee, Tin L; Yu, Jun; Wong, Nathalie; Yip, Kevin Y; To, Ka-Fai; Cheng, Alfred S L

    2016-04-01

    Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

  2. Regulation of p53, nuclear factor {kappa}B and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin

    SciTech Connect

    Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

    2008-01-01

    Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-{kappa}B), we also investigated the effect of bromelain on Cox-2 and NF-{kappa}B expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-{kappa}B by blocking phosphorylation and subsequent degradation of I{kappa}B{alpha}. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-{kappa}B-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.

  3. Regulation of p53, nuclear factor kappaB and cyclooxygenase-2 expression by bromelain through targeting mitogen-activated protein kinase pathway in mouse skin.

    PubMed

    Kalra, Neetu; Bhui, Kulpreet; Roy, Preeti; Srivastava, Smita; George, Jasmine; Prasad, Sahdeo; Shukla, Yogeshwer

    2008-01-01

    Bromelain is a pharmacologically active compound, present in stems and immature fruits of pineapples (Ananas cosmosus), which has been shown to have anti-edematous, anti-inflammatory, anti-thrombotic and anti-metastatic properties. In the present study, antitumorigenic activity of bromelain was recorded in 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted 2-stage mouse skin model. Results showed that bromelain application delayed the onset of tumorigenesis and reduced the cumulative number of tumors, tumor volume and the average number of tumors/mouse. To establish a cause and effect relationship, we targeted the proteins involved in the cell death pathway. Bromelain treatment resulted in upregulation of p53 and Bax and subsequent activation of caspase 3 and caspase 9 with concomitant decrease in antiapoptotic protein Bcl-2 in mouse skin. Since persistent induction of cyclooxygenase-2 (Cox-2) is frequently implicated in tumorigenesis and is regulated by nuclear factor-kappa B (NF-kappaB), we also investigated the effect of bromelain on Cox-2 and NF-kappaB expression. Results showed that bromelain application significantly inhibited Cox-2 and inactivated NF-kappaB by blocking phosphorylation and subsequent degradation of IkappaBalpha. In addition, bromelain treatment attenuated DMBA-TPA-induced phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), mitogen-activated protein kinase (MAPK) and Akt. Taken together, we conclude that bromelain induces apoptosis-related proteins along with inhibition of NF-kappaB-driven Cox-2 expression by blocking the MAPK and Akt/protein kinase B signaling in DMBA-TPA-induced mouse skin tumors, which may account for its anti-tumorigenic effects.

  4. Dynamic Imaging of Pancreatic Nuclear Factor κB (NF-κB) Activation in Live Mice Using Adeno-associated Virus (AAV) Infusion and Bioluminescence*

    PubMed Central

    Orabi, Abrahim I.; Sah, Swati; Javed, Tanveer A.; Lemon, Kathryn L.; Good, Misty L.; Guo, Ping; Xiao, Xiangwei; Prasadan, Krishna; Gittes, George K.; Jin, Shunqian; Husain, Sohail Z.

    2015-01-01

    Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 μg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo. PMID:25802340

  5. Review of EPRI Nuclear Human Factors Program

    SciTech Connect

    Hanes, L.F.; O`Brien, J.F.

    1996-03-01

    The Electric Power Research Institute (EPRI) Human Factors Program, which is part of the EPRI Nuclear Power Group, was established in 1975. Over the years, the Program has changed emphasis based on the shifting priorities and needs of the commercial nuclear power industry. The Program has produced many important products that provide significant safety and economic benefits for EPRI member utilities. This presentation will provide a brief history of the Program and products. Current projects and products that have been released recently will be mentioned.

  6. Dose-rate effects of protons on in vivo activation of nuclear factor-kappa B and cytokines in mouse bone marrow cells

    SciTech Connect

    Rithidech, K.N.; Rusek, A.; Reungpatthanaphong, P.; Honikel, L.; Simon, S.R.

    2010-05-28

    The objective of this study was to determine the kinetics of nuclear factor-kappa B (NF-{kappa}B) activation and cytokine expression in bone marrow (BM) cells of exposed mice as a function of the dose rate of protons. The cytokines included in this study are pro-inflammatory [i.e., tumor necrosis factor-alpha (TNF-{alpha}), interleukin-1beta (IL-1{beta}), and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and IL-10). We gave male BALB/cJ mice a whole-body exposure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons, delivered at 5 or 10 mGy min{sup -1}, the dose and dose rates found during solar particle events in space. As a reference radiation, groups of mice were exposed to 0 (sham-controls) or 1 Gy of {sup 137}Cs {gamma} rays (10 mGy min{sup -1}). After irradiation, BM cells were collected at 1.5, 3, 24 h, and 1 month for analyses (five mice per treatment group per harvest time). The results indicated that the in vivo time course of effects induced by a single dose of 1 Gy of 100 MeV protons or {sup 137}Cs {gamma} rays, delivered at 10 mGy min{sup -1}, was similar. Although statistically significant levels of NF-{kappa}B activation and pro-inflammatory cytokines in BM cells of exposed mice when compared to those in the corresponding sham controls (Student's t-test, p < 0.05 or < 0.01) were induced by either dose rate, these levels varied over time for each protein. Further, only a dose rate of 5 mGy min{sup -1} induced significant levels of anti-inflammatory cytokines. The results indicate dose-rate effects of protons.

  7. The activity of the carbamoyl phosphate synthase 1 promoter in human liver-derived cells is dependent on hepatocyte nuclear factor 3-beta.

    PubMed

    Chen, Zhanfei; Tang, Nanhong; Wang, Xiaoqian; Chen, Yanling

    2017-03-08

    Carbamoyl phosphate synthase 1 (CPS1) is the rate-limiting enzyme in the first step of the urea cycle and an indispensable enzyme in the metabolism of human liver. However, CPS1 epigenetic regulation involves promoter analysis and the role of liver-enriched transcription factors (LETFs), which is not fully elucidated. In this work, the promoter region of hCPS1 gene was cloned, and its activity was investigated. An LETF, hepatocyte nuclear factor 3-beta (HNF3β), was found to promote the transcriptional expression of CPS1 in liver-derived cell lines. In addition, dual-luciferase reporter assay shows that the essential binding sites of the HNF3β may exist in the oligonucleotide -70 nt to +73 nt. Two putative binding sites are available for HNF3β. Mutation analysis results show that the binding site 2 of HNF3β was effective, and the transcriptional activity of CPS1 promoter significantly decreased after mutation. Electrophoretic mobile shift assay (EMSA) and ChIP assay confirmed that HNF3β can interact with the binding site in the CPS1 promoter region of -70 nt to +73 nt promoter region in vivo and in vitro to regulate the transcription of CPS1. Moreover, HNF3β overexpression enhanced the transcription of CPS1 and consequently improved the mRNA and protein levels of CPS1, whereas the knockdown of HNF3β showed the opposite effects. Finally, urea production in cells was measured, and ammonia detoxification improved significantly in cells after transfection with HNF3β. HNF3β plays a vital role in regulation of CPS1 gene and could promote the metabolism of ammonia by regulating CPS1 expression.

  8. High Estradiol Concentrations Induce Heat Shock Protein 70 Expression and Suppress Nuclear Factor Kappa B Activation in Human Endometrial Epithelial Cells.

    PubMed

    Chen, Chin-Der; Chen, Shee-Uan; Chou, Chia-Hung; Chen, Mei-Jou; Wen, Wen-Fen; Wu, Szu-Yuan; Yang, Yu-Shih; Yang, Jehn-Hsiahn

    2016-09-07

    The high serum estradiol (E2) concentrations induced during in vitro fertilization are detrimental to endometrial receptivity and may result in lower embryo implantation rates. We have previously found that high E2 concentrations inhibit the activation of nuclear factor kappa B (NF-kappa B), which led to endometrial epithelial cells (EECs) apoptosis. The objective of this study is to investigate the signaling pathways through which high E2 results in NF-kappa B downregulation in EECs. Isolated human EECs were cultured in different concentrations of E2 (10(-10), 10(-9), 10(-8), 10(-7) M). The expression of heat shock protein 70 (Hsp70) and heat shock factor 1 (HSF-1) were upregulated under supraphysiological E2 (10(-7) M) concentration, whereas phosphorylated inhibitory kappa B-alpha (pI kappa B-alpha) and NF-kappa B p65 subunits were downregulated. Immunohistochemistry of C57BL/6 mouse EECs, that were exposed in vivo to high serum E2 from the administration of 20 IUs of equine chorionic gonadotropin, also demonstrated the same increase in HSF-1 and Hsp70 expression, and decrease in NF-kappa B. Immunoprecipitation of the induced Hsp70 proteins was achieved with the addition of inhibitory kappa B kinase gamma (IKK-gamma) antibodies, and elimination of this reaction occurred after addition of hsp70 siRNA. In conclusion, high E2 concentrations enhance HSF-1 and Hsp70 expression in EECs. The induced Hsp70 forms a complex with IKK-gamma and inhibits pI kappa B-alpha, which consequently suppresses NF-kappa B activation.

  9. Luteolin is a bioflavonoid that attenuates adipocyte-derived inflammatory responses via suppression of nuclear factor-κB/mitogen-activated protein kinases pathway

    PubMed Central

    Nepali, Sarmila; Son, Ji-Seon; Poudel, Barun; Lee, Ji-Hyun; Lee, Young-Mi; Kim, Dae-Ki

    2015-01-01

    Background: Inflammation of adipocytes has been a therapeutic target for treatment of obesity and metabolic disorders which cause insulin resistance and hence lead to type II diabetes. Luteolin is a bioflavonoid with many beneficial properties such as antioxidant, antiproliferative, and anti-cancer. Objectives: To elucidate the potential anti-inflammatory response and the underlying mechanism of luteolin in 3T3-L1 adipocytes. Materials and Methods: We stimulated 3T3-L1 adipocytes with the mixture of tumor necrosis factor-α, lipopolysaccharide, and interferon-γ (TLI) in the presence or absence of luteolin. We performed Griess’ method for nitric oxide (NO) production and measure mRNA and protein expressions by real-time polymerase chain reaction and western blotting, respectively. Results: Luteolin opposed the stimulation of inducible nitric oxide synthase and NO production by simultaneous treatment of adipocytes with TLI. Furthermore, it reduced the pro-inflammatory genes such as cyclooxygenase-2, interleukin-6, resistin, and monocyte chemoattractant protein-1. Furthermore, luteolin improved the insulin sensitivity by enhancing the expression of insulin receptor substrates (IRS1/2) and glucose transporter-4 via phosphatidylinositol-3K signaling pathway. This inhibition was associated with suppression of Iκ-B-α degradation and subsequent inhibition of nuclear factor-κB (NF-κB) p65 translocation to the nucleus. In addition, luteolin blocked the phosphorylation of ERK1/2, c-Jun N-terminal Kinases and also p38 mitogen-activated protein kinases (MAPKs). Conclusions: These results illustrate that luteolin attenuates inflammatory responses in the adipocytes through suppression of NF-κB and MAPKs activation, and also improves insulin sensitivity in 3T3-L1 cells, suggesting that luteolin may represent a therapeutic agent to prevent obesity-associated inflammation and insulin resistance. PMID:26246742

  10. Neuroepithelial Transforming Gene 1 (Net1) Binds to Caspase Activation and Recruitment Domain (CARD)- and Membrane-associated Guanylate Kinase-like Domain-containing (CARMA) Proteins and Regulates Nuclear Factor κB Activation*

    PubMed Central

    Vessichelli, Mariangela; Ferravante, Angela; Zotti, Tiziana; Reale, Carla; Scudiero, Ivan; Picariello, Gianluca; Vito, Pasquale; Stilo, Romania

    2012-01-01

    The molecular complexes containing CARMA proteins have been recently identified as a key components in the signal transduction pathways that regulate activation of nuclear factor κB (NF-κB) transcription factor. Here, we used immunoprecipitation coupled with mass spectrometry to identify cellular binding partners of CARMA proteins. Our data indicate that the Rho guanine nucleotide exchange factor Net1 binds to CARMA1 and CARMA3 in resting and activated cells. Net1 expression induces NF-κB activation and cooperates with BCL10 and CARMA proteins in inducing NF-κB activity. Conversely, shRNA-mediated abrogation of Net1 results in impaired NF-κB activation following stimuli that require correct CARMA-BCL10-MALT1 complex formation and functioning. Microarray expression data are consistent with a positive role for Net1 on NF-κB activation. Thus, this study identifies Net1 as a CARMA-interacting molecule and brings important information on the molecular mechanisms that control NF-κB transcriptional activity. PMID:22343628

  11. Neuroepithelial transforming gene 1 (Net1) binds to caspase activation and recruitment domain (CARD)- and membrane-associated guanylate kinase-like domain-containing (CARMA) proteins and regulates nuclear factor κB activation.

    PubMed

    Vessichelli, Mariangela; Ferravante, Angela; Zotti, Tiziana; Reale, Carla; Scudiero, Ivan; Picariello, Gianluca; Vito, Pasquale; Stilo, Romania

    2012-04-20

    The molecular complexes containing CARMA proteins have been recently identified as a key components in the signal transduction pathways that regulate activation of nuclear factor κB (NF-κB) transcription factor. Here, we used immunoprecipitation coupled with mass spectrometry to identify cellular binding partners of CARMA proteins. Our data indicate that the Rho guanine nucleotide exchange factor Net1 binds to CARMA1 and CARMA3 in resting and activated cells. Net1 expression induces NF-κB activation and cooperates with BCL10 and CARMA proteins in inducing NF-κB activity. Conversely, shRNA-mediated abrogation of Net1 results in impaired NF-κB activation following stimuli that require correct CARMA-BCL10-MALT1 complex formation and functioning. Microarray expression data are consistent with a positive role for Net1 on NF-κB activation. Thus, this study identifies Net1 as a CARMA-interacting molecule and brings important information on the molecular mechanisms that control NF-κB transcriptional activity.

  12. The effect of P2X7 receptor activation on nuclear factor-κB phosphorylation induced by status epilepticus in the rat hippocampus.

    PubMed

    Kim, Ji-Eun; Kim, Duk-Soo; Jin Ryu, Hea; Il Kim, Won; Kim, Min-Ju; Won Kim, Dae; Young Choi, Soo; Kang, Tea-Cheon

    2013-06-01

    Nuclear factor-kappa B (NFκB) signal is essential for neuronal survival and its activation may protect neuron against various stimuli. Since purinergic signals activate NFκB through the P2X7 receptor, we investigated the distinct pattern of NF-κB phosphorylation in neurons by P2X7 receptor activation following status epilepticus (SE) in an effort to understand the role of P2X7 receptor in epileptogenic insult. In non-SE animals, 2'(3')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (BzATP, a P2X7R agonist) treatment increased only p52-Ser869 NF-κB phosphorylation in neuron. Following SE, p52-Ser865, p52-Ser869, p65-Ser276, p65-Ser311, p65-Ser468, and p65-Ser529 NF-κB phosphorylation was significantly decreased in CA1 and CA3 neurons. However, BzATP treatment prevented reductions in p65-Ser276, p65-Ser311, p65-Ser529, and p52-Ser869 NF-κB phosphorylations in CA1 and/or CA3 neurons induced by SE. Furthermore, BzATP treatment reduced SE-induced p65-Ser311, p65-Ser468, p65-Ser536, and p52-Ser869 NF-κB phosphorylations in astrocytes. These findings indicate that P2X7 functions may be involved in the regulation of SE-induced reactive astrocytes and neuronal degeneration via NF-κB phosphorylations in response to pilocarpine-induced SE in the rat hippocampus.

  13. The alpha-methylene-gamma-butyrolactone moiety in dehydrocostus lactone is responsible for cytoprotective heme oxygenase-1 expression through activation of the nuclear factor E2-related factor 2 in HepG2 cells.

    PubMed

    Jeong, Gil-Saeng; Pae, Hyun-Ock; Jeong, Sun-Oh; Kim, Youn-Chul; Kwon, Tae-Oh; Lee, Ho Sub; Kim, Nam-Song; Park, Seok Don; Chung, Hun-Taeg

    2007-06-22

    Inducible heme oxygenase (HO)-1 acts against oxidants that are thought to play a major role in the pathogenesis of several diseases. The alpha-methylene-gamma-butyrolactone (CH2-BL) structural unit, which characterizes a group of naturally occurring sesquiterpene lactones, is known to possess numerous biological activities. In the present study, we evaluated dehydrocostus lactone possessing CH2-BL moiety, one of the bioactive constituents of the medicinal plant Saussurea lappa, as an inducer of cytoprotective HO-1. In HepG2 cells, treatment with dehydrocostus lactone induced HO-1 expression and increased HO activity in a concentration-dependent manner. Similar results were also observed when the cells were incubated with CH2-BL, a parent structure of dehydrocostus lactone. In contrast, mokko lactone, a reduced product of dehydrocostus lactone, and alpha-methyl-gamma-butyrolactone (CH3-BL), a parent structure of mokko lactone, did not induce HO-1 expression. Pretreatment with either dehydrocostus lactone or CH2-BL for 6 h protected the cells from hydrogen peroxide-mediated toxicity, whereas mokko lactone or CH3-BL failed to exert a cytoprotective action. Inhibition of HO-1 expression by HO-1 small interfering RNA (siRNA) abrogated cellular protection afforded by dehydrocostus lactone or CH2-BL. In addition, dehydrocostus lactone caused the nuclear accumulation of the nuclear factor E2-related factor 2 (Nrf2) and increased the promoter activity of antioxidant response element (ARE). Using Nrf2 siRNA, Nrf2 activation was confirmed to contribute to cytoprotective HO-1 expression by dehydrocostus lactone or CH2-BL. Collectively, our findings suggest that CH2-BL moiety in dehydrocostus lactone increases cellular resistance to oxidant injury in HepG2 cells, presumably through Nrf2/ARE-dependent HO-1 expression.

  14. Identification of UV-protective activators of nuclear factor erythroid-derived 2-related factor 2 (Nrf2) by combining a chemical library screen with computer-based virtual screening.

    PubMed

    Lieder, Franziska; Reisen, Felix; Geppert, Tim; Sollberger, Gabriel; Beer, Hans-Dietmar; auf dem Keller, Ulrich; Schäfer, Matthias; Detmar, Michael; Schneider, Gisbert; Werner, Sabine

    2012-09-21

    Nuclear factor erythroid-derived 2-related factor 2 (Nrf2) is a master regulator of cellular antioxidant defense systems, and activation of this transcription factor is a promising strategy for protection of skin and other organs from environmental insults. To identify efficient Nrf2 activators in keratinocytes, we combined a chemical library screen with computer-based virtual screening. Among 14 novel Nrf2 activators, the most potent compound, a nitrophenyl derivative of 2-chloro-5-nitro-N-phenyl-benzamide, was characterized with regard to its molecular mechanism of action. This compound induced the expression of cytoprotective genes in keratinocytes isolated from wild-type but not from Nrf2-deficient mice. Most importantly, it showed low toxicity and protected primary human keratinocytes from UVB-induced cell death. Therefore, it represents a potential lead compound for the development of drugs for skin protection under stress conditions. Our study demonstrates that chemical library screening combined with advanced computational similarity searching is a powerful strategy for identification of bioactive compounds, and it points toward an innovative therapeutic approach against UVB-induced skin damage.

  15. Age-Related Nuclear Translocation of P2X6 Subunit Modifies Splicing Activity Interacting with Splicing Factor 3A1

    PubMed Central

    Díaz-Hernández, Juan Ignacio; Sebastián-Serrano, Álvaro; Gómez-Villafuertes, Rosa

    2015-01-01

    P2X receptors are ligand-gated ion channels sensitive to extracellular nucleotides formed by the assembling of three equal or different P2X subunits. In this work we report, for the first time, the accumulation of the P2X6 subunit inside the nucleus of hippocampal neurons in an age-dependent way. This location is favored by its anchorage to endoplasmic reticulum through its N-terminal domain. The extracellular domain of P2X6 subunit is the key to reach the nucleus, where it presents a speckled distribution pattern and is retained by interaction with the nuclear envelope protein spectrin α2. The in vivo results showed that, once inside the nucleus, P2X6 subunit interacts with the splicing factor 3A1, which ultimately results in a reduction of the mRNA splicing activity. Our data provide new insights into post-transcriptional regulation of mRNA splicing, describing a novel mechanism that could explain why this process is sensitive to changes that occur with age. PMID:25874565

  16. Nuclear Factor of κB1 Is a Key Regulator for the Transcriptional Activation of Milk Synthesis in Bovine Mammary Epithelial Cells.

    PubMed

    Huang, Xin; Zang, Yanli; Zhang, Minghui; Yuan, Xiaohan; Li, Meng; Gao, Xuejun

    2017-02-03

    The nuclear factor of κB (NFκB) family has been well known for its significant role in regulating the expression of numerous genes that control many biological processes. However, it is unclear whether NFκB could regulate milk synthesis. In this study, we identified NFκB1 as a critical regulator for milk synthesis in bovine mammary epithelial cells (BMECs). Gene function study revealed that NFκB1 modulates the expression of mammalian target of rapamycin (mTOR), sterol response element-binding protein (SREBP)-1c, and β4Gal-T2 for milk synthesis. Furthermore, chromatin immunoprecipitation assays showed that both methionine (Met) and estrogen (E) triggered NFκB1 to bind to gene promoters of mTOR, SREBP-1c, and β4Gal-T2 in BMECs. In addition, we confirmed that Met and E triggered NFκB1 expression and phosphorylation via phosphatidylinositol-3-kinase (PI3K) but not mTOR signaling pathway. Taken together, our study reveals that NFκB1 acts as a PI3K but not mTOR-dependent critical mediator for the transcriptional activation of signaling molecules regulating milk synthesis in BMECs.

  17. Effect of Lead Nanoparticles Inhalation on Bone Calcium Sensing Receptor, Hydroxyapatite Crystal and Receptor Activator of Nuclear Factor-Kappa B in Rats

    PubMed Central

    Leonas, Rendra; Noor, Zairin; Rasyid, Hermawan Nagar; Madjid, Tita Husnitawati; Tanjung, Fachry Ambia

    2016-01-01

    This study aimed to investigate whether Pb nanoparticle exposure affects the bone calcium sensing receptor (CaSR), hydroxyapatite crystal, and receptor activator of nuclear factor-kappa B (RANK) in rats exposed to subchronic and chronic inhalation. Thirty two rats were randomly divided into eight groups. One group is a non-exposed group. While three groups were exposed to nanoparticles Pb at the following doses 6.25; 12.5; or 25 mg/m3 an hour daily for 28 days. Another three groups were exposed to nanoparticles Pb at following doses 6.25; 12.5; and 25 mg/m3 one hour daily for 6 months. The expression of trabecular CaSR was significantly decreased at the all doses subchronic exposure compared to the control group (P < 0.05). The CaSR expression significantly decreased in second and third doses subchronic exposure groups compared to the control groups (P < 0.05). With subchronic exposure, the crystal size was increased in second dose group and decreased in lowest and highest doses compared to the control (untreated) group. The crystal size and c-axis were decreased in all dose chronic exposures compared to the control (untreated) group. The expression of cortical RANK was significantly lower at the two lowest dose chronic exposures compared to the control group (P < 0.05). In conclusion, Pb nanoparticle inhibit hydroxyapatite crystal growth at least a part via down regulation of CaSR and RANK. PMID:28077890

  18. The nuclear-encoded sigma factor SIG4 directly activates transcription of chloroplast psbA and ycf17 genes in the unicellular red alga Cyanidioschyzon merolae.

    PubMed

    Fujii, Gaku; Imamura, Sousuke; Era, Atsuko; Miyagishima, Shin-ya; Hanaoka, Mitsumasa; Tanaka, Kan

    2015-05-01

    The plant organelle chloroplast originated from the endosymbiosis of a cyanobacterial-like photosynthetic bacterium, and still retains its own genome derived from this ancestor. We have been focusing on a unicellular red alga, Cyanidioschyzon merolae, as a model photosynthetic eukaryote. In this study, we analyzed the transcriptional specificity of SIG4, which is one of four nuclear-encoded chloroplast RNA polymerase sigma factors in this alga. Accumulation of the SIG4 protein was observed in response to nitrogen depletion or high light conditions. By comparing the chloroplast transcriptomes under nitrogen depletion and SIG4-overexpressing conditions, we identified several candidate genes as SIG4 targets. Together with the results of chromatin immunoprecipitation analysis, the promoters of the psbA (encoding the D1 protein of the photosystem II reaction center) and ycf17 (encoding a protein of the early light-inducible protein family) genes were shown to be direct activation targets. The phycobilisome (PBS) CpcB protein was decreased by SIG4 overexpression, which suggests the negative involvement of SIG4 in PBS accumulation.

  19. Molecular Profiling of Giant Cell Tumor of Bone and the Osteoclastic Localization of Ligand for Receptor Activator of Nuclear Factor κB

    PubMed Central

    Morgan, Teresa; Atkins, Gerald J.; Trivett, Melanie K.; Johnson, Sandra A.; Kansara, Maya; Schlicht, Stephen L.; Slavin, John L.; Simmons, Paul; Dickinson, Ian; Powell, Gerald; Choong, Peter F.M.; Holloway, Andrew J.; Thomas, David M.

    2005-01-01

    Giant cell tumor of bone (GCT) is a generally benign, osteolytic neoplasm comprising stromal cells and osteoclast-like giant cells. The osteoclastic cells, which cause bony destruction, are thought to be recruited from normal monocytic pre-osteoclasts by stromal cell expression of the ligand for receptor activator of nuclear factor κB (RANKL). This model forms the foundation for clinical trials in GCTs of novel cancer therapeutics targeting RANKL. Using expression profiling, we identified both osteoblast and osteoclast signatures within GCTs, including key regulators of osteoclast differentiation and function such as RANKL, a C-type lectin, osteoprotegerin, and the wnt inhibitor SFRP4. After ex vivo generation of stromal- and osteoclast-enriched cultures, we unexpectedly found that RANKL mRNA and protein were more highly expressed in osteoclasts than in stromal cells, as determined by expression profiling, flow cytometry, immunohistochemistry, and reverse transcriptase-polymerase chain reaction. The expression patterns of molecules implicated in signaling between stromal cells and monocytic osteoclast precursors were analyzed in both primary and fractionated GCTs. Finally, using array-based comparative genomic hybridization, neither GCTs nor the derived stromal cells demonstrated significant genomic gains or losses. These data raise questions regarding the role of RANKL in GCTs that may be relevant to the development of molecularly targeted therapeutics for this disease. PMID:15972958

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

    PubMed

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

    2011-04-01

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

  1. Effects of vitamin E on receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) in rats treated with nicotine.

    PubMed

    Norazlina, M; Maizatul-Neza, J; Azarina, A; Nazrun, A S; Norliza, M; Ima-Nirwana, S

    2010-03-01

    Vitamin E is found to reverse the effects of nicotine on bone and this study aimed to determine its mechanism. Male Sprague Dawley rats were divided into four groups and treated for 3 months: Group 1 was the control group (RC). Groups 2 (N), 3 (N+TT) and 4 (N+ATF) received nicotine 7 mg/kg throughout the treatment period. In addition, groups 3 and 4 received tocotrienol 60 mg/kg and alpha-tocopherol 60 mg/kg respectively during months 2 and 3. Parameters measured were serum osteoprotegerin (OPG), serum receptor activator of nuclear factor kappa B ligand (RANKL), femoral and lumbar bone calcium content and body weight. Nicotine did not affect OPG or RANKL levels but reduced bone calcium content suggesting the calcium loss is not due to increase osteoclastogenesis. OPG was increased in N+ATF while RANKL was slightly increased in N+TT. Both vitamin E supplements restored bone calcium loss induced by nicotine. Nicotine impaired weight gain in all treatment groups starting week 4 however, N+TT group was comparable to RC from week 6 onwards. Bone protective effects of ATF, but not TT, may be partly due to inhibition of osteoclastogenesis.

  2. Histone deacetylase 3 inhibition re-establishes synaptic tagging and capture in aging through the activation of nuclear factor kappa B

    PubMed Central

    Sharma, Mahima; Shivarama Shetty, Mahesh; Arumugam, Thiruma Valavan; Sajikumar, Sreedharan

    2015-01-01

    Aging is associated with impaired plasticity and memory. Altered epigenetic mechanisms are implicated in the impairment of memory with advanced aging. Histone deacetylase 3 (HDAC3) is an important negative regulator of memory. However, the role of HDAC3 in aged neural networks is not well established. Late long-term potentiation (late-LTP), a cellular correlate of memory and its associative mechanisms such as synaptic tagging and capture (STC) were studied in the CA1 area of hippocampal slices from 82–84 week old rats. Our findings demonstrate that aging is associated with deficits in the magnitude of LTP and impaired STC. Inhibition of HDAC3 augments the late-LTP and re-establishes STC. The augmentation of late-LTP and restoration of STC is mediated by the activation of nuclear factor kappa B (NFκB) pathway. We provide evidence for the promotion of associative plasticity in aged neural networks by HDAC3 inhibition and hence propose HDAC3 and NFκB as the possible therapeutic targets for treating age -related cognitive decline. PMID:26577291

  3. Nuclear Factor {kappa}-Light Chain-Enhancer of Activated B Cells is Activated by Radiotherapy and is Prognostic for Overall Survival in Patients With Rectal Cancer Treated With Preoperative Fluorouracil-Based Chemoradiotheraphy

    SciTech Connect

    O'Neil, Bert H.; Funkhouser, William K.; Calvo, Benjamin F.; Meyers, Michael O.; Kim, Hong Jin; Goldberg, Richard M.; Bernard, Stephen A.; Caskey, Laura; Deal, Allison M.; Wright, Fred; Baldwin, Albert S.; Tepper, Joel E.

    2011-07-01

    Purpose: Rectal cancer is often clinically resistant to radiotherapy (RT) and identifying molecular markers to define the biologic basis for this phenomenon would be valuable. The nuclear factor {kappa}-light chain-enhancer of activated B cells (NF-{kappa}B) is a potential anti-apoptotic transcription factor that has been associated with resistance to RT in model systems. The present study was designed to evaluate NF-{kappa}B activation in patients with rectal cancer undergoing chemoradiotherapy to determine whether NF-{kappa}B activity correlates with the outcome in rectal cancer patients. Methods and Materials: A total of 22 patients underwent biopsy at multiple points in a prospective study and the data from another 50 were analyzed retrospectively. The pretreatment tumor tissue was analyzed for multiple NF-{kappa}B subunits by immunohistochemistry. Serial tumor biopsy cores were analyzed for NF-{kappa}B-regulated gene expression using reverse transcriptase polymerase chain reaction and for NF-{kappa}B subunit nuclear localization using immunohistochemistry. Results: Several NF-{kappa}B target genes (Bcl-2, cellular inhibitor of apoptosis protein [cIAP]2, interleukin-8, and tumor necrosis factor receptor-associated-1) were significantly upregulated by a single fraction of RT at 24 h, demonstrating for the first time that NF-{kappa}B is activated by RT in human rectal tumors. The baseline NF-{kappa}B p50 nuclear expression did not correlate with the pathologic response to RT. However, an increasing baseline p50 level was prognostic for overall survival (hazard ratio, 2.15; p = .040). Conclusion: NF-{kappa}B nuclear expression at baseline in rectal cancer was prognostic for overall survival but not predictive of the response to RT. Larger patient numbers are needed to assess the effect of NF-{kappa}B target gene upregulation on the response to RT. Our results suggest that NF-{kappa}B might play an important role in tumor metastasis but not to the resistance to

  4. Mitogen-activated protein kinase 3/mitogen-activated protein kinase 1 activates apoptosis during testicular ischemia-reperfusion injury in a nuclear factor-kappaB-independent manner.

    PubMed

    Minutoli, Letteria; Antonuccio, Pietro; Polito, Francesca; Bitto, Alessandra; Squadrito, Francesco; Di Stefano, Vincenzo; Nicotina, Piero Antonio; Fazzari, Carmine; Maisano, Daniele; Romeo, Carmelo; Altavilla, Domenica

    2009-02-14

    Nuclear factor kappa-B (NF-kappaB), mitogen-activated protein kinase3/MAPK1 and MAPK8 are involved in testicular ischemia reperfusion injury (testicular-I/R). NF-kappaB knock-out mice (KO) subjected to testicular-I/R have a reduced testicular damage, blunted MAPK8 activation and enhanced MAPK3/MAPK1 activity. To better understand the role of MAPK3/MAPK1 up-regulation during testicular-I/R, we investigated the effects of PD98059, an inhibitor of MAPK3/MAPK1, in KO mice during testicular-I/R. KO and wild-type (WT) animals underwent 1 h testicular ischemia followed by 24 h reperfusion or a sham testicular-I/R. Animals received either PD98059 (5 mg/kg/ip) or its vehicle. MAPK3/MAPK1, BAX, caspase-3 and -9 and TNF-alpha expression were assessed along with histological examination and an immunostaining for protein of apoptosis. Testicular-I/R caused a greater increase in MAPK3/MAPK1 in KO than in WT animals in both testes. KO mice had a lower expression of the apoptotic proteins and TNF-alpha as well as reduced histological damage compared to WT. Immunostaining confirmed the lower expression of BAX in the Leydig cells of KO mice. Administration of PD98059, abrogated MAPK3/MAPK1 expression and slightly reduced TNF-alpha but did not improve or reverse the histological damage in KO. PD98059 significantly reduced the histological damage in WT mice and markedly reduced the apoptotic proteins in KO and WT mice. These results suggest that testicular-I/R triggers also a pathway of organ damage involving MAPK3/MAPK1, TNF-alpha, BAX, caspase-3 and -9 that activates an apoptotic machinery in an NF-kappaB independent manner. These findings should contribute to better understand testicular torsion-induced damage.

  5. Sesamin attenuates mast cell-mediated allergic responses by suppressing the activation of p38 and nuclear factor-κB.

    PubMed

    Li, Liang Chang; Piao, Hong Mei; Zheng, Ming Yu; Lin, Zhen Hua; Li, Guangzhao; Yan, Guang Hai

    2016-01-01

    Establishing therapeutic agents for the treatment of allergic diseases is an important focus of human health research. Sesamin, a lignan in sesame oil, exhibits a diverse range of pharmacological properties. However, to the best of our knowledge, the effect of sesamin on mast cell‑mediated allergic responses has not yet been investigated. Thus, the aim of the present study was to investigate the effect of sesamin on mast cell‑mediated allergic responses and the underlying mechanisms by which it produces this effect. In rats, oral administration of sesamin inhibited passive cutaneous anaphylaxis. Sesamin exposure attenuated immunoglobulin E‑induced histamine release from rat peritoneal mast cells, which was indicated to be mediated by the modulation of intracellular calcium. In human mast cells, sesamin reduced the stimulatory effects of phorbol 12‑myristate 13‑acetate and calcium ionophore A23187 on the production and secretion of pro‑inflammatory cytokines, including tumor necrosis factor‑α and interleukin‑6. The inhibitory effect of sesamin on pro‑inflammatory cytokine production was dependent on nuclear factor κ‑light‑chain‑enhancer of activated B cells (NF‑κB) and p38 mitogen‑activated protein kinase (MAPK). The present study demonstrates that sesamin inhibits mast cell‑derived inflammatory allergic reactions by blocking histamine release, and pro‑inflammatory cytokine production and secretion. In addition, the findings indicate that the effect of sesamin is mediated by its effect on p38 MAPK/NF‑κB signaling. Furthermore, the in vivo and in vitro anti‑allergic effects of sesamin reported in the present study suggest that it is a promising therapeutic agent for the treatment of inflammatory allergic diseases.

  6. Effects of globularifolin on cell survival, nuclear factor-κB activity, neopterin production, tryptophan breakdown and free radicals in vitro.

    PubMed

    Sipahi, Hande; Becker, Kathrin; Gostner, Johanna M; Charehsaz, Mohammad; Kirmizibekmez, Hasan; Schennach, Harald; Aydin, Ahmet; Fuchs, Dietmar

    2014-01-01

    The potential effects of globularifolin, an acylated iridoid glucoside, on cell survival, inflammation markers and free radicals scavenging were investigated. Viability assay on human myelomomonocytic cell line THP-1 and human peripheral blood mononuclear cells (PBMC) using the Cell-Titer Blue assay proved that globularifolin had no toxic effect at the tested concentrations. Conversely, it is proportional to the dose globularifolin increased growth of THP-1 cells (p <0.01). On human PBMC, globularifolin at 6.25 and 12.5 μM concentrations showed a stimulatory effect, while at 12.5-200 μM it suppressed response of PBMC to stimulation with phytohemagglutinin (PHA). Globularifolin (50-200 μM) enhanced neopterin formation dose-dependently, whereas tryptophan breakdown was not influenced. At 50-200 μM in unstimulated PBMC in THP-1 cells, globularifolin induced a significant expression of nuclear factor-κB (NF-κB) as was quantified by Quanti-Blue assay. By contrast, in lipopolysaccharide (LPS)-stimulated cells, the higher concentrations of globularifolin suppressed NF-κB expression dose-dependently and a significant decrease was observed at 200 μM concentration. A positive correlation was found between increased neopterin and NF-κB activity (p <0.01). Similarly, a positive correlation was observed between neopterin levels in mitogen-induced cells and NF-κB activity in LPS-stimulated cells after treatment with globularifolin (p=0.001). The free radical scavenging capacity of globularifolin evaluated by Oxygen Radical Absorbance Capacity (ORAC) assay showed relative ORAC values of 0.36±0.05 μmol Trolox equivalent/μmol. All together, results show that natural antioxidant globularifolin might represent a potential immunomodulatory as well as proliferative agent, which deserves further in vitro and in vivo studies.

  7. Comparison the relationship between the levels of insulin resistance, hs-CRP, percentage of body fat and serum osteoprotegerin/receptor activator of nuclear factor κβ ligand in prediabetic patients.

    PubMed

    Bilgir, Oktay; Yavuz, Mehmet; Bilgir, Ferda; Akan, Ozden Y; Bayindir, Aslı G; Calan, Mehmet; Bozkaya, Giray; Yuksel, Arif

    2017-01-31

    BACKGROUND Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPN) are soluble members of the tumor necrosis factor superfamily. Growing evidence suggest that there is link between inflammation, insulin resistance and OPG, sRANKL. We aimed to ascertain whether OPG and sRANKL levels are altered in prediabetic subjects and there is association between OPG, sRANKL and metabolic parameters.

  8. Ethanol Extract of Cirsium japonicum var. ussuriense Kitamura Exhibits the Activation of Nuclear Factor Erythroid 2-Related Factor 2-dependent Antioxidant Response Element and Protects Human Keratinocyte HaCaT Cells Against Oxidative DNA Damage

    PubMed Central

    Yoo, Ok-Kyung; Choi, Bu Young; Park, Jin-Oh; Lee, Ji-Won; Park, Byoung-Kwon; Joo, Chul Gue; Heo, Hyo-Jung; Keum, Young-Sam

    2016-01-01

    Keratinocytes are constantly exposed to extracellular insults, such as ultraviolet B, toxic chemicals and mechanical stress, all of which can facilitate the aging of keratinocytes via the generation of intracellular reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that plays a critical role in protecting keratinocytes against oxidants and xenobiotics by binding to the antioxidant response element (ARE), a cis-acting element existing in the promoter of most phase II cytoprotective genes. In the present study, we have attempted to find novel ethanol extract(s) of indigenous plants of Jeju island, Korea that can activate the Nrf2/ARE-dependent gene expression in human keratinocyte HaCaT cells. As a result, we identified that ethanol extract of Cirsium japonicum var. ussuriense Kitamura (ECJUK) elicited strong stimulatory effect on the ARE-dependent gene expression. Supporting this observation, we found that ECJUK induced the expression of Nrf2, hemoxygenase-1, and NAD(P)H:quinone oxidoreductase-1 and this event was correlated with Akt1 phosphorylation. We also found that ECJUK increased the intracellular reduced glutathione level and suppressed 12-O-tetradecanoylphorbol acetate-induced 8-hydroxyguanosine formation without affecting the overall viability. Collectively, our results provide evidence that ECJUK can protect against oxidative stress-mediated damages through the activation of Nrf2/ARE-dependent phase II cytoprotective gene expression. PMID:27051652

  9. Graphene quantum dots induce apoptosis, autophagy, and inflammatory response via p38 mitogen-activated protein kinase and nuclear factor-κB mediated signaling pathways in activated THP-1 macrophages.

    PubMed

    Qin, Yiru; Zhou, Zhi-Wei; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Qiu, Jia-Xuan; Duan, Wei; Yang, Tianxin; Zhou, Shu-Feng

    2015-01-02

    The biomedical application of graphene quantum dots (GQDs) is a new emerging area. However, their safety data are still in scarcity to date. Particularly, the effect of GQDs on the immune system remains unknown. This study aimed to elucidate the interaction of GQDs with macrophages and the underlying mechanisms. Our results showed that GQDs slightly affected the cell viability and membrane integrity of macrophages, whereas GQDs significantly increased reactive oxygen species (ROS) generation and apoptotic and autophagic cell death with an increase in the expression level of Bax, Bad, caspase 3, caspase 9, beclin 1, and LC3-I/II and a decrease in that of Bcl-2. Furthermore, low concentrations of GQDs significantly increased the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-8, whereas high concentrations of GQDs elicited opposite effects on the cytokines production. SB202190, a selective inhibitor of p38 mitogen-activated protein kinase (MAPK), abolished the cytokine-inducing effect of GQDs in macrophages. Moreover, GQDs significantly increased the phosphorylation of p38 MAPK and p65, and promoted the nuclear translocation of nuclear factor-κB (NF-κB). Taken together, these results show that GQDs induce ROS generation, apoptosis, autophagy, and inflammatory response via p38MAPK and NF-κB mediated signaling pathways in THP-1 activated macrophages.

  10. A high-resolution peak fractionation approach for streamlined screening of nuclear-factor-E2-related factor-2 activators in Salvia miltiorrhiza.

    PubMed

    Zhang, Hui; Luo, Li-Ping; Song, Hui-Peng; Hao, Hai-Ping; Zhou, Ping; Qi, Lian-Wen; Li, Ping; Chen, Jun

    2014-01-24

    Generation of a high-purity fraction library for efficiently screening active compounds from natural products is challenging because of their chemical diversity and complex matrices. In this work, a strategy combining high-resolution peak fractionation (HRPF) with a cell-based assay was proposed for target screening of bioactive constituents from natural products. In this approach, peak fractionation was conducted under chromatographic conditions optimized for high-resolution separation of the natural product extract. The HRPF approach was automatically performed according to the predefinition of certain peaks based on their retention times from a reference chromatographic profile. The corresponding HRPF database was collected with a parallel mass spectrometer to ensure purity and characterize the structures of compounds in the various fractions. Using this approach, a set of 75 peak fractions on the microgram scale was generated from 4mg of the extract of Salvia miltiorrhiza. After screening by an ARE-luciferase reporter gene assay, 20 diterpene quinones were selected and identified, and 16 of these compounds were reported to possess novel Nrf2 activation activity. Compared with conventional fixed-time interval fractionation, the HRPF approach could significantly improve the efficiency of bioactive compound discovery and facilitate the uncovering of minor active components.

  11. Receptor activator of nuclear factor kappa-B ligand (RANKL) but not sclerostin or gene polymorphisms is related to joint destruction in early rheumatoid arthritis.

    PubMed

    Boman, Antonia; Kokkonen, Heidi; Ärlestig, Lisbeth; Berglin, Ewa; Rantapää-Dahlqvist, Solbritt

    2017-02-11

    The aim of this study was to analyze relationships between receptor activator of nuclear factor kappa-B (RANKL), sclerostin and their gene polymorphisms with radiological progression in patients with early rheumatoid arthritis (RA). Patients with early RA (n = 407, symptomatic <1 year) (ARA criteria) examined radiologically at inclusion and after 24 months were consecutively included. Disease activity score and C-reactive protein were regularly recorded. Sclerostin, RANKL, and anti-CCP2 antibodies were analyzed in plasma at baseline using ELISAs. Data on gene polymorphism for sclerostin and RANKL were extracted from Immunochip analysis. Sex- and age-matched controls (n = 71) were identified from the Medical Biobank of Northern Sweden. The concentration of RANKL was significantly higher in patients compared with controls, median (IQR) 0.56 (0.9) nmol/L and 0.20 (0.25) nmol/L (p < 0.001), and in anti-CCP2-positive patients compared with sero-negative individuals. Sclerostin was significantly increased in female patients 0.59 (0.47-0.65) ng/mL compared with female controls 0.49 (0.4-0.65) ng/mL (p < 0.02). RANKL concentration was related to the Larsen score at baseline (p < 0.01), after 24 months (p < 0.001), and to radiological progression at 24 months (p < 0.001). Positivity of RANKL and anti-CCP2 yielded significant risk for progression with negativity for both as reference. No single nucleotide polymorphism encoding TNFSF11 or SOST was associated with increased concentrations of the factors. The concentration of RANKL was related to the Larsen score at baseline, at 24 months, and radiological progression at 24 months particularly in anti-CCP2-positive patients, while the concentration of sclerostin was unrelated to radiological findings.

  12. Nuclear transport factors: global regulation of mitosis.

    PubMed

    Forbes, Douglass J; Travesa, Anna; Nord, Matthew S; Bernis, Cyril

    2015-08-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator - the γ-TuRC complex - and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

  13. Nuclear Transport Factors: Global Regulation of Mitosis

    PubMed Central

    Forbes, Douglass J.; Travesa, Anna; Nord, Matthew; Bernis, Cyril

    2015-01-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear transport receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator the γ-TuRC complex and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores towards the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic. PMID:25982429

  14. Role of Osteoprotegerin and Receptor Activator of Nuclear Factor-κB Ligand in Bone Loss Related to Advanced Chronic Obstructive Pulmonary Disease

    PubMed Central

    Ugay, Ludmila; Kochetkova, Evgenia; Nevzorova, Vera; Maistrovskaia, Yuliya

    2016-01-01

    Background: Osteoporosis is a common complication of chronic obstructive pulmonary disease (COPD). Recent clinical and biological researches have increasingly delineated the biomolecular pathways of bone metabolism regulation in COPD. We extended this work by examining the specific association and potential contribution of the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) axis to the pathogenesis of osteoporosis in advanced COPD. The aim of this study was to assess the relationships of serum OPG, RANKL, and tumor necrosis factor-alpha (TNF-α) with bone turnover in men with very severe COPD. Methods: Pulmonary function, T-score at the lumbar spine (LS) and femoral neck (FN), serum OPG, RANKL, soluble receptor of tumor necrosis factor-alpha-I and II (sTNFR-I, sTNFR-II), osteocalcin (OC), and β-CrossLaps (βCL) levels were measured in 45 men with very severe stage COPD and 36 male non-COPD volunteers. COPD patients and healthy controls were compared using an independent t-test and Mann–Whitney U-test. The Pearson coefficient was used to assess the relationships between variables. Results: OPG and OC were lower in male COPD patients than in control subjects whereas RANKL, serum βCL, TNF-α, and its receptors were higher. OPG directly correlated with forced expiratory volume in 1 s (FEV1) % predicted (r = 0.46, P < 0.005), OC (r = 0.34, P < 0.05), LS (r = 0.56, P < 0.001), and FN T-score (r = 0.47, P < 0.01). In contrast, serum RANKL inversely associated with LS and FN T-score (r = −0.62, P < 0.001 and r = −0.48, P < 0.001) but directly correlated with βCL (r = 0.48, P < 0.001). In addition, OPG was inversely correlated with RANKL (r = −0.39, P < 0.01), TNF-α (r = −0.56, P < 0.001), and sTNFR-I (r = −0.40, P < 0.01). Conclusion: Our results suggest that serum OPG and RANKL levels are inversely associated with bone loss in men with advanced stage COPD. PMID:27411457

  15. Multiple nuclear localization signals function in the nuclear import of the transcription factor Nrf2.

    PubMed

    Theodore, Melanie; Kawai, Yumiko; Yang, Jianqi; Kleshchenko, Yuliya; Reddy, Sekhar P; Villalta, Fernando; Arinze, Ifeanyi J

    2008-04-04

    Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494-511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins alpha5 and beta1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin alpha-beta heterodimer nuclear import receptor system plays a critical role in the import process.

  16. Building Public Confidence in Nuclear Activities

    SciTech Connect

    Isaacs, T

    2002-02-13

    Achieving public acceptance has become a central issue in discussions regarding the future of nuclear power and associated nuclear activities. Effective public communication and public participation are often put forward as the key building blocks in garnering public acceptance. A recent international workshop in Finland provided insights into other features that might also be important to building and sustaining public confidence in nuclear activities. The workshop was held in Finland in close cooperation with Finnish stakeholders. This was most appropriate because of the recent successes in achieving positive decisions at the municipal, governmental, and Parliamentary levels, allowing the Finnish high-level radioactive waste repository program to proceed, including the identification and approval of a proposed candidate repository site Much of the workshop discussion appropriately focused on the roles of public participation and public communications in building public confidence. It was clear that well constructed and implemented programs of public involvement and communication and a sense of fairness were essential in building the extent of public confidence needed to allow the repository program in Finland to proceed. It was also clear that there were a number of other elements beyond public involvement that contributed substantially to the success in Finland to date. And, in fact, it appeared that these other factors were also necessary to achieving the Finnish public acceptance. In other words, successful public participation and communication were necessary but not sufficient. What else was important? Culture, politics, and history vary from country to country, providing differing contexts for establishing and maintaining public confidence. What works in one country will not necessarily be effective in another. Nonetheless, there appear to be certain elements that might be common to programs that are successful in sustaining public confidence, and some of

  17. Building Public Confidence in Nuclear Activities

    SciTech Connect

    Isaacs, T

    2002-03-27

    Achieving public acceptance has become a central issue in discussions regarding the future of nuclear power and associated nuclear activities. Effective public communication and public participation are often put forward as the key building blocks in garnering public acceptance. A recent international workshop in Finland provided insights into other features that might also be important to building and sustaining public confidence in nuclear activities. The workshop was held in Finland in close cooperation with Finnish stakeholders. This was most appropriate because of the recent successes in achieving positive decisions at the municipal, governmental, and Parliamentary levels, allowing the Finnish high-level radioactive waste repository program to proceed, including the identification and approval of a proposed candidate repository site. Much of the workshop discussion appropriately focused on the roles of public participation and public communications in building public confidence. It was clear that well constructed and implemented programs of public involvement and communication and a sense of fairness were essential in building the extent of public confidence needed to allow the repository program in Finland to proceed. It was also clear that there were a number of other elements beyond public involvement that contributed substantially to the success in Finland to date. And, in fact, it appeared that these other factors were also necessary to achieving the Finnish public acceptance. In other words, successful public participation and communication were necessary but not sufficient. What else was important? Culture, politics, and history vary from country to country, providing differing contexts for establishing and maintaining public confidence. What works in one country will not necessarily be effective in another. Nonetheless, there appear to be certain elements that might be common to programs that are successful in sustaining public confidence and some of

  18. Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus.

    PubMed

    Niinuma, Akiko; Higuchi, Masaya; Takahashi, Masahiko; Oie, Masayasu; Tanaka, Yuetsu; Gejyo, Fumitake; Tanaka, Nobuyuki; Sugamura, Kazuo; Xie, Li; Green, Patrick L; Fujii, Masahiro

    2005-09-01

    Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.

  19. Effect of lifestyle interventions with or without metformin therapy on serum levels of osteoprotegerin and receptor activator of nuclear factor kappa B ligand in patients with prediabetes.

    PubMed

    Arslan, Muyesser Sayki; Tutal, Esra; Sahin, Mustafa; Karakose, Melia; Ucan, Bekir; Ozturk, Gulfer; Cakal, Erman; Biyikli Gencturk, Zeynep; Ozbek, Mustafa; Delibasi, Tuncay

    2017-02-01

    Osteoprotegerin has been shown to be increased in cardiovascular disorders and type 2 diabetes mellitus. Prediabetes represents a high risk condition for diabetes and diabetic complications. Therefore, we aimed to find the relationship between prediabetes and osteoprotegerin with nuclear factor-B ligand, carotid intima media thickness, and metabolic markers. A total of 54 participants with prediabetes including impaired fasting glucose (n = 21), impaired glucose tolerance (n = 8), impaired fasting glucose and impaired glucose tolerance (n = 25), and 60 healthy individuals as a control were admitted to the study. Metabolic and anthropometric parameters, insulin resistance variables, osteoprotegerin, and nuclear factor-B ligand markers, carotid intima media thickness were examined at baseline for all participants. To evaluate the effect of therapy we determined the same parameters after the end of the study. Measurements of waist circumference, body mass index, body fat percentage and levels of fasting blood glucose, fasting insulin, homeostatic model assessment of insulin resistance, triglyceride levels and hsCRP and carotid intima media thickness were significantly higher in patients with prediabetes (p < 0.05). We also found higher osteoprotegerin and lower nuclear factor-B ligand levels in patients than in controls however, the value was non-significant (p > 0.05). Patients with prediabetes were under lifestyle interventions with (group 1, n = 33) or without metformin (group 2, n = 21) therapy. Baseline anthropometric and metabolic characteristics were not found statistically different in group 1 and group 2. Mean follow up period of the patients were 7.9 ± 2.2 month (min-max: 6-12 months). After the follow up period we evaluated the same parameters and found significant differences between waist circumference, body mass index, body fat percentage, fasting insulin, homeostatic model assessment of insulin resistance, and

  20. Receptor activator of nuclear factor-kappaB ligand-induced mouse osteoclast differentiation is associated with switching between NADPH oxidase homologues.

    PubMed

    Sasaki, Hideyuki; Yamamoto, Hironori; Tominaga, Kumiko; Masuda, Kiyoshi; Kawai, Tomoko; Teshima-Kondo, Shigetada; Matsuno, Kuniharu; Yabe-Nishimura, Chihiro; Rokutan, Kazuhito

    2009-07-15

    Reactive oxygen species (ROS) have been suggested to regulate receptor activator of nuclear factor-kappaB ligand (RANKL)-stimulated osteoclast differentiation. Stimulation of wild-type mouse bone marrow monocyte/macrophage lineage (BMM) cells by RANKL down-regulated NADPH oxidase 2 (Nox2) mRNA expression by half. RANKL reciprocally increased Nox1 mRNA levels and newly induced Nox4 transcript expression. BMM cells from Nox1 knockout (Nox1(-/-)) as well as Nox2(-/-) mice generated ROS in response to RANKL and differentiated into osteoclasts in the same way as wild-type BMM cells, which was assessed by the appearance of tartrate-resistant acid phosphatase-positive, multinucleated cells having the ability to form resorption pits and by the expression of osteoclast marker genes. A small interfering RNA (siRNA) targeting Nox1 or Nox2 failed to inhibit the RANKL-stimulated ROS generation and osteoclast formation in wild-type cells, whereas Nox1 and Nox2 siRNAs significantly suppressed the ROS generation and osteoclast formation in Nox2(-/-) and Nox1(-/-) cells, respectively. We also confirmed that Nox4 siRNA did not affect the RANKL-dependent events in Nox2(-/-) cells, whereas p22(phox) siRNA suppressed the events in both wild-type and Nox1(-/-) cells. Collectively, our results suggest that there may be a flexible compensatory mechanism between Nox1 and Nox2 for RANKL-stimulated ROS generation to facilitate osteoclast differentiation.

  1. Serum levels of osteoprotegerin and receptor activator of nuclear factor -κB ligand in children with early juvenile idiopathic arthritis: a 2-year prospective controlled study

    PubMed Central

    2010-01-01

    Background The clinical relevance of observations of serum levels of osteoprotegerin (OPG) and receptor activator of nuclear factor -κB ligand (RANKL) in juvenile idiopathic arthritis (JIA) is not clear. To elucidate the potential role of OPG and RANKL in JIA we determined serum levels of OPG and RANKL in patients with early JIA compared to healthy children, and prospectively explored changes in relation to radiographic score, bone and lean mass, severity of the disease, and treatment. Methods Ninety children with early oligoarticular or polyarticular JIA (ages 6-18 years; mean disease duration 19.4 months) and 90 healthy children individually matched for age, sex, race, and county of residence, were examined at baseline and 2-year follow-up. OPG and RANKL were quantified by enzyme-immunoassay. Data were analyzed with the use of t-tests, ANOVA, and multiple regression analyses. Results Serum OPG was significantly lower in patients than controls at baseline, and there was a trend towards higher RANKL and a lower OPG/RANKL ratio. Patients with polyarthritis had significantly higher increments in RANKL from baseline to follow-up, compared to patients with oligoarthritis. RANKL was a significant negative predictor for increments in total body lean mass. Patients who were receiving corticosteroids (CS) or disease-modifying antirheumatic drugs (DMARDs) at follow-up had higher OPG/RANKL ratio compared with patients who did not receive this medication. Conclusions The data supports that levels of OPG are lower in patients with JIA compared to healthy children, and higher levels of RANKL is associated with more serious disease. RANKL was a significant negative predictor of lean mass in patients with JIA. The OPG/RANKL ratio was higher in patients on DMARDs or CS treatment. PMID:21134287

  2. Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-γ/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells.

    PubMed

    Luo, Zaiming; Aslam, Shakil; Welch, William J; Wilcox, Christopher S

    2015-04-01

    Dimethylarginine dimethylaminohydrolase (DDAH) degrades asymmetric dimethylarginine, which inhibits nitric oxide (NO) synthase (NOS). Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcriptional factor that binds to antioxidant response elements and transcribes many antioxidant genes. Because the promoters of the human DDAH-1 and DDAH-2, endothelial NOS (eNOS) and PPAR-γ genes contain 2 to 3 putative antioxidant response elements, we hypothesized that they were regulated by Nrf2/antioxidant response element. Incubation of human renal glomerular endothelial cells with the Nrf2 activator tert-butylhydroquinone (20 μmol·L(-1)) significantly (P<0.05) increased NO and activities of NOS and DDAH and decreased asymmetric dimethylarginine. It upregulated genes for hemoxygenase-1, eNOS, DDAH-1, DDAH-2, and PPAR-γ and partitioned Nrf2 into the nucleus. Knockdown of Nrf2 abolished these effects. Nrf2 bound to one antioxidant response element on DDAH-1 and DDAH-2 and PPAR-γ promoters but not to the eNOS promoter. An increased eNOS and phosphorylated eNOS (P-eNOSser-1177) expression with tert-butylhydroquinone was prevented by knockdown of PPAR-γ. Expression of Nrf2 was reduced by knockdown of PPAR-γ, whereas PPAR-γ was reduced by knockdown of Nrf2, thereby demonstrating 2-way positive interactions. Thus, Nrf2 transcribes HO-1 and other genes to reduce reactive oxygen species, and DDAH-1 and DDAH-2 to reduce asymmetric dimethylarginine and PPAR-γ to increase eNOS and its phosphorylation and activity thereby coordinating 3 pathways that enhance endothelial NO generation.

  3. Enhancing laboratory activities in nuclear medicine education.

    PubMed

    Grantham, Vesper; Martin, Chris; Schmitz, Casey

    2009-12-01

    Hands-on or active learning is important in nuclear medicine education. As more curricula start to require greater standards and as distance education expands, the effective use of laboratories in nuclear medicine education remains important in physics, instrumentation, and imaging but is often overlooked or underutilized. Laboratory exercises are a unique opportunity for nuclear medicine educators to facilitate students' critical thinking and problem-solving skills in a manner that often cannot occur in lectures or during online education. Given the lack of current laboratory tools and publications, there exists a requirement for nuclear medicine educators to develop, enhance, and monitor educational tools for laboratory exercises. Expanding technologies, variations in imaging and measurement systems, and the need to ensure that the taught technology is relevant to nuclear medicine students are issues faced by nuclear medicine educators. This article, based on principles of instructional design, focuses on the components and development of effective and enhanced nuclear medicine laboratories in our current educational environment.

  4. Curcumin attenuates chronic ethanol-induced liver injury by inhibition of oxidative stress via mitogen-activated protein kinase/nuclear factor E2-related factor 2 pathway in mice

    PubMed Central

    Xiong, Zhang E; Dong, Wei Guo; Wang, Bao Ying; Tong, Qiao Yun; Li, Zhong Yan

    2015-01-01

    Objective: This study aimed to investigate the protective effect of curcumin on chronic ethanol-induced liver injury in mice and to explore its underlying mechanisms. Materials and Methods: Ethanol-exposed Balb/c mice were simultaneously treated with curcumin for 6 weeks. Liver injury was evaluated by biochemical and histopathological examination. Lipid peroxidation and anti-oxidant activities were measured by spectrophotometric method. Anti-oxidative genes expression such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and superoxide dismutase (SOD) were determined by real-time polymerase chain reaction. The nuclear factor E2-related factor 2 (Nrf2) and the phosphorylation states of specific proteins central to intracellular signaling cascades were measured by western blotting. Results: Curcumin treatment protected liver from chronic ethanol-induced injury through reducing serum alanine aminotransferase and aspartate aminotransferase activities, improving liver histological architecture, and reversing lipid disorders indicated by decrease of triglyceride, total cholesterol and low-density lipoprotein-cholesterol levels and increase of High-density lipoprotein-cholesterol levels. Meanwhile, curcumin administration attenuated oxidative stress via up-regulating SOD and glutathione peroxidase activities, leading to a reduction of lipid hydroperoxide production. In addition, curcumin increased Nrf2 activation and anti-oxidative genes expressions such as NQO1, HO-1, and SOD through inducing extracellular signal-regulated kinase (ERK) and p38 phosphorylation. Conclusion: Our data suggested that curcumin protected the liver from chronic-ethanol induced injury through attenuating oxidative stress, at least partially, through ERK/p38/Nrf2-mediated anti-oxidant signaling pathways. PMID:26600714

  5. Nuclear factor-I and activator protein-2 bind in a mutually exclusive way to overlapping promoter sequences and trans-activate the human growth hormone gene.

    PubMed Central

    Courtois, S J; Lafontaine, D A; Lemaigre, F P; Durviaux, S M; Rousseau, G G

    1990-01-01

    Transcription of the human growth hormone (hGH) gene and its regulation are controlled by trans-acting factors that bind to hGH gene promoter sequences. Several DNase I footprints have been described within 500 bp of this promoter, one of which (-289 to -267) has not yet been ascribed to a defined factor. By DNase I footprinting, gel mobility shift, and methylation interference assays with extracts from HeLa cells and GH-producing pituitary tumor (GC) cells, we show that this factor belongs to the NF-I family. When NF-I was competed out of the cell extracts, the trans-acting factor AP-2 bound to the same site as NF-I. AP-2 was present not only in HeLa cells, but also in GC cells albeit at a much lower concentration. Consistent with the mutually exclusive binding of NF-I and AP-2, their methylation interference patterns included four guanine residues that were crucial for binding of both NF-I and AP-2. Cell-free transcription from the hGH gene promoter showed that these two factors can transactivate this gene. Images PMID:2308836

  6. Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

    PubMed Central

    van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

    1992-01-01

    The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

  7. Agonist-induced activation releases peroxisome proliferator-activated receptor beta/delta from its inhibition by palmitate-induced nuclear factor-kappaB in skeletal muscle cells.

    PubMed

    Jové, Mireia; Laguna, Juan C; Vázquez-Carrera, Manuel

    2005-05-01

    The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood, but there is a strong correlation between insulin resistance and intramyocellular lipid accumulation in skeletal muscle. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. The aim of this work was to study whether the exposure of skeletal muscle cells to palmitate affected peroxisome proliferator-activated receptor (PPAR) beta/delta activity. Here, we report that exposure of C2C12 skeletal muscle cells to 0.75 mM palmitate reduced (74%, P<0.01) the mRNA levels of the PPARbeta/delta-target gene pyruvatedehydrogenase kinase 4 (PDK-4), which is involved in fatty acid utilization. This reduction was not observed in the presence of the PPARbeta/delta agonist L-165041. This drug prevented palmitate-induced nuclear factor (NF)-kappaB activation. Increased NF-kappaB activity after palmitate exposure was associated with enhanced protein-protein interaction between PPARbeta/delta and p65. Interestingly, treatment with the PPARbeta/delta agonist L-165041 completely abolished this interaction. These results indicate that palmitate may reduce fatty acid utilization in skeletal muscle cells by reducing PPARbeta/delta signaling through increased NF-kappaB activity.

  8. A diarylheptanoid from lesser galangal (Alpinia officinarum) inhibits proinflammatory mediators via inhibition of mitogen-activated protein kinase, p44/42, and transcription factor nuclear factor-kappa B.

    PubMed

    Yadav, Prem N; Liu, Zhihua; Rafi, Mohamed M

    2003-06-01

    The diarylheptanoid 7-(4'-hydroxy-3'-methoxyphenyl)-1-phenylhept-4-en-3-one (HMP) is a naturally occurring phytochemical found in lesser galangal (Alpinia officinarum). In the present study, we have demonstrated the anti-inflammatory properties of this compound on mouse macrophage cell line (RAW 264.7) and human peripheral blood mononuclear cells (PBMCs) in vitro. Treatment of RAW 264.7 cells with HMP (6.25-25 microM) significantly inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production. This compound also inhibited the release of LPS-induced proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) from human PB-MCs in vitro. In addition, Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated that HMP decreased LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression in RAW 264.7 cells. Furthermore, HMP treatment also reduced nuclear factor-kappa B (NF-kappa B) DNA binding induced by LPS in RAW 264.7 cells. To elucidate the molecular mechanism for inhibition of proinflammatory mediators by HMP (25 microM), we have studied the effect of HMP on LPS-induced p38 and p44/42 mitogen-activated protein kinase (MAPK). We observed that the phosphorylation of p44/42 MAPK in LPS-stimulated RAW 264.7 cells was markedly inhibited by HMP, whereas activation of p38 MAPK was not affected. These results suggested that HMP from lesser galangal suppressed the LPS-induced production of NO, IL-1 beta, and TNF-alpha and expression of iNOS and COX-2 gene expression by inhibiting NF-kappa B activation and phosphorylation of p44/42 MAPK.

  9. Activation of the transcription factor nuclear factor-kappa B in uterine luminal epithelial cells by interleukin 1 Beta 2: a novel interleukin 1 expressed by the elongating pig conceptus.

    PubMed

    Mathew, Daniel J; Newsom, Emily M; Guyton, Jennifer M; Tuggle, Christopher K; Geisert, Rodney D; Lucy, Matthew C

    2015-04-01

    Conceptus mortality is greatest in mammals during the peri-implantation period, a time when conceptuses appose and attach to the uterine surface epithelium while releasing proinflammatory molecules. Interleukin 1 beta (IL1B), a master proinflammatory cytokine, is released by the primate, rodent, and pig blastocyst during the peri-implantation period and is believed to be essential for establishment of pregnancy. The gene encoding IL1B has duplicated in the pig, resulting in a novel gene. Preliminary observations indicate that the novel IL1B is specifically expressed by pig conceptuses during the peri-implantation period. To verify this, IL1B was cloned from mRNA isolated from Day 12 pig conceptuses and compared with IL1B cloned from mRNA isolated from pig peripheral blood leukocytes (PBLs). The pig conceptuses, but not the PBLs, expressed a novel IL1B, referred to here as interleukin 1 beta 2 (IL1B2). Porcine endometrium was treated with recombinant porcine interleukin 1 beta 1 (IL1B1), the prototypical cytokine, and IL1B2 proteins. Immunohistochemistry and real-time RT-PCR were used to measure activation of nuclear factor-kappa B (NFKB) and NFKB-regulated transcripts, respectively, within the endometrium. Both IL1B1 and IL1B2 activated NFKB in the uterine luminal epithelium within 4 h. The NFKB activation and related gene expression, however, were lower in endometrium treated with IL1B2, suggesting that the conceptus-derived cytokine may have reduced activity within the uterus. In conclusion, the peri-implantation pig conceptus expresses a novel IL1B that can activate NFKB within the uterine surface epithelium, likely creating a proinflammatory microenvironment during establishment of pregnancy in the pig.

  10. Calcineurin/nuclear factors of activated T cells (NFAT)-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP), a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway.

    PubMed

    Yang, Jianhua; Hu, Guanghui; Wang, Shen-Wu; Li, Yucheng; Martin, Rachel; Li, Kang; Yao, Zhengbin

    2003-05-09

    We report in this study the identification and characterization of a novel protein that we designated as calcineurin/NFAT-activating and immunoreceptor tyrosine-based activation motif (ITAM)-containing protein (CNAIP). The predicted 270-amino acid sequence contains an N-terminal signal peptide, an immunoglobin domain in the extracellular region, a transmembrane domain and an ITAM in the cytoplasmic tail. Quantitative reverse transcription-PCR showed that CNAIP was preferentially expressed in neutrophils, monocytes, mast cells, and other immune-related cells. Co-transfection of CNAIP expression constructs with luciferase reporter plasmids in HMC-1 cells resulted in activation of interleukin-13 and tumor necrosis factor-alpha promoters, which was mediated through the calcineurin/NFAT-signaling pathway. Mutation of either or both tyrosines in the ITAM abolished transcriptional activation induced by CNAIP, indicating that the ITAM is indispensable for CNAIP function in activating cytokine gene promoters. Thus, it is concluded that CNAIP is a novel ITAM-containing protein that activates the calcineurin/NFAT-signaling pathway and the downstream cytokine gene promoters.

  11. Antiosteoclastogenesis activity of a CO2 laser antagonizing receptor activator for nuclear factor kappaB ligand-induced osteoclast differentiation of murine macrophages

    NASA Astrophysics Data System (ADS)

    Kuo, Chun-Liang; Kao, Chia-Tze; Fang, Hsin-Yuan; Huang, Tsui-Hsien; Chen, Yi-Wen; Shie, Ming-You

    2015-03-01

    Macrophage cells are the important effector cells in the immune reaction which are indispensable for osteoclastogenesis; their heterogeneity and plasticity renders macrophages a primer target for immune system modulation. In recent years, there have been very few studies about the effects of macrophage cells on laser treatment-regulated osteoclastogenesis. In this study, RAW 264.7 macrophage cells were treated with RANKL to regulate osteoclastogenesis. We used a CO2 laser as a model biostimulation to investigate the role of osteoclastogenic. We also evaluated cell viability, cell death and cathepsin K expression. The CO2 laser inhibited a receptor activator of the NF-ĸB ligand (RANKL)-induced formation of osteoclasts during the osteoclast differentiation process. It was also found that irradiation for two times reduced RANKL-enhanced TRAP activity in a dose-dependent manner. Furthermore, CO2 laser-treatment diminished the expression and secretion of cathepsin K elevated by RANKL and was concurrent with the inhibition of TRAF6 induction and NF-ĸB activation. The current report demonstrates that CO2 laser abrogated RANKL-induced osteoclastogenesis by retarding osteoclast differentiation. The CO2 laser can modulate every cell through dose-dependent in vitro RANKL-mediated osteoclastogenesis, such as the proliferation and fusion of preosteoclasts and the maturation of osteoclasts. Therefore, the current results serve as an improved explanation of the cellular roles of macrophage cell populations in osteoclastogenesis as well as in alveolar bone remodeling by CO2 laser-treatment.

  12. Mechanism of nuclear factor of activated T-cells mediated FasL expression in corticosterone -treated mouse Leydig tumor cells

    PubMed Central

    Chai, Wei-Ran; Chen, Yong; Wang, Qian; Gao, Hui-Bao

    2008-01-01

    Background Fas and FasL is important mediators of apoptosis. We have previously reported that the stress levels of corticosterone (CORT, glucocorticoid in rat) increase expression of Fas/FasL and activate Fas/FasL signal pathway in rat Leydig cells, which consequently leads to apoptosis. Moreover, our another study showed that nuclear factor of activated T-cells (NFAT) may play a potential role in up-regulation of FasL during CORT-treated rat Leydig cell. It is not clear yet how NFAT is involved in CORT-induced up-regulation of FasL. The aim of the present study is to investigate the molecular mechanisms of NFAT-mediated FasL expression in CORT-treated Leydig cells. Results Western blot analysis showed that NFAT2 expression is present in mouse Leydig tumor cell (mLTC-1). CORT-induced increase in FasL expression in mLTC-1 was ascertained by Western Blot analysis and CORT-induced increase in apoptotic frequency of mLTC-1 cells was detected by FACS with annexin-V labeling. Confocal imaging of NFAT2-GFP in mLTC-1 showed that high level of CORT stimulated NFAT translocation from the cytoplasm to the nucleus. RNA interference-mediated knockdown of NFAT2 significantly attenuated CORT-induced up-regulation of FasL expression in mLTC. These results corroborated our previous finding that NFAT2 is involved in CORT-induced FasL expression in rat Leydig cells and showed that mLTC-1 is a suitable model for investigating the mechanism of CORT-induced FasL expression. The analysis of reporter constructs revealed that the sequence between -201 and +71 of mouse FasL gene is essential for CORT-induced FasL expression. The mutation analysis demonstrated that CORT-induced FasL expression is mediated via an NFAT binding element located in the -201 to +71 region. Co-transfection studies with an NFAT2 expression vector and reporter construct containing -201 to +71 region of FasL gene showed that NFAT2 confer a strong inducible activity to the FasL promoter at its regulatory region. In

  13. [Influence of work factors on health state in personnel servicing military nuclear technical objects].

    PubMed

    Poluboiarinov, V N; Iusov, I G; Ivanchenko, A V; Turlakov, Iu S

    2014-01-01

    Complex of occupational studies and medical, statistical research helped to reveal climate, geographic and other factors influencing health state of personnel servicing military nuclear technical objects. Considering peculiarities of occupational activities in various specialists, the authors specified measures to improve medical service for nuclear technical military officers directly working with nuclear ammunition. Practical application of the measures helped to gain 1.5-1.7 times improvement in morbidity parameters among nuclear technical military officers.

  14. Constitutive activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor κB signaling in glioblastoma cancer stem cells regulates the Notch pathway.

    PubMed

    Garner, Jo Meagan; Fan, Meiyun; Yang, Chuan He; Du, Ziyun; Sims, Michelle; Davidoff, Andrew M; Pfeffer, Lawrence M

    2013-09-06

    Malignant gliomas are locally aggressive, highly vascular tumors that have a dismal prognosis, and present therapies provide little improvement in the disease course and outcome. Many types of malignancies, including glioblastoma, originate from a population of cancer stem cells (CSCs) that are able to initiate and maintain tumors. Although CSCs only represent a small fraction of cells within a tumor, their high tumor-initiating capacity and therapeutic resistance drives tumorigenesis. Therefore, it is imperative to identify pathways associated with CSCs to devise strategies to selectively target them. In this study, we describe a novel relationship between glioblastoma CSCs and the Notch pathway, which involves the constitutive activation of STAT3 and NF-κB signaling. Glioma CSCs were isolated and maintained in vitro using an adherent culture system, and the biological properties were compared with the traditional cultures of CSCs grown as multicellular spheres under nonadherent culture conditions. Interestingly, both adherent and spheroid glioma CSCs show constitutive activation of the STAT3/NF-κB signaling pathway and up-regulation of STAT3- and NF-κB-dependent genes. Gene expression profiling also identified components of the Notch pathway as being deregulated in glioma CSCs, and the deregulated expression of these genes was sensitive to treatment with STAT3 and NF-κB inhibitors. This finding is particularly important because Notch signaling appears to play a key role in CSCs in a variety of cancers and controls cell fate determination, survival, proliferation, and the maintenance of stem cells. The constitutive activation of STAT3 and NF-κB signaling pathways that leads to the regulation of Notch pathway genes in glioma CSCs identifies novel therapeutic targets for the treatment of glioma.

  15. Vitamin A-dependent transcriptional activation of the nuclear factor of activated T cells c1 (NFATc1) is critical for the development and survival of B1 cells.

    PubMed

    Maruya, Mikako; Suzuki, Keiichiro; Fujimoto, Hanae; Miyajima, Michio; Kanagawa, Osami; Wakayama, Teruhiko; Fagarasan, Sidonia

    2011-01-11

    B1 cells represent a distinct subset of B cells that produce most of the natural serum IgM and much of the gut IgA and function as an important component of early immune responses to pathogens. The development of B1 cells depends on the nuclear factor of activated T cells c1 (NFATc1), a transcription factor abundantly expressed by B1 cells but not by conventional B2 cells. However, the factors that regulate the expression of NFATc1 in B1 cells remain unknown. Here we show that a vitamin A-deficient diet results in reduction of NFATc1 expression in B1 cells and almost complete loss of the B1 cell compartment. As a consequence, vitamin A-deficient mice have reduced serum IgM and are unable to mount T cell-independent antibody responses against bacterial antigens. We demonstrate that injection of all-trans retinoic acid induces the expression of NFATc1, particularly from the constitutive P2 promoter, and leads to the increase of the B1 cells. Thus, the retinoic acid-dependent pathway is critical for regulating NFATc1 expression and for maintenance of the natural memory B cell compartment.

  16. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    PubMed

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities.

  17. Nuclear Control of Respiratory Chain Expression by Nuclear Respiratory Factors and PGC-1-Related Coactivator

    PubMed Central

    Scarpulla, Richard C.

    2010-01-01

    Expression of the respiratory apparatus depends on both nuclear and mitochondrial genes. Although these genes are sequestered in distinct cellular organelles, their transcription relies on nucleus-encoded factors. Certain of these factors are directed to the mitochondria, where they sponsor the bi-directional transcription of mitochondrial DNA. Others act on nuclear genes that encode the majority of the respiratory subunits and many other gene products required for the assembly and function of the respiratory chain. The nuclear respiratory factors, NRF-1 and NRF-2, contribute to the expression of respiratory subunits and mitochondrial transcription factors and thus have been implicated in nucleo-mitochondrial interactions. In addition, coactivators of the PGC-1 family serve as mediators between the environment and the transcriptional machinery governing mitochondrial biogenesis. One family member, peroxisome proliferator-activated receptor γ coactivator PGC-1-related coactivator (PRC), is an immediate early gene product that is rapidly induced by mitogenic signals in the absence of de novo protein synthesis. Like other PGC-1 family members, PRC binds NRF-1 and activates NRF-1 target genes. In addition, PRC complexes with NRF-2 and HCF-1 (host cell factor-1) in the activation of NRF-2-dependent promoters. HCF-1 functions in cell-cycle progression and has been identified as an NRF-2 coactivator. The association of these factors with PRC is suggestive of a role for the complex in cell growth. Finally, shRNA-mediated knock down of PRC expression results in a complex phenotype that includes the inhibition of respiratory growth on galactose and the loss of respiratory complexes. Thus, PRC may help integrate the expression of the respiratory apparatus with the cell proliferative program. PMID:19076454

  18. Palmitate induces tumor necrosis factor-alpha expression in C2C12 skeletal muscle cells by a mechanism involving protein kinase C and nuclear factor-kappaB activation.

    PubMed

    Jové, Mireia; Planavila, Anna; Sánchez, Rosa M; Merlos, Manuel; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2006-01-01

    The mechanisms responsible for increased expression of TNF-alpha in skeletal muscle cells in diabetic states are not well understood. We examined the effects of the saturated acid palmitate on TNF-alpha expression. Exposure of C2C12 skeletal muscle cells to 0.75 mm palmitate enhanced mRNA (25-fold induction, P < 0.001) and protein (2.5-fold induction) expression of the proinflammatory cytokine TNF-alpha. This induction was inversely correlated with a fall in GLUT4 mRNA levels (57% reduction, P < 0.001) and glucose uptake (34% reduction, P < 0.001). PD98059 and U0126, inhibitors of the ERK-MAPK cascade, partially prevented the palmitate-induced TNF-alpha expression. Palmitate increased nuclear factor (NF)-kappaB activation and incubation of the cells with the NF-kappaB inhibitors pyrrolidine dithiocarbamate and parthenolide partially prevented TNF-alpha expression. Incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C (PKC), abolished palmitate-induced TNF-alpha expression, and restored GLUT4 mRNA levels. Palmitate treatment enhanced the expression of phospho-PKCtheta, suggesting that this PKC isoform was involved in the changes reported, and coincubation of palmitate-treated cells with the PKC inhibitor chelerythrine prevented the palmitate-induced reduction in the expression of IkappaBalpha and insulin-stimulated Akt activation. These findings suggest that enhanced TNF-alpha expression and GLUT4 down-regulation caused by palmitate are mediated through the PKC activation, confirming that this enzyme may be a target for either the prevention or the treatment of fatty acid-induced insulin resistance.

  19. Nuclear Factor of Activated T Cells-dependent Down-regulation of the Transcription Factor Glioma-associated Protein 1 (GLI1) Underlies the Growth Inhibitory Properties of Arachidonic Acid*

    PubMed Central

    Comba, Andrea; Almada, Luciana L.; Tolosa, Ezequiel J.; Iguchi, Eriko; Marks, David L.; Vara Messler, Marianela; Silva, Renata; Fernandez-Barrena, Maite G.; Enriquez-Hesles, Elisa; Vrabel, Anne L.; Botta, Bruno; Di Marcotulio, Lucia; Ellenrieder, Volker; Eynard, Aldo R.; Pasqualini, Maria E.; Fernandez-Zapico, Martin E.

    2016-01-01

    Numerous reports have demonstrated a tumor inhibitory effect of polyunsaturated fatty acids (PUFAs). However, the molecular mechanisms modulating this phenomenon are in part poorly understood. Here, we provide evidence of a novel antitumoral mechanism of the PUFA arachidonic acid (AA). In vivo and in vitro experiments showed that AA treatment decreased tumor growth and metastasis and increased apoptosis. Molecular analysis of this effect showed significantly reduced expression of a subset of antiapoptotic proteins, including BCL2, BFL1/A1, and 4-1BB, in AA-treated cells. We demonstrated that down-regulation of the transcription factor glioma-associated protein 1 (GLI1) in AA-treated cells is the underlying mechanism controlling BCL2, BFL1/A1, and 4-1BB expression. Using luciferase reporters, chromatin immunoprecipitation, and expression studies, we found that GLI1 binds to the promoter of these antiapoptotic molecules and regulates their expression and promoter activity. We provide evidence that AA-induced apoptosis and down-regulation of antiapoptotic genes can be inhibited by overexpressing GLI1 in AA-sensitive cells. Conversely, inhibition of GLI1 mimics AA treatments, leading to decreased tumor growth, cell viability, and expression of antiapoptotic molecules. Further characterization showed that AA represses GLI1 expression by stimulating nuclear translocation of NFATc1, which then binds the GLI1 promoter and represses its transcription. AA was shown to increase reactive oxygen species. Treatment with antioxidants impaired the AA-induced apoptosis and down-regulation of GLI1 and NFATc1 activation, indicating that NFATc1 activation and GLI1 repression require the generation of reactive oxygen species. Collectively, these results define a novel mechanism underlying AA antitumoral functions that may serve as a foundation for future PUFA-based therapeutic approaches. PMID:26601952

  20. Using Nuclear Receptor Activity to Stratify Hepatocarcinogens

    PubMed Central

    Shah, Imran; Houck, Keith; Judson, Richard S.; Kavlock, Robert J.; Martin, Matthew T.; Reif, David M.; Wambaugh, John; Dix, David J.

    2011-01-01

    Background Nuclear receptors (NR) are a superfamily of ligand-activated transcription factors that control a range of cellular processes. Persistent stimulation of some NR is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. Here we report on a systematic analysis of new in vitro human NR activity data on 309 environmental chemicals in relationship to their liver cancer-related chronic outcomes in rodents. Results The effects of 309 environmental chemicals on human constitutive androstane receptors (CAR/NR1I3), pregnane X receptor (PXR/NR1I2), aryl hydrocarbon receptor (AhR), peroxisome proliferator-activated receptors (PPAR/NR1C), liver X receptors (LXR/NR1H), retinoic X receptors (RXR/NR2B) and steroid receptors (SR/NR3) were determined using in vitro data. Hepatic histopathology, observed in rodents after two years of chronic treatment for 171 of the 309 chemicals, was summarized by a cancer lesion progression grade. Chemicals that caused proliferative liver lesions in both rat and mouse were generally more active for the human receptors, relative to the compounds that only affected one rodent species, and these changes were significant for PPAR (p0.001), PXR (p0.01) and CAR (p0.05). Though most chemicals exhibited receptor promiscuity, multivariate analysis clustered them into relatively few NR activity combinations. The human NR activity pattern of chemicals weakly associated with the severity of rodent liver cancer lesion progression (p0.05). Conclusions The rodent carcinogens had higher in vitro potency for human NR relative to non-carcinogens. Structurally diverse chemicals with similar NR promiscuity patterns weakly associated with the severity of rodent liver cancer progression. While these results do not prove the role of NR activation in human liver cancer, they do have implications for nuclear receptor chemical biology and provide insights into putative toxicity pathways. More importantly, these findings suggest the

  1. Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1.

    PubMed Central

    vom Baur, E; Zechel, C; Heery, D; Heine, M J; Garnier, J M; Vivat, V; Le Douarin, B; Gronemeyer, H; Chambon, P; Losson, R

    1996-01-01

    Using a yeast two-hybrid system we report the isolation of a novel mouse protein, mSUG1, that interacts with retinoic acid receptor alpha (RAR alpha) both in yeast cells and in vitro in a ligand- and AF-2 activating domain (AF-2 AD)-dependent manner and show that it is a structural and functional homologue of the essential yeast protein SUG1. mSUG1 also efficiently interacts with other nuclear receptors, including oestrogen (ER), thyroid hormone (TR), Vitamin D3 (VDR) and retinoid X (RXR) receptors. By comparing the interaction properties of these receptors with mSUG1 and TIF1, we demonstrate that: (i) RXR alpha efficiently interacts with TIF1, but not with mSUG1, whereas TR alpha interacts much more efficiently with mSUG1 than with TIF1, and RAR alpha, VDR and ER efficiently interact with mSUG1 and TIF1; (ii) the amphipathic alpha-helix core of the AF-2 AD is differentially involved in interactions of RAR alpha with mSUG1 and TIF1; (iii) the AF-2 AD cores of RAR alpha and ER are similarly involved in their interaction with TIF1, but not with mSUG1. Thus, the interaction interfaces between the different receptors and either mSUG1 or TIF1 may vary depending on the nature of the receptor and the putative mediator of its AF-2 function. We discuss the possibility that mSUG1 and TIF1 may mediate the transcriptional activity of the AF-2 of nuclear receptors through different mechanisms. Images PMID:8598193

  2. Factor XII Contact Activation.

    PubMed

    Naudin, Clément; Burillo, Elena; Blankenberg, Stefan; Butler, Lynn; Renné, Thomas

    2017-03-27

    Contact activation is the surface-induced conversion of factor XII (FXII) zymogen to the serine protease FXIIa. Blood-circulating FXII binds to negatively charged surfaces and this contact to surfaces triggers a conformational change in the zymogen inducing autoactivation. Several surfaces that have the capacity for initiating FXII contact activation have been identified, including misfolded protein aggregates, collagen, nucleic acids, and platelet and microbial polyphosphate. Activated FXII initiates the proinflammatory kallikrein-kinin system and the intrinsic coagulation pathway, leading to formation of bradykinin and thrombin, respectively. FXII contact activation is well characterized in vitro and provides the mechanistic basis for the diagnostic clotting assay, activated partial thromboplastin time. However, only in the past decade has the critical role of FXII contact activation in pathological thrombosis been appreciated. While defective FXII contact activation provides thromboprotection, excess activation underlies the swelling disorder hereditary angioedema type III. This review provides an overview of the molecular basis of FXII contact activation and FXII contact activation-associated disease states.

  3. Persistent activation of nuclear factor-kappa B and expression of pro-inflammatory cytokines in bone marrow cells after exposure of mice to protons

    NASA Astrophysics Data System (ADS)

    Rithidech, Kanokporn; Reungpatthanaphong, Paiboon; Honikel, Louise; Whorton, Elbert

    Protons are the most abundant component of solar particle events (SPEs) in space. Information is limited on early-and late-occurring in vivo biological effects of exposure to protons at doses and dose rates that are similar to what astronauts encounter in space. We conducted a study series to fill this knowledge gap. We focused on the biological effects of 100 MeV/n protons, which are one of the most abundant types of protons induced during SPEs. We gave BALB/cJ mice a whole-body exposure to 0.5 or 1.0 Gy of 100 MeV/n protons, delivered at 0.5 or 1.0 cGy/min. These doses and dose rates of protons were selected because they are comparable to those of SPEs taking place in space. For each dose and dose rate of 100 MeV/n protons, mice exposed to 0 Gy of protons served as sham controls. Mice included in this study were also part of a study series conducted to examine the extent and the mechanisms involved in in vivo induction of genomic instability (expressed as late-occurring chromosome instability) by 100 MeV/n protons. Bone marrow (BM) cells were collected from groups of mice for analyses at different times post-exposure, i.e. early time-points (1.5, 3, and 24 hr) and late time-points (1 and 6 months). At each harvest time, there were five mice per treatment group. Several endpoints were used to investigate the biological effects of 100 MeV/n protons in BM cells from irradiated and sham control mice. The scope of this study was to determine the dose-rate effects of 0.5 Gy of 100 MeV/n protons in BM cells on the kinetics of nuclear factor-kappa B (NF-kappa B) activation and the expression of selected NF-kappa B target proteins known to be involved in inflammatory response, i.e. pro-inflammatory cytokines (TNF-alpha, IL-1 beta, and IL-6). Significantly high levels (p values ranging from p¡0.01 and p¡0.05) of activated NF-kappa B were observed in BM cells collected from irradiated mice, relative to those obtained from the corresponding sham controls, at all time

  4. Study of Arsenic Sulfide in Solid Tumor Cells Reveals Regulation of Nuclear Factors of Activated T-cells by PML and p53

    PubMed Central

    Ding, Wenping; Tong, Yingying; Zhang, Xiuli; Pan, Minggui; Chen, Siyu

    2016-01-01

    Arsenic sulfide (AS) has excellent cytotoxic activity in acute promyelocytic leukemia (APL) but its activity in solid tumors remains to be explored. Here we show that AS and cyclosporine A (CsA) exerted synergistic inhibitory effect on cell growth and c-Myc expression in HCT116 cells. AS inhibited the expression of PML, c-Myc, NFATc1, NFATc3, and NFATc4, while stimulating the expression of p53 and NFATc2. Knockdown of PML reduced NFATc1, NFATc2, NFATc3 and NFATc4 expression while overexpression of p53 stimulated NFATc2-luciferase activity that was further augmented by AS by binding to a set of p53 responsive elements (PREs) on the NFATc2 promoter. Additionally, overexpression of p53 suppressed NFATc3 and NFATc4. Reciprocally, NFATc3 knockdown enhanced p53 while reducing MDM2 expression indicating that NFATc3 is a negative regulator of p53 while a positive regulator of MDM2, consistent with its tumor-promoting property as knockdown of NFATc3 retarded cell growth in vitro and tumor growth in xenograft. In patients with colon cancer, tumor expression of NFATc2 correlated with superior survival, while nuclear NFATc1 with inferior survival. These results indicate that AS differentially regulates NFAT pathway through PML and p53 and reveal an intricate reciprocal regulatory relationship between NFAT proteins and p53 pathway. PMID:26795951

  5. Wogonin prevents lipopolysaccharide-induced acute lung injury and inflammation in mice via peroxisome proliferator-activated receptor gamma-mediated attenuation of the nuclear factor-kappaB pathway

    PubMed Central

    Yao, Jing; Pan, Di; Zhao, Yue; Zhao, Li; Sun, Jie; Wang, Yu; You, Qi-Dong; Xi, Tao; Guo, Qing-Long; Lu, Na

    2014-01-01

    Acute lung injury (ALI) from a variety of clinical disorders, characterized by diffuse inflammation, is a cause of acute respiratory failure that develops in patients of all ages. Previous studies reported that wogonin, a flavonoid-like chemical compound which was found in Scutellaria baicalensis, has anti-inflammatory effects in several inflammation models, but not in ALI. Here, the in vivo protective effect of wogonin in the amelioration of lipopolysaccharide (LPS) -induced lung injury and inflammation was assessed. In addition, the in vitro effects and mechanisms of wogonin were studied in the mouse macrophage cell lines Ana-1 and RAW264.7. In vivo results indicated that wogonin attenuated LPS-induced histological alterations. Peripheral blood leucocytes decreased in the LPS-induced group, which was ameliorated by wogonin. In addition, wogonin inhibited the production of several inflammatory cytokines, including tumour necrosis factor-α, interleukin-1β (IL-1β) and IL-6, in the bronchoalveolar lavage fluid and lung tissues after LPS challenge, while the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor GW9662 reversed these effects. In vitro results indicated that wogonin significantly decreased the secretion of IL-6, IL-1β and tumour necrosis factor-α in Ana-1 and RAW264.7 cells, which was suppressed by transfection of PPARγ small interfering RNA and GW9662 treatment. Moreover, wogonin activated PPARγ, induced PPARγ-mediated attenuation of the nuclear translocation and the DNA-binding activity of nuclear factor-κB in vivo and in vitro. In conclusion, all of these results showed that wogonin may serve as a promising agent for the attenuation of ALI-associated inflammation and pathology by regulating the PPARγ-involved nuclear factor-κB pathway. PMID:24766487

  6. Nuclear Receptor Activity and Liver Cancer Lesion Progression

    EPA Science Inventory

    Nuclear receptors (NRs) are ligand-activated transcription factors that control diverse cellular processes. Chronic stimulation of some NRs is a non-genotoxic mechanism of rodent liver cancer with unclear relevance to humans. We explored this question using human CAR, PXR, PPARα,...

  7. Examination of psychological variables related to nuclear attitudes and nuclear activism

    SciTech Connect

    Roy, P.J.

    1985-01-01

    It was hypothesized that knowledge about nuclear arms developments would not be correlated with nuclear attitudes, that sense of efficacy would be positively correlated with magnitude of nuclear activism, and that death anxiety would be correlated with high level of nuclear knowledge and anti-nuclear attitudes, but not with sense of power. It was also hypothesized that positive correlations would be found between nuclear activism and political activism, knowledge of nuclear facts, and degree of adherence to anti-nuclear attitudes. One hundred and forty three women and 90 men participated in this questionnaire study. Major findings are as follows. In general, the more people knew about nuclear developments, the more anti-nuclear were their attitudes. Also, regardless of nuclear attitudes, a positive correlation was found between knowledge of nuclear facts and nuclear activism. Death anxiety and powerlessness were not correlated. There was a positive correlation between anxiety and both nuclear knowledge and anti-nuclear attitudes. A strong positive correlation was found between nuclear activism and anti-nuclear attitudes, and between political activism and nuclear activism. Internal locus of control did not correlate significantly with high sense of power or with high degree of nuclear activism.

  8. Dendrobium moniliforme Exerts Inhibitory Effects on Both Receptor Activator of Nuclear Factor Kappa-B Ligand-Mediated Osteoclast Differentiation in Vitro and Lipopolysaccharide-Induced Bone Erosion in Vivo.

    PubMed

    Baek, Jong Min; Kim, Ju-Young; Ahn, Sung-Jun; Cheon, Yoon-Hee; Yang, Miyoung; Oh, Jaemin; Choi, Min Kyu

    2016-03-01

    Dendrobium moniliforme (DM) is a well-known plant-derived extract that is widely used in Oriental medicine. DM and its chemical constituents have been reported to have a variety of pharmacological effects, including anti-oxidative, anti-inflammatory, and anti-tumor activities; however, no reports discuss the beneficial effects of DM on bone diseases such as osteoporosis. Thus, we investigated the relationship between DM and osteoclasts, cells that function in bone resorption. We found that DM significantly reduced receptor activator of nuclear factor kappa-B ligand (RANKL)-induced tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation; DM directly induced the down-regulation of c-Fos and nuclear factor of activated T cells c1 (NFATc1) without affecting other RANKL-dependent transduction pathways. In the later stages of osteoclast maturation, DM negatively regulated the organization of filamentous actin (F-actin), resulting in impaired bone-resorbing activity by the mature osteoclasts. In addition, micro-computed tomography (μ-CT) analysis of the murine model revealed that DM had a beneficial effect on lipopolysaccharide (LPS)-mediated bone erosion. Histological analysis showed that DM attenuated the degradation of trabecular bone matrix and formation of TRAP-positive osteoclasts in bone tissues. These results suggest that DM is a potential candidate for the treatment of metabolic bone disorders such as osteoporosis.

  9. Manassantin B isolated from Saururus chinensis inhibits cyclooxygenase-2-dependent prostaglandin D2 generation by blocking Fyn-mediated nuclear factor-kappaB and mitogen activated protein kinase pathways in bone marrow derived-mast cells.

    PubMed

    Lu, Yue; Hwang, Seung-Lark; Son, Jong Keun; Chang, Hyeun Wook

    2013-01-01

    The authors investigated the effect of manassantin B (Man B) isolated from Saururus chinensis (S. chinensis) on cyclooxygenase-2 (COX-2)-dependent prostaglandin D2 (PGD2) generation in mouse bone marrow derived-mast cells (BMMCs). Man B inhibited the generation of PGD2 dose-dependently by inhibiting COX-2 expression in immunoglobulin E (IgE)/Ag-stimulated BMMCs. To elucidate the mechanism responsible for the inhibition of COX-2 expression by Man B, the effects of Man B on the activation of nuclear factor-kappaB (NF-κB), a transcription factor essential and mitogen-activated protein kinases (MAPKs) for COX-2 induction, were examined. Man B attenuated the nuclear translocation of NF-κB p65 and its DNA-binding activity by inhibiting inhibitors of kappa Bα (IκBα) degradation and concomitantly suppressing IκB kinase (IKK) phosphorylation. In addition, Man B suppressed phosphorylation of MAPKs including extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK) and p38. It was also found that Man B suppressed Fyn kinase activation and consequent downstream signaling processes, including those involving Syk, Gab2, and Akt. Taken together, the present results suggest that Man B suppresses COX-2 dependent PGD2 generation by primarily inhibiting Fyn kinase in FcεRI-mediated mast cells.

  10. Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes paragraph sign.

    PubMed

    Afaq, Farrukh; Malik, Arshi; Syed, Deeba; Maes, Daniel; Matsui, Mary S; Mukhtar, Hasan

    2005-01-01

    Excessive exposure of solar ultraviolet (UV) radiation, particularly its UV-B component, to humans causes many adverse effects that include erythema, hyperplasia, hyperpigmentation, immunosuppression, photoaging and skin cancer. In recent years, there is increasing use of botanical agents in skin care products. Pomegranate derived from the tree Punica granatum contains anthocyanins (such as delphinidin, cyanidin and pelargonidin) and hydrolyzable tannins (such as punicalin, pedunculagin, punicalagin, gallagic and ellagic acid esters of glucose) and possesses strong antioxidant and anti-inflammatory properties. Recently, we have shown that pomegranate fruit extract (PFE) possesses antitumor promoting effects in a mouse model of chemical carcinogenesis. To begin to establish the effect of PFE for humans in this study, we determined its effect on UV-B-induced adverse effects in normal human epidermal keratinocytes (NHEK). We first assessed the effect of PFE on UV-B-mediated phosphorylation of mitogen-activated protein kinases (MAPK) pathway in NHEK. Immunoblot analysis demonstrated that the treatment of NHEK with PFE (10-40 microg/mL) for 24 h before UV-B (40 mJ/cm(2)) exposure dose dependently inhibited UV-B-mediated phosphorylation of ERKl/2, JNK1/2 and p38 protein. We also observed that PFE (20 microg/mL) inhibited UV-B-mediated phosphorylation of MAPK in a time-dependent manner. Furthermore, in dose- and time-dependent studies, we evaluated the effect of PFE on UV-B-mediated activation of nuclear factor kappa B (NF-kappaB) pathway. Using Western blot analysis, we found that PFE treatment of NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated degradation and phosphorylation of IkappaBalpha and activation of IKKalpha. Using immunoblot analysis, enzyme-linked immunosorbent assay and electrophoretic mobility shift assay, we found that PFE treatment to NHEK resulted in a dose- and time-dependent inhibition of UV-B-mediated nuclear translocation and

  11. Gastro-duodenal fluid induced nuclear factor-κappaB activation and early pre-malignant alterations in murine hypopharyngeal mucosa

    PubMed Central

    Vageli, Dimitra P.; Prasad, Manju L.; Sasaki, Clarence T.

    2016-01-01

    We recently described the role of gastro-duodenal fluids (GDFs) in generating changes consistent with hypopharyngeal neoplasia through activation of NF-κB pathway, using an in vitro model of human hypopharyngeal normal keratinocytes. Here, we further provide evidence that gastro-duodenal reflux is a risk factor for early pre-malignant alterations in hypopharyngeal mucosa (HM) related to an activated NF-κB oncogenic pathway, using both an in vitro and a novel in vivo model of C57Bl/6J mice. Histological, immunohistochemical and automated quantitative analysis documents significant NF-κB activation and early pre-malignant alterations in HM topically exposed to GDFs, compared to acid alone and other controls. Early pre-malignant histologic lesions exhibited increased Ki67, CK14 and ΔNp63, cell proliferation markers, changes of cell adhesion molecules, E-Cadherin and β-catenin, and STAT3 activation. The in vivo effect of NF-κB activation is positively correlated with p-STAT3, Ki67, CK14 or β-catenin expression, while GDFs induce significant transcriptional activation of RELA(p65), bcl-2, TNF-α, STAT3, EGFR and wnt5A, in vivo. Our in vivo model demonstrates selectively activated NF-κB in response to topically administrated GDFs, leading to early pre-malignant events in HM. PMID:26745676

  12. Public opinion factors regarding nuclear power

    SciTech Connect

    Benson, B.

    1991-12-31

    This paper is an effort to identify, as comprehensively as possible, public concerns about nuclear power, and to assess, where possible, the relative importance of these concerns as they relate to government regulation of and policy towards nuclear power. It is based on some two dozen in-depth interviews with key communicators representing the nuclear power industry, the environmental community, and government, as well as on the parallel efforts in our research project: (1) review of federal court case law, (2) a selective examination of the Nuclear Regulatory Commission (NRC) administrative process, and (3) the preceding George Mason University research project in this series. The paper synthesizes our findings about public attitudes towards nuclear power as expressed through federal court case law, NRC administrative law, public opinion surveys, and direct personal interviews. In so doing, we describe the public opinion environment in which the nuclear regulatory process must operate. Our premise is that public opinion ultimately underlies the approaches government agencies take towards regulating nuclear power, and that, to the degree that the nuclear power industry`s practices are aligned with public opinion, a more favorable regulatory climate is possible.

  13. Public opinion factors regarding nuclear power

    SciTech Connect

    Benson, B.

    1991-01-01

    This paper is an effort to identify, as comprehensively as possible, public concerns about nuclear power, and to assess, where possible, the relative importance of these concerns as they relate to government regulation of and policy towards nuclear power. It is based on some two dozen in-depth interviews with key communicators representing the nuclear power industry, the environmental community, and government, as well as on the parallel efforts in our research project: (1) review of federal court case law, (2) a selective examination of the Nuclear Regulatory Commission (NRC) administrative process, and (3) the preceding George Mason University research project in this series. The paper synthesizes our findings about public attitudes towards nuclear power as expressed through federal court case law, NRC administrative law, public opinion surveys, and direct personal interviews. In so doing, we describe the public opinion environment in which the nuclear regulatory process must operate. Our premise is that public opinion ultimately underlies the approaches government agencies take towards regulating nuclear power, and that, to the degree that the nuclear power industry's practices are aligned with public opinion, a more favorable regulatory climate is possible.

  14. Functional interaction of hepatic nuclear factor-4 and peroxisome proliferator-activated receptor-gamma coactivator 1alpha in CYP7A1 regulation is inhibited by a key lipogenic activator, sterol regulatory element-binding protein-1c.

    PubMed

    Ponugoti, Bhaskar; Fang, Sungsoon; Kemper, Jongsook Kim

    2007-11-01

    Insulin inhibits transcription of cholesterol 7alpha-hydroxylase (Cyp7a1), a key gene in bile acid synthesis, and the hepatic nuclear factor-4 (HNF-4) site in the promoter was identified as a negative insulin response sequence. Using a fasting/feeding protocol in mice and insulin treatment in HepG2 cells, we explored the inhibition mechanisms. Expression of sterol regulatory element-binding protein-1c (SREBP-1c), an insulin-induced lipogenic factor, inversely correlated with Cyp7a1 expression in mouse liver. Interaction of HNF-4 with its coactivator, peroxisome proliferator-activated receptor-gamma coactivator 1alpha (PGC-1alpha), was observed in livers of fasted mice and was reduced after feeding. Conversely, HNF-4 interaction with SREBP-1c was increased after feeding. In vitro studies suggested that SREBP-1c competed with PGC-1alpha for direct interaction with the AF2 domain of HNF-4. Reporter assays showed that SREBP-1c, but not of a SREBP-1c mutant lacking the HNF-4 interacting domain, inhibited HNF-4/PGC-1alpha transactivation of Cyp7a1. SREBP-1c also inhibited PGC-1alpha-coactivation of estrogen receptor, constitutive androstane receptor, pregnane X receptor, and farnesoid X receptor, implying inhibition of HNF-4 by SREBP-1c could extend to other nuclear receptors. In chromatin immunoprecipitation studies, HNF-4 binding to the promoter was not altered, but PGC-1alpha was dissociated, SREBP-1c and histone deacetylase-2 (HDAC2) were recruited, and acetylation of histone H3 was decreased upon feeding. Adenovirus-mediated expression of a SREBP-1c dominant-negative mutant, which blocks the interaction of SREBP-1c and HNF-4, partially but significantly reversed the inhibition of Cyp7a1 after feeding. Our data show that SREBP-1c functions as a non-DNA-binding inhibitor and mediates, in part, suppression of Cyp7a1 by blocking functional interaction of HNF-4 and PGC-1alpha. This mechanism may be relevant to known repression of many other HNF-4 target genes upon

  15. Factors to be Considered in Long-Term Monitoring of a Former Nuclear Test Site in a Geophysically Active and Water-rich Environment

    NASA Astrophysics Data System (ADS)

    Eichelberger, J.; Hill, G.; Patrick, M.; Freymueller, J.; Barnes, D.; Kelley, J.; Layer, P.

    2001-12-01

    The US Department of Energy (USDOE) is currently undertaking an ambitious program of environmental remediation of the surface of Amchitka Island in the western Aleutians, where three underground nuclear tests were conducted during 1963-1971. Among these tests was Cannikin, at approximately 5 megatons the largest nuclear device ever exploded underground by the United States and equivalent in seismic energy release to a magnitude 7 earthquake. The blast caused about 1 m of uplift of the Bering Sea coastline in the 3-km-wide fault-bounded block within which it was detonated. The impending final transfer of stewardship of this area to the US Fish and Wildlife Service as part of the Alaska Maritime National Wildlife Refuge raises anew the question of the potential for transport of radionuclides from the shot cavity, located at 1791 m depth in mafic laharic breccias, into the accessible environment. In particular, there is concern about whether such contaminants could become concentrated in the marine food chain that is used for subsistence by Alaskan Natives (and by the broader international community through the North Pacific and Bering Fisheries). Both possible transport pathways in the form of faults and transport medium in the form of abundant water are present. Since the pre-plate tectonics paradigm days of active testing, the scientific community's understanding of the tectonic context of the Aleutian Islands has grown tremendously. Recently, the first direct measurements of motion within the arc have been made. How this new understanding should guide plans for long-term monitoring of the site is an important question. Convergence due to subduction of the North Pacific plate beneath North America ranges from near-normal at the Alaska Peninsula and eastern Aleutian islands to highly oblique in the west. Amchitka itself can be seen as a subaerial portion of a 200-km-long Rat Island arc crest segment. This fragment has torn from the Andreanof Islands to the east at

  16. The Arabidopsis thaliana Nuclear Factor Y Transcription Factors

    PubMed Central

    Zhao, Hang; Wu, Di; Kong, Fanying; Lin, Ke; Zhang, Haishen; Li, Gang

    2017-01-01

    Nuclear factor Y (NF-Y) is an evolutionarily conserved trimeric transcription factor complex present in nearly all eukaryotes. The heterotrimeric NF-Y complex consists of three subunits, NF-YA, NF-YB, and NF-YC, and binds to the CCAAT box in the promoter regions of its target genes to regulate their expression. Yeast and mammal genomes generally have single genes with multiple splicing isoforms that encode each NF-Y subunit. By contrast, plant genomes generally have multi-gene families encoding each subunit and these genes are differentially expressed in various tissues or stages. Therefore, different subunit combinations can lead to a wide variety of NF-Y complexes in various tissues, stages, and growth conditions, indicating the potentially diverse functions of this complex in plants. Indeed, many recent studies have proved that the NF-Y complex plays multiple essential roles in plant growth, development, and stress responses. In this review, we highlight recent progress on NF-Y in Arabidopsis thaliana, including NF-Y protein structure, heterotrimeric complex formation, and the molecular mechanism by which NF-Y regulates downstream target gene expression. We then focus on its biological functions and underlying molecular mechanisms. Finally, possible directions for future research on NF-Y are also presented. PMID:28119722

  17. The Arabidopsis thaliana Nuclear Factor Y Transcription Factors.

    PubMed

    Zhao, Hang; Wu, Di; Kong, Fanying; Lin, Ke; Zhang, Haishen; Li, Gang

    2016-01-01

    Nuclear factor Y (NF-Y) is an evolutionarily conserved trimeric transcription factor complex present in nearly all eukaryotes. The heterotrimeric NF-Y complex consists of three subunits, NF-YA, NF-YB, and NF-YC, and binds to the CCAAT box in the promoter regions of its target genes to regulate their expression. Yeast and mammal genomes generally have single genes with multiple splicing isoforms that encode each NF-Y subunit. By contrast, plant genomes generally have multi-gene families encoding each subunit and these genes are differentially expressed in various tissues or stages. Therefore, different subunit combinations can lead to a wide variety of NF-Y complexes in various tissues, stages, and growth conditions, indicating the potentially diverse functions of this complex in plants. Indeed, many recent studies have proved that the NF-Y complex plays multiple essential roles in plant growth, development, and stress responses. In this review, we highlight recent progress on NF-Y in Arabidopsis thaliana, including NF-Y protein structure, heterotrimeric complex formation, and the molecular mechanism by which NF-Y regulates downstream target gene expression. We then focus on its biological functions and underlying molecular mechanisms. Finally, possible directions for future research on NF-Y are also presented.

  18. Human factors design guidelines for maintainability of Department of Energy nuclear facilities

    SciTech Connect

    Bongarra, J.P. Jr.; VanCott, H.P.; Pain, R.F.; Peterson, L.R.; Wallace, R.I.

    1985-06-18

    Intent of these guidelines is to provide design and design review teams of DOE nuclear facilities with human factors principles to enhance the design and aid in the inspection of DOE nuclear facilities, systems, and equipment. These guidelines are concerned with design features of DOE nuclear facilities which can potentially affect preventive and corrective maintenance of systems within DOE nuclear facilities. Maintenance includes inspecting, checking, troubleshooting, adjusting, replacing, repairing, and servicing activities. Other factors which influence maintainability such as repair and maintenance suport facilities, maintenance information, and various aspects of the environment are also addressed.

  19. Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

    PubMed

    Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

    2014-01-01

    Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

  20. Factors regulating microglia activation

    PubMed Central

    Kierdorf, Katrin; Prinz, Marco

    2013-01-01

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

  1. Kpna7 interacts with egg-specific nuclear factors in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nuclear proteins are required for initiation of transcription in early embryos before embryonic genome activation. The regulation of transportation of nuclear proteins is mediated by transport factors known as importins (karyopherins). Kpna7 is a newly discovered member of the importin a family, whi...

  2. Homeobox A9 directly targeted by miR-196b regulates aggressiveness through nuclear Factor-kappa B activity in non-small cell lung cancer cells.

    PubMed

    Yu, Seong-Lan; Lee, Dong Chul; Sohn, Hyun Ahm; Lee, Soo Young; Jeon, Hyo Sung; Lee, Joon H; Park, Chang Gyo; Lee, Hoi Young; Yeom, Young Il; Son, Ji Woong; Yoon, Yoo Sang; Kang, Jaeku

    2016-12-01

    MicroRNAs (miRNAs) are recognized as crucial posttranscriptional regulators of gene expression, and play critical roles as oncogenes or tumor suppressors in various cancers. Here, we show that miR-196b is upregulated in mesenchymal-like-state non-small cell lung cancer (NSCLC) cells and lung cancer tissues. Moreover, miR-196b upregulation stimulates cell invasion and a change in cell morphology to a spindle shape via loss of cell-to-cell contacts. We identified homeobox A9 (HOXA9) as a target gene of miR-196b by using public databases such as TargetScan, miRDB, and microRNA.org. HOXA9 expression is inversely correlated with miR-196b levels in clinical NSCLC samples as compared to that in corresponding control samples, and with the migration and invasion of NSCLC cells. Ectopic expression of HOXA9 resulted in a suppression of miR-196b-induced cell invasion, and HOXA9 reexpression increased E-cadherin expression. Furthermore, HOXA9 potently attenuated the expression of snail family zinc finger 2 (SNAI2/SLUG) and matrix metallopeptidase 9 (MMP9) by controlling the binding of nuclear factor-kappa B to the promoter of SLUG and MMP9 genes, respectively. Therefore, we suggest that HOXA9 plays a central role in controlling the aggressive behavior of lung cancer cells and that miR-196b can serve as a potential target for developing anticancer agents. © 2015 Wiley Periodicals, Inc.

  3. Nuclear Science Teaching Aids and Activities.

    ERIC Educational Resources Information Center

    Woodburn, John H.

    This publication is a sourcebook for science teachers. It provides guides for basic laboratory work in nuclear energy, suggesting various teacher and student demonstrations. Ideas for science clubs, science fairs, and project research seminars are presented. Problem-solving activities for both science and mathematics classes are included, as well…

  4. Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation

    PubMed Central

    Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

    2016-01-01

    Background: Rice (Oryza sativa) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. Materials and Methods: In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Results: We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. Conclusion: This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. SUMMARY Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin

  5. Variance Reduction Factor of Nuclear Data for Integral Neutronics Parameters

    SciTech Connect

    Chiba, G. Tsuji, M.; Narabayashi, T.

    2015-01-15

    We propose a new quantity, a variance reduction factor, to identify nuclear data for which further improvements are required to reduce uncertainties of target integral neutronics parameters. Important energy ranges can be also identified with this variance reduction factor. Variance reduction factors are calculated for several integral neutronics parameters. The usefulness of the variance reduction factors is demonstrated.

  6. Heat shock protein 70 protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus via inhibition of nuclear factor-κB activation-induced nitric oxide synthase II expression.

    PubMed

    Chang, Chiung-Chih; Chen, Shang-Der; Lin, Tsu-Kung; Chang, Wen-Neng; Liou, Chia-Wei; Chang, Alice Y W; Chan, Samuel H H; Chuang, Yao-Chung

    2014-02-01

    Status epilepticus induces subcellular changes that may eventually lead to neuronal cell death in the hippocampus. Based on an animal model of status epilepticus, our laboratory showed previously that sustained hippocampal seizure activity activates nuclear factor-κB (NF-κB) and upregulates nitric oxide synthase (NOS) II gene expression, leading to apoptotic neuronal cell death in the hippocampus. The present study examined the potential modulatory role of heat shock protein 70 (HSP70) on NF-κB signaling in the hippocampus following experimental status epilepticus. In Sprague-Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Expression of HSP70 was elevated as early as 1h after the elicitation of sustained seizure activity, followed by a progressive elevation that peaked at 24h. Pretreatment with an antisense oligonucleotide against hsp70 decreased the HSP70 expression, and significantly augmented IκB kinase (IKK) activity and phosphorylation of IκBα, alongside enhanced nuclear translocation and DNA binding activity of NF-κB in the hippocampal CA3 neurons and glial cells. These cellular events were followed by enhanced upregulation of NOS II and peroxynitrite expression 3h after sustained seizure activity that led to an increase of caspase-3 and DNA fragmentation in the hippocampal CA3 neurons 7days after experimental status epilepticus. We concluded that HSP70 protects against apoptotic cell death induced by NF-κB activation and NOS II-peroxynitrite signaling cascade in the hippocampal CA3 and glial cells following experimental status epilepticus via suppression of IKK activity and deactivation of IκBα.

  7. A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.

    PubMed

    Hayashi, Y; Yamagishi, M; Nishimoto, Y; Taguchi, O; Matsukage, A; Yamaguchi, M

    1999-12-03

    The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene.

  8. A 116-kDa phytoglycoprotein inhibits aberrant crypt foci formation through modulation of manganese superoxide dismutase, inducible nitric oxide synthase, cyclooxygenase-2, nuclear factor-kappa B, activator protein-1, and proliferating cell nuclear antigen in 1,2-dimethylhydrazine/dextran sodium sulfate-treated ICR mice.

    PubMed

    Lee, Sei-Jung; Lim, Kye-Taek

    2008-11-01

    The 116-kDa Ulmus davidiana Nakai (UDN) glycoprotein is a naturally occurring phytoglycoprotein found in the stem of UDN. In this study, we investigated the chemopreventive effect of UDN glycoprotein on inflammation-mediated colorectal carcinogenesis induced by 10 mg/kg 1,2-dimethylhydrazine and 2% dextran sodium sulfate in ICR mice. Consumption of UDN glycoprotein (0.01 and 0.02%) significantly reduced the frequency of colonic aberrant crypt foci, the expression of colonic proliferating cell nuclear antigen, and the release of plasma lactate dehydrogenase without any cytotoxic activity at the initiation stage of colorectal carcinogenesis in 1,2-dimethylhydrazine/dextran sodium sulfate-treated mice. In addition, UDN glycoprotein has antioxidative effects on the formation of plasma thiobarbituric acid reactive substances and on the production of plasma inducible nitric oxide, accompanying the normalizing effects on the activity of colonic antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in the mice. UDN glycoprotein intake also remarkably attenuated the expression of inflammation-related factors (inducible nitric oxide synthase and cyclooxygenase-2) and the DNA-binding activity of redox-sensitive transcription factors (nuclear factor-kappa B and activator protein-1) in the mice. Collectively, the results suggest that UDN glycoprotein has chemopreventive potential at the initiation stage of colorectal cancer by reducing the factors responsible for oxidative stress, inflammation, and carcinogenesis.

  9. Inhibitor of nuclear factor-Kappa B activation attenuates venular constriction, leukocyte rolling-adhesion and microvessel rupture induced by ethanol in intact rat brain microcirculation: relation to ethanol-induced brain injury.

    PubMed

    Altura, Burton M; Gebrewold, Asefa

    2002-12-06

    The present study was designed to test the hypothesis that acute, local administration of a specific inhibitor of nuclear factor-Kappa B activation (which prevents rapid proteolysis of IKB-alpha) will attenuate cerebral (cortical) venular constrictions, leukocyte-endothelial wall interactions and postcapillary damage induced by medium to high concentrations of ethanol in the intact rat brain. Perivascular or i.p. administration of ethanol (100, 250 mg/dl) to the intact rat brain resulted in concentration-dependent venular vasospasm, rolling and adherence of leukocytes to venular walls and rupture of postcapillary venules with focal hemorrhages. Superfusion of the in-situ brain with N(alpha)-L-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a specific inhibitor of IKB-alpha proteolysis, attenuated greatly the spasmogenic, leukocyte rolling-endothelial cell adhesion and postcapillary hemorrhages induced by ethanol. These new data suggest that inhibition of alcohol-inducible degradation of IKB-alpha by TPKC can prevent much of the adverse microvascular actions of ethanol in the intact rat brain. Moreover, these new in-situ results suggest that activation of nuclear factor-Kappa B seems to play a major modulatory role in the adverse cerebral vascular actions of concentrations of alcohol found in the blood of alcohol-intoxicated subjects and human stroke victims.

  10. Palmitate-induced interleukin 6 production is mediated by protein kinase C and nuclear-factor kappaB activation and leads to glucose transporter 4 down-regulation in skeletal muscle cells.

    PubMed

    Jové, Mireia; Planavila, Anna; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2005-07-01

    The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. Here, we report that exposure of C2C12 skeletal muscle cells to 0.5 mm palmitate results in increased mRNA levels (3.5-fold induction; P < 0.05) and secretion (control 375 +/- 57 vs. palmitate 1129 +/- 177 pg/ml; P < 0.001) of the proinflammatory cytokine IL-6. Palmitate increased nuclear factor-kappaB activation and coincubation of the cells with palmitate and the nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate prevented both IL-6 expression and secretion. Furthermore, incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C, and phorbol myristate acetate, that down-regulates protein kinase C in long-term incubations, abolished induction of IL-6 production. Finally, exposure of skeletal muscle cells to palmitate caused a fall in the mRNA levels of glucose transporter 4 and insulin-stimulated glucose uptake, whereas in the presence of anti-IL-6 antibody, which neutralizes the biological activity of mouse IL-6 in cell culture, these reductions were prevented. These findings suggest that IL-6 may mediate several of the prodiabetic effects of palmitate.

  11. Nuclear Decay Factors Crack Up mRNA.

    PubMed

    Tudek, Agnieszka; Schmid, Manfred; Jensen, Torben Heick

    2017-03-02

    In this issue of Molecular Cell, Bresson et al. (2017) show that the nuclear RNA decay factors Nab3 and Mtr4 reshape the coding transcriptome during glucose starvation in budding yeast, placing nuclear mRNA metabolism as an important contributor of gene expression regulation.

  12. Cloning and functional analysis of spliced isoforms of human nuclear factor I-X: interference with transcriptional activation by NFI/CTF in a cell-type specific manner.

    PubMed Central

    Apt, D; Liu, Y; Bernard, H U

    1994-01-01

    Previous studies of the epithelial specificity of the human papillomavirus type 16 (HPV-16) enhancer pointed out an important role of nuclear factor I (NFI). In epithelial cells, NFI proteins are derived from the NFI-C gene and referred to as NFI/CTF. In contrast, fibroblasts, where the enhancer is inactive, express high levels of NFI from the NFI-X gene. To compare NFI-C and NFI-X derived transcription factors, we cloned and functionally investigated two differentially spliced forms of NFI-X from human fibroblasts. NFI-X1 has 95% homology with a transcript previously identified in hamster liver cells. NFI-X2, a spliced variant, misses 41 amino acids of the proline-rich activation domain. NFI-X expression, examined by Northern blots, shows strong cell-type specific variation in comparison with NFI/CTF. While the transcriptional activation domain of NFI-X2, functionally tested as GAL4-fusion protein in epithelial and fibroblast cells, activates transcription from promoter as well as enhancer position similar to NFI/CTF-1, the activation domain of NFI-X1 fails to activate transcription from enhancer position. In Drosophila cells, void of endogenous NFI proteins, full length NFI/CTF-1 and NFI-X2 activate a reporter construct containing only NFI sites as well as the NFI dependent HPV-16 enhancer. In contrast, NFI-X1 fails to activate the HPV-16 enhancer. Furthermore, overexpression of NFI-X1 in epithelial cells down-regulates the HPV-16 enhancer. Our findings suggest that the family of NFI transcription factors should not be viewed as constitutive activators, but rather, that NFI-C and NFI-X have divergent functions after binding in promoter or enhancer position. This property, combined with the differential expression of NFI-X, can achieve cell-type specificity of NFI dependent promoters and enhancers. Images PMID:7937100

  13. Experimental Nuclear Physics Activity in Italy

    NASA Astrophysics Data System (ADS)

    Chiavassa, E.; de Marco, N.

    2003-04-01

    The experimental Nuclear Physics activity of the Italian researchers is briefly reviewed. The experiments, that are financially supported by the INFN, are done in strict collaboration by more than 500 INFN and University researchers. The experiments cover all the most important field of the modern Nuclear Physics with probes extremely different in energy and interactions. Researches are done in all the four National Laboratories of the INFN even if there is a deeper involvement of the two national laboratories expressly dedicated to Nuclear Physics: the LNL (Laboratorio Nazionale di Legnaro) and LNS (Laboratorio Nazionale del Sud) where nuclear spectroscopy and reaction dynamics are investigated. All the activities with electromagnetic probes develops in abroad laboratories as TJNAF, DESY, MAMI, ESFR and are dedicated to the studies of the spin physics and of the nucleon resonance; hypernuclear and kaon physics is investigated at LNF. A strong community of researchers work in the relativistic and ultra-relativistic heavy ions field in particular at CERN with the SPS Pb beam and in the construction of the ALICE detector for heavy-ion physics at the LHC collider. Experiments of astrophysical interest are done with ions of very low energy; in particular the LUNA accelerator facility at LNGS (Laboratorio Nazionale del Gran Sasso) succeeded measuring cross section at solar energies, below or near the solar Gamow peak. Interdisciplinary researches on anti-hydrogen atom spectroscopy and on measurements of neutron cross sections of interest for ADS development are also supported.

  14. Morris Water Maze Training in Mice Elevates Hippocampal Levels of Transcription Factors Nuclear Factor (Erythroid-derived 2)-like 2 and Nuclear Factor Kappa B p65

    PubMed Central

    Snow, Wanda M.; Pahlavan, Payam S.; Djordjevic, Jelena; McAllister, Danielle; Platt, Eric E.; Alashmali, Shoug; Bernstein, Michael J.; Suh, Miyoung; Albensi, Benedict C.

    2015-01-01

    Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory. PMID:26635523

  15. Human factors aspects of advanced instrumentation in the nuclear industry

    SciTech Connect

    Carter, R.J.

    1989-01-01

    An important consideration in regards to the use of advanced instrumentation in the nuclear industry is the interface between the instrumentation system and the human. A survey, oriented towards identifying the human factors aspects of digital instrumentation, was conducted at a number of United States (US) and Canadian nuclear vendors and utilities. Human factors issues, subsumed under the categories of computer-generated displays, controls, organizational support, training, and related topics were identified. 20 refs., 2 tabs.

  16. Examining Factors Affecting Attitudes toward Nuclear Power in Taiwan

    NASA Astrophysics Data System (ADS)

    Chan, Tzu-Jen

    Nuclear power has become a major issue in Taiwan for several decades. The objective of the present study is to obtain evidence about the major determinants contributing to attitudes toward nuclear power, by investigating socioeconomic factors, environmental attitudes, knowledge of issues, trust, and risk perception, in shaping nuclear attitudes. A face-to-face survey was conducted using paper-based questionnaires from July 2014 to September 2014. Finally, 364 surveys were collected, of which 356 met validation requirements. The findings showed (1) knowledge of issues, trust in university scientists, trust in environmental groups, and risk perception directly influence attitudes toward nuclear power. (2) Risk perception is directly influenced by trust in nuclear authorities, trust in environmental groups, environmental attitudes, and party preference. (3) Gender, age, and party preference directly influence knowledge, trust in nuclear authorities, or trust in university scientists. The potential explanations and implications of findings are discussed.

  17. The Exposure Rate Conversion Factor for Nuclear Fallout

    SciTech Connect

    Spriggs, G D

    2009-02-11

    Nuclear fallout is comprised of approximately 2000 radionuclides. About 1000 of these radionuclides are either primary fission products or activated fission products that are created during the burn process. The exposure rate one meter above the surface produced by this complex mixture of radionuclides varies rapidly with time since many of the radionuclides are short-lived and decay numerous times before reaching a stable isotope. As a result, the mixture of radionuclides changes rapidly with time. Using a new code developed at the Lawrence Livermore National Laboratory, the mixture of radionuclides at any given point in time can be calculated. The code also calculates the exposure rate conversion factor (ECF) for all 3864 individual isotopes contained in its database based on the total gamma energy released per decay. Based on the combination of isotope mixture and individual ECFs, the time-dependent variation of the composite exposure rate conversion factor for nuclear fallout can be easily calculated. As example of this new capability, a simple test case corresponding to a 10 kt, uranium-plutonium fuel has been calculated. The results for the time-dependent, composite ECF for this test case are shown in Figure 1. For comparison, we also calculated the composite exposure rate conversion factor using the conversion factors found in Federal Guidance Report No.12 (FGR-12) published by ORNL, which contains the conversion factors for approximately 1000 isotopes. As can be noted from Figure 1, the two functions agree reasonably well at times greater than about 30 minutes. However, they do not agree at early times since FGR-12 does not include all of the short-lived isotopes that are produced in nuclear fallout. It should also be noted that the composite ECF at one hour is 19.7 R/hr per Ci/m{sup 2}. This corresponds to 3148 R/hr per 1 kt per square mile, which agrees reasonably well with the value of 3000 R/hr per 1 kt per square mile as quoted by Glasstone. We have

  18. Toll-Like Receptor 4–Mediated Nuclear Factor Kappa B Activation Is Essential for Sensing Exogenous Oxidants to Propagate and Maintain Oxidative/Nitrosative Cellular Stress

    PubMed Central

    Karki, Rajendra; Igwe, Orisa J.

    2013-01-01

    The mechanism(s) by which cells can sense exogenous oxidants that may contribute to intracellular oxidative/nitrosative stress is not clear. The objective of this study was to determine how cells might respond to exogenous oxidants to potentially initiate, propagate and/or maintain inflammation associated with many human diseases through NF-κB activation. First, we used HEK-Blue cells that are stably transfected with mouse toll-like receptor 4 (mTLR4) or mouse TLR2. These cells also express optimized secreted embryonic alkaline phosphatase (SEAP) reporter gene under the control of a promoter inducible by NF-κB transcription factor. These cells were challenged with their respective receptor-specific ligands, different pro-oxidants and/or inhibitors that act at different levels of the receptor signaling pathways. A neutralizing antibody directed against TLR4 inhibited responses to both TLR4-specific agonist and a prooxidant, which confirmed that both agents act through TLR4. We used the level of SEAP released into the culture media due to NF-κB activation as a measure of TLR4 or TLR2 stimulation. Pro-oxidants evoked increased release of SEAP from HEK-Blue mTLR4 cells at a much lower concentration compared with release from the HEK-Blue mTLR2 cells. Specific TLR4 signaling pathway inhibitors and oxidant scavengers (anti-oxidants) significantly attenuated oxidant-induced SEAP release by TLR4 stimulation. Furthermore, a novel pro-oxidant that decays to produce the same reactants as activated phagocytes induced inflammatory pain responses in the mouse orofacial region with increased TLR4 expression, and IL-1β and TNFα tissue levels. EUK-134, a synthetic serum-stable scavenger of oxidative species decreased these effects. Our data provide in vitro and related in vivo evidence that exogenous oxidants can induce and maintain inflammation by acting mainly through a TLR4-dependent pathway, with implications in many chronic human ailments. PMID:24058497

  19. Activation of human factor IX (Christmas factor).

    PubMed Central

    Di Scipio, R G; Kurachi, K; Davie, E W

    1978-01-01

    Human Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXabeta, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt approximately equal to 11,000) from the internal region of the precursor molecule and the generation of Factor IXabeta (mol wt approximately equal to 46,000). Factor IXabeta is composed of a light chain (mol wt approximately equal to 18,000) and a heavy chain (mol wt approximately equal to 28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXabeta also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXaalpha by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IXabeta was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its

  20. Cell penetrating peptide inhibitors of Nuclear Factor-kappa B

    PubMed Central

    Orange, J. S.; May, M. J.

    2010-01-01

    The nuclear factor kappa B (NF-κB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-κB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-κB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-κB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-κB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-κB signaling. Remarkably, several of these NF-κB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting. PMID:18668204

  1. Galaxy interactions and strength of nuclear activity

    NASA Technical Reports Server (NTRS)

    Simkin, S. M.

    1990-01-01

    Analysis of data in the literature for differential velocities and projected separations of nearby Seyfert galaxies with possible companions shows a clear difference in projected separations between type 1's and type 2's. This kinematic difference between the two activity classes reinforces other independent evidence that their different nuclear characteristics are related to a non-nuclear physical distinction between the two classes. The differential velocities and projected separations of the galaxy pairs in this sample yield mean galaxy masses, sizes, and mass to light ratios which are consistent with those found by the statistical methods of Karachentsev. Although the galaxy sample discussed here is too small and too poorly defined to provide robust support for these conclusions, the results strongly suggest that nuclear activity in Seyfert galaxies is associated with gravitational perturbations from companion galaxies, and that there are physical distinctions between the host companions of Seyfert 1 and Seyfert 2 nuclei which may depend both on the environment and the structure of the host galaxy itself.

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

    SciTech Connect

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

    2014-07-18

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

  3. Reconstruction of adenovirus replication origins with a human nuclear factor I binding site.

    PubMed

    Adhya, S; Shneidman, P S; Hurwitz, J

    1986-03-05

    Nuclear factor I is a host-coded DNA-binding protein that stimulates initiation of adenovirus DNA replication. To understand the mechanism of action of nuclear factor I, we have constructed, by recombinant DNA techniques, origins of replication in which the adenovirus type 5 nuclear factor I binding site (FIB site) has been replaced by a FIB site isolated from human genomic DNA (Gronostajski, R. M., Nagata, K., and Hurwitz, J. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 4013-4017). Assays of such recombinants for initiation and elongation in vitro showed that nuclear factor I was active only when the FIB site was relatively close to the DNA terminus, i.e. the FIB site was centered at nucleotides 30-36 from the end of the DNA. Nuclear factor I was active in either orientation within this distance range. The presence of one or two additional FIB sites in the downstream region had no effect. The implications of these results for the mechanism of nuclear factor I action are discussed.

  4. Determining Yankee Nuclear Power Station neutron activation

    SciTech Connect

    Heider, K.J.; Morrissey, K.J. )

    1993-01-01

    The Yankee nuclear power station located in Rowe, Massachusetts, permanently ceased power operations on February 26, 1992, after 31 yr of operation. Yankee has since initiated decommissioning planning activities. A significant component of these activities is a determination of the extent of radiological contamination of the Yankee site. Included in this effort was determination of the extent of neutron activation of plant components. This paper describes the determination of the neutron activation of the Yankee reactor vessel, associated internals, and surrounding structures. The Yankee reactor vessel is a 600-MW(thermal) stainless steel-lined, carbon steel vessel with stainless steel internal components designed by Westinghouse. The reactor vessel is surrounded and supported by a carbon steel neutron shield tank that was filled with chromated water during plant operation. A 5-ft-thick concrete biological shield wall surrounds the neutron shield tank. A project is under way to remove the reactor vessel internals from the reactor vessel.

  5. Expression of constitutive androstane receptor, hepatic nuclear factor 4 alpha, and P450 oxidoreductase genes determines interindividual variability in basal expression and activity of a broad scope of xenobiotic metabolism genes in the human liver.

    PubMed

    Wortham, Matthew; Czerwinski, Maciej; He, Lin; Parkinson, Andrew; Wan, Yu-Jui Yvonne

    2007-09-01

    Identification of genetic variation predictive of clearance rate of a wide variety of prescription drugs could lead to cost-effective personalized medicine. Here we identify regulatory genes whose variable expression level among individuals may have widespread effects upon clearance rate of a variety of drugs. Twenty liver samples with variable CYP3A activity were profiled for expression level and activity of xenobiotic metabolism genes as well as genes involved in the regulation thereof. Regulatory genes whose expression level accounted for the highest degree of collinearity among expression levels of xenobiotic metabolism genes were identified as possible master regulators of drug clearance rate. Significant linear correlations (p < 0.05) were identified among mRNA levels of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, MRP2, OATP2, P450 oxidoreductase (POR), and UDP-glucuronosyltranferase 1A1, suggesting that these xenobiotic metabolism genes are coregulated at the transcriptional level. Using partial regression analysis, constitutive androstane receptor (CAR) and hepatic nuclear factor 4 alpha (HNF4 alpha) were identified as the nuclear receptors whose expression levels are most strongly associated with expression of coregulated xenobiotic metabolism genes. POR expression level, which is also associated with CAR and HNF4 alpha expression level, was found to be strongly associated with the activity of many cytochromes P450. Thus, interindividual variation in the expression level of CAR, HNF4 alpha, and POR probably determines variation in expression and activity of a broad scope of xenobiotic metabolism genes and, accordingly, clearance rate of a variety of xenobiotics. Identification of polymorphisms in these candidate master regulator genes that account for their variable expression among individuals may yield readily detectable biomarkers that could serve as predictors of xenobiotic clearance rate.

  6. Activation of the canonical nuclear factor-κB pathway is involved in isoflurane-induced hippocampal interleukin-1β elevation and the resultant cognitive deficits in aged rats

    SciTech Connect

    Li, Zheng-Qian; Rong, Xiao-Ying; Liu, Ya-Jie; Ni, Cheng; Tian, Xiao-Sheng; Mo, Na; Chui, De-Hua; Guo, Xiang-Yang

    2013-09-06

    Highlights: •Isoflurane induces hippocampal IL-1β elevation and cognitive deficits in aged rats. •Isoflurane transiently activates the canonical NF-κB pathway in aged rat hippocampus. •NF-κB inhibitor mitigates isoflurane-induced IL-1β elevation and cognitive deficits. •We report a linkage between NF-κB signaling, IL-1β expression, and cognitive changes. -- Abstract: Although much recent evidence has demonstrated that neuroinflammation contributes to volatile anesthetic-induced cognitive deficits, there are few existing mechanistic explanations for this inflammatory process. This study was conducted to investigate the effects of the volatile anesthetic isoflurane on canonical nuclear factor (NF)-κB signaling, and to explore its association with hippocampal interleukin (IL)-1β levels and anesthetic-related cognitive changes in aged rats. After a 4-h exposure to 1.5% isoflurane in 20-month-old rats, increases in IκB kinase and IκB phosphorylation, as well as a reduction in the NF-κB inhibitory protein (IκBα), were observed in the hippocampi of isoflurane-exposed rats compared with control rats. These events were accompanied by an increase in NF-κB p65 nuclear translocation at 6 h after isoflurane exposure and hippocampal IL-1β elevation from 1 to 6 h after isoflurane exposure. Nevertheless, no significant neuroglia activation was observed. Pharmacological inhibition of NF-κB activation by pyrrolidine dithiocarbamate markedly suppressed the IL-1β increase and NF-κB signaling, and also mitigated the severity of cognitive deficits in the Morris water maze task. Overall, our results demonstrate that isoflurane-induced cognitive deficits may stem from upregulation of hippocampal IL-1β, partially via activation of the canonical NF-κB pathway, in aged rats.

  7. A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK).

    PubMed Central

    Yanagisawa, Ken; Tago, Kenji; Hayakawa, Morisada; Ohki, Motomichi; Iwahana, Hiroyuki; Tominaga, Shin-Ichi

    2003-01-01

    Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions. PMID:12418963

  8. LANSCE nuclear science facilities and activities

    SciTech Connect

    Nelson, Ronald O

    2010-01-01

    Nuclear science activities at the Los Alamos Neutron Science Center (LANSCE) encompass measurements spanning the neutron energy range from thermal to 600 MeV. The neutron sources use spallation of the LANSCE 800 MeV pulsed proton beam with the time-of-flight technique to measure properties of neutron-induced reactions as a function of energy over this large energy range. Current experiments are conducted at the Lujan Center moderated neutron source, the unmoderated WNR target, and with a lead-slowing-down spectrometer. Instruments in use include the DANCE array of BaF{sub 2} scintillators for neutron capture studies, the FIGARO array of liquid scintillator neutron detectors, the GEANIE array of high-resolution HPGe x-ray and gamma-ray detectors, and a number of fission chambers, and other detectors. The LANL capabilities for production and handling of radioactive materials coupled with the neutron sources and detectors at LANSCE are enabling new and challenging measurements for a variety of applications including nuclear energy and nuclear astrophysics. An overview of recent research and examples of results is presented.

  9. Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

    PubMed

    Davison, James M; Lickwar, Colin R; Song, Lingyun; Breton, Ghislain; Crawford, Gregory E; Rawls, John F

    2017-04-06

    Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish hnf4a activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.

  10. Artemisinin triggers a G1 cell cycle arrest of human Ishikawa endometrial cancer cells and inhibits cyclin-dependent kinase-4 promoter activity and expression by disrupting nuclear factor-κB transcriptional signaling.

    PubMed

    Tran, Kalvin Q; Tin, Antony S; Firestone, Gary L

    2014-03-01

    Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

  11. Low extracellular magnesium ions induce lipid peroxidation and activation of nuclear factor-kappa B in canine cerebral vascular smooth muscle: possible relation to traumatic brain injury and strokes.

    PubMed

    Altura, Burton M; Gebrewold, Asefa; Zhang, Aimin; Altura, Bella T

    2003-05-08

    The present study was designed to test the hypothesis that administration of low extracellular levels of magnesium ions ([Mg(2+)](o)) to primary cultured cerebral vascular smooth muscle cells will cause lipid peroxidation, degradation of IkappaB-alpha, and activation of nuclear transcription factor kappa B (NF-kappaB) in cultured cerebral vascular smooth muscle cells. Low [Mg(2+)](o) (0, 0.15, 0.3 and 0.48 mM) resulted in concentration-dependent rises in malondialdehyde (MDA) in as little as 3 h after exposure to low [Mg(2+)](o), rising to levels 3-12xnormal after 18-24 h; the lower the [Mg(2+)](o), the higher the MDA level. Using electrophoretic mobility shift assays and specific antibodies, low [Mg(2+)](o) caused two DNA-binding proteins (p50, p65) to rise in nuclear extracts in a concentration-dependent manner. High [Mg(2+)](o) (i.e. 4.8 mM) downregulated p50 and p65. Using a rabbit antibody, IkappaB phosphorylation (and degradation) was stimulated by low [Mg(2+)](o) (in a concentration-dependent manner) and inhibited by a low concentration of the NF-kappaB inhibitor, pyrrolidine dithiocarbamate. These new biochemical and molecular data indicate that low [Mg(2+)](o), in concentrations found in the blood of patients, after traumatic brain injury (TBI) and diverse types of strokes, can elicit rapid lipid peroxidation and activation of NF-kappaB in cerebral vascular smooth muscle cells. The present results, when viewed in light of other recently published data, suggest that low [Mg(2+)](o)-induced lipid peroxidation and activation of NF-kappaB play important roles in TBI and diverse types of strokes.

  12. L-arginine attenuates Interleukin-1β (IL-1β) induced Nuclear Factor Kappa-Beta (NF-κB) activation in Caco-2 cells

    PubMed Central

    Meng, Qinghe; Cooney, Mitchell; Yepuri, Natesh; Cooney, Robert N.

    2017-01-01

    Background Specific nutrients like L-arginine (L-Arg) ameliorate intestinal inflammation, however the exact mechanisms of this effect are unclear. We hypothesized the anti-inflammatory effects of L-Arg require active transport and metabolism by inducible nitric oxide synthase (iNOS) to generate nitric oxide (NO). To test this hypothesis we examined the effects of L-Arg, L-Arg transport activity, NO production and iNOS inhibitor on IL-1β-mediated NF-κB-activation in Caco-2 cells. Methods Caco-2 cells were cultured, transfected with a NF-κB promoter luciferase vector, incubated ± L-Arg, ± IL-1β and luciferase activity was measured. Using siRNA we inhibited the L-Arg cationic amino acid transporter system y+ (CAT1) expression and examined its effects on L-Arg transport activity and IL-1β-mediated NF-κB-activation. Finally, the effects of sodium nitroprusside (SNP, a NO donor) and Nω-nitro-L-arginine (NNA, an iNOS inhibitor) on IL-1β-mediated NF-κB-activation were examined. Results IL-1β increased NF-κB luciferase activity (8-fold) and NF-κB expression (mRNA and protein), both of these were significantly decreased by L-Arg. System y+ CAT1 siRNA decreased CAT1 expression, L-Arg transport activity and attenuated the inhibitory effects of L-Arg on NF- κB activity. SNP attenuated the IL-1β-induced increase in NF-κB luciferase activity and expression, whereas NNA diminished the inhibitory effects of L-Arg on IL-1β-inducible NF- κB luciferase activity. Conclusion The inhibitory effects of L-Arg on IL-1β-mediated NF-κB-activation in Caco-2 cells involve L-Arg transport activity by CAT1, regulation of IL-1β-mediated increases in NF-κB expression, changes in iNOS expression and NO production. Our data suggest the inhibitory effects of L-Arg on NF-κB activation are mediated in part by iNOS since SNP preserves and NNA attenuates the effects of L-Arg on IL-1β-mediated NF-κB-activation and expression. PMID:28334039

  13. Nuclear factor-κ B inducing kinase is required for graft-versus-host disease

    PubMed Central

    Sánchez-Valdepeñas, Carmen; Casanova, Lucía; Colmenero, Isabel; Arriero, Mar; González, África; Lozano, Nieves; González-Vicent, Marta; Díaz, Miguel A.; Madero, Luís; Fresno, Manuel; Ramírez, Manuel

    2010-01-01

    Background Donor T lymphocytes are directly responsible for graft-versus-host disease. Molecules important in T-cell function may, therefore, be appropriate targets for graft-versus-host disease therapy and/or prophylaxis. Here we analyzed whether nuclear factor-κ B inducing kinase might have a role in graft-versus-host disease. Design and Methods We studied the expression of nuclear factor-κ B inducing kinase in human samples from patients with graft-versus-host disease. We also explored the effect of nuclear factor-κ B inducing kinase in a murine model of graft-versus-host disease using donor cells from aly/aly mice (deficient in nuclear factor-κ B inducing kinase) and C57BL/6 mice (control). Results We detected expression of nuclear factor-κ B inducing kinase in T-lymphocytes in the pathological lesions of patients with acute graft-versus-host disease. Mice transplanted with aly/aly T lymphocytes did not develop graft-versus-host disease at all, while mice receiving C57BL/6 cells died of a lethal form of the disease. Deficiency of nuclear factor-κ B inducing kinase did not affect the engrafting ability of donor T cells, but severely impaired their expansion capacity early after transplantation, and aly/aly T cells showed a higher proportion of apoptosis than did C57BL/6 T cells. Effector T lymphocytes were the T-cell subset most affected by nuclear factor-κ B inducing kinase deficiency. We also detected lower amounts of inflammatory cytokines in the serum of mice receiving aly/aly T cells than in the serum of mice receiving C57BL/6 T cells. Conclusions Our results show that nuclear factor-κ B inducing kinase has a role in graft-versus-host disease by maintaining the viability of activated alloreactive T lymphocytes. PMID:20823135

  14. The ligand-bound thyroid hormone receptor in macrophages ameliorates kidney injury via inhibition of nuclear factor-κB activities

    PubMed Central

    Furuya, Fumihiko; Ishii, Toshihisa; Tamura, Shogo; Takahashi, Kazuya; Kobayashi, Hidetoshi; Ichijo, Masashi; Takizawa, Soichi; Kaneshige, Masahiro; Suzuki-Inoue, Katsue; Kitamura, Kenichiro

    2017-01-01

    In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor α (TRα) in macrophages and examined the role of ligand-bound TRα in macrophage polarization-mediated anti-inflammatory effects. TRα-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRα displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRα-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1β levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRα on macrophages plays a protective role in kidney inflammation through the inhibition of NF-κB pathways, possibly by affecting the pro- and anti-inflammatory balance that controls the development of CKD. PMID:28272516

  15. The ligand-bound thyroid hormone receptor in macrophages ameliorates kidney injury via inhibition of nuclear factor-κB activities.

    PubMed

    Furuya, Fumihiko; Ishii, Toshihisa; Tamura, Shogo; Takahashi, Kazuya; Kobayashi, Hidetoshi; Ichijo, Masashi; Takizawa, Soichi; Kaneshige, Masahiro; Suzuki-Inoue, Katsue; Kitamura, Kenichiro

    2017-03-08

    In chronic kidney disease (CKD) patients, inflammation plays a pivotal role in the progression of renal fibrosis. Hypothyroidism is associated with an increased occurrence of atherosclerosis and inflammation, suggesting protective roles of thyroid hormones and their receptors against inflammatory processes. The contribution of thyroid hormone receptors to macrophage differentiation has not been well documented. Here, we focused on the endogenous thyroid hormone receptor α (TRα) in macrophages and examined the role of ligand-bound TRα in macrophage polarization-mediated anti-inflammatory effects. TRα-deficient irradiated chimeric mice showed exacerbated tubulointerstitial injury in a unilateral ureteral obstruction model. Compared with wild-type macrophages, macrophages isolated from the obstructed kidneys of mice lacking TRα displayed increased expression of proinflammatory cytokines that was accompanied by enhanced nuclear translocation of p65. Comparison of TRα-deficient bone marrow-derived macrophages with wild-type macrophages confirmed the propensity of the former cells to produce excessive IL-1β levels. Co-culture of these macrophages with renal epithelial cells induced more severe damage to the epithelial cells via the IL-1 receptor. Our findings indicate that ligand-bound TRα on macrophages plays a protective role in kidney inflammation through the inhibition of NF-κB pathways, possibly by affecting the pro- and anti-inflammatory balance that controls the development of CKD.

  16. Autophagosomal IkappaB alpha degradation plays a role in the long term control of tumor necrosis factor-alpha-induced nuclear factor-kappaB (NF-kappaB) activity.

    PubMed

    Colleran, Amy; Ryan, Aideen; O'Gorman, Angela; Mureau, Coralie; Liptrot, Catherine; Dockery, Peter; Fearnhead, Howard; Egan, Laurence J

    2011-07-01

    Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect.

  17. Autophagosomal IκBα Degradation Plays a Role in the Long Term Control of Tumor Necrosis Factor-α-induced Nuclear Factor-κB (NF-κB) Activity*

    PubMed Central

    Colleran, Amy; Ryan, Aideen; O'Gorman, Angela; Mureau, Coralie; Liptrot, Catherine; Dockery, Peter; Fearnhead, Howard; Egan, Laurence J.

    2011-01-01

    Transcription factor NF-κB is persistently activated in many chronic inflammatory diseases and cancers. The short term regulation of NF-κB is well understood, but little is known about the mechanisms of its long term activation. We studied the effect of a single application of TNF-α on NF-κB activity for up to 48 h in intestinal epithelial cells. Results show that NF-κB remained persistently activated up to 48 h after TNF-α and that the long term activation of NF-κB was accompanied by a biphasic degradation of IκBα. The first phase of IκBα degradation was proteasome-dependent, but the second was not. Further investigation showed that TNF-α stimulated formation of autophagosomes in intestinal epithelial cells and that IκBα co-localized with autophagosomal vesicles. Pharmacological or genetic blockade of autophagosome formation or the inhibition of lysosomal proteases decreased TNF-α-induced degradation of IκBα and lowered NF-κB target gene expression. Together, these findings indicate a role of autophagy in the control of long term NF-κB activity. Because abnormalities in autophagy have been linked to ineffective innate immunity, we propose that alterations in NF-κB may mediate this effect. PMID:21454695

  18. Summary of nuclear fuel reprocessing activities around the world

    SciTech Connect

    Mellinger, P.J.; Harmon, K.M.; Lakey, L.T.

    1984-11-01

    This review of international practices for nuclear fuel reprocessing was prepared to provide a nontechnical summary of the current status of nuclear fuel reprocessing activities around the world. The sources of information are widely varied.

  19. Economic Conditions and Factors Affecting New Nuclear Power Deployment

    SciTech Connect

    Harrison, Thomas J.

    2014-10-01

    This report documents work performed in support of the US Department of Energy Office of Nuclear Energy’s Advanced Small Modular Reactor (AdvSMR) program. The report presents information and results from economic analyses to describe current electricity market conditions and those key factors that may impact the deployment of AdvSMRs or any other new nuclear power plants. Thus, this report serves as a reference document for DOE as it moves forward with its plans to develop advanced reactors, including AdvSMRs. For the purpose of this analysis, information on electricity markets and nuclear power plant operating costs will be combined to examine the current state of the nuclear industry and the process required to successfully move forward with new nuclear power in general and AdvSMRs in particular. The current electricity market is generally unfavorable to new nuclear construction, especially in deregulated markets with heavy competition from natural gas and subsidized renewables. The successful and profitable operation of a nuclear power plant (or any power plant) requires the rate at which the electricity is sold to be sufficiently greater than the cost to operate. The wholesale rates in most US markets have settled into values that provide profits for most operating nuclear power plants but are too low to support the added cost of capital recovery for new nuclear construction. There is a strong geographic dependence on the wholesale rate, with some markets currently able to support new nuclear construction. However, there is also a strong geographic dependence on pronuclear public opinion; the areas where power prices are high tend to have unfavorable views on the construction of new nuclear power plants. The use of government-backed incentives, such as subsidies, can help provide a margin to help justify construction projects that otherwise may not seem viable. Similarly, low interest rates for the project will also add a positive margin to the economic

  20. Nuclear Factor-Kappa B Activity Regulates Brain Expression of P-Glycoprotein in the Kainic Acid-Induced Seizure Rats

    PubMed Central

    Yu, Nian; Di, Qing; Liu, Hao; Hu, Yong; Jiang, Ying; Yan, Yu-kui; Zhang, Yan-fang; Zhang, Ying-dong

    2011-01-01

    This study was aimed to investigate the effect of NF-κB activity on the seizure susceptibility, brain damage, and P-gp expression in kainic acid- (KA-) induced seizure rats. Male SD rats were divided into saline control group (NS group), KA induced epilepsy group (EP group), and epilepsy group intervened with NF-κB inhibitor-pyrrolidine dithiocarbamate salt (PDTC group) or with dexamethasone (DEX group). No seizures were observed in the rats of NS group. Compared with NS group, increased P-gp expression and NF-κB activation in the rat brain of the EP group were observed after KA micro-injection. Both PDTC and DEX pre-treatment significantly increased the latency to grade III or V seizure onset compared to EP group but failed to show neuron-protective effect as the number of survival neurons didn't significantly differ from that in EP group. Furthermore, PDTC pre-treatment significantly decreased P-gp expression along with NF-κB activation in the hippocampus CA3 area and amygdala complex of rats compared with the EP group, implying that NF-κB activation involved in the seizure susceptibility and seizure induced brain P-gp over-expression. Additionally, DEX pre-treatment only decreased P-gp expression level without inhibition of NF-κB activation, suggesting NF-κB independent pathway may also participate in regulating seizure induced P-gp over-expression. PMID:21403895

  1. The unit cost factors and calculation methods for decommissioning - Cost estimation of nuclear research facilities

    SciTech Connect

    Kwan-Seong Jeong; Dong-Gyu Lee; Chong-Hun Jung; Kune-Woo Lee

    2007-07-01

    Available in abstract form only. Full text of publication follows: The uncertainties of decommissioning costs increase high due to several conditions. Decommissioning cost estimation depends on the complexity of nuclear installations, its site-specific physical and radiological inventories. Therefore, the decommissioning costs of nuclear research facilities must be estimated in accordance with the detailed sub-tasks and resources by the tasks of decommissioning activities. By selecting the classified activities and resources, costs are calculated by the items and then the total costs of all decommissioning activities are reshuffled to match with its usage and objectives. And the decommissioning cost of nuclear research facilities is calculated by applying a unit cost factor method on which classification of decommissioning works fitted with the features and specifications of decommissioning objects and establishment of composition factors are based. Decommissioning costs of nuclear research facilities are composed of labor cost, equipment and materials cost. Of these three categorical costs, the calculation of labor costs are very important because decommissioning activities mainly depend on labor force. Labor costs in decommissioning activities are calculated on the basis of working time consumed in decommissioning objects and works. The working times are figured out of unit cost factors and work difficulty factors. Finally, labor costs are figured out by using these factors as parameters of calculation. The accuracy of decommissioning cost estimation results is much higher compared to the real decommissioning works. (authors)

  2. The In Vitro Effect of Acidic-Pepsin on Nuclear Factor KappaB Activation and Its Related Oncogenic Effect on Normal Human Hypopharyngeal Cells

    PubMed Central

    Sasaki, Clarence T.; Toman, Julia; Vageli, Dimitra

    2016-01-01

    Background Extra-esophageal carcinogenesis has been widely discussed in relation to the chronic effects of laryngopharyngeal reflux and most prominently with pepsin historically central to this discussion. With refluxate known to include gastric (pepsin) and duodenal (bile) fluids, we recently demonstrated the mechanistic role of NF-κB in mediating the preneoplastic effects of acidic-bile. However, the role of pepsin in promoting hypopharyngeal premalignant events remains historically unclear. Here, we investigate the in vitro effect of acidic-pepsin on the NF-κB oncogenic pathway to better define its potential role in hypopharyngeal neoplasia. Methods Human hypopharyngeal primary cells (HHPC) and keratinocytes (HHK) were repetitively exposed to physiologic pepsin concentrations (0.1 mg/ml) at pH 4.0, 5.0 and 7.0. Cellular localization of phospho-NF-κB and bcl-2 was determined using immunofluorescence and western blotting. NF-κB transcriptional activity was tested by luc reporter and qPCR. Analysis of DNA content of pepsin treated HHK and HHPC was performed using Fluorescence-activated-cell sorting assay. To explore a possible dose related effect, pepsin concentration was reduced from 0.1 to 0.05 and 0.01 mg/ml. Results At physiologic concentration, acidic-pepsin (0.1 mg/ml at pH 4.0) is lethal to most normal hypopharyngeal cells. However, in surviving cells, no NF-κB transcriptional activity is noted. Acidic-pepsin fails to activate the NF-κB or bcl-2, TNF-α, EGFR, STAT3, and wnt5α but increases the Tp53 mRNAs, in both HHPC and HHK. Weakly acidic-pepsin (pH 5.0) and neutral-pepsin (pH 7.0) induce mild activation of NF-κB with increase in TNF-α mRNAs, without oncogenic transcriptional activity. Lower concentrations of pepsin at varying pH do not produce NF-κB activity or transcriptional activation of the analyzed genes. Conclusion Our findings in vitro do not support the role of acidic-pepsin in NF-κB related hypopharyngeal carcinogenesis. PMID:27973541

  3. Inhibitory effects of the standardized extract (DA-9601) of Artemisia asiatica Nakai on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase-2 expression, inducible nitric oxide synthase expression and nuclear transcription factor kappa B activation in mouse skin.

    PubMed

    Seo, Hyo-Joung; Park, Kwang-Kyun; Han, Seong Su; Chung, Won-Yoon; Son, Mi-Won; Kim, Won-Bae; Surh, Young-Joon

    2002-08-01

    Artemisia asiatica Nakai has been used in traditional Asian medicine for the treatment of inflammatory and other disorders. Previous studies have revealed that the formulated ethanol extract (DA-9601) of A. asiatica has pronounced antioxidative and antiinflammatory activities and exhibits cytoprotective effects against experimentally induced gastrointestinal, hepatic and pancreatic damage. In the present study, we assessed the inhibitory effect of DA-9601 on tumor promotion, which is closely linked to inflammatory tissue damage. As an initial approach to evaluating the possible antitumor-promoting potential of DA-9601, its effects on TPA-induced ear edema were examined in female ICR mice. Pretreatment of the inner surface of the mouse ear with DA-9601 30 min prior to topical application of TPA inhibited ear edema at 5 hr. TPA-stimulated expression of epidermal COX-2 and iNOS was also mitigated by topical application of the same extract. Moreover, DA-9601 abrogated the TPA-mediated activation of NF-kappa B/Rel and AP-1 in mouse epidermis. Suppression of epidermal NF-kappa B by DA-9601 appeared to be mediated in part through inhibition of I kappa B alpha degradation, thereby blocking the nuclear translocation of p65, the functional subunit of NF-kappa B. DA-9601 also significantly suppressed TPA-induced ODC activity and papilloma formation in mouse skin. Taken together, these findings suggest that DA-9601 derived from A. asiatica possesses potential chemopreventive activities.

  4. Butrin, Isobutrin, and Butein from Medicinal Plant Butea monosperma Selectively Inhibit Nuclear Factor-κB in Activated Human Mast Cells: Suppression of Tumor Necrosis Factor-α, Interleukin (IL)-6, and IL-8

    PubMed Central

    Rasheed, Zafar; Akhtar, Nahid; Khan, Abubakar; Khan, Khursheed A.

    2010-01-01

    Activation of mast cells in rheumatoid synovial tissue has often been associated with tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-8 production and disease pathogenesis by adjacent cell types. Butea monosperma (BM) is a well known medicinal plant in India and the tropics. The aim of this study was to examine whether a standardized extract of BM flower (BME) could inhibit inflammatory reactions in human mast cells (HMC) using activated HMC-1 cells as a model. Four previously characterized polyphenols—butrin, isobutrin, isocoreopsin, and butein—were isolated from BME by preparative thin layer chromatography, and their purity and molecular weights were determined by liquid chromatography/mass spectrometry analysis. Our results showed that butrin, isobutrin, and butein significantly reduced the phorbol 12-myristate 13-acetate and calcium ionophore A23187-induced inflammatory gene expression and production of TNF-α, IL-6, and IL-8 in HMC-1 cells by inhibiting the activation of NF-κB. In addition, isobutrin was most potent in suppressing the NF-κB p65 activation by inhibiting IκBα degradation, whereas butrin and butein were relatively less effective. In vitro kinase activity assay revealed that isobutrin was a potent inhibitor of IκB kinase complex activity. This is the first report identifying the molecular basis of the reported anti-inflammatory effects of BME and its constituents butrin, isobutrin, and butein. The novel pharmacological actions of these polyphenolic compounds indicate potential therapeutic value for the treatment of inflammatory and other diseases in which activated mast cells play a role. PMID:20164300

  5. Fn14 is regulated via the RhoA pathway and mediates nuclear factor-kappaB activation by Angiotensin II

    PubMed Central

    Li, Zhengwei; Shen, Zhida; Du, Lailing; He, Jialin; Chen, Shengyu; Zhang, Jiefang; Luan, Yi; Fu, Guosheng

    2016-01-01

    Angiotesin II (Ang II) plays an important role in cardiac remodeling. Fibroblast growth factor inducible-14 (Fn14) is the smallest member of the tumor necrosis factor superfamily of receptors. Currently, little is known about the functional role of Fn14 in the heart. Chiefly, we observe the up-regulation of extracellular matrix in in vivo model. We therefore assess the expression and regulation of Fn14 in cardiomyocytes and in vivo models induced by Ang II. In order to study the regulation of Fn14, cardiac remodeling was established in rats and neonatal cardiomyocytes were used in in vitro model. As well, Ang II is able to strongly induce Fn14 expression in in vivo and in vitro models. Fn14 is mediated via RhoA pathways, since siRNA against RhoA prevented the expression of Fn14 in cardiomyocytes. Pretreatment of cardiomyoctes with siRNA against NF-κB and IκBα also decreased Fn14 expression induced by Ang II. We here describe for the first time Ang II regulation of Fn14 in in vivo and in vitro models via RhoA, NF-κB and NF-κB driven gene signaling pathway. In conclusion, Fn14 may be important in regulating the process of cardiac remodeling induced by Ang II. PMID:28078010

  6. Nuclear factor of activated T cells regulates the expression of interleukin-4 in Th2 cells in an all-or-none fashion.

    PubMed

    Köck, Juliana; Kreher, Stephan; Lehmann, Katrin; Riedel, René; Bardua, Markus; Lischke, Timo; Jargosch, Manja; Haftmann, Claudia; Bendfeldt, Hanna; Hatam, Farahnaz; Mashreghi, Mir-Farzin; Baumgrass, Ria; Radbruch, Andreas; Chang, Hyun-Dong

    2014-09-26

    Th2 memory lymphocytes have imprinted their Il4 genes epigenetically for expression in dependence of T cell receptor restimulation. However, in a given restimulation, not all Th cells with a memory for IL-4 expression express IL-4. Here, we show that in reactivated Th2 cells, the transcription factors NFATc2, NF-kB p65, c-Maf, p300, Brg1, STAT6, and GATA-3 assemble at the Il4 promoter in Th2 cells expressing IL-4 but not in Th2 cells not expressing it. NFATc2 is critical for assembly of this transcription factor complex. Because NFATc2 translocation into the nucleus occurs in an all-or-none fashion, dependent on complete dephosphorylation by calcineurin, NFATc2 controls the frequencies of cells reexpressing Il4, translates analog differences in T cell receptor stimulation into a digital decision for Il4 reexpression, and instructs all reexpressing cells to express the same amount of IL-4. This analog-to-digital conversion may be critical for the immune system to respond to low concentrations of antigens.

  7. H2 inhibits TNF-α-induced lectin-like oxidized LDL receptor-1 expression by inhibiting nuclear factor κB activation in endothelial cells.

    PubMed

    Song, Guohua; Tian, Hua; Liu, Jia; Zhang, Hongle; Sun, Xuejun; Qin, Shucun

    2011-09-01

    H(2) is a therapeutic antioxidant that can reduce oxidative stress. Oxidized low-density lipoprotein, which plays roles in atherosclerosis, may promote endothelial dysfunction by binding the cell-surface receptor LOX-1. LOX-1 expression can be upregulated by various stimuli, including TNF-α. Thus, we aimed to examine whether the upregulation of LOX-1 by different stimuli could be blocked by H(2) in endothelial cells. H(2) significantly abolished the upregulation of LOX-1 by different stimuli, including TNF-α, at the protein and mRNA levels. The TNF-α-induced upregulation of LOX-1 was also attenuated by the NF-κB inhibitor N-acetyl-L-cysteine. H(2) inhibited the TNF-α-induced activation of NF-κB and the phosphorylation of IκB-α. Furthermore, H(2) inhibited the expression of LOX-1 and the activation of NF-κB in apolipoprotein E knockout mice, an animal model of atherosclerosis. Thus, H(2) probably inhibits cytokine-induced LOX-1 gene expression by suppressing NF-κB activation.

  8. Inhibition of nuclear factor-κB activation in pancreatic β-cells has a protective effect on allogeneic pancreatic islet graft survival.

    PubMed

    Eldor, Roy; Abel, Roy; Sever, Dror; Sadoun, Gad; Peled, Amnon; Sionov, Ronit; Melloul, Danielle

    2013-01-01

    Pancreatic islet transplantation, a treatment for type 1 diabetes, has met significant challenges, as a substantial fraction of the islet mass fails to engraft, partly due to death by apoptosis in the peri- and post-transplantation periods. Previous evidence has suggested that NF-κB activation is involved in cytokine-mediated β-cell apoptosis and regulates the expression of pro-inflammatory and chemokine genes. We therefore sought to explore the effects of β-cell-specific inhibition of NF-κB activation as a means of cytoprotection in an allogeneic model of islet transplantation. To this end, we used islets isolated from the ToI-β transgenic mouse, where NF-κB signalling can specifically and conditionally be inhibited in β-cells by expressing an inducible and non-degradable form of IκBα regulated by the tet-on system. Our results show that β-cell-specific blockade of NF-κB led to a prolonged islet graft survival, with a relative higher preservation of the engrafted endocrine tissue and reduced inflammation. Importantly, a longer delay in allograft rejection was achieved when mice were systemically treated with the proteasome inhibitor, Bortezomib. Our findings emphasize the contribution of NF-κB activation in the allograft rejection process, and suggest an involvement of the CXCL10/IP-10 chemokine. Furthermore, we suggest a potential, readily available therapeutic agent that may temper this process.

  9. Protective effect of ellagic acid on concanavalin A-induced hepatitis via toll-like receptor and mitogen-activated protein kinase/nuclear factor κB signaling pathways.

    PubMed

    Lee, Jae Hong; Won, Jong Hoon; Choi, Jong Min; Cha, Hye Hyeon; Jang, Yeo Jin; Park, Seohyeon; Kim, Han Gyeol; Kim, Hyung Chul; Kim, Dae Kyong

    2014-10-15

    Ellagic acid (EA) is present in certain fruits and nuts, including raspberries, pomegranates, and walnuts, and has anti-inflammatory and antioxidant properties. The aims of this study were to examine the protective effect of EA on concanavalin A (Con A)-induced hepatitis and to elucidate its underlying molecular mechanisms in mice. Mice were orally administered EA at different doses before the intravenous delivery of Con A; the different experimental groups were as follows: (i) vehicle control, (ii) Con A alone without EA, (iii) EA at 50 mg/kg, (iv) EA at 100 mg/kg, and (v) EA at 200 mg/kg. We found that EA pretreatment significantly reduced the levels of plasma aminotransferase and liver necrosis in Con A-induced hepatitis. Also, EA significantly decreased the expression levels of the toll-like receptor 2 (TLR2) and TLR4 mRNA and protein in liver tissues. Further, EA decreased the phosphorylation of JNK, ERK1/2, and p38. EA-treated groups showed suppressions of nuclear factor κB (NF-κB) and IκB-α degradation levels in liver tissues. In addition, EA pretreatment decreased the expression of pro-inflammatory cytokines, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β). These results suggest that EA protects against T-cell-mediated hepatitis through TLR and mitogen-activated protein kinase (MAPK)/NF-κB signaling pathways.

  10. Small heterodimer partner attenuates hydrogen peroxide-induced expression of cyclooxygenase-2 and inducible nitric oxide synthase by suppression of activator protein-1 and nuclear factor-κB in renal proximal tubule epithelial cells.

    PubMed

    Park, Jung Sun; Choi, Hoon In; Bae, Eun Hui; Ma, Seong Kwon; Kim, Soo Wan

    2017-03-01

    The orphan nuclear receptor, small heterodimer partner (SHP), plays a negative regulatory role in innate immune responses and is involved in various inflammatory signaling pathways. In the present study, we aimed to ascertain whether SHP is effective in preventing hydrogen peroxide (H2O2)-induced kidney tubular inflammation and explored the molecular mechanisms underlying the protective effects of SHP. Renal ischemia/reperfusion (I/R) injury was induced in mice by clamping both renal pedicles for 30 min. The effects of H2O2 on cell viability in human renal proximal tubule (HK-2) cells were determined using MTT assays. 2',7'-DCF-DA was used to determine intracellular reactive oxygen species (ROS). SHP, cyclooxygenase-2 (COX-2) levels, and inducible nitric oxide synthase (iNOS) expression levels were determined by semi-quantitative immunoblotting and real-time polymerase chain reaction. In addition, SHP, nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) promoter activities were determined by luciferase assays. SHP mRNA and protein expression levels were reduced, whereas COX-2 and iNOS levels were increased in mice subjected to renal I/R. H2O2 treatment in HK-2 cells decreased cell viability, increased ROS production, and induced COX-2 and iNOS expression. These changes were counteracted by transient transfection with SHP. H2O2 treatment decreased SHP luciferase activity, which was recovered by treatment with the NF-κB inhibitor Bay11-7082, transfection with dominant-negative c-Jun or treatment with N-acetyl cysteine (NAC). AP-1 and NF-κB promoter activities were increased by H2O2 and this increase was blocked by SHP transfection. To conclude, SHP protected HK-2 cells from H2O2-induced tubular injury by inhibition of COX-2 and iNOS through suppression of AP-1 and NF-κB promoter activities.

  11. Role of zinc finger structure in nuclear localization of transcription factor Sp1

    SciTech Connect

    Ito, Tatsuo; Azumano, Makiko; Uwatoko, Chisana; Itoh, Kohji Kuwahara, Jun

    2009-02-27

    Transcription factor Sp1 is localized in the nucleus and regulates gene expression. Our previous study demonstrated that the carboxyl terminal region of Sp1 containing 3-zinc finger region as DNA binding domain can also serve as nuclear localization signal (NLS). However, the nuclear transport mechanism of Sp1 has not been well understood. In this study, we performed a gene expression study on mutant Sp1 genes causing a set of amino acid substitutions in zinc finger domains to elucidate nuclear import activity. Nuclear localization of the GFP-fused mutant Sp1 proteins bearing concomitant substitutions in the first and third zinc fingers was highly inhibited. These mutant Sp1 proteins had also lost the binding ability as to the GC box sequence. The results suggest that the overall tertiary structure formed by the three zinc fingers is essential for nuclear localization of Sp1 as well as dispersed basic amino acids within the zinc fingers region.

  12. C-type lectin receptor dectin-3 mediates trehalose 6,6'-dimycolate (TDM)-induced Mincle expression through CARD9/Bcl10/MALT1-dependent nuclear factor (NF)-κB activation.

    PubMed

    Zhao, Xue-Qiang; Zhu, Le-Le; Chang, Qing; Jiang, Changying; You, Yun; Luo, Tianming; Jia, Xin-Ming; Lin, Xin

    2014-10-24

    Previous studies indicate that both Dectin-3 (also called MCL or Clec4d) and Mincle (also called Clec4e), two C-type lectin receptors, can recognize trehalose 6,6'-dimycolate (TDM), a cell wall component from mycobacteria, and induce potent innate immune responses. Interestingly, stimulation of Dectin-3 by TDM can also induce Mincle expression, which may enhance the host innate immune system to sense Mycobacterium infection. However, the mechanism by which Dectin-3 induces Mincle expression is not fully defined. Here, we show that TDM-induced Mincle expression is dependent on Dectin-3-mediated NF-κB, but not nuclear factor of activated T-cells (NFAT), activation, and Dectin-3 induces NF-κB activation through the CARD9-BCL10-MALT1 complex. We found that bone marrow-derived macrophages from Dectin-3-deficient mice were severely defective in the induction of Mincle expression in response to TDM stimulation. This defect is correlated with the failure of TDM-induced NF-κB activation in Dectin-3-deficient bone marrow-derived macrophages. Consistently, inhibition of NF-κB, but not NFAT, impaired TDM-induced Mincle expression, whereas NF-κB, but not NFAT, binds to the Mincle promoter. Dectin-3-mediated NF-κB activation is dependent on the CARD9-Bcl10-MALT1 complex. Finally, mice deficient for Dectin-3 or CARD9 produced much less proinflammatory cytokines and keyhole limpet hemocyanin (KLH)-specific antibodies after immunization with an adjuvant containing TDM. Overall, this study provides the mechanism by which Dectin-3 induces Mincle expression in response to Mycobacterium infection, which will have significant impact to improve adjuvant and design vaccine for antimicrobial infection.

  13. Regulation of constitutive androstane receptor and its target genes by fasting, cAMP, hepatocyte nuclear factor alpha, and the coactivator peroxisome proliferator-activated receptor gamma coactivator-1alpha.

    PubMed

    Ding, Xunshan; Lichti, Kristin; Kim, Insook; Gonzalez, Frank J; Staudinger, Jeff L

    2006-09-08

    Animal studies reveal that fasting and caloric restriction produce increased activity of specific metabolic pathways involved in resistance to weight loss in liver. Evidence suggests that this phenomenon may in part occur through the action of the constitutive androstane receptor (CAR, NR1I3). Currently, the precise molecular mechanisms that activate CAR during fasting are unknown. We show that fasting coordinately induces expression of genes encoding peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), CAR, cytochrome P-450 2b10 (Cyp2b10), UDP-glucuronosyltransferase 1a1 (Ugt1a1), sulfotransferase 2a1 (Sult2a1), and organic anion-transporting polypeptide 2 (Oatp2) in liver in mice. Treatments that elevate intracellular cAMP levels also produce increased expression of these genes in cultured hepatocytes. Our data show that PGC-1alpha interaction with hepatocyte nuclear factor 4alpha (HNF4alpha, NR2A1) directly regulates CAR gene expression through a novel and evolutionarily conserved HNF4-response element (HNF4-RE) located in its proximal promoter. Expression of PGC-1alpha in cells increases CAR expression and ligand-independent CAR activity. Genetic studies reveal that hepatic expression of HNF4alpha is required to produce fasting-inducible CAR expression and activity. Taken together, our data show that fasting produces increased expression of genes encoding key metabolic enzymes and an uptake transporter protein through a network of interactions involving cAMP, PGC-1alpha, HNF4alpha, CAR, and CAR target genes in liver. Given the recent finding that mice lacking CAR exhibit a profound decrease in resistance to weight loss during extended periods of caloric restriction, our findings have important implications in the development of drugs for the treatment of obesity and related diseases.

  14. SKLB023 Blocks Joint Inflammation and Cartilage Destruction in Arthritis Models via Suppression of Nuclear Factor-Kappa B Activation in Macrophage

    PubMed Central

    Liu, Juan; Li, Xiuxia; Pei, Heying; Xiang, Mingli; Chen, Lijuan

    2013-01-01

    Rheumatoid arthritis (RA) is the most common arthritis and is mainly characterized by symmetric polyarticular joint disorders. Our previous study demonstrated a novel small molecule compound (Z)-N-(3-Chlorophenyl)-2-(4-((2,4-dioxothiazolidin-5-ylidene) methyl) phenoxy) acet-amide (SKLB023) showed potently anti-arthritic effects in a rat arthritis model, however, the underlying mechanisms for this are largely unknown. Both NF-κB and macrophages were reported to play important roles in the pathologic processes of RA. The purposes of this study were to indicate whether NF-κB and macrophages contributed to anti-arthritic effects of SKLB023 in two experimental arthritis models. Our results showed that SKLB023 could significantly improve joint inflammation and cartilage destruction both in adjuvant induced arthritis (AIA) and collagen-induced arthritis (CIA) models. We further found that the binding activation of NF-κB to DNA in joint tissues and RAW264.7 macrophages were suppressed by SKLB023. SKLB023 also inhibited the NF-κB activity in peritoneal macrophages by luciferase assay. Furthermore, the number of macrophages in synovial tissues was decreased after the treatment of different doses of SKLB023. The levels of TNF-α, IL-1β, and IL-6 in plasma, and the levels of TNF-α, NO, and IL-1β in peritoneal macrophages were down-regulated by SKLB023. Finally, SKLB023 attenuated the expression of iNOS and COX-2 in vivo and suppressed the phosphorylations of components of the mitogen-activated protein kinases (MAPKs). These observations identify a novel function for SKLB023 as an inhibitor of NF-κB in macrophages of RA, highlighting that SKLB023 was a potential therapeutic strategy for RA. PMID:23431370

  15. GATA transcription factors associate with a novel class of nuclear bodies in erythroblasts and megakaryocytes.

    PubMed Central

    Elefanty, A G; Antoniou, M; Custodio, N; Carmo-Fonseca, M; Grosveld, F G

    1996-01-01

    The nuclear distribution of GATA transcription factors in murine haemopoietic cells was examined by indirect immunofluorescence. Specific bright foci of GATA-1 fluorescence were observed in erythroleukaemia cells and primary murine erythroblasts and megakaryocytes, in addition to diffuse nucleoplasmic localization. These foci, which were preferentially found adjacent to nucleoli or at the nuclear periphery, did not represent sites of active transcription or binding of GATA-1 to consensus sites in the beta-globin loci. Immunoelectron microscopy demonstrated the presence of intensely labelled structures likely to represent the GATA-1 foci seen by immunofluorescence. The GATA-1 nuclear bodies differed from previously described nuclear structures and there was no co-localization with nuclear antigens involved in RNA processing or other ubiquitous (Spl, c-Jun and TBP) or haemopoietic (NF-E2) transcription factors. Interestingly, GATA-2 and GATA-3 proteins also localized to the same nuclear bodies in cell lines co-expressing GATA-1 and -2 or GATA-1 and -3 gene products. This pattern of distribution is, thus far, unique to the GATA transcription factors and suggests a protein-protein interaction with other components of the nuclear bodies via the GATA zinc finger domain. Images PMID:8617207

  16. Personality Factors and Nuclear Power Plant Operators: Initial License Success

    NASA Astrophysics Data System (ADS)

    DeVita-Cochrane, Cynthia

    Commercial nuclear power utilities are under pressure to effectively recruit and retain licensed reactor operators in light of poor candidate training completion rates and recent candidate failures on the Nuclear Regulatory Commission (NRC) license exam. One candidate failure can cost a utility over $400,000, making the successful licensing of new operators a critical path to operational excellence. This study was designed to discover if the NEO-PI-3, a 5-factor measure of personality, could improve selection in nuclear utilities by identifying personality factors that predict license candidate success. Two large U.S. commercial nuclear power corporations provided potential participant contact information and candidate results on the 2014 NRC exam from their nuclear power units nation-wide. License candidates who participated (n = 75) completed the NEO-PI-3 personality test and results were compared to 3 outcomes on the NRC exam: written exam, simulated operating exam, and overall exam result. Significant correlations were found between several personality factors and both written and operating exam outcomes on the NRC exam. Further, a regression analysis indicated that personality factors, particularly Conscientiousness, predicted simulated operating exam scores. The results of this study may be used to support the use of the NEO-PI-3 to improve operator selection as an addition to the current selection protocol. Positive social change implications from this study include support for the use of a personality measure by utilities to improve their return-on-investment in candidates and by individual candidates to avoid career failures. The results of this study may also positively impact the public by supporting the safe and reliable operation of commercial nuclear power utilities in the United States.

  17. Advanced Glycation End Products (AGE) Potently Induce Autophagy through Activation of RAF Protein Kinase and Nuclear Factor κB (NF-κB).

    PubMed

    Verma, Neeharika; Manna, Sunil K

    2016-01-15

    Advanced glycation end products (AGE) accumulate in diabetic patients and aging people because of high amounts of three- or four-carbon sugars derived from glucose, thereby causing multiple consequences, including inflammation, apoptosis, obesity, and age-related disorders. It is important to understand the mechanism of AGE-mediated signaling leading to the activation of autophagy (self-eating) that might result in obesity. We detected AGE as one of the potent inducers of autophagy compared with doxorubicin and TNF. AGE-mediated autophagy is inhibited by suppression of PI3K and potentiated by the autophagosome maturation blocker bafilomycin. It increases autophagy in different cell types, and that correlates with the expression of its receptor, receptor for AGE. LC3B, the marker for autophagosomes, is shown to increase upon AGE stimulation. AGE-mediated autophagy is partially suppressed by inhibitor of NF-κB, PKC, or ERK alone and significantly in combination. AGE increases sterol regulatory element binding protein activity, which leads to an increase in lipogenesis. Although AGE-mediated lipogenesis is affected by autophagy inhibitors, AGE-mediated autophagy is not influenced by lipogenesis inhibitors, suggesting that the turnover of lipid droplets overcomes the autophagic clearance. For the first time, we provide data showing that AGE induces several cell signaling cascades, like NF-κB, PKC, ERK, and MAPK, that are involved in autophagy and simultaneously help with the accumulation of lipid droplets that are not cleared effectively by autophagy, therefore causing obesity.

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

    PubMed

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

    2010-01-01

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

  19. Electronic and Nuclear Factors in Charge and Excitation Transfer

    SciTech Connect

    Piotr Piotrowiak

    2004-09-28

    We report the and/or state of several subprojects of our DOE sponsored research on Electronic and Nuclear Factors in Electron and Excitation Transfer: (1) Construction of an ultrafast Ti:sapphire amplifier. (2) Mediation of electronic interactions in host-guest molecules. (3) Theoretical models of electrolytes in weakly polar media. (4) Symmetry effects in intramolecular excitation transfer.

  20. Small GTPase Rho signaling is involved in {beta}1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor {kappa}B ligand on osteoblasts and osteoclast maturation

    SciTech Connect

    Hirai, Fumihiko; Nakayamada, Shingo; Okada, Yosuke; Saito, Kazuyoshi; Kurose, Hitoshi; Mogami, Akira; Tanaka, Yoshiya . E-mail: tanaka@med.uoeh-u.ac.jp

    2007-04-27

    We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. {beta}1 Integrin were highly expressed on osteoblasts. Engagement of {beta}1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor {kappa}B ligand (RANKL) on osteoblasts. Rho was activated by {beta}1 stimulation in osteoblasts. {beta}1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of {beta}1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in {beta}1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.

  1. Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2.

    PubMed

    Martin, Lynn; Pingle, Sandeep C; Hallam, Daniel M; Rybak, Leonard P; Ramkumar, Vickram

    2006-01-01

    Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-alpha and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-alpha release in human UB939 macrophages by activating an adenosine A(3) receptor (A(3)AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A(3)AR-dependent inhibition of TNF-alpha release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 mug/ml) increased TNF-alpha release, which was reduced in a dose-dependent manner by adenosine analogs N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A(3)AR blockade. The increase in TNF-alpha release was preceded by an increase in intracellular Ca(2+) levels. Inhibition of intracellular Ca(2+) release by IB-MECA, a selective agonist of the A(3)AR, or with BAPTA-AM, an intracellular Ca(2+) chelator, reduced LPS-stimulated TNF-alpha release. Activation of the A(3)AR or inhibition of intracellular Ca(2+) release also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A(3)AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-alpha and NO via the A(3)AR, involves suppression of intracellular Ca(2+)signaling, leading to suppression of NF-kappaB and ERK1/2 pathways.

  2. Occupational Employment in Nuclear-Related Activities, 1981.

    ERIC Educational Resources Information Center

    Baker, Joe G.; Olsen, Kathryn

    Employment in nuclear- and nuclear energy-related activities in 1981 was examined and compared to that in previous years. Survey instruments were returned by 784 establishments. Total 1981 nuclear-related employment was estimated to be 249,500--a growth of 22,600 (10%) workers over the 1977 total. Government-owned, contractor-operated (GOCO)…

  3. Combined glutamine and arginine decrease proinflammatory cytokine production by biopsies from Crohn's patients in association with changes in nuclear factor-kappaB and p38 mitogen-activated protein kinase pathways.

    PubMed

    Lecleire, Stéphane; Hassan, Aktham; Marion-Letellier, Rachel; Antonietti, Michel; Savoye, Guillaume; Bôle-Feysot, Christine; Lerebours, Eric; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

    2008-12-01

    Glutamine (Gln) and arginine (Arg) are conditionally essential amino acids with immunomodulatory properties. The aim of the study was to assess the effects of Gln and Arg alone or in combination on cytokine release by cultured colonic biopsies from patients with active Crohn's disease (CD). Ten consecutive patients [mean (range) age 26 (18-39) y] with active colonic CD (mean CD activity index: 383.7 +/- 129.8) were prospectively included in the study. Eight colonic biopsies were obtained via a colonoscopy and incubated during 18 h with low (physiological) or high (pharmacological) doses of Arg (0.1 or 2 mmol/L designated as Arg(low) or Arg(high), respectively) and Gln (0.6 or 10 mmol/L designated as Gln(low) or Gln(high), respectively). The concentrations of cytokines [interleukin (IL)-4, IL-10, IL-8, IL-6, tumor necrosis factor-alpha (TNFalpha), IL-1beta, interferon-gamma) were assessed by ELISA, and nitric oxide (NO) production was evaluated by Griess assay. Nuclear factor (NF)-kappaB p65 subunit, inhibitor of NFkappaB-alpha, and p38 mitogen-activated protein kinase (MAPK) were assessed by immunoblotting. Arg(high)/Gln(high) decreased the production of TNFalpha, IL-1beta, IL-8, and IL-6 (each P < 0.01). Arg(low)/Gln(high) decreased IL-6 and IL-8 production (both P < 0.01), whereas Arg(high)/Gln(low) did not affect cytokine and NO production. Arg(low)/Gln(high) and Arg(high)/Gln(high) decreased NF-kappaB p65 subunit expression, whereas p38 MAPK was decreased only by Arg(high)/Gln(high). Combined pharmacological doses of Arg and Gln decreased TNFalpha and the main proinflammatory cytokines release in active colonic CD biopsies via NF-kappaB and p38 MAPK pathways. These results could be the basis of prospective studies evaluating the effects of enteral supply of combined Arg and Gln during active CD.

  4. Cloning and characterization of the human beta2-glycoprotein I (beta2-GPI) gene promoter: roles of the atypical TATA box and hepatic nuclear factor-1alpha in regulating beta2-GPI promoter activity.

    PubMed Central

    Wang, Hsueh-Hsiao; Chiang, An-Na

    2004-01-01

    Beta2-glycoprotein I (beta2-GPI) is a plasma glycoprotein primarily synthesized in the liver. The interindividual variability of beta2-GPI expression in subjects with various metabolic syndromes and disease states suggests that it may have clinical importance. However, the regulation of beta2-GPI gene expression has not been clarified. To gain more insight into the control of beta2-GPI gene expression, we cloned the 4.1-kb 5'-flanking region and characterized the proximal promoter of the beta2- GPI gene in this study. Cis -acting elements required for beta2-GPI promoter activity were identified with transient transfection assays in the hepatoma cell lines HepG2 and Huh7 and in non-hepatic HeLa cells. Serial deletion analyses of the beta2-GPI 5'-flanking sequence revealed that the region from -197 to +7 had strong promoter activity in hepatoma cells but not in HeLa cells. Truncation and site-directed mutagenesis of putative cis -elements within this region showing an atypical TATA box and a HNF-1 (hepatic nuclear factor-1) element were both essential for the beta2-GPI promoter activity. Subsequent gel mobility shift assays confirmed the interaction of HNF-1alpha with the HNF-1 site residing downstream of the TATA box. Co-transfection of beta2-GPI promoter-luciferase vector with HNF-1alpha expression vector in Huh7 and HNF-1-deficient HeLa cells demonstrated the transactivation effect of HNF-1alpha on beta2-GPI promoter activity. In addition, overexpression of HNF-1alpha enhanced the endogenous beta2-GPI expression. These results suggest that the atypical TATA box and HNF-1 cis-element are critical for beta2-GPI transcription and HNF-1alpha may play an important role in cell-specific regulation of beta2-GPI gene expression. PMID:14984368

  5. Bay11-7082 attenuates neuropathic pain via inhibition of nuclear factor-kappa B and nucleotide-binding domain-like receptor protein 3 inflammasome activation in dorsal root ganglions in a rat model of lumbar disc herniation

    PubMed Central

    Zhang, Ailiang; Wang, Kun; Ding, Lianghua; Bao, Xinnan; Wang, Xuan; Qiu, Xubin; Liu, Jinbo

    2017-01-01

    Lumbar disc herniation (LDH) is an important cause of radiculopathy, but the underlying mechanisms are incompletely understood. Many studies suggested that local inflammation, rather than mechanical compression, results in radiculopathy induced by LDH. On the molecular and cellular level, nuclear factor-kappa B (NF-κB) and nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasome have been implicated in the regulation of neuroinflammation formation and progression. In this study, the autologous nucleus pulposus (NP) was implanted in the left L5 dorsal root ganglion (DRG) to mimic LDH in rats. We investigated the expression of NF-κB and the components of NLRP3 inflammasome in the DRG neurons in rats. Western blotting and immunofluorescence for the related molecules, including NLRP3, apoptosis-associated speck-like protein containing caspase-1 activator domain (ASC), caspase-1, interleukin (IL)-1β, IL-18, IκBα, p-IκBα, p65, p-p65, and calcitonin gene-related peptide (CGRP) were examined. In the NP-treated group, the activations of NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65 in DRG neurons in rats were elevated at 1 day after surgery, and the peak occurred at 7 days. Treatment with Bay11-7082, an inhibitor of the actions of IKK-β, was able to inhibit expression and activation of the molecules (NLRP3, ASC, caspase-1, IL-1β, IL-18, p-IκBα, and p-p65) and relieve the pain in rats. Our study shows that NF-κB and NLRP3 inflammasome are involved in the maintenance of NP-induced pain, and that Bay11-7082 could alleviate mechanical allodynia and thermal hyperalgesia by inhibiting NF-κB and NLRP3 inflammasome activation. PMID:28243141

  6. Dual-energy precursor and nuclear erythroid-related factor 2 activator treatment additively improve redox glutathione levels and neuron survival in aging and Alzheimer mouse neurons upstream of reactive oxygen species.

    PubMed

    Ghosh, Debolina; LeVault, Kelsey R; Brewer, Gregory J

    2014-01-01

    To determine whether glutathione (GSH) loss or increased reactive oxygen species (ROS) are more important to neuron loss, aging, and Alzheimer's disease (AD), we stressed or boosted GSH levels in neurons isolated from aging 3xTg-AD neurons compared with those from age-matched nontransgenic (non-Tg) neurons. Here, using titrating with buthionine sulfoximine, an inhibitor of γ-glutamyl cysteine synthetase (GCL), we observed that GSH depletion increased neuronal death of 3xTg-AD cultured neurons at increasing rates across the age span, whereas non-Tg neurons were resistant to GSH depletion until old age. Remarkably, the rate of neuron loss with ROS did not increase in old age and was the same for both genotypes, which indicates that cognitive deficits in the AD model were not caused by ROS. Therefore, we targeted for neuroprotection activation of the redox sensitive transcription factor, nuclear erythroid-related factor 2 (Nrf2) by 18 alpha glycyrrhetinic acid to stimulate GSH synthesis through GCL. This balanced stimulation of a number of redox enzymes restored the lower levels of Nrf2 and GCL seen in 3xTg-AD neurons compared with those of non-Tg neurons and promoted translocation of Nrf2 to the nucleus. By combining the Nrf2 activator together with the NADH precursor, nicotinamide, we increased neuron survival against amyloid beta stress in an additive manner. These stress tests and neuroprotective treatments suggest that the redox environment is more important for neuron survival than ROS. The dual neuroprotective treatment with nicotinamide and an Nrf2 inducer indicates that these age-related and AD-related changes are reversible.

  7. Aloperine attenuated neuropathic pain induced by chronic constriction injury via anti-oxidation activity and suppression of the nuclear factor kappa B pathway

    SciTech Connect

    Xu, Ya-Qiong; Jin, Shao-Ju; Liu, Ning; Li, Yu-Xiang; Zheng, Jie; Ma, Lin; Du, Juan; Zhou, Ru; Zhao, Cheng-Jun; Niu, Yang; Sun, Tao; Yu, Jian-Qiang

    2014-09-05

    Highlights: • Aloperine has anti-nociceptive effects on neuropathic pain induced CCI. • Aloperine reduces ROS in neuropathic pain mice. • Aloperine down-regulates the expression of NF-κB and its downstream pro-inflammatory cytokines in neuropathic pain mice. - Abstract: Objective: To investigate whether aloperine (ALO) has antinociceptive effects on neuropathic pain induced by chronic constriction injury, whether ALO reduces ROS against neuropathic pain, and what are the mechanisms involved in ALO attenuated neuropathic pain. Methods: Mechanical and cold allodynia, thermal and mechanical hyperalgesia and spinal thermal hyperalgesia were estimated by behavior methods such as Von Frey filaments, cold-plate, radiant heat, paw pressure and tail immersion on one day before surgery and days 7, 8, 10, 12 and 14 after surgery, respectively. In addition, T-AOC, GSH-PX, T-AOC and MDA in the spinal cord (L4/5) were measured to evaluate anti-oxidation activity of ALO on neuropathic pain. Expressions of NF-κB and pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in the spinal cord (L4/5) were analyzed by using Western blot. Results: Administration of ALO (80 mg/kg and 40 mg/kg, i.p.) significantly increased paw withdrawal threshold, paw pressure, paw withdrawal latencies, tail-curling latencies, T-AOC, GSH-PX and T-SOD concentration, reduced the numbers of paw lifts and MDA concentration compared to CCI group. ALO attenuated CCI induced up-regulation of expressions of NF-κB, TNF-α, IL-6, IL-1β at the dose of 80 mg/kg (i.p.). Pregabalin produced similar effects serving as positive control at the dose of 10 mg/kg (i.p.). Conclusion: ALO has antinociceptive effects on neuropathic pain induced by CCI. The antinociceptive effects of ALO against neuropathic pain is related to reduction of ROS, via suppression of NF-κB pathway.

  8. Sevoflurane Inhibits Nuclear Factor-κB Activation in Lipopolysaccharide-Induced Acute Inflammatory Lung Injury via Toll-Like Receptor 4 Signaling

    PubMed Central

    Sun, Xi Jia; Li, Xiao Qian; Wang, Xiao Long; Tan, Wen Fei; Wang, Jun Ke

    2015-01-01

    Background Infection is a common cause of acute lung injury (ALI). This study was aimed to explore whether Toll-like receptors 4 (TLR4) of airway smooth muscle cells (ASMCs) play a role in lipopolysaccharide (LPS)-induced airway hyperresponsiveness and potential mechanisms. Methods In vivo: A sensitizing dose of LPS (50 µg) was administered i.p. to female mice before anesthesia with either 3% sevoflurane or phenobarbital i.p. After stabilization, the mice were challenged with 5 µg of intratracheal LPS to mimic inflammatory attack. The effects of sevoflurane were assessed by measurement of airway responsiveness to methacholine, histological examination, and IL-1, IL-6, TNF-α levels in bronchoalveolar lavage fluid (BALF). Protein and gene expression of TLR4 and NF-κB were also assessed. In vitro: After pre-sensitization of ASMCs and ASM segments for 24h, levels of TLR4 and NF-κB proteins in cultured ASMCs were measured after continuous LPS exposure for 1, 3, 5, 12 and 24h in presence or absence of sevoflurane. Constrictor and relaxant responsiveness of ASM was measured 24 h afterwards. Results The mRNA and protein levels of NF-κB and TLR4 in ASM were increased and maintained at high level after LPS challenge throughout 24h observation period, both in vivo and in vitro. Sevoflurane reduced LPS-induced airway hyperresponsiveness, lung inflammatory cell infiltration and proinflammatory cytokines release in BALF as well as maximal isometric contractile force of ASM segments to acetylcholine, but it increased maximal relaxation response to isoproterenol. Treatment with specific NF-κB inhibitor produced similar protections as sevoflurane, including decreased expressions of TLR4 and NF-κB in cultured ASMCs and improved pharmacodynamic responsiveness of ASM to ACh and isoproterenol. Conclusions This study demonstrates the crucial role of TLR4 activation in ASMCs during ALI in response to LPS. Sevoflurane exerts direct relaxant and anti-inflammatory effects in vivo

  9. Penetration Factor for Nuclear Fusion Reaction in Nonthermal Astrophysical Plasmas

    NASA Astrophysics Data System (ADS)

    Ki, Dai-Han; Jung, Young-Dae

    2011-02-01

    The nonthermal effects on the nuclear fusion reaction process are investigated in Lorentzian astrophysical plasmas. The closed expression of the classical turning point in Lorentzian plasmas is obtained by the Lambert W-function. Using the WKB analysis with the effective screening length, the closed expressions of the fusion penetration factor and the cross section for the nuclear fusion reaction in Lorentzian plasmas are obtained as functions of the spectral index, relative kinetic energy, and plasma parameters. It is shown that the nonthermal character of the Lorentzian plasma enhances the fusion penetration factor. In addition, the nonthermal effect on the penetration factor is found to be more significant in plasmas with higher densities. It would be expected that the fusion reaction rates of the p-p chain and the CNO cycle in nonthermal plasmas are always greater than those in thermal Maxwellian plasmas.

  10. Nuclear fear and children: the impact of parental nuclear activism, responsivity, and fear

    SciTech Connect

    LaGuardia, M.R.

    1986-01-01

    This study examines the extent to which parental nuclear fear, parental activism, and parental responsivity is associated with children's (age 10) nuclear fear. Other associated variables investigated include: nuclear denial, general anxiety and fear, and the personal characteristics of sex, socio-economic status, and academic aptitude. Findings indicate that children attend to nuclear issues when their parents attend to a significant degree. Children's hopelessness about the arms race is increased as parents' worry about nuclear war increases. Children's fear about not surviving a nuclear war increases as parents' worry about survivability decreases. Children who have more general fears also indicated that they have a high level of hopelessness, pervasive worry, and much concern about being able to survive a nuclear war. Children with a high degree of general anxiety did not indicate high degrees of nuclear fears. Children with high academic aptitude were more knowledgeable about nuclear issues and expressed more fears about the nuclear threat. Boys demonstrated more knowledge about nuclear issues than girls, and girls expressed much more frequent fear and worry about the nuclear threat than boys. Parents of lower socio-economic statues (SES) expressed more denial about the nuclear threat and were more pro-military than the higher SES parents.

  11. Humic acid in drinking well water induces inflammation through reactive oxygen species generation and activation of nuclear factor-κB/activator protein-1 signaling pathways: a possible role in atherosclerosis.

    PubMed

    Hseu, You-Cheng; Senthil Kumar, K J; Chen, Chih-Sheng; Cho, Hsin-Ju; Lin, Shu-Wei; Shen, Pei-Chun; Lin, Cheng-Wen; Lu, Fung-Jou; Yang, Hsin-Ling

    2014-01-15

    Humic acid (HA) has been implicated as one of the etiological factors in the peripheral vasculopathy of blackfoot disease (BFD) in Taiwan. However, the underlying pathophysiological mechanisms of BFD are not well defined. In this study, we used an in vitro and in vivo model, in which HA (25-200μg/mL) activated macrophages to produce pro-inflammatory molecules by activating their transcriptional factors. HA exposure induced NO and PGE2 production followed by induction of iNOS and COX-2 through NF-κB/AP-1 transactivation in macrophages. In addition, the production of TNF-α and IL-1β was significantly increased by HA. Moreover, HA-induced iNOS and COX-2 expression were down-regulated by the NF-κB and AP-1 inhibitors pyrrolidine dithiocarbamate (PDTC) and Tanshinone, respectively. Furthermore, generations of ROS and nitrotyrosine, as well as activation of the AKT and MAPKs signaling cascades were observed after HA exposure. Specifically, HA-induced NF-κB activation was mediated by ROS and AKT, and that HA-induced AP-1 activation was mediated by JNK and ERK. Notably, HA-mediated AKT, JNK, and ERK activation was ROS-independent. The inflammatory potential of HA was correlated with increased expression of HO-1 and Nrf2. Furthermore, an in vivo study confirms that mice exposed to HA, the serum levels of TNF-α and IL-1β was significantly increased in a dose-dependent manner. This report marks the first confirmation that environmental exposure of HA induces inflammation in macrophages, which may be one of the main causes of early atherogenesis in blackfoot disease.

  12. An overview of a nuclear reprocessing plant Human Factors programme.

    PubMed

    Kirwan, Barry

    2003-09-01

    This paper presents a case study of a large Human Factors programme applied in the nuclear fuel reprocessing industry (1987-1991). The paper outlines the key Human Factors issues addressed, as well as the impacts achieved, and gives an indication of the resources utilised (approximately 15 person-years of effort). It also considers the starting point of the programme, in terms of the factors that led to the need for such an extensive programme. Some general lessons learned are given at the end of the paper.

  13. Regulation of the Drosophila hypoxia-inducible factor alpha Sima by CRM1-dependent nuclear export.

    PubMed

    Romero, Nuria M; Irisarri, Maximiliano; Roth, Peggy; Cauerhff, Ana; Samakovlis, Christos; Wappner, Pablo

    2008-05-01

    Hypoxia-inducible factor alpha (HIF-alpha) proteins are regulated by oxygen levels through several different mechanisms that include protein stability, transcriptional coactivator recruitment, and subcellular localization. It was previously reported that these transcription factors are mainly nuclear in hypoxia and cytoplasmic in normoxia, but so far the molecular basis of this regulation is unclear. We show here that the Drosophila melanogaster HIF-alpha protein Sima shuttles continuously between the nucleus and the cytoplasm. We identified the relevant nuclear localization signal and two functional nuclear export signals (NESs). These NESs are in the Sima basic helix-loop-helix (bHLH) domain and promote CRM1-dependent nuclear export. Site-directed mutagenesis of either NES provoked Sima nuclear retention and increased transcriptional activity, suggesting that nuclear export contributes to Sima regulation. The identified NESs are conserved and probably functional in the bHLH domains of several bHLH-PAS proteins. We propose that rapid nuclear export of Sima regulates the duration of cellular responses to hypoxia.

  14. 5-Hydroxytrytophan inhibits tert-butylhydroperoxide (t-BHP)-induced oxidative damage via the suppression of reactive species (RS) and nuclear factor-kappaB (NF-kappaB) activation on human fibroblast.

    PubMed

    Bae, Sung Jin; Lee, Jun Sik; Kim, Ji Min; Lee, Eun Kyeong; Han, Yu Kyeong; Kim, Hyun Jung; Choi, Jehun; Ha, Young Mi; No, Jae-Kyung; Kim, Yun Hee; Yu, Byung Pal; Chung, Hae Young

    2010-05-26

    5-Hydroxytryptophan (5HTP), an analogue of tryptophan, is a precursor of serotonin that also has effective antioxidative and anti-apoptotic properties (1) . However, the cellular mechanisms underlying these properties of 5HTP have not been explored. In this study, we tested the hypothesis that 5HTP exerts its antioxidative action against oxidative stress and inflammation by suppressing the activation of the key pro-inflammatory transcriptional pathways, p38 mitogen-activated protein kinase (p38MAPK) and nuclear factor-kappaB (NF-kappaB). The study was carried out using human fibroblast cells that were challenged by tert-butylhydroperoxide (t-BHP)-induced oxidative damage. Results show that 5HTP significantly reduced t-BHP-induced oxidative damage in human fibroblast cells, as determined by cell cytotoxicity, intracellular reactive species (RS) and peroxynitrite (ONOO(-)) generation, and inducible nitric oxide synthase expression. Moreover, 5HTP protected human fibroblast cells against t-BHP-induced oxidative DNA damage, as determined by 4,6-diamidino-2-phenlylindole (DAPI) staining. Pretreatment of human fibroblast cells with 5HTP also dose-dependently inhibited glutathione (GSH) depletion, indicating that it protects cells against t-BHP-induced oxidative damage. Western blot analysis revealed that 5HTP also markedly increased Bcl-2 expression and suppressed both p38MAPK and NF-kappaB activation in the t-BHP-treated human fibroblast cells. When these results are taken together, they strongly indicate that 5HTP has beneficial and protective effects against t-BHP-induced cell death in vitro, as demonstrated by its antioxidative and anti-inflammatory actions. Data further showed that the protective mechanisms underlying the actions of 5HTP against oxidative stress-induced damage are associated with RS/ONOO(-) scavenging and the inhibition of lipid peroxidation and GSH depletion.

  15. Linoleic acid-induced expression of inducible nitric oxide synthase and cyclooxygenase II via p42/44 mitogen-activated protein kinase and nuclear factor-kappaB pathway in retinal pigment epithelial cells.

    PubMed

    Fang, I-Mo; Yang, Chang-Hao; Yang, Chung-May; Chen, Muh-Shy

    2007-11-01

    High linoleic acid (LA) intake is known to correlate with age-related macular degeneration (AMD), but the molecular mechanisms remain unclear. This study was conducted to investigate the effects of LA on expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase II (COX-2) and their associated signaling pathways in human retinal pigment epithelial (RPE) cells. ARPE-19 cells were treated with different concentrations of LA. Expressions of iNOS and COX-2 were examined using semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Concentrations of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) in the culture medium were determined by enzyme-link immunosorbent assay (ELISA). Activation of p42/44, p38, JNK mitogen-activated protein kinase (MAPK) and nuclear factors (NF)-kappaB were evaluated by Western blot analysis and electrophoretic mobility shift assay (EMSA). We found that LA induced expression of iNOS and COX-2 in RPE cells at the mRNA and protein levels in a time-and dose-dependent manner. Upregulation of iNOS and COX-2 resulted in increased production of NO and PGE(2). Moreover, LA caused degradation of IkappaB and increased NF-kappaB DNA binding activity. Effects of LA-induced iNOS and COX-2 expression were inhibited by a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). LA activated p42/44, but not p38 or JNK MAPK. Inhibition of p42/44 activity by PD98059 significantly reduced LA-induced activation of NF-kappaB. Linoleic acid-induced expression of iNOS and COX-2 as well as PGE(2) and NO release in RPE cells were sequentially mediated through activation of p42/p44, MAPK, then NF-kappaB. These results may provide new insights into both mechanisms of LA action on RPE cells and pathogenesis of age-related macular degeneration.

  16. Follistatin‐like protein 1 contributes to dendritic cell and T‐lymphocyte activation in nasopharyngeal carcinoma patients by altering nuclear factor κb and Jun N‐terminal kinase expression

    PubMed Central

    Wang, Hong; Wu, Senyong; Huang, Shiping; Yin, Shaolin; Zou, Guilong; Huang, Kuan'en; Zhang, Zhe

    2016-01-01

    Follistatin‐like protein 1 (FSTL1) is a newly characterized protein that can regulate the immune response in various ways. Dendritic cells (DCs) are central to immune regulation. In this study, we explored the impact of FSTL1 on DC activity in nasopharyngeal carcinoma (NPC) patients. The surface expression of CD40, CD86, and HLA‐DR on DCs was analyzed and showed significantly elevated expression levels, indicating DC maturity. After FSTL1 was added to DCs collected from NPC patients (n = 50), controls (n = 47), and healthy donors (n = 10), interferon γ secretion and T‐cell receptor expression in cytotoxic T lymphocytes were also investigated. In the experimental groups, the expression of the critical immune protein nuclear factor (NF)‐κb was upregulated, whereas Jun N‐terminal kinase (JNK) was downregulated. Our findings demonstrate that FSTL1 plays a critical role in immune regulation, enhancing the antigen presentation ability of DCs by up‐regulating NF‐κb expression and down‐regulating JNK expression. PMID:27859422

  17. Hodgkin's lymphoma cells are efficiently engrafted and tumor marker CD30 is expressed with constitutive nuclear factor-kappaB activity in unconditioned NOD/SCID/gammac(null) mice.

    PubMed

    Dewan, Md Zahidunnabi; Watanabe, Mariko; Ahmed, Sunjida; Terashima, Kazuo; Horiuchi, Sankichi; Sata, Tetsutaro; Honda, Mitsuo; Ito, Mamoru; Watanabe, Toshiki; Horie, Ryouichi; Yamamoto, Naoki

    2005-08-01

    As there are very few reproducible animal models without conditioning available for the study of human B-cell-type Hodgkin's lymphoma (HL), we investigated the ability of HL cells to induce tumors using novel NOD/SCID/gammac(null) (NOG) mice. Four human Epstein-Barr virus-negative cell lines (KM-H2 and L428 originated from B cells, L540 and HDLM2 originated from T cells) were inoculated either subcutaneously in the postauricular region or intravenously in the tail of unmanipulated NOG mice. All cell lines successfully engrafted and produced tumors with infiltration of cells in various organs of all mice. Tumor cells had classical histomorphology as well as expression patterns of the tumor marker CD30, which is a cell surface antigen expressed on HL. Tumor progression in mice inoculated with B-cell-type, but not T-cell-type, HL cells correlated with an elevation in serum human interleukin-6 levels. Tumor cells from the mice also retained strong nuclear factor (NF)-kappaB DNA binding activity, and the induced NF-kappaB components were indistinguishable from those cultured in vitro. The reproducible growth behavior and preservation of characteristic features of both B-cell-type and T-cell-type HL in the mice suggest that this new xenotransplant model can provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth, and to develop novel anticancer therapies.

  18. Suplatast tosilate alleviates nasal symptoms through the suppression of nuclear factor of activated T-cells-mediated IL-9 gene expression in toluene-2,4-diisocyanate-sensitized rats.

    PubMed

    Mizuguchi, Hiroyuki; Orimoto, Naoki; Kadota, Takuya; Kominami, Takahiro; Das, Asish K; Sawada, Akiho; Tamada, Misaki; Miyagi, Kohei; Adachi, Tsubasa; Matsumoto, Mayumi; Kosaka, Tomoya; Kitamura, Yoshiaki; Takeda, Noriaki; Fukui, Hiroyuki

    2016-03-01

    Histamine H1 receptor (H1R) gene is upregulated in patients with pollinosis; its expression level is highly correlated with the nasal symptom severity. Antihistamines are widely used as allergy treatments because they inhibit histamine signaling by blocking H1R or suppressing H1R signaling as inverse agonists. However, long-term treatment with antihistamines does not completely resolve toluene-2,4-diisocyanate (TDI)-induced nasal symptoms, although it can decrease H1R gene expression to the basal level, suggesting additional signaling is responsible for the pathogenesis of the allergic symptoms. Here, we show that treatment with suplatast tosilate in combination with antihistamines markedly alleviates nasal symptoms in TDI-sensitized rats. Suplatast suppressed TDI-induced upregulation of IL-9 gene expression. Suplatast also suppressed ionomycin/phorbol-12-myristate-13-acetate-induced upregulation of IL-2 gene expression in Jurkat cells, in which calcineurin (CN)/nuclear factor of activated T-cells (NFAT) signaling is known to be involved. Immunoblot analysis demonstrated that suplatast inhibited binding of NFAT to DNA. Furthermore, suplatast suppressed ionomycin-induced IL-9 mRNA upregulation in RBL-2H3 cells, in which CN/NFAT signaling is also involved. These data suggest that suplatast suppressed NFAT-mediated IL-9 gene expression in TDI-sensitized rats and this might be the underlying mechanism of the therapeutic effects of combined therapy of suplatast with antihistamine.

  19. ATP1B3 Protein Modulates the Restriction of HIV-1 Production and Nuclear Factor κ Light Chain Enhancer of Activated B Cells (NF-κB) Activation by BST-2*

    PubMed Central

    Nishitsuji, Hironori; Sugiyama, Ryuichi; Abe, Makoto; Takaku, Hiroshi

    2016-01-01

    Here, we identify ATP1B3 and fibrillin-1 as novel BST-2-binding proteins. ATP1B3 depletion in HeLa cells (BST-2-positive cells), but not 293T cells (BST-2-negative cells), induced the restriction of HIV-1 production in a BST-2-dependent manner. In contrast, fibrillin-1 knockdown reduced HIV-1 production in 293T and HeLa cells in a BST-2-independent manner. Moreover, NF-κB activation was enhanced by siATP1B3 treatment in HIV-1- and HIV-1ΔVpu-infected HeLa cells. In addition, ATP1B3 silencing induced high level BST-2 expression on the surface of HeLa cells. These results indicate that ATP1B3 is a co-factor that accelerates BST-2 degradation and reduces BST-2-mediated restriction of HIV-1 production and NF-κB activation. PMID:26694617

  20. Humic acid in drinking well water induces inflammation through reactive oxygen species generation and activation of nuclear factor-κB/activator protein-1 signaling pathways: A possible role in atherosclerosis

    SciTech Connect

    Hseu, You-Cheng; Senthil Kumar, K.J.; Chen, Chih-Sheng; Cho, Hsin-Ju; Lin, Shu-Wei; Shen, Pei-Chun; Lin, Cheng-Wen; Lu, Fung-Jou; Yang, Hsin-Ling

    2014-01-15

    Humic acid (HA) has been implicated as one of the etiological factors in the peripheral vasculopathy of blackfoot disease (BFD) in Taiwan. However, the underlying pathophysiological mechanisms of BFD are not well defined. In this study, we used an in vitro and in vivo model, in which HA (25–200 μg/mL) activated macrophages to produce pro-inflammatory molecules by activating their transcriptional factors. HA exposure induced NO and PGE{sub 2} production followed by induction of iNOS and COX-2 through NF-κB/AP-1 transactivation in macrophages. In addition, the production of TNF-α and IL-1β was significantly increased by HA. Moreover, HA-induced iNOS and COX-2 expression were down-regulated by the NF-κB and AP-1 inhibitors pyrrolidine dithiocarbamate (PDTC) and Tanshinone, respectively. Furthermore, generations of ROS and nitrotyrosine, as well as activation of the AKT and MAPKs signaling cascades were observed after HA exposure. Specifically, HA-induced NF-κB activation was mediated by ROS and AKT, and that HA-induced AP-1 activation was mediated by JNK and ERK. Notably, HA-mediated AKT, JNK, and ERK activation was ROS-independent. The inflammatory potential of HA was correlated with increased expression of HO-1 and Nrf2. Furthermore, an in vivo study confirms that mice exposed to HA, the serum levels of TNF-α and IL-1β was significantly increased in a dose-dependent manner. This report marks the first confirmation that environmental exposure of HA induces inflammation in macrophages, which may be one of the main causes of early atherogenesis in blackfoot disease. - Highlights: • Humic acid (HA) induce pro-inflammatory cytokines and mediators in macrophages. • HA-induced inflammation is mediated by ROS and NF-κB/AP-1 signaling pathways. • The inflammatory potential of HA correlated with activation of Nrf2/HO-1 genes. • HA exposure to mice increased pro-inflammatory cytokines production in vivo. • HA may be one of the main causes of early

  1. Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1

    PubMed Central

    Liang, Ke; Ding, Xue-qin; Lin, Chen

    2015-01-01

    The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus. PMID:25687434

  2. Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1.

    PubMed

    Liang, Ke; Ding, Xue-Qin; Lin, Chen; Kang, Y James

    2015-11-01

    The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus.

  3. Galaxy interactions and the stimulation of nuclear activity

    NASA Technical Reports Server (NTRS)

    Heckman, Timothy M.

    1990-01-01

    The author discusses the idea that interactions between galaxies can lead to enhanced galactic activity. He discusses whether, apart from the observational evidence, there is a strong theoretical or heuristic motivation for investigating galaxy interactions as stimulators of nuclear activity in galaxies. Galactic interactions as mechanisms for triggering nuclear starbursts are covered.

  4. Methylation status of a single CpG locus 3 bases upstream of TATA-box of receptor activator of nuclear factor-kappaB ligand (RANKL) gene promoter modulates cell- and tissue-specific RANKL expression and osteoclastogenesis.

    PubMed

    Kitazawa, Riko; Kitazawa, Sohei

    2007-01-01

    Receptor activator of nuclear factor-kappaB ligand (RANKL) expression is tissue specific and limited to certain subsets of T-lymphocytes and stromal/osteoblastic cells. Even among osteoblasts, RANKL is expressed on about 20% of osteoblasts of the normal mouse. To clarify the mechanism of population-specific RANKL expression, we analyzed the effect of CpG methylation on its transcription, mRNA and protein expression as well as on osteoclastogenesis. Subpopulations of ST2 cells were used: P9, which expresses RANKL and supports osteoclastogenesis, and P16, which does not. By sodium bisulfite mapping, the rate of CpG methylation of the -65/+350 region, especially of CpG locus no. 1 three bases upstream of the TATA-box, was higher in P16 than in P9 ST2 cells. ChIP and gel shift assay showed that methylated CpG locus no. 1 was a target of MeCP2 binding that, in turn, blocked the binding of the TATA-box binding protein to the TATA-box. In vitro methylation by SssI of the promoter construct reduced its transcriptional activity at the steady state and its response to 1alpha,25(OH)2 vitamin D3. Conversely, treatment with DNA methylase inhibitor, 5-aza-2'-deoxycytidine, significantly restored RANKL expression and osteoclastogenesis in P16 cells. Except for primary cultured osteoblasts, CpG locus no. 1 was frequently methylated in various normal mouse tissues. We propose that the methylation status of the CpG locus three bases upstream of the TATA-box modulates the control of cell- and tissue-specific expression of RANKL gene and osteoclastogenesis. The heterogeneity of stromal/ osteoblastic cells in response to bone-resorbing stimuli may be attributed, in part, to the methylation status of the RANKL gene promoter.

  5. Inhibition of Hageman factor activation

    PubMed Central

    Nossel, H. L.; Rubin, H.; Drillings, M.; Hsieh, R.

    1968-01-01

    A method for studying inhibitors of the contact stages of blood coagulation is described. A number of positively charged substances were shown to inhibit the contact stages. The inhibitory substances include spermine, cytochrome c, ribonuclease, and lysozyme. The inhibitory effect of these substances was neutralized by the addition of an activated plasma thromboplastin antecedent, factor XI, (PTA) fraction. Other positively charged substances including protamine, hexadimethrine, polylysine, polyornithine, methylene blue, and ortho-toluidine blue also inhibited the contact stages of coagulation, but the inhibitory effect on coagulation was not neutralized by the activated PTA fraction. Negatively charged substances such as heparin and insulin did not inhibit the contact stages of coagulation. Cytochrome c inhibited Celite adsorption of a partially purified Hageman factor fraction, and cytochrome, ribonuclease, spermine, and lysozome inhibited the adsorption of Hageman factor from PTA-deficient plasma. Very much smaller quantities of Celite completely adsorbed Hageman factor from the fraction rather than from whole plasma, which suggested the possibility that plasma contains an inhibitor or inhibitors of Hageman factor adsorption. Furthermore cytochrome c, spermine, ribonuclease, and lysozyme inhibited the coagulant activity of the following activators of the Hageman and PTA factors: Celite, kaolin, sodium stearate, ellagic acid, and skin. It is suggested that negatively charged sites on these activators are critical for adsorption and activation and that inhibition results from neutralization of the negatively charged sites by the adsorbed inhibtor. Tests with polylysine polymers indicate that inhibitory activity is directly related to molecular size over the molecular weight range of 4000 to 100,000. PMID:5645860

  6. Tumor necrosis factor alpha promotes the proliferation of human nucleus pulposus cells via nuclear factor-κB, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase.

    PubMed

    Wang, Xiao-Hu; Hong, Xin; Zhu, Lei; Wang, Yun-Tao; Bao, Jun-Ping; Liu, Lei; Wang, Feng; Wu, Xiao-Tao

    2015-04-01

    Although tumor necrosis factor alpha (TNF-α) is known to play a critical role in intervertebral disc (IVD) degeneration, the effect of TNF-α on nucleus pulposus (NP) cells has not yet been elucidated. The aim of this study was to explore the effect of TNF-α on proliferation of human NP cells. NP cells were treated with different concentrations of TNF-α. Cell proliferation was determined by cell counting kit-8 (CCK-8) analysis and Ki67 immunofluorescence staining, and expression of cyclin B1 was studied by quantitative real-time RT-PCR. Cell cycle was measured by flow cytometry and cell apoptosis was analyzed using an Annexin V-fluorescein isothiocyanate (FITC) & propidium iodide (PI) apoptosis detection kit. To identify the mechanism by which TNF-α induced proliferation of NP cells, selective inhibitors of major signaling pathways were used and Western blotting was carried out. Treatment with TNF-α increased cell viability (as determined by CCK-8 analysis) and expression of cyclin B1 and the number of Ki67-positive and S-phase NP cells, indicating enhancement of proliferation. Consistent with this, NP cell apoptosis was suppressed by TNF-α treatment. Moreover, inhibition of NF-κB, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) blocked TNF-α-stimulated proliferation of NP cells. In conclusion, the current findings suggest that the effect of TNF-α on IVD degeneration involves promotion of the proliferation of human NP cells via the NF-κB, JNK, and p38 MAPK pathways.

  7. Key Response Planning Factors for the Aftermath of Nuclear Terrorism

    SciTech Connect

    Buddemeier, B R; Dillon, M B

    2009-01-21

    Despite hundreds of above-ground nuclear tests and data gathered from Hiroshima and Nagasaki, the effects of a ground-level, low-yield nuclear detonation in a modern urban environment are still the subject of considerable scientific debate. Extensive review of nuclear weapon effects studies and discussions with nuclear weapon effects experts from various federal agencies, national laboratories, and technical organizations have identified key issues and bounded some of the unknowns required to support response planning for a low-yield, ground-level nuclear detonation in a modern U.S. city. This study, which is focused primarily upon the hazards posed by radioactive fallout, used detailed fallout predictions from the advanced suite of three-dimensional (3-D) meteorology and plume/fallout models developed at Lawrence Livermore National Laboratory (LLNL), including extensive global Key Response Planning Factors for the Aftermath of Nuclear Terrorism geographical and real-time meteorological databases to support model calculations. This 3-D modeling system provides detailed simulations that account for complex meteorology and terrain effects. The results of initial modeling and analysis were presented to federal, state, and local working groups to obtain critical, broad-based review and feedback on strategy and messaging. This effort involved a diverse set of communities, including New York City, National Capitol Regions, Charlotte, Houston, Portland, and Los Angeles. The largest potential for reducing casualties during the post-detonation response phase comes from reducing exposure to fallout radiation. This can be accomplished through early, adequate sheltering followed by informed, delayed evacuation.B The response challenges to a nuclear detonation must be solved through multiple approaches of public education, planning, and rapid response actions. Because the successful response will require extensive coordination of a large number of organizations, supplemented by

  8. Nuclear transportation of exogenous epidermal growth factor receptor and androgen receptor via extracellular vesicles.

    PubMed

    Read, Jolene; Ingram, Alistair; Al Saleh, Hassan A; Platko, Khrystyna; Gabriel, Kathleen; Kapoor, Anil; Pinthus, Jehonathan; Majeed, Fadwa; Qureshi, Talha; Al-Nedawi, Khalid

    2017-01-01

    Epidermal growth factor receptor (EGFR) plays a central role in the progression of several human malignancies. Although EGFR is a membrane receptor, it undergoes nuclear translocation, where it has a distinct signalling pathway. Herein, we report a novel mechanism by which cancer cells can directly transport EGFR to the nucleus of other cells via extracellular vesicles (EVs). The transported receptor is active and stimulates the nuclear EGFR pathways. Interestingly, the translocation of EGFR via EVs occurs independently of the nuclear localisation sequence that is required for nuclear translocation of endogenous EGFR. Also, we found that the mutant receptor EGFRvIII could be transported to the nucleus of other cells via EVs. To assess the role of EVs in the regulation of an actual nuclear receptor, we studied the regulation of androgen receptor (AR). We found that full-length AR and mutant variant ARv7 are secreted in EVs derived from prostate cancer cell lines and could be transported to the nucleus of AR-null cells. The EV-derived AR was able to bind the androgen-responsive promoter region of prostate specific antigen, and recruit RNA Pol II, an indication of active transcription. The nuclear-translocated AR via EVs enhanced the proliferation of acceptor cells in the absence of androgen. Finally, we provide evidence that nuclear localisation of AR could occur in vivo via orthotopically-injected EVs in male SCID mice prostate glands. To our knowledge, this is the first study showing the nuclear translocation of nuclear receptors via EVs, which significantly extends the role of EVs as paracrine transcriptional regulators.

  9. Reprint of "Nuclear transport factors: global regulation of mitosis".

    PubMed

    Forbes, Douglass J; Travesa, Anna; Nord, Matthew S; Bernis, Cyril

    2015-06-01

    The unexpected repurposing of nuclear transport proteins from their function in interphase to an equally vital and very different set of functions in mitosis was very surprising. The multi-talented cast when first revealed included the import receptors, importin alpha and beta, the small regulatory GTPase RanGTP, and a subset of nuclear pore proteins. In this review, we report that recent years have revealed new discoveries in each area of this expanding story in vertebrates: (a) The cast of nuclear import receptors playing a role in mitotic spindle regulation has expanded: both transportin, a nuclear import receptor, and Crm1/Xpo1, an export receptor, are involved in different aspects of spindle assembly. Importin beta and transportin also regulate nuclear envelope and pore assembly. (b) The role of nucleoporins has grown to include recruiting the key microtubule nucleator – the γ-TuRC complex – and the exportin Crm1 to the mitotic kinetochores of humans. Together they nucleate microtubule formation from the kinetochores toward the centrosomes. (c) New research finds that the original importin beta/RanGTP team have been further co-opted by evolution to help regulate other cellular and organismal activities, ranging from the actual positioning of the spindle within the cell perimeter, to regulation of a newly discovered spindle microtubule branching activity, to regulation of the interaction of microtubule structures with specific actin structures. (d) Lastly, because of the multitudinous roles of karyopherins throughout the cell cycle, a recent large push toward testing their potential as chemotherapeutic targets has begun to yield burgeoning progress in the clinic.

  10. Methamphetamine oxidative stress, neurotoxicity, and functional deficits are modulated by nuclear factor-E2-related factor 2.

    PubMed

    Ramkissoon, Annmarie; Wells, Peter G

    2015-12-01

    Activation of redox-sensitive transcription factors like nuclear factor-E2-related factor 2 (Nrf2) can enhance the transcription of cytoprotective genes during oxidative stress. We investigated whether Nrf2 is activated by methamphetamine (METH) thereby altering neurotoxicity in Nrf2 +/+ and -/- adult mouse brain. A single dose of METH can induce the mRNA levels of Nrf2-regulated antioxidant and cytoprotective proteins in mouse brain. Multiple-day dosing with METH enhanced DNA oxidation and decreased tyrosine hydroxylase and dopamine transporter staining in the striatum, indicating dopaminergic nerve terminal toxicity, which was more severe in -/- mice, as were deficits in motor coordination and olfactory discrimination. These Nrf2-dependent effects were independent of changes in METH metabolism or the induction of hyperthermia. Similarly, METH increased striatal glial fibrillary acidic protein, indicating neurotoxicity. METH neurotoxicity was also observed in the glial cells and in the GABAergic system of the olfactory bulbs and was enhanced in -/- mice, whereas dopaminergic parameters were unaffected. With one-day dosing of METH, there were no differences between +/+ and -/- mice in either basal or METH-enhanced DNA oxidation and neurotoxicity markers. Nrf2-mediated pathways accordingly may protect against the neurodegenerative effects and functional deficits initiated by METH and perhaps other reactive oxygen species-enhancing neurotoxicants, when there is time for transcriptional activation and protein induction. In human users of METH, this mechanism may be essential when differences in drug abuse patterns may alter the induction and duration of Nrf2 activation thereby modulating susceptibility to the neurotoxic effects of METH.

  11. Nuclear Factor-Y is an adipogenic factor that regulates leptin gene expression

    PubMed Central

    Lu, Yi-Hsueh; Dallner, Olof Stefan; Birsoy, Kivanc; Fayzikhodjaeva, Gulya; Friedman, Jeffrey M.

    2015-01-01

    Objective Leptin gene expression is highly correlated with cellular lipid content in adipocytes but the transcriptional mechanisms controlling leptin expression in vivo are poorly understood. In this report, we set out to identify cis- and trans-regulatory elements controlling leptin expression. Methods Leptin-BAC luciferase transgenic mice combining with other computational and molecular techniques were used to identify transcription regulatory elements including a CCAAT-binding protein Nuclear Factor Y (NF-Y). The function of NF-Y in adipocyte was studied in vitro with 3T3-L1 cells and in vivo with adipocyte-specific knockout of NF-Y. Results Using Leptin-BAC luciferase mice, we showed that DNA sequences between −22 kb and +8.8 kb can confer quantitative expression of a leptin reporter. Computational analysis of sequences and gel shift assays identified a 32 bp sequence (chr6: 28993820–2899385) consisting a CCAAT binding site for Nuclear Factor Y (NF-Y) and this was confirmed by a ChIP assay in vivo. A deletion of this 32 bp sequence in the −22 kb to +8.8 kb leptin-luciferase BAC reporter completely abrogates luciferase reporter activity in vivo. RNAi mediated knockdown of NF-Y interfered with adipogenesis in vitro and adipocyte-specific knockout of NF-Y in mice reduced expression of leptin and other fat specific genes in vivo. Further analyses of the fat specific NF-Y knockout revealed that these animals develop a moderately severe lipodystrophy that is remediable with leptin therapy. Conclusions These studies advance our understanding of leptin gene expression and show that NF-Y controls the expression of leptin and other adipocyte genes and identifies a new form of lipodystrophy. PMID:25973387

  12. Dietary bitter melon seed increases peroxisome proliferator-activated receptor-γ gene expression in adipose tissue, down-regulates the nuclear factor-κB expression, and alleviates the symptoms associated with metabolic syndrome.

    PubMed

    Gadang, Vidya; Gilbert, William; Hettiararchchy, Navam; Horax, Ronny; Katwa, Laxmansa; Devareddy, Latha

    2011-01-01

    The objective of this study was to examine the extent to which bitter melon seed (BMS) alleviates the symptoms associated with metabolic syndrome and elucidate the mechanism by which BMS exerts beneficial effects. Three-month-old female Zucker rats were assigned to following groups: lean control (L-Ctrl), obese control (O-Ctrl), and obese + BMS (O-BMS). The control groups were fed AIN-93M purified rodent diet, and the O-BMS group was fed AIN-93M diet modified to contain 3.0% (wt/wt) ground BMS for 100 days. After 100 days of treatment, BMS supplementation in the obese rats lowered the total serum cholesterol by 38% and low-density lipoprotein-cholesterol levels by about 52% and increased the ratio of serum high-density lipoprotein-cholesterol to total cholesterol compared to the O-Ctrl group. The percentage of total liver lipids was about 32% lower and serum triglyceride levels were 71% higher in the O-BMS group compared to the O-Ctrl group. Serum glucose levels were significantly lowered partly because of the increase in the serum insulin levels in the BMS-based diet groups. BMS supplementation increased the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) in the white adipose tissue of the obese rats significantly (P < .05) and down-regulated the expression of PPAR-γ, nuclear factor-κB (NF-κB), and interferon-γ mRNA in heart tissue of the obese rats. The findings of this study suggest that BMS improves the serum and liver lipid profiles and serum glucose levels by modulating PPAR-γ gene expression. To our knowledge, this study for the first time shows that BMS exerts cardioprotective effects by down-regulating the NF-κB inflammatory pathway.

  13. Down-regulation of mitogen-activated protein kinases and nuclear factor-κB signaling is involved in rapamycin suppression of TLR2-induced inflammatory response in monocytic THP-1 cells.

    PubMed

    Sun, Ruili; Zhang, Yi; Ma, Shijiang; Qi, Hengtian; Wang, Mingyong; Duan, Juhong; Ma, Shujun; Zhu, Xiaofei; Li, Guancheng; Wang, Hui

    2015-10-01

    Tripalmitoyl-S-glycero-Cys-(Lys) 4 (Pam3CSK4) interacted with TLR2 induces inflammatory responses through the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signal pathway. Rapamycin can suppress TLR-induced inflammatory responses; however, the detailed molecular mechanism is not fully understood. Here, the mechanism by which rapamycin suppresses TLR2-induced inflammatory responses was investigated. It was found that Pam3CSK4-induced pro-inflammatory cytokines were significantly down-regulated at both the mRNA and protein levels in THP-1 cells pre-treated with various concentrations of rapamycin. Inhibition of phosphatidylinositol 3-kinase/protein kinase-B (PI3K/AKT) signaling did not suppress the expression of pro-inflammatory cytokines, indicating that the immunosuppression mediated by rapamycin in THP1 cells is independent of the PI3K/AKT pathway. RT-PCR showed that Erk and NF-κB signal pathways are related to the production of pro-inflammatory cytokines. Inhibition of Erk or NF-κB signaling significantly down-regulated production of pro-inflammatory cytokines. Additionally, western blot showed that pre-treatment of THP-1 cells with rapamycin down-regulates MAPKs and NF-κB signaling induced by Pam3CSK4 stimulation, suggesting that rapamycin suppresses Pam3CSK4-induced pro-inflammatory cytokines via inhibition of TLR2 signaling. It was concluded that rapamycin suppresses TLR2-induced inflammatory responses by down-regulation of Erk and NF-κB signaling.

  14. Human Factors Engineering Review Model for advanced nuclear power reactors

    SciTech Connect

    O'Hara, J.; Higgins, J. ); Goodman, C.; Galletti, G.: Eckenrode, R. )

    1993-01-01

    One of the major issues to emerge from the initial design reviews under the certification process was that detailed human-systems interface (HSI) design information was not available for staff review. To address the lack of design detail issue. The Nuclear Regulatory Commission (NRC) is performing the design certification reviews based on a design process plan which describes the human factors engineering (HFE) program elements that are necessary and sufficient to develop an acceptable detailed design specification. Since the review of a design process is unprecedented in the nuclear industry. The criteria for review are not addressed by current regulations or guidance documents and. therefore, had to be developed. Thus, an HFE Program Review Model was developed. This paper will describe the model's rationale, scope, objectives, development, general characteristics. and application.

  15. Human Factors Engineering Review Model for advanced nuclear power reactors

    SciTech Connect

    O`Hara, J.; Higgins, J.; Goodman, C.; Galletti, G.: Eckenrode, R.

    1993-05-01

    One of the major issues to emerge from the initial design reviews under the certification process was that detailed human-systems interface (HSI) design information was not available for staff review. To address the lack of design detail issue. The Nuclear Regulatory Commission (NRC) is performing the design certification reviews based on a design process plan which describes the human factors engineering (HFE) program elements that are necessary and sufficient to develop an acceptable detailed design specification. Since the review of a design process is unprecedented in the nuclear industry. The criteria for review are not addressed by current regulations or guidance documents and. therefore, had to be developed. Thus, an HFE Program Review Model was developed. This paper will describe the model`s rationale, scope, objectives, development, general characteristics. and application.

  16. Nuclear factor kappa B role in inflammation associated gastrointestinal malignancies.

    PubMed

    Gambhir, Sahil; Vyas, Dinesh; Hollis, Michael; Aekka, Apporva; Vyas, Arpita

    2015-03-21

    Nuclear factor kappa B (NF-κB) has an established role in the regulation of innate immunity and inflammation. NF-κB is also involved in critical mechanisms connecting inflammation and cancer development. Recent investigations suggest that the NF-κB signaling cascade may be the central mediator of gastrointestinal malignancies including esophageal, gastric and colorectal cancers. This review will explore NF-κB's function in inflammation-associated gastrointestinal malignancies, highlighting its oncogenic contribution to each step of carcinogenesis. NF-κB's role in the inflammation-to-carcinoma sequence in gastrointestinal malignancies warrants stronger emphasis upon targeting this pathway in achieving greater therapeutic efficacy.

  17. Hepatocyte nuclear factor 4α regulation of bile acid and drug metabolism

    PubMed Central

    Chiang, John YL

    2013-01-01

    The hepatocyte nuclear factor 4α (HNF4α) is a liver-enriched nuclear receptor that plays a critical role in early morphogenesis, fetal liver development, liver differentiation and metabolism. Human HNF4α gene mutations cause maturity on-set diabetes of the young type 1, an autosomal dominant non-insulin-dependent diabetes mellitus. HNF4α is an orphan nuclear receptor because of which the endogenous ligand has not been firmly identified. The trans-activating activity of HNF4α is enhanced by interacting with co-activators and inhibited by corepressors. Recent studies have revealed that HNF4α plays a central role in regulation of bile acid metabolism in the liver. Bile acids are required for biliary excretion of cholesterol and metabolites, and intestinal absorption of fat, nutrients, drug and xenobiotics for transport and distribution to liver and other tissues. Bile acids are signaling molecules that activate nuclear receptors to control lipids and drug metabolism in the liver and intestine. Therefore, HNF4α plays a central role in coordinated regulation of bile acid and xenobiotics metabolism. Drugs that specifically activate HNF4α could be developed for treating metabolic diseases such as diabetes, dyslipidemia and cholestasis, as well as drug metabolism and detoxification. PMID:19239393

  18. A Pro-Inflammatory Role for Nuclear Factor Kappa B in Childhood Obstructive Sleep Apnea Syndrome

    PubMed Central

    Israel, Lee P.; Benharoch, Daniel; Gopas, Jacob; Goldbart, Aviv D.

    2013-01-01

    Study Objectives: Childhood obstructive sleep apnea syndrome (OSAS) is associated with an elevation of inflammatory markers such as C-reactive protein (CRP) that correlates with specific morbidities and subsides following intervention. In adults, OSAS is associated with activation of the transcription factor nuclear factor kappa B (NF-kB). We explored the mechanisms underlying NF-kB activation, based on the hypothesis that specific NF-kB signaling is activated in children with OSAS. Design: Adenoid and tonsillar tissues from children with OSAS and matched controls were immunostained against NF-kB classical (p65 and p50) and alternative (RelB and p52) pathway subunits, and NF-kB-dependent cytokines: interleukin (IL)- 1α, IL-1β, tumor necrosis factor-α, and IL-8). Serum CRP levels were measured in all subjects. NF-kB induction was evaluated by a luciferase-NF-kB reporter assay in L428 cells constitutively expressing NF-kB and in Jurkat cells with inducible NF-kB expression. p65 translocation to the nucleus, reflecting NF-kB activation, was measured in cells expressing fluorescent NF-kB-p65-GFP (green fluorescent protein). Setting: Sleep research laboratory. Patients or Participants: Twenty-five children with OSAS and 24 without OSAS. Interventions: N/A. Measurements and Results: Higher expression of IL-1α and classical NF-kB subunits p65 and p50 was observed in adenoids and tonsils of children with OSAS. Patient serum induced NF-kB activity, as measured by a luciferase-NF-kB reporter assay and by induction of p65 nuclear translocation in cells permanently transfected with GFP-p65 plasmid. IL-1β showed increased epithelial expression in OSAS tissues. Conclusions: Nuclear factor kappa B is locally and systemically activated in children with obstructive sleep apnea syndrome. This observation may motivate the search for new anti-inflammatory strategies for controlling nuclear factor kappa B activation in obstructive sleep apnea syndrome. Citation: Israel LP

  19. Human Factors Considerations in New Nuclear Power Plants: Detailed Analysis.

    SciTech Connect

    OHara,J.; Higgins, J.; Brown, W.; Fink, R.

    2008-02-14

    This Nuclear Regulatory Commission (NRC) sponsored study has identified human-performance issues in new and advanced nuclear power plants. To identify the issues, current industry developments and trends were evaluated in the areas of reactor technology, instrumentation and control technology, human-system integration technology, and human factors engineering (HFE) methods and tools. The issues were organized into seven high-level HFE topic areas: Role of Personnel and Automation, Staffing and Training, Normal Operations Management, Disturbance and Emergency Management, Maintenance and Change Management, Plant Design and Construction, and HFE Methods and Tools. The issues where then prioritized into four categories using a 'Phenomena Identification and Ranking Table' methodology based on evaluations provided by 14 independent subject matter experts. The subject matter experts were knowledgeable in a variety of disciplines. Vendors, utilities, research organizations and regulators all participated. Twenty issues were categorized into the top priority category. This Brookhaven National Laboratory (BNL) technical report provides the detailed methodology, issue analysis, and results. A summary of the results of this study can be found in NUREG/CR-6947. The research performed for this project has identified a large number of human-performance issues for new control stations and new nuclear power plant designs. The information gathered in this project can serve as input to the development of a long-term strategy and plan for addressing human performance in these areas through regulatory research. Addressing human-performance issues will provide the technical basis from which regulatory review guidance can be developed to meet these challenges. The availability of this review guidance will help set clear expectations for how the NRC staff will evaluate new designs, reduce regulatory uncertainty, and provide a well-defined path to new nuclear power plant licensing.

  20. Delineating role of ubiquitination on nuclear factor-kappa B pathway by a computational modeling approach

    SciTech Connect

    Lee, Jungsul; Choi, Kyungsun; Choi, Chulhee

    2010-01-01

    Mutant ubiquitin found in neurodegenerative diseases has been thought to hamper activation of transcription factor nuclear factor-kappa B (NF-{kappa}B) by inhibiting ubiquitin-proteasome system (UPS). It has been reported that ubiquitin also is involved in signal transduction in an UPS-independent manner. We used a modeling and simulation approach to delineate the roles of ubiquitin on NF-{kappa}B activation. Inhibition of proteasome complex increased maximal activation of IKK mainly by decreasing the UPS efficiency. On the contrary, mutant ubiquitin decreased maximal activity of IKK. Computational modeling showed that the inhibition effect of mutant ubiquitin is mainly attributed to decreased activity of UPS-independent function of ubiquitin. Collectively, our results suggest that mutant ubiquitin affects NF-{kappa}B activation in an UPS-independent manner.

  1. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

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

    PubMed

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

    2016-10-01

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

  3. Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways

    PubMed Central

    2014-01-01

    Introduction Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. Methods A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. Results Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N

  4. Serum osteoprotegerin and soluble receptor activator of nuclear factor kappaB ligand levels in patients with a history of differentiated thyroid carcinoma: a case-controlled cohort study.

    PubMed

    Giusti, Massimo; Cecoli, Francesca; Fazzuoli, Laura; De Franchis, Vincenzina; Ceresola, Enrica; Ferone, Diego; Mussap, Michele; Minuto, Francesco

    2007-05-01

    Overt hyperthyroidism is associated with changes in bone metabolism, whereas the effect of levothyroxine (L-T4) load in patients with differentiated thyroid carcinoma (DTC) is controversial. The aim of our study was to evaluate osteoprotegerin (OPG) and soluble receptor activator