Activation of the NF-kappaB transcription factor in a T-lymphocytic cell line by hypochlorous acid.
ABSTRACT: Reactive oxygen species (ROS) such as hydrogen peroxide serve as second messengers in the induction of the transcription factor NF-kappaB, and hence in the activation and replication of human immunodeficiency virus type 1 (HIV-1) in human cells. During inflammatory reactions, many oxidative species are produced, one of which is hypochlorous acid (HOCl), which is responsible for the microbicidal effects of activated human polymorphonuclear leukocytes. Treatment of a T-lymphocytic cell line with micromolar concentrations of HOCl promoted the appearance of transcription factor NF-kappaB (the heterodimer p50/p65) in the nucleus of the cells, even in the absence of de novo protein synthesis. Western blot analysis of the NF-kappaB inhibitory subunits (IkappaB) demonstrated that both IkappaB-alpha proteolysis and p105 processing were induced by the treatment. NF-kappaB activation was very effective when cells were subjected to hyperthermia before being treated with HOCl. Various antioxidants, such as pyrrolidine dithiocarbamate, p-bromophenacyl-bromide and nordihydroguaiaretic acid could strongly reduce NF-kappaB translocation, demonstrating the importance of oxidative species in the transduction mechanism. Moreover, ACH-2 cells treated with HOCl or H2O2 released tumour necrosis factor-alpha (TNF-alpha) in the supernatants. The importance of TNF-alpha release in NF-kappaB induction by HOCl or H2O2 was demonstrated by the fact that: (1) the nuclear appearance of NF-kappaB was promoted in untreated cells; and (2) synergism between TNF-alpha and HOCl was detected. Collectively, these results suggest that HOCl should be considered as an oxidative species capable of inducing NF-kappaB in a T-lymphocytic cell line through a transduction mechanism involving ROS, and having a long-distance effect through subsequent TNF-alpha release.
Project description:Treatment of cancer with tumor necrosis factor-alpha (TNF-alpha) is hindered by resistance and toxicity. The flexible heteroarotinoid, SHetA2, sensitizes resistant ovarian cancer cells to TNF-alpha-induced extrinsic apoptosis, and also induces intrinsic apoptosis as a single agent. This study tested the hypothesis that nuclear factor-kappaB (NF-kappaB) is involved in SHetA2-regulated intrinsic and extrinsic apoptosis. SHetA2 inhibited basal and TNF-alpha-induced or hydrogen peroxide-induced NF-kappaB activity through counter-regulation of upstream kinase (IkappaB kinase) activity, inhibitor protein (IkappaB-alpha) phosphorylation, and p-65 NF-kappaB subunit nuclear translocation, but independently of reactive oxygen species generation. Ectopic over-expression of p-65, or treatment with TNF-alpha receptor 1 (TNFR1) small interfering RNA or a caspase-8 inhibitor, each attenuated synergistic apoptosis by SHetA2 and TNF-alpha, but did not affect intrinsic apoptosis caused by SHetA2. In conclusion, NF-kappaB repression is involved in SHetA2 circumvention of resistance to TNF-alpha-induced extrinsic apoptosis, but not in SHetA2 induction of intrinsic apoptosis.
Project description:Inflammation is beneficial when it is part of the innate immune response, but harmful when it occurs in an unregulated, chronic manner. We now report that IkappaB-beta, a member of the classical IkappaB family, serves a dual role of both inhibiting and facilitating the inflammatory response. IkappaB-beta degradation releases NF-kappaB dimers which upregulate proinflammatory target genes such as TNF-alpha. Suprisingly absence of IkappaB-beta results in a dramatic reduction of TNF-alpha in response to LPS even though the activation of NF-kappaB is normal. The inhibition of TNF-alpha mRNA expression can be correlated to the absence of nuclear, hypophosphorylated-IkappaB-beta bound to p65:cRel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaB-beta acts through p65:cRel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaB-beta knockout mice are resistant to LPS induced septic shock and collagen-induced arthritis, and therefore blocking IkappaB-beta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha producting during the inflammatory response. Overall design: Wild type and IkappaB-beta knockout BMDM cells were stimulated with LPS(1ug/ml) for 0, 1, and 5 hours. RNA isolated from the cells was analyzed on Affymetrix Mouse Genome 430A 2.0 gene expression chip.
Project description:Chromaffin cells of the adrenal medulla elaborate and secrete catecholamines and neuropeptides for hormonal and paracrine signaling in stress and during inflammation. We have recently documented the action of the cytokine TNF-alpha on neuropeptide secretion and biosynthesis in isolated bovine chromaffin cells. Here, we demonstrate that the type 2 TNF-alpha receptor (TNF-R2) mediates TNF-alpha signaling in chromaffin cells via activation of nuclear factor (NF)-kappaB. Microarray and suppression subtractive hybridization have been used to identify TNF-alpha target genes in addition to those encoding the neuropeptides galanin, vasoactive intestinal polypeptide, and secretogranin II in chromaffin cells. TNF-alpha, acting through the TNF-R2, causes an early up-regulation of NF-kappaB, long-lasting induction of the NF-kappaB target gene inhibitor kappaB (IkappaB), and persistent stimulation of other NF-kappaB-associated genes including mitogen-inducible gene-6 (MIG-6), which acts as an IkappaB signaling antagonist, and butyrate-induced transcript 1. Consistent with long-term activation of the NF-kappaB signaling pathway, delayed induction of neuropeptide gene transcription by TNF-alpha in chromaffin cells is blocked by an antagonist of NF-kappaB signaling. TNF-alpha-dependent signaling in neuroendocrine cells thus leads to a unique, persistent mode of NF-kappaB activation that features long-lasting transcription of both IkappaB and MIG-6, which may play a role in the long-lasting effects of TNF-alpha in regulating neuropeptide output from the adrenal, a potentially important feedback station for modulating long-term cytokine effects in inflammation.
Project description:ROS (reactive oxygen species) play important roles in the progression of a number of human pathologies. ROS promote cell death, but can also induce gene transcription. The transcription factor NF-kappaB (nuclear factor kappaB) plays a critical role in oxidative stress responses. One of the proteins regulated by NF-kappaB is the zinc-finger protein A20. In TNF (tumour necrosis factor)-alpha signalling, NF-kappaB induction of A20 leads to increased cell survival. In the present paper, we show that in response to oxidative stress, A20 actually enhances cell death by necrosis, but not by apoptosis. Exposure of cells to ROS leads to the up-regulation of A20 which acts via a negative-feedback loop to block NF-kappaB activation and cellular survival. Silencing of A20 by RNAi (RNA interference) increases both the induction of NF-kappaB and the subsequent survival of cells exposed to high doses of oxidative stress, which, in untreated cells, promotes death by necrosis. Cells which express high basal levels of A20 are less protected from oxidative-stress-induced cell death when compared with cells with lower A20 expression. We also show that A20 regulates NF-kappaB by blocking the degradation of IkappaB (inhibitory protein kappaB) alpha. These data highlight a novel role for A20 in oxidative stress responses by terminating NF-kappaB-dependent survival signalling and thus sensitizing cells to death by necrosis.
Project description:Mammalian signaling networks contain an abundance of negative feedback regulators that may have overlapping ("fail-safe") or specific functions. Within the NF-kappaB signaling module, IkappaB alpha is known as a negative feedback regulator, but the newly characterized inhibitor IkappaB delta is also inducibly expressed in response to inflammatory stimuli. To examine IkappaB delta's roles in inflammatory signaling, we mathematically modeled the 4-IkappaB-containing NF-kappaB signaling module and developed a computational phenotyping methodology of general applicability. We found that IkappaB delta, like IkappaB alpha, can provide negative feedback, but each functions stimulus-specifically. Whereas IkappaB delta attenuates persistent, pathogen-triggered signals mediated by TLRs, the more prominent IkappaB alpha does not. Instead, IkappaB alpha, which functions more rapidly, is primarily involved in determining the temporal profile of NF-kappaB signaling in response to cytokines that serve intercellular communication. Indeed, when removing the inducing cytokine stimulus by compound deficiency of the tnf gene, we found that the lethality of ikappab alpha(-/-) mouse was rescued. Finally, we found that IkappaB delta provides signaling memory owing to its long half-life; it integrates the inflammatory history of the cell to dampen NF-kappaB responsiveness during sequential stimulation events.
Project description:Inflammation is beneficial when it is part of the innate immune response, but harmful when it occurs in an unregulated, chronic manner. We now report that IkappaB-beta, a member of the classical IkappaB family, serves a dual role of both inhibiting and facilitating the inflammatory response. IkappaB-beta degradation releases NF-kappaB dimers which upregulate proinflammatory target genes such as TNF-alpha. Suprisingly absence of IkappaB-beta results in a dramatic reduction of TNF-alpha in response to LPS even though the activation of NF-kappaB is normal. The inhibition of TNF-alpha mRNA expression can be correlated to the absence of nuclear, hypophosphorylated-IkappaB-beta bound to p65:cRel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaB-beta acts through p65:cRel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaB-beta knockout mice are resistant to LPS induced septic shock and collagen-induced arthritis, and therefore blocking IkappaB-beta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha producting during the inflammatory response. Wild type and IkappaB-beta knockout BMDM cells were stimulated with LPS(1ug/ml) for 0, 1, and 5 hours. RNA isolated from the cells was analyzed on Affymetrix Mouse Genome 430A 2.0 gene expression chip.
Project description:The activation of pro-inflammatory gene programs by nuclear factor-kappaB (NF-kappaB) is primarily regulated through cytoplasmic sequestration of NF-kappaB by the inhibitor of kappaB (IkappaB) family of proteins. IkappaBbeta, a major isoform of IkappaB, can sequester NF-kappaB in the cytoplasm, although its biological role remains unclear. Although cells lacking IkappaBbeta have been reported, in vivo studies have been limited and suggested redundancy between IkappaBalpha and IkappaBbeta. Like IkappaBalpha, IkappaBbeta is also inducibly degraded; however, upon stimulation by lipopolysaccharide (LPS), it is degraded slowly and re-synthesized as a hypophosphorylated form that can be detected in the nucleus. The crystal structure of IkappaBbeta bound to p65 suggested this complex might bind DNA. In vitro, hypophosphorylated IkappaBbeta can bind DNA with p65 and c-Rel, and the DNA-bound NF-kappaB:IkappaBbeta complexes are resistant to IkappaBalpha, suggesting hypophosphorylated, nuclear IkappaBbeta may prolong the expression of certain genes. Here we report that in vivo IkappaBbeta serves both to inhibit and facilitate the inflammatory response. IkappaBbeta degradation releases NF-kappaB dimers which upregulate pro-inflammatory target genes such as tumour necrosis factor-alpha (TNF-alpha). Surprisingly, absence of IkappaBbeta results in a dramatic reduction of TNF-alpha in response to LPS even though activation of NF-kappaB is normal. The inhibition of TNF-alpha messenger RNA (mRNA) expression correlates with the absence of nuclear, hypophosphorylated-IkappaBbeta bound to p65:c-Rel heterodimers at a specific kappaB site on the TNF-alpha promoter. Therefore IkappaBbeta acts through p65:c-Rel dimers to maintain prolonged expression of TNF-alpha. As a result, IkappaBbeta(-/-) mice are resistant to LPS-induced septic shock and collagen-induced arthritis. Blocking IkappaBbeta might be a promising new strategy for selectively inhibiting the chronic phase of TNF-alpha production during the inflammatory response.
Project description:The role of azadirachtin, an active component of a medicinal plant Neem (Azadirachta indica), on TNF-induced cell signaling in human cell lines was investigated. Azadirachtin blocks TNF-induced activation of nuclear factor kappaB (NF-kappaB) and also expression of NF-kappaB-dependent genes such as adhesion molecules and cyclooxygenase 2. Azadirachtin inhibits the inhibitory subunit of NF-kappaB (IkappaB alpha) phosphorylation and thereby its degradation and RelA (p65) nuclear translocation. It blocks IkappaB alpha kinase (IKK) activity ex vivo, but not in vitro. Surprisingly, azadirachtin blocks NF-kappaB DNA binding activity in transfected cells with TNF receptor-associated factor (TRAF)2, TNF receptor-associated death domain (TRADD), IKK, or p65, but not with TNFR, suggesting its effect is at the TNFR level. Azadirachtin blocks binding of TNF, but not IL-1, IL-4, IL-8, or TNF-related apoptosis-inducing ligand (TRAIL) with its respective receptors. Anti-TNFR antibody or TNF protects azadirachtin-mediated down-regulation of TNFRs. Further, in silico data suggest that azadirachtin strongly binds in the TNF binding site of TNFR. Overall, our data suggest that azadirachtin modulates cell surface TNFRs thereby decreasing TNF-induced biological responses. Thus, azadirachtin exerts an anti-inflammatory response by a novel pathway, which may be beneficial for anti-inflammatory therapy.
Project description:The NF-kappaB pathway plays a pivotal role in proliferation, differentiation, apoptosis, and immune responses in mammals. The NF-kappaB inhibitor, IkappaB, has classically been characterized for its ability to sequester NF-kappaB transcription factors in the cytoplasm. Nevertheless, a nuclear fraction of IkappaBalpha has consistently been detected and associated with repression of nuclear NF-kappaB. Now we show that IkappaBalpha physically associates with different repression elements such as nuclear corepressors and histone acetyltransferases and deacetylases (HDACs). More remarkably, chromatin immunoprecipitation experiments demonstrate that IkappaBalpha is recruited to the promoter regions of the Notch-target gene, hes1, together with HDAC1 and -5, whereas we did not detect IkappaBalpha associated with classical NF-kappaB target genes such as IL6 and RANTES. TNF-alpha treatment results in a temporary release of IkappaBalpha from the hes1 promoter that correlates with increased histone acetylation and transcriptional activation. In addition, we demonstrate that both IkappaB kinase-alpha and -beta are simultaneously recruited to the hes1 promoter in response to TNF-alpha, coinciding with a maximum of IkappaBalpha release and gene activation. Moreover, TNF-alpha-dependent histone H3 acetylation, release of IkappaBalpha from the hes1 promoter, and hes1 mRNA synthesis are affected in IKK-alpha(-/-) mouse embryonic fibroblasts. We propose that IkappaBalpha plays a previously undescribed role in regulating the recruitment of repression elements to specific promoters. Recruitment of IKKs to the nucleus in response to TNF-alpha may induce chromatin-associated IkappaBalpha release and gene activation. These findings provide additional insight in the cross-talk between NF-kappaB and other signaling pathways.
Project description:IkappaB-zeta [inhibitor of NF-kappaB (nuclear factor kappaB) zeta] is a nuclear protein that is induced upon stimulation of TLRs (Toll-like receptors) and IL (interleukin)-1 receptor. IkappaB-zeta harbours C-terminal ankyrin repeats that interact with NF-kappaB. Our recent studies have shown that, upon stimulation, IkappaB-zeta is essential for the induction of a subset of inflammatory genes, represented by IL-6, whereas it inhibits the expression of TNF (tumour necrosis factor)-alpha. In the present study, we investigated mechanisms that determine the different functions of IkappaB-zeta. We found that co-expression of IkappaB-zeta and the NF-kappaB subunits synergistically activates transcription of the hBD-2 (human beta-defensin 2) and NGAL (neutrophil gelatinase-associated lipocalin) genes, whereas it inhibits transcription of E-selectin. Reporter analyses indicated that, in addition to an NF-kappaB-binding site, a flanking C/EBP (CCAAT/enhancer-binding protein)-binding site in the promoters is essential for the IkappaB-zeta-mediated transcriptional activation. Using an artificial promoter consisting of the NF-kappaB- and C/EBP-binding sites, transcriptional activation was observed upon co-transfection with IkappaB-zeta and NF-kappaB, indicating that these sequences are minimal elements that confer the IkappaB-zeta-mediated transcriptional activation. Chromatin immunoprecipitation assays and knockdown experiments showed that both IkappaB-zeta and the NF-kappaB subunits were recruited to the NGAL promoter and were essential for the transcriptional activation of the hBD-2 and NGAL promoters on stimulation with IL-1beta. The activation of the NGAL promoter by transfection of IkappaB-zeta and NF-kappaB was suppressed in C/EBPbeta-depleted cells. Thus IkappaB-zeta acts as an essential transcriptional activator by forming a complex with NF-kappaB on promoters harbouring the NF-kappaB- and C/EBP-binding sites, upon stimulation of TLRs or IL-1 receptor.