No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
NF-ϰB: A Multifaceted Transcription Factor Regulated at Several Levels
Abstract
NF-kappaB is a generic name for an evolutionarily conserved transcription-factor system that contributes to the mounting of an effective immune response but is also involved in the regulation of cell proliferation, development, and apoptosis. The implication of NF-kappaB in central biological processes and its extraordinary connectivity to other signaling pathways raise a need for highly controlled regulation of NF-kappaB activity at several levels. While all NF-kappaB activation pathways share a central and critical proteasome-mediated step that leads to the degradation of inhibitory proteins and the release of DNA-binding subunits, there is evidence for a downstream level of NF-kappaB regulation that employs several mechanisms. These include promoter-specific exchange of dimers and modification of the transactivating p65 subunit by phosphorylation, acetylation, ubiquitination, or prolyl isomerization. The signaling pathways and enzymes controlling this second level of regulation and their potential use as therapeutic targets for the treatment of NF-kappaB-associated pathologies are discussed here.
... ). Ces protéines sont des facteurs de transcription qui fonctionnent en dimères, capables de se fixer l'ADN et qui possèdent un domaine RHD (NF-κB/Rel homology domain)(Schmitz, M. L. et al., 2004).Toutefois, seules les protéines p65, c-Rel et RelB possèdent un domaine trans-activateur. L'association p65/p50 est le dimère NF-κB le plus abondant et qui possède la plus grande activité trans-activatrice. ...
... L'association p65/p50 est le dimère NF-κB le plus abondant et qui possède la plus grande activité trans-activatrice. Au repos, les dimères sont séquestrés dans le cytoplasme par une protéine de la famille des IκB (inhibitor of κB) comprenant IκBα et IκBβ exprimées de façon ubiquitaire, IκBγ et IκBε dont l'expression se limite aux cellules lymphoïdes(Mercurio, F. and Manning, A., 1999;Schmitz, M. L. et al., 2004). Sous l'action de signaux multiples en amont de la voie, les IκBs vont être dégradées par le protéasome, libérant ainsi le domaine NLS (nuclear localisation sequence) des sous-unités du complexe NF-κB. ...
... Sous l'action de signaux multiples en amont de la voie, les IκBs vont être dégradées par le protéasome, libérant ainsi le domaine NLS (nuclear localisation sequence) des sous-unités du complexe NF-κB. Cette dégradation est induite majoritairement par trois voies d'activation : (i) la voie classique (NEMO dépendante), (ii) la voie apoptotique et (iii) la voie non-classique (NEMO indépendante)(Schmitz, M. L. et al., 2004;Viatour, P. et al., 2005).La voie classique est la voie majoritaire d'activation du dimère p65/p50 et fait intervenir le complexe IKK (IκB kinase). Ce complexe cytoplasmique d'activation se compose des kinases IKKα et IKKβ associées à un dimère de protéines NEMO (=IKKε)(Joyce, D. et al., 2001;Viatour, P. et al., 2005) (Figure 18). ...
L’oncogène K-ras est une petite GTPase de la super famille RAS, fréquemment impliquée dans les cancers, en particulier celui du pancréas, l’un des plus mortels dans les pays occidentaux. Les mutations de l’oncogène K-ras sont considérées comme l’un des événements initiateurs de la cancérogenèse pancréatique et son activité oncogénique est un élément indispensable à la progression tumorale. Cependant, le ciblage thérapeutique de K-ras reste inefficace, à ce jour. Se focaliser sur les cibles précoces de son activité oncogénique semble ainsi être une stratégie alternative intéressante. La mucine membranaire MUC4 est une glycoprotéine de haut poids moléculaire fréquemment dérégulée dans les cancers. Dans le cancer du pancréas, MUC4 est néo-exprimée dès les stades prénéoplasiques et est impliquée dans les propriétés biologiques permettant la progression tumorale et la chimiorésistance. La régulation de MUC4 par K-ras dans la cancérogenèse pancréatique reste à décrypter. En utilisant le modèle murin de cancérogenèse pancréatique Pdx1-Cre; LStopL-K-rasG12D, nous avons mis en évidence que la néo-expression précoce de la mucine Muc4, dans les lésions pancréatiques prénéoplasiques intraépithéliales (PanINs) formées suite à la présence du K-ras muté, est corrélée avec l’activation des voies de signalisation ERK, JNK and NF-κB. In vitro, la transfection de mutants constitutivement activés de K-rasG12V dans les cellules tumorales humaines induit une augmentation de l’expression de MUC4. Cette activation intervient au niveau transcriptionel par le recrutement des facteurs de transcription AP-1 et NF-κB via les voies MAPK, JNK and NF-κB et au niveau post-transcriptionel par un mécanisme impliquant la RalB GTPase. Ensemble, ces résultats démontre que MUC4 est une cible transcriptionelle et post-transcriptionelle de l’activité oncogénique de K-ras dans le cancer du pancréas. Ces résultats ouvrent de nouvelles pistes pour développer des stratégies ciblant les étapes précoces de ce cancer.
... While posttranslational phosphorylation of p65 may be required for docking and expression of one gene, other posttranslational modifications of the transcription factor can prevent its docking on gene promoters [81][82][83][84] and transactivation despite the promoter regions of these genes containing the requisite kappa-B nucleotide motif. This phosphorylative coding helps shape p65-p50 transactivation selectivity after the transcription factor has translocated into 4 the nucleus [83,85] guiding it to highly specific genomic targets [86][87][88]. Despite the fact that the transcription factor potentially expresses as many as 150 different genes this phosphorylative coding, as will be further detailed, limits transactivation to the expression of genes which produce an appropriately measured behavioural response to the stimulus that started in motion the NF-kB activity [89][90][91][92][93]. ...
... As previously discussed, phosphorylation of various p65 residues affords the NF-kB transcription factor the phosphorylative coding required to dock on some promoters for transactivation and not others once it has translocated. For example, phosphorylation of p65 serine 536 is expected to be necessary for nuclear translocation but is also required for transcription of IL-6 [85]. However, lack of p65 serine 536 phosphorylation abrogates p65 affinity for the IL-8 gene promotor [83]. ...
... The variable trigger points for inflammatory activity result in variations of the same p65 protein [85] that differ in their phosphorylation and heterodimer configurations; the outcome of which is multiple condition-specific responses from one transcription factor. Nevertheless, curcumin inhibits the different IKK isoforms involved in these different pathways and the different NF-kB heterodimer configurations that rise out of these different trigger points [23,154,155]. ...
Various parts of the turmeric plant have been used as medicinal treatment for various conditions from ulcers and arthritis to cardiovascular disease and neuroinflammation. The rhizome’s curcumin extract is the most studied active constituent, which exhibits an expansive polypharmacology with influence on many key inflammatory markers. Despite the expansive reports of curcucmin’s therapeutic value, clinical reliability and research repeatability with curcumin treatment are still poor. The pharmacology must be better understood and reliably mapped if curcumin is to be accepted and used in modern medical applications. Although the polypharmacology of this extract has been considered, in mainstream medicine, to be a drawback, a perspective change reveals a comprehensive and even synergistic shaping of the NF-kB pathway, including transactivation. Much of the inconsistent research data and unreliable clinical outcomes may be due to a lack of standardization which also pervades research standard samples. The possibility of other well-known curcumin by-products contributing in the polypharmacology is also discussed. A new flowchart of crosstalk in transduction pathways that lead to shaping of nuclear NF-kB transactivation is generated and a new calibration or standardization protocol for the extract is proposed which could lead to more consistent data extraction and improved reliability in therapy.
... The mechanism of NF-κB activation in vivo remains contentious. The initiation and execution of apoptosis depend on activation of the extrinsic and intrinsic death pathways, including NF-κB pathway, JNK pathway (Catz and Johnson 2001).The NF-κB family of transcription factors have pivotal role in regulating this process (Schmitz et al. 2004). And the most common form of NF-κB is RelA(P65). ...
... Although the pro-apoptotic function of NF-κB has been characterized, the underlined mechanisms have yet to be fully resolved. Previous studies indicate that in response to cell stress, NF-κB promotes apoptosis through the nucleolar accumulation of RelA(P65) (Loveridge et al. 2008;Schmitz et al. 2004;Stark and Dunlop 2005), which is demonstrated to cause cell death by downregulating NF-κBdependent, anti-apoptotic gene transcription. Furthermore, some researchers also unraveled NF-κB-independent transcriptional, pro-apoptotic pathway that the relocalization of NPM induced by RelA(P65) facilitated the mitochondrial accumulation of BAX (Khandelwal et al. 2011). ...
Myocardial ischemia/reperfusion (I/R) injury induces cardiomyocyte apoptosis to deteriorate heart function. Thus, how to inhibit cardiomyocyte apoptosis is the focus of recent researches. Proteasome family member PSMB4 (proteasome subunit beta type-4) promotes cell survival. The relationship between PSMB4 and cardiomyocyte apoptosis during myocardial I/R is unknown. In this study, PSMB4 expression increased in rat myocardial I/R model, positively correlated with cleaved caspase-3 expression, negatively correlated with Bcl-2 expression. In vitro, neonatal ventricle cardiomyocyte hypoxia/reoxygenation (H/R) model was constructed to mimic myocardial I/R. PSMB4 silence promoted cardiomyocyte apoptosis and IκBα expression, inhibited the activation of NF-κB. On the contrary, PSMB4 overexpession inhibited cardiomyocyte apoptosis and IκBα expression, promoted the activation of NF-κB. Additionally, PSMB4-IκBα interaction was identified, suggesting that PSMB4 might participate in the proteasome dependent degradation of IκBα. The data indicates that PSMB4 inhibits cardiomyocyte apoptosis via activating NF-κB signaling pathway during myocardial I/R, which can supply novel molecular target for the treatment of ischemic heart disease.
... Importantly, there is a second level of regulation of NF-κB/Rel proteins mediated by post-translational modification, such as ubiquitination, prolyl isomerization, phosphorylation, and acetylation of NF-κB subunits [21,22]. This regulation controls the duration and amplitude of NF-κB activation through regulation of nuclear entry, DNA binding, transactivation, and the interaction of NF-κB proteins with other transactivators. ...
... Uncontrolled regulation of NF-κB is linked to disease states such as chronic inflammation and cancer [44]. The regulation of NF-κB by IκB is well recognized as one of the major regulatory mechanisms that modulate NF-κB activity [16,21,22,45]. Studies also indicate that an IκBα-independent termination of NF-κB signal transduction by ubiquitin-and proteasomedependent degradation of the p65 subunit in the nucleus is critical for the efficient termination of NF-κB activation [22,45]. ...
Background: Spectrin, beta, non-erythrocytic 1 (SPTBN1), an adapter protein for transforming growth factor beta (TGF-β) signaling, is recognized as a tumor suppressor in the development of hepatocellular carcinoma (HCC); however, the underlying molecular mechanisms of this tumor suppression remain obscure.
Methods: The effects on expression of pro-inflammatory cytokines upon the inhibition or impairment of SPTBN1 in HCC cell lines and liver tissues of Sptbn1+/- and wild-type (WT) mice were assessed by analyses of quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), enzyme linked immunosorbent assay (ELISA), Western blotting and gene array databases from HCC patients. We investigated the detailed molecular mechanisms underlying the inflammatory responses by immunoprecipitation-Western blotting, luciferase reporter assay, chromatin immunoprecipitation quantitative real time PCR (ChIP-qPCR), immunohistochemistry (IHC) and electrophoretic mobility shift assay (EMSA). The proportion of myeloid-derived suppressor cells in liver, spleen, bone marrow and peripheral blood samples from WT and Sptbn1+/- mice were measured by fluorescence-activated cell sorting (FACS) analysis. Further, the hepatocacinogenesis and its correlation with inflammatory microenvironment by loss of SPTBN1/SOCS1 and induction of p65 were analyzed by treating WT and Sptbn1+/- mice with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC).
Results: Loss of SPTBN1 in HCC cells upregulated the expression of pro-inflammatory cytokines including interleukin-1α (IL-1α), IL-1β, and IL-6, and enhanced NF-κB transcriptional activation. Mechanistic analyses revealed that knockdown of SPTBN1 by siRNA downregulated the expression of suppressor of cytokine signaling 1 (SOCS1), an E3 ligase of p65, and subsequently upregulated p65 accumulation in the nucleus of HCC cells. Restoration of SOCS1 abrogated this SPTBN1 loss-associated elevation of p65 in HCC cells. In human HCC tissues, SPTBN1 gene expression was inversely correlated with gene expression of IL-1α, IL-1β and IL-6. Furthermore, a decrease in the levels of SPTBN1 gene, as well as an increase in the gene expression of IL-1β or IL-6 predicted shorter relapse free survival in HCC patients, and that HCC patients with low expression of SPTBN1 or SOCS1 protein is associated with poor survival. Heterozygous loss of SPTBN1 (Sptbn1+/-) in mice markedly upregulated hepatic expression of IL-1α, IL-1β and IL-6, and elevated the proportion of myeloid-derived suppressor cells (MDSCs) and CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Foxp3⁺Treg) cells in the liver, promoting hepatocarcinogenesis of mouse fed by DDC.
Conclusions: Our findings provided evidence that loss of SPTBN1 in HCC cells increases p65 protein stability via the inhibition of SOCS1 and enhances NF-κB activation, stimulating the release of inflammatory cytokines, which are critical molecular mechanisms for the loss of SPTBN1-induced liver cancer formation. Reduced SPTBN1 and SOCS1 predict poor outcome in HCC patients.
... (2) the non-canonical one, with an important function in B lymphocytes and (3) the activation induced by DNA damage [39,40]. A second level of regulation is post-translational modifications of NF-κB subunits, carried out by various proteins, including the IκB kinase (IKK) complex. ...
... In quiescent cells, NF-κB is located in the cytoplasm, associated with inhibitory proteins (IκB-α, IκB-β, IκB-γ, IκBNS, Bcl-3) and some precursor proteins such as p100 and p105 (which, once cleaved, give rise to p52 and p50 subunits, respectively) [40]. In the canonical activation pathway, upon arrival of a stimulus to the cell, a phosphorylation occurs, followed by ubiquitination and degradation of its inhibitory proteins, in a proteasome dependent-manner. ...
Inflammation is an adaptive response in pursuit of homeostasis reestablishment triggered by harmful conditions or stimuli, such as an infection or tissue damage. Liver diseases cause approximately 2 million deaths per year worldwide and hepatic inflammation is a common factor to all of them, being the main driver of hepatic tissue damage and causing progression from non-alcoholic fatty liver disease (NAFLD) to non-alcoholic steatohepatitis (NASH), cirrhosis and, ultimately, hepatocellular carcinoma (HCC). The metabolic sensor SIRT1, a class III histone deacetylase with strong expression in metabolic tissues such as the liver, and transcription factor NF-κB, a master regulator of inflammatory response, show an antagonistic relationship in controlling inflammation. For this reason, SIRT1 targeting is emerging as a potential strategy to improve different metabolic and/or inflammatory pathologies. In this review, we explore diverse upstream regulators and some natural/synthetic activators of SIRT1 as possible therapeutic treatment for liver diseases.
... Receptor proteins first activate IκB kinase (IKK) upon stimulation (24). IKK then phosphorylates serine at the regulatory site of the IκB subunit on the intracellular NF-κB/IκB compound, which allows the IκB subunit to be ubiquitinated and degraded by the proteasome to release the NF-κB dimer (25)(26)(27)(28)(29). With the degradation of IκB, free P65 is phosphorylated by protein kinase A at serine 276 in the cytoplasm, and then phosphorylated P65 enters the nucleus and binds to corresponding binding sites on genes, which initiates transcription (30). ...
... Western blotting. Western blotting was performed according to standard methods (26). Total protein from cell lines was extracted using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and quantified using the Bradford method (36). ...
Thyroid hormone receptor-interacting protein 6 (TRIP6), a member of the zyxin family of Lin-Isl-Mec (LIM) proteins, is an adaptor protein primarily expressed in epithelial cells. TRIP6 can regulate a variety of cellular responses, such as actin cytoskeletal reorganization and cell adhesion. However, to the best of our knowledge, the role of TRIP6 in osteosarcoma (Os) has not been previously reported. Therefore, the present study investigated the role of TRIP6 in the occurrence and development of Os, and the potential of utilizing TRIP6 as a therapeutic target in Os. The present results suggested that the expression levels of TRIP6 were significantly increased in Os cells and clinical tissue specimens compared with normal osteoblasts and adjacent non-tumor tissue. Moreover, the present results suggested that overexpressing TRIP6 significantly increased proliferation, migration and invasion, while inhibiting apoptosis in Os cells. However, silencing TRIP6 decreased proliferation, migration and invasion, while activating apoptosis in Os cells. The present results suggested that overexpression of TRIP6 increased NF-κB activation by decreasing the protein expression levels of inhibitor of κBα, and increasing total and phosphorylated P65 levels. The present results indicated that TRIP6 silencing decreased NF-κB activation. Collectively, the present results suggested that TRIP6 may play a role in promoting Os cell proliferation, migration and invasion, while inhibiting cell apoptosis. Furthermore, TRIP6 may be utilized as a novel prognostic biomarker and therapeutic target in Os.
... NF-κB resides in the cytoplasm, where it binds with inhibitory κB (IκB). However, stimuli that activate HSCs lead to dissociation of NF-κB from IκB, and allow it to translocate into the nucleus, where it transactivates genes related to the survival of activated HSCs (Luedde and Schwabe, 2011;Schmitz et al., 2004). In addition to sequestration of NF-κB from IκB, covalent modification of NF-κB (e.g., phosphorylation) is required to regulate NF-κB activity in HSCs (Luedde and Schwabe, 2011;Oakley et al., 2009). ...
... Our results indicated that HsA decreased the phosphorylation of IKKε, but not TBK1, in primary activated HSCs in a concentration-dependent manner. Several upstream kinases, such as IKKα, IKKβ, IKKε, TBK1, and ribosomal S6 kinase, are known to directly phosphorylate Ser 536 of p65 Schmitz et al., 2004). Moreover, IKKα/ β inhibition by sulfasalazine ameliorates CCl 4 -induced liver fibrosis by inducing apoptosis in activated HSCs (Oakley et al., 2005). ...
Hemistepsin A (HsA), isolated from Hemistepta lyrata (Bunge) Bunge, has the ability to ameliorate hepatitis in mice. However, the effects of H. lyrata and HsA on other types of liver disease have not been explored. In this report, we investigated the effects of H. lyrata and HsA on liver fibrosis and the underlying molecular mechanisms in activated hepatic stellate cells (HSCs). Based on cell viability-guided isolation, we found HsA was the major natural product responsible for H. lyrata-mediated cytotoxicity in LX-2 cells. HsA significantly decreased the viability of LX-2 cells and primary activated HSCs, increased the binding of Annexin V, and altered the expression of apoptosis-related proteins, suggesting that HsA induces apoptosis in activated HSCs. HsA reduced the phosphorylation of IKKε and the transactivation of nuclear factor-κB (NF-κB). Moreover, HsA decreased the phosphorylation of Akt and its downstream signaling molecules. Transfection experiments suggested that inhibition of NF-κB or Akt is essential for HsA-induced apoptosis of HSCs. In a CCl4-induced liver fibrosis model, HsA administration significantly decreased ALT and AST activities. Furthermore, HsA attenuated CCl4-mediated collagen deposits and profibrogenic genes expression in hepatic tissue. Thus, HsA may serve as a natural product for managing liver fibrosis through inhibition of NF-κB/Akt-dependent signaling.
... This inspired us to ask whether these drugs enhance endocrine differentiation by modulating NF-κB signaling. In quiescent cells, the NF-κB complex is sequestered in the cytoplasm and associates with inhibitory IκB proteins (Schmitz et al., 2004). The 23 Hit I drugs are listed. ...
... DOI: 10.7554/eLife.08261.010 Activation of NF-κB signaling leads to kinase-dependent phosphorylation and degradation of IκB, allowing NF-κB to translocate to the nucleus and regulate target-gene transcription (Schmitz et al., 2004). Despite having different molecular targets, thioctic acid and parthenolide both block NF-κB nuclear translocation (Ying et al., 2010;Ghantous et al., 2013). ...
... In quiescent cells, the NF-κB complex is isolated to the cytoplasm and interacts with inhibitory IκB proteins. The activation of NF-κB signaling leads to kinase-dependent phosphorylation and the degradation of the IκB proteins causing NF-κB to be translocated to the nucleus and regulate target-gene transcription [18]. The zebrafish has unique conserved and compatible NF-kB/IkB proteins among vertebrates and shows the importance of the NF-kB pathway in mesoderm formation during early embryogenesis [19]. ...
NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The Casper(roy−/−, nacre−/−) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent transgenic zebrafish cellular phenotype to study inflammatory processes in vivo. We outline the experimental setup to generate a transparent transgenic NF-kB/Casper strain of zebrafish through the cross-breeding of Casper and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the transgenic transparent progeny of Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent transgenic strain of the NF-kB reporter line has been coined as “Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−)gmc1”. We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed Tg(6xNF-kB:EGFP); Casper(roy−/−, nacre−/−)gmc1 homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.
... It is well known that NF-κB is an essential transcriptional regulator of inflammatory response in the liver (Luedde and Schwabe, 2011). NF-κB is located in the cytoplasm and combines with inhibitory proteins and some precursor proteins normally (Schmitz et al., 2004). When the stimulus activates IKK, NF-κB is released from its inhibitory proteins and translocates to the nucleus, followed by activating gene transcription (Karin and Ben-Neriah, 2000). ...
Liver fibrosis is considered a sustained wound healing response and metabolic syndrome, and its therapy is of great significance for chronic liver disease. Schizandrin C, as one lignan from hepatic protectant Schisandra chinensis, can depress the oxidative effect and lipid peroxidation, and protect against liver injury. In this study, C57BL/6J mice were used to estimate a liver fibrosis model by CCl4, and Schizandrin C exerted an anti-hepatic fibrosis effect, as evidenced by decreased alanine aminotransferase, aspartate aminotransferase and total bilirubin activities in serum, lower hydroxyproline content, recuperative structure and less collagen accumulation in the liver. In addition, Schizandrin C reduced the expressions of alpha-smooth muscle actin and type Ι collagen in the liver. In vitro experiments also revealed that Schizandrin C attenuated hepatic stellate cell activation in both LX-2 and HSC-T6 cells. Furthermore, lipidomics and quantitative real-time PCR analysis revealed that Schizandrin C regulated the lipid profile and related metabolic enzymes in the liver. In addition, the mRNA levels of inflammation factors were downregulated by Schizandrin C treatment, accompanied by lower protein levels of IκB-Kinase-β, nuclear factor kappa-B p65, and phospho-nuclear factor kappa-B p65. Finally, Schizandrin C inhibited the phosphorylation of p38 MAP kinase and extracellular signal-regulated protein kinase, which were activated in the CCl4 fibrotic liver. Taken together, Schizandrin C can regulate lipid metabolism and inflammation to ameliorate liver fibrosis by nuclear factor kappa-B and p38/ERK MAPK signaling pathways. These findings supported Schizandrin C as a potential drug for liver fibrosis.
... Post-translation modifications, including phosphorylation, are known to play an important role in regulating the trans-activation activities of RelA/p65 (18)(19)(20). Previously, it was shown that phosphorylation of serine-276 is critical for p65-mediated repression of BRMS1 (breast cancer metastasis suppressor 1) (21). We asked if serine-276 is essential for repression of GPRC5A. ...
Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulations play a critical role. Lung tumor suppressor GPRC5A is repressed in most non-small cell lung cancer (NSCLC), however the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induces GPRC5A repression both in vitro and in vivo. Intriguingly, trans-activation of NF-κB downstream targets is not required, but the trans-activation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in GPRC5A promoter. Instead, p65 was complexed with RARα/β, and recruited to the RA-response element (RARE) site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complex, and suppressed transcription. Noticeably, phosphorylation on Serine276 of p65 is required for interaction with RARα/β and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression is through suppression of histone H3K9ac, but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a HDAC inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via complex with RARα/β and mediates epigenetic repression via suppression of H3K9ac.
... Evidence in multiple cell types suggests that phosphorylation of p65 at Ser276 is mediated by protein kinase A and ribosomal protein S6 kinase alpha-5 (MSK1) kinase in the cytosolic and nuclear compartments, respectively. IκB kinase (IKK), TANK-binding kinase 1 (TBK1), and 90 kDa ribosomal S6 kinase (RSK1) have been identified as putative kinases that control the phosphorylation of p65 at the S536 residue [27][28][29][30][31][32]. Lastly, it is widely felt that phosphorylation at S276 promotes half-life of p65, while activation of S536 results in increased proteasomal degradation of NFκB; based on these conclusions, it is postulated that phosphorylation of p65 at S276 contributes to cell survival, whereas phosphorylation at S536 accelerates cell death via apoptosis [33]. ...
Background/aims:
We recently reported increased phosphorylation (at S536) of the p65 subunit of NFκB (Rel A) in pancreatic beta (INS-1 832/13) cells following exposure to hyperglycemic (HG) conditions. We also demonstrated that HG-induced S536 phosphorylation of p65 is downstream to the regulatory effects of CARD9 since deletion of CARD9 expression significantly attenuated HG-induced S536 phosphorylation of p65 in beta cells. The overall objective of the current investigation is to identify putative mechanisms underlying HG-induced phosphorylation of p65 in islet beta cells following exposure to HG conditions.
Methods:
INS-1 832/13 cells were incubated in low glucose (LG; 2.5 mM) or high glucose (HG; 20 mM) containing media for 24 hours in the absence or presence of small molecule inhibitors of G protein prenylation and activation. Non-nuclear and nuclear fractions were isolated from INS-1 832/13 cells using a commercially available (NE-PER) kit. Degree of S536 phosphorylation of the p65 subunit was quantified by western blotting and densitometry.
Results:
HG-induced p65 phosphorylation was significantly attenuated by inhibitors of protein prenylation (e.g., simvastatin and L-788,123). Pharmacological inhibition of Tiam1-Rac1 (e.g., NSC23766) and Vav2-Rac1 (e.g., Ehop-016) signaling pathways exerted minimal effects on HG-induced p65 phosphorylation. However, EHT-1864, a small molecule compound, which binds to Rac1 thereby preventing GDP/GTP exchange, markedly suppressed HG-induced p65 phosphorylation, suggesting that Rac1 activation is requisite for HG-mediated p65 phosphorylation. Lastly, EHT-1864 significantly inhibited nuclear association of STAT3, but not total p65, in INS-1 832/13 cells exposed to HG conditions.
Conclusion:
Activation of Rac1, a step downstream to HG-induced activation of CARD9, might represent a requisite signaling step in the cascade of events leading to HG-induced S536 phosphorylation of p65 and nuclear association of STAT3 in pancreatic beta cells. Data from these investigations further affirm the role(s) of Rac1 as a mediator of metabolic stress- induced dysfunction of the islet beta cell.
... Other inhibitory mechanisms are possible. PPARα can bind to p300, the histone acetyltransferase responsible for the acetylation of Lys310 on subunit p65 NF-κB [57][58][59], a post-translational modification fundamental for NF-κB activation [60]. Moreover, the activation of PPARα promotes the expression and activity of Sirtuin 1 (SIRT1), which inhibits NF-κB by deacetylation [61,62]. ...
The strong relationship between metabolic alterations and non-alcoholic steatohepatitis (NASH) suggests a pathogenic interplay. However, many aspects have not yet been fully clarified. Nowadays, NASH is becoming the main cause of liver-associated morbidity and mortality. Therefore, an effort to understand the mechanisms underlying the pathogenesis of NASH is critical. Among the nuclear receptor transcription factors, peroxisome-proliferator-activated receptor alpha (PPARα) is highly expressed in the liver, where it works as a pivotal transcriptional regulator of the intermediary metabolism. In this context, PPARα’s function in regulating the lipid metabolism is essential for proper liver functioning. Here, we review metabolic liver genes under the control of PPARα and discuss how this aspect can impact the inflammatory condition and pathogenesis of NASH.
... NF-κB transcription factor plays an important role in the pathways that lead to inflammation, angiogenesis, metastasis, antiapoptosis, and multidrug resistance [74,75]. Irregularity in NF-κB activity leads to prolonged inflammation and ultimately cancer. ...
Withaferin A (WA) is a pivotal withanolide that has conquered a conspicuous place in research, owning to its multidimensional biological properties. It is an abundant constituent in Withania somnifera Dunal. (Ashwagandha, WS) that is one of the prehistoric pivotal remedies in Ayurveda. This article reviews the literature about the pharmacological profile of WA with special emphasis on its anticancer aspect. We reviewed research publications concerning WA through four databases and provided a descriptive analysis of literature without statistical or qualitative analysis. WA has been found as an effective remedy with multifaceted mechanisms and a broad spectrum of pharmacological profiles. It has anticancer, anti-inflammatory, antiherpetic, antifibrotic, antiplatelet, profibrinolytic, immunosuppressive, antipigmentation, antileishmanial, and healing potentials. Evidence for wide pharmacological actions of WA has been established by both in vivo and in vitro studies. Further, the scientific literature accentuates the role of WA harboring a variable therapeutic spectrum for integrative cancer chemoprevention and cure. WA is a modern drug from traditional medicine that is necessary to be advanced to clinical trials for advocating its utility as a commercial drug.
... Fig. 3 demonstrates that CO causes elevations of the p65 subunit of NF-κB and phospho-Ser 536 p65 in brain. Inducible phosphorylation of NF-κB is considered a key mechanism in the positive regulation of NF-κB activity (Schmitz et al., 2004). Phosphorylation at serine 536 of the p65 subunit represents the site with the most potent inducible phosphorylation in response to inflammatory stimuli, and it is highly conserved among different species (Ghosh and Karin, 2002;Sakurai et al., 1999). ...
We hypothesized that carbon monoxide (CO) establishes an inflammatory cycle mediated by microparticles (MPs). Mice exposed to a CO protocol (1000 ppm for 40 min and then 3000 ppm for 20 min) that causes neuroinflammation exhibit NF-κB activation in astrocytes leading to generation of MPs expressing thrombospondin-1(TSP-1) that collect in deep cervical lymph nodes draining the brain glymphatic system. TSP-1 bearing MPs gain access to the blood stream where they activate neutrophils to generate a new family of MPs, and also stimulate endothelial cells as documented by leakage of intravenous 2000 kDa dextran. At the brain microvasculature, neutrophil and MPs sequestration, and myeloperoxidase activity result in elevations of the p65 subunit of NF-κB, serine 536 phosphorylated p65, CD36, and loss of astrocyte aquaporin-4 that persist for at least 7 days. Knock-out mice lacking the CD36 membrane receptor are resistant to all CO inflammatory changes. Events triggered by CO are recapitulated in naïve wild type mice injected with cervical node MPs from CO-exposed mice, but not control mice. All MPs-mediated events are inhibited with a NF-κB inhibitor, a myeloperoxidase inhibitor, or anti-TSP-1 antibodies. We conclude that astrocyte-derived MPs expressing TSP-1 establish a feed-forward neuroinflammatory cycle involving endothelial CD36-to-astrocyte NF-κB crosstalk. As there is currently no treatment for CO-induced neurological sequelae, these findings pose several possible sites for therapeutic interventions.
... IκB: inhibitor of κB; P-IκB: phosphorylated inhibitor of κB; NF-κB p65:nuclear transcription factor-κB p65; P-NF-κB p65: phosphorylated nuclear transcription factor-κB p65 for the production of pro-inflammatory cytokines (Schmitz & Baeuerle, 1991). It has been confirmed that IκB effectively prevented p65 translocation to the nucleus via the interaction with the NF-κB N-terminal Rel homology domain (Schmitz et al., 2004). ...
Chemical liver injury is a common cause of liver disease primarily characterized by oxidative stress and inflammation. Sialoglycoproteins isolated from the eggs of Carassius auratus (Ca‐SGP) have been proved to exhibit the antioxidant effect. However, the effect of Ca‐SGP on liver injury remains unclear. Thus, this study was aimed to determine the effect of Ca‐SGP on CCL4‐induced chronic chemical liver injury and explore the underlying molecular mechanism. Results showed that Ca‐SGP mitigated the elevated levels of serum alanine aminotransferase and aspartate aminotransferase, inhibited the systemic oxidative stress, and reduced the levels of pro‐inflammatory factors TNF‐α and IL‐1β. Histologic results showed that Ca‐SGP supplements alleviated hepatocyte necrosis and liver macrophage infiltration. Further, Ca‐SGP supplement decreased endoplasmic reticulum stress‐related proteins expression, including BiP, IRE‐α, p‐IRE‐α, and TRAF2, and further inhibited the trigger of the NF‐κB pathway. In summary, Ca‐SGP might be a novel agent for liver injury treatment, and its potential mechanism was related to the inhibition of liver inflammation induced by the endoplasmic reticulum.
Practical application
The fish egg is an important by‐product in fish processing. Carassius auratus is a common freshwater fish with large catches and low prices. However, the eggs of C. auratus are usually direct discard or processed into salted roe products, and the quality and value of these salted products are unsatisfactory. In this current study, we confirmed that sialoglycoproteins isolated from the C. auratus eggs have the potential for the treatment of liver injury and determined that its mechanism is related to the endoplasmic reticulum and inflammation, which put forward a new idea for solving the by‐product of fish processing.
... The present study also showed the increased levels of Rel A gene expression in the treated cells of all the cancer cell lines which indicates that the Ficus latex extracts possess the ability of inducing apoptosis and inhibiting the cell proliferation (Fig. 15). The NF-kB proteins, family of transcriptional factors, play a vital role in regulating the apoptotic process in cell [25,26]. Rel A (P65), the most common form of NF-kB, is generally rendered stable in cytoplasm by Ikappa B (IkB), an inhibitory protein [27]. ...
Background: An extract of Ficus religiosa latex has been previously found to possess potent pharmacological activity with high antioxidant content phytochemical. The present research was conducted to investigate the chemo-preventive efficacy of latex extract on human breast adenocarcinoma MDA MB 231, human neroblastoma IMR 32, and human colorectal HCT 116 cell lines.
... The present study also showed the increased levels of Rel A gene expression in the treated cells of all the cancer cell lines which indicates that the Ficus latex extracts possess the ability of inducing apoptosis and inhibiting the cell proliferation (Fig. 15). The NF-kB proteins, family of transcriptional factors, play a vital role in regulating the apoptotic process in cell [25,26]. Rel A (P65), the most common form of NF-kB, is generally rendered stable in cytoplasm by Ikappa B (IkB), an inhibitory protein [27]. ...
Background
An extract of Ficus religiosa latex has been previously found to possess potent pharmacological activity with high antioxidant content phytochemical. The present research was conducted to investigate the chemo-preventive efficacy of latex extract on human breast adenocarcinoma MDA MB 231, human neroblastoma IMR 32, and human colorectal HCT 116 cell lines.
Results
The results showed that the latex crude extract induced cytotoxicity in all the selected cell lines with IC 50 value 4.8 ± 1.13 μg/ml against the IMR 32 cell line. The cell cycle analysis results indicated the arrest and accumulation of cells at G1 phase in case of MDA MB 231 cells and HCT 116 cells whereas in the case of IMR 32 cells the arrest was in G2/M phase. The clear bands of fragments observed in DNA ladder experiments showed that apoptosis is induced by extracts in the cell lines. This could be correlated with the gene level expression studies on selected pro-apoptotic (p53 and caspase-3) and anti-apoptotic (Bcl-2, AKT) genes, which got upregulated and downregulated, respectively.
Conclusion
Based on the experimental evidence, Ficus religiosa contains phytochemicals with potent antitumor activities.
... tRF/miR -1308 is another non-coding RNA, that targets the apoptotic pathway through an NF KB inhibitor (NKIRA 1) (16). Nuclear factor-kappa B (NFκB) is playing a great role in regulating cell differentiation, proliferation, immune response and blocking apoptosis (17,18). Recently the scienti c are witness the reevaluation of the miRNA markers in the miRbase database. ...
Background
Colorectal cancer (CRC), which is one of lethal and invasive cancer with metastasis at the time of diagnosis. Despite, advanced therapy and surgical techniques, Patients with metastases CRC have a poor survival rate. Enhanced predictive biomarkers such as non-coding RNAs (ncRNAs) are needed at the time of diagnosis to better therapies. The tRNA- derived fragments (tRF) are non-protein-coding sequences that probably function as oncogenes or tumor suppressor genes, tRFs produced under physiological or stress conditions from enzymatically cleavage in tRNAs.
Methods
Documenting the tRNA-derived fragments (tRF) in colorectal cancer and showed that it is significantly down-regulated in colorectal tissue tumors compared to normal tissue samples. Since data distribution was not normal, Mann-Whitney, Kruskal-Wallis, and Spearman correlation coefficient tests were used for analysis.
Results
Among several tRF, quantitative real-time PCR analyses declared that tRF/miR-1280, tRNA-derived fragment-Asp, tRNA-derived fragment-Val down-regulated in CRC. Notably, its expression was correlated with invasive stage and metastases (p < 0.05).
Conclusions
This research can elucidate the association between tRFs with the invasion stage and CRC clinical pathology, our results not only expand better understanding about tRNA-derived fragments species in CRC but also highlight the potential usages of tRFs as a promising biomarker in the prognosis of metastatic cancer.
... Nuclear factor-κB (NF-κβ) is an inducible transcription factor that is involved in the modulation of several cell processes, including cell growth and apoptosis (Schmitz et al. 2004). LoTempio et al. (2005 tested curcumin in head and neck squamous carcinoma (HNSCC) cell lines, CCL 23, CAL 27, and UM-SCC1 in a dose-dependent manner, which resulted in reduced nuclear expression of NF-κβ. ...
Every cell requires energy which they obtain from oxidative phosphorylation to perform major metabolic functions. This energy requirement is dependent on their metabolic rate, and it is expected that higher the metabolic rate, higher is the amount of energy expended. However, cancer cells have a unique metabolic behavior where they require only a minimal amount of energy, exhibiting a condition called the Warburg effect to survive and proliferate. They undergo glycolysis despite the availability of adequate oxygen (aerobic glycolysis) and obtain less energy as compared to normal cells from this process. This finding has helped many researchers to look for an alternative in treating cancer which is one of the leading causes of death today. This chapter will elucidate on the role of Warburg effect in modulating cancer cell metabolism and describe recent findings on this unique pathway employed by cancer cells. The chapter will also shed light on an alternative model called the reverse Warburg effect that provides an environment rich in energy for tumor growth and discuss the potential of using this pathway for therapeutically targeting cancer cells.
... Nuclear factor-κB (NF-κβ) is an inducible transcription factor that is involved in the modulation of several cell processes, including cell growth and apoptosis (Schmitz et al. 2004). LoTempio et al. (2005 tested curcumin in head and neck squamous carcinoma (HNSCC) cell lines, CCL 23, CAL 27, and UM-SCC1 in a dose-dependent manner, which resulted in reduced nuclear expression of NF-κβ. ...
Cancer cell proliferation and their adaptation to tough environments depend critically on metabolic pathways. The pathological, phenotypic, and structural level understanding of metabolic reprogramming that actually takes place in the tumor microenvironment is known to provide an essential understanding of the strategies of anticancer drug development. Here, the different metabolic pathways, their key targets, and treatments are highlighted for the implementation of interdisciplinary approaches to understand the cancer metabolism pathways as well as the discovery-to-development process of anticancer drugs. In recent years, there have been advancements in metabolomics, proteomics, and in silico tools and techniques having higher sensitivity and prediction accuracy for the determination of various pharmacodynamic, pharmacokinetic properties, and metabolic pathways in the early drug discovery stages. Hence, they are more efficient to identify and validate the cancer proteins and characterize their structural–dynamical–functional relationships in an economically, fast, and less tedious process. Here, we review the available pathways, therapeutic targets, and the therapeutically active compounds reported to date which are known to modulate cancerous targets and reached into the clinical trials and/or approved by the FDA are highlighted. Some novel anticancer modulating mechanisms and targets reported recently and streamlined in the discovery phase are also discussed.
... Interestingly, Lys-122 and -123 are the only residues contacting DNA in the minor grove (Chen FE 1998) and their acetylation reduces p65 binding to DNA (Kiernan R 2003). On the other hand, acetylation of Lys-310 enhances p65 transcriptional activity (Quivy V 2004;Schmitz ML 2004). It has been suggested that acetylated-p65 is more easily exported to the cytoplasm by IkBα (Kiernan R 2003). ...
Endothelial injury often causes intimal hyperplasia, a disease characterised by local inflammation and critical narrowing or restenosis of the blood vessel. Endothelial cells (EC) grown on collagen particles are highly effective in inhibiting intimal hyperplasia in various animal models, and this effect appears to be, at least in part, the result of EC-derived soluble factors that suppress local vascular inflammation. To test this hypothesis, we produced EC on collagen particles-conditioned medium (ECPCM), which was expected to contain soluble anti-inflammatory factors. Indeed, EC treated in vitro with ECPCM together with pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) displayed reduced gene expression of the inflammation-related adhesion molecules E-selectin and VCAM-1. Investigation of the molecular mechanism of action for the anti-inflammatory activity excluded mRNA stability of E-selectin and VCAM-1, activation of signalling cascades via the NF-kB and Stat3 pathways, and nuclear localization of transcription factors. ECPCM did affect the TNFα-induced binding of p65, a subunit of the NF-kB transcription factor, to the E-selectin and VCAM-1 promoters. These results suggest that inhibition of gene transcription is responsible for the ECPCM-mediated suppression of inflammatory responses in EC. The therapeutic effects of ECPCM were supported by in vivo experiments performed on the mutant mouse strain JR5558, which develops spontaneous choroidal neovascularization (CNV) lesions associated with inflammatory cell recruitment and expression of inflammatory adhesion molecules. The CNV lesion area and recruitment of activated macrophages were both decreased in JR5558 mice given intraperitoneal injections of ECPCM. ECPCM might therefore have therapeutic potential in treating inflammatory vascular diseases.
... Schmitz, M. L., Mattioli, I., Buss, H., and Kracht, M. (2004) Chembiochem 5(10), 1348-1358 39. ...
... In unstimulated cells, NF-κB is sequestered in the cytoplasm as an inactive transcription factor complex by its physical association with one of the several inhibitors of NF-κB (IκBs). These proteins also comprise a structurally and functionally related family of molecules, which include IκBα (37kDa), IκBβ (43kDa), IκBγ, IκBε, p105/p50 (C terminus), p100/p52 (C terminus), 38]. ...
Der Nukleare Faktor kappa B (NF-κB) spielt eine zentrale Rolle in vielen Entzündungs- und Nerven-zerstörenden Krankheiten, aber auch in der Entstehung von Krebs. NF-κB gehört zu einer Familie von induzierbaren Transkriptionsfaktoren, welche hauptverantwortlich sind für die Regulation von Genen, die in der Immunabwehr und in Entzündungsprozessen eine wichtige Rolle spielen. Mehr als 500 verschiedene NF-κB abhängige Gene konnten bis jetzt identifiziert werden. PARP-1 ist ein Chromatin-assoziiertes Protein, welches in hohen Mengen im Zellkern vorkommt und zu einer grossen Familie von Enzymen gehört, welche ADP-Ribose Einheiten von NAD als Substrat auf sich selber oder andere Chromatin-assoziierte Proteine übertragen. PARP-1 Knockout-Mäuse zeigen eine ausgesprochen hohe Resistenz gegenüber experimentell induziertem Septischem Schock, Diabetes Typ 1 und Parkinson Krankheit. Dieser Phänotyp ist ein klares Indiz dafür, dass PARP-1 in die Pathogenese dieser Prozesse involviert ist. In der Tat fungiert PARP-1 als wichtiger Koaktivator von NF-κB. Darüber hinaus wurde kürzlich in einer Studie gezeigt, dass die proteolytische Spaltung von PARP-1 durch Caspasen möglicherweise wichtig ist für die PARP-1 Aktivität in NF-κB abhängigen Krankheitsprozessen. Neben PARP-1 sind einige andere Koaktivatoren wie zum Beispiel die Koaktivator assoziierte Arginin Methyltransferase-1 (CARM-1) ebenfalls essentiell für die NF-κB abhängige transkriptionelle Aktivierung von Genen. Ziel dieser Studie war NF-κB abhängige Gene in Abhängigkeit von PARP-1 und CARM-1 zu identifizieren und darüber hinaus solche, die von der proteolytischen Spaltung von PARP-1 durch Caspasen abhängig sind. Zu diesem Zweck wurden die Expressionsstärken von Wildtyp Zellen (embryonale Mausfibroblasten, respektive Lungenfibroblasten und Raw264) mit den entsprechenden Knock-out oder Knock-in Zellen nach Stimulation verglichen. Es wurden 16 Gene von den über 500 von NF-κB abhängigen Genen untersucht. Die Resultate zeigten, dass immer nur ein Teil von den NF-κB abhängigen Genen durch die untersuchten Koaktivatoren aktiviert wird. Weiter konnte gezeigt werden, dass die Expression von NF-κB abhängigen Genen durch PARP Hemmer unter den getesteten Konditionen nicht beeinflusst wird. Andererseits scheinen einige Gene durch Caspase Aktivität, z.B. Spaltung von PARP-1, reguliert zu werden. All diese Resultate sprechen für einen starken Beweis, dass PARP-1 und CARM-1 eine essentielle Rolle in NF-κB abhängiger transkriptioneller Aktivierung von Genen spielt. Nuclear factor kappa B (NF-κB) had been suggested to play important roles in inflammatory and neurodegenerative disorders as well as carcinogenesis. NF-κB encompasses a family of inducible and widely expressed transcription factors which played a crucial role in the regulation of genes involved in immune and inflammatory responses. Over 500 target genes depending on NF-κB had been identified so far. PARP-1, an abundant nuclear chromatin associated protein belongs to a large family of enzymes that catalyze the transfer of ADP-ribose units from its substrate ?- nicotinamide adenine dinucleotide covalently to itself and other chromatin associated proteins. PARP-1 deficient mice were protected against experimentally induced septic shock, diabetes type I and Parkinsonism. This phenotype indicated that PARP-1 is important for the pathogenesis of these events. Indeed, PARP-1 was shown to be an important coactivator of NF-κB. Interestingly, recent studies had shown that the cleavage of PARP-1 by caspases could be important for the activity of PARP-1 in NF-κB- dependent disease processes. In addition to PARP-1, many other coactivators such as the coactivator-associated arginine methyltransferase-1 (CARM-1) are also required for NF-κB-dependent gene expression. Aim of this thesis was to investigate NF-κB-dependent gene expression in regard to their PARP-1 or CARM-1 dependency and furthermore to the caspase-mediated cleavage of PARP-1. For this purpose, gene expression levels of wild-type mouse lung fibroblasts, mouse embryonic fibroblasts and Raw264 cells were compared with the corresponding knock-out or knock-in cells upon stimulation. From over 500 target genes of NF-κB, 16 selected genes were analyzed. The results indicated that only a subset of the target genes were activated by the certain investigated coactivators. Moreover, PARP inhibitors did not influence NF-κB-dependent gene expression under the tested conditions. On the other hand, a small subset of NF-κB-dependent genes seemed to be regulated by caspase activities, potentially through the cleavage of PARP- 1. Together these results provide strong evidence for a crucial role of PARP-1 and CARM- 1 in NF-κB gene expression.
... After degradation of the IκB, p65 NF-κB is subject to acetylation of the Lys 310 p65 NF-κB by p300. This modification is very important with respect to the proper functioning of p65 NF-κB [138,139]. PPARα [30,140] and PPARγ [141] bind p300. Assembly of these complexes leads to loss of enzymatic properties of p300 and, as a consequence, to inhibition of activation of p65 NF-κB through reduced of acetylation of this NF-κB subunit. ...
The peroxisome proliferator-activated receptor (PPAR) family includes three transcription factors: PPARα, PPARβ/δ, and
PPARγ. PPAR are nuclear receptors activated by oxidised and nitrated fatty acid derivatives as well as by cyclopentenone
prostaglandins (PGA 2 and 15d-PGJ 2 ) during the inflammatory response. This results in the modulation of the pro-inflam-
matory response, preventing it from being excessively activated. Other activators of these receptors are nonsteroidal anti-
inflammatory drug (NSAID) and fatty acids, especially polyunsaturated fatty acid (PUFA) (arachidonic acid, ALA, EPA,
and DHA). The main function of PPAR during the inflammatory reaction is to promote the inactivation of NF-κB. Possible
mechanisms of inactivation include direct binding and thus inactivation of p65 NF-κB or ubiquitination leading to proteolytic
degradation of p65 NF-κB. PPAR also exert indirect effects on NF-κB. They promote the expression of antioxidant enzymes,
such as catalase, superoxide dismutase, or heme oxygenase-1, resulting in a reduction in the concentration of reactive oxygen
species (ROS), i.e., secondary transmitters in inflammatory reactions. PPAR also cause an increase in the expression of IκBα,
SIRT1, and PTEN, which interferes with the activation and function of NF-κB in inflammatory reactions.
... Among these, Cucurmin from turmeric has been revealed to suppress cellular proliferation in oral, colon, breast and other cancers (ElAttar and Virji, 1999). Nuclear factor causes the activation of various cell processes like apoptosis and cell growth (Schmitz et al., 2004). This factor is activated in many cells such as inflammatory cytokines, i.e., tumor necrosis factor (TNF) and interleukin-1 and mitogens (Aggarwal et al., 2003;Duarte et al., 2010). ...
... Subsequently, inhibitory protein IκB-α and p65 are phosphorylated, meanwhile, inhibitory molecule is degraded by proteasome-mediated. Then, the heterodimer p50/p65 is released and migrats from the cytoplasm to the nucleus to regulate the expression of multiple target genes, such as TNF-α, IL-6, iNOS and so on [9,10]. ...
Background:
Epimedium sagittatum (Sieb.et Zucc.) Maxim., Ying-Yang-Huo in Chinese has been used as a traditional Chinese medicine and is deemed to "reinforce the kidney Yang". Previous studies showed that E. sagittatum could modulate the immune system and treat some chronic disease such as rheumatic arthritis, cardiovascular diseases and osteoporosis. The aim of this study is to evaluate the anti-inflammatory effects of ethyl acetate extracts (YYHs) of E. sagittatum and its mechanisms of action.
Methods:
In order to explore the composition of YYHs, YYHs was analyzed using high performance liquid chromatography-mass spectrometry-mass spectrometry (HPLC-MS/MS) and in comparison with reference standards. Anti-inflammatory model was established in LPS-induced RAW264.7 cells. The levels of nitric oxide (NO) were measured with the Griess reagent. Production of tumor necrosis factor-alpha (TNF-α) and interleukin-2 (IL-2) were measured by enzyme-linked immunosorbent assays (ELISA). In addition, expression of p-p65 protein and TLR4/MD-2 complex was detected by western blots and flow cytometric, respectively. Nuclear factor kappa B (NF-κB) nuclear translocation was observed by fluorescence microscope.
Results:
A total of eight compounds were identified, of which icariside II was the most abundant compound. YYHs (12.5-50 μg/mL) had no obvious cytotoxic effect on cells, and remarkably inhibited LPS-induced production of NO, TNF-α and IL-2 with a dose-dependent manner. Additionally, YYHs up-regulated expression of p-p65 and TLR4/MD-2 complex. Further research showed that YYHs significantly suppressed NF-κB p65 nuclear translocation.
Conclusion:
In brief, YYHs contributed to the inhibition of LPS-induced inflammatory response through the TLR4/MD-2-mediated NF-κB pathway and may be a potential choice to combat inflammation diseases. It includes a schema of pathways at the end of the paper.
... The transcriptional activity of the transcription factor NF-κB can be regulated at multiple levels: The transcriptional activity of NF-κB is activated by degradation of IκBs, whereas the nuclear transcription potential of p65 is further regulated by post-translational modifications, such as phosphorylation, ubiquitination or acetylation (9). Notable, phosphorylation of p65 at Ser276, Ser529, and or Ser536 is essential for the promotion of gene expression (10). ...
Although enteral nutrition therapy for inflammatory bowel disease has been confirmed to be an effective treatment method, the exact mechanism responsible for the effects of enteral nutrition remains unclear. The aim of the present study was to investigate the protective effect of exclusive enteral nutrition (EEN) against colitis, and to elucidate the potential mechanisms by inhibiting p65 activation via regulating the p38/mitogen‑ and stress‑activated protein kinase‑1 (MSK1) pathway. Experiments were performed by establishing dextran sulfate sodium (DSS)‑mice colitis and picrylsulfonic acid solution (TNBS)‑induced rat colitis, and the results demonstrated that EEN treatment attenuated body weight loss, colon length shortening and colonic pathological damage caused by colitis. EEN also inhibited inflammatory cells infiltration and decreased myeloperoxidase and inducible nitric oxide synthase activities. Furthermore, EEN significantly reduced the production of pro‑inflammatory mediators in serum and the colon. Mechanically, EEN suppressed activation of p65 by inhibiting the p38/MSK1 pathway. In conclusion, the present study demonstrated that EEN attenuated DSS‑ and TNBS‑induced colitis by inhibiting p65 activation via regulating the p38/MSK1 pathway, thus suggesting that EEN is effective in the treatment of colitis.
... The anti-inflammatory effects of RSV are not cell type specific and are performed by different mechanisms: RSV suppresses IKK activity and phosphorylation, IκB degradation, NF-κB subunits nuclear translocation and DNA binding activity, RelA phosphorylation, and transcriptional coactivation and repression (38). However, some studies have not reported any effects of RSV on NF-κB cascade (24,39). ...
Background: Resveratrol (RSV) provides several important biological functions in wide variety of cells. In this study, we investigated the molecular mechanisms underlying anti-inflammatory effect of RSV on HepG2 cells by assessing the gene expression
of RelA and c-Jun- subunits of NF-κB and AP-1 transcription factors.
Methods: HepG2 cells were settled in a serum- free medium with high concentrations of glucose (30 mM) and insulin (1 µM) overnight and were then incubated with RSV (5, 10, and 20 µM) for 24 and 48 hours. Real time quantitative polymerase chain reaction (qRT-PCR) was used to determine RelA and c-Jun expression.
Results: RSV diminished hyperglycemia/hyperinsulinemia stimulated expression of c-Jun dose- dependently after 24 and 48 hours (p<0.05). In addition, RelA gene expression was decreased dose-dependently in all RSV doses after 48-hour incubation (p<0.05). Our results indicated that RSV may reduce NF-κB and AP-1 activity via RelA and c-Jun gene regulation.
Conclusion: The findings of the present study demonstrated that RSV may be considered as a preventative and therapeutic agent for antagonizing inflammation in Hepatocellular carcinoma (HCC).
... Amongst others, the IκBα protein (and its related family members, IκBβ and IκBε) can be regarded as one of the most powerful and universal negative regulators of NF-κB [45]. A plethora of infectious and inflammatory conditions, including cytokines such as interleukin(IL)-1 or tumor necrosis factor(TNF)α trigger the active and rapid destruction of IκBα by the well-characterized phosphorylation-dependent proteasomal degradation pathway [46,47]. This allows NF-κB subunits to translocate to the nucleus and to induce transcription of numerous target genes, including the NFKBIA gene that encodes IκBα [48]. ...
Stressful conditions occuring during cancer, inflammation or infection activate adaptive responses that are controlled by the unfolded protein response (UPR) and the nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. These systems can be triggered by chemical compounds but also by cytokines, toll-like receptor ligands, nucleic acids, lipids, bacteria and viruses. Despite representing unique signaling cascades, new data indicate that the UPR and NF-κB pathways converge within the nucleus through ten major transcription factors (TFs), namely activating transcription factor (ATF)4, ATF3, CCAAT/enhancer-binding protein (CEBP) homologous protein (CHOP), X-box-binding protein (XBP)1, ATF6α and the five NF-κB subunits. The combinatorial occupancy of numerous genomic regions (enhancers and promoters) coordinates the transcriptional activation or repression of hundreds of genes that collectively determine the balance between metabolic and inflammatory phenotypes and the extent of apoptosis and autophagy or repair of cell damage and survival. Here, we also discuss results from genetic experiments and chemical activators of endoplasmic reticulum (ER) stress that suggest a link to the cytosolic inhibitor of NF-κB (IκB)α degradation pathway. These data show that the UPR affects this major control point of NF-κB activation through several mechanisms. Taken together, available evidence indicates that the UPR and NF-κB interact at multiple levels. This crosstalk provides ample opportunities to fine-tune cellular stress responses and could also be exploited therapeutically in the future.
... It has been shown that SIRT1 participates in the reduction of neuroinflammation during aging by deacetylating non-histone proteins, specifically NF-κB, an ubiquitary master transcriptional regulator, hub to endless inflammatory and immune signaling cascades in different organs and systems including the brain (Xie et al., 2013). The transcriptional activation domain of NF-κB is located in the p65 subunit (Chen and Greene, 2003;Kaltschmidt et al., 1994), a subunit critical for transactivation of proinflammatory genes, which is modulated by posttranslational modifications such as phosphorylation and acetylation (Chen and Greene, 2003;Oeckinghaus and Ghosh, 2009;Schmitz et al., 2004;Perkins, 2007;Yeung et al., 2004;Perkins, 2007). p65 acetylation increases the duration and effectiveness of the activation of NF-κB, fostering inflammation (Chen and Greene, 2003) (Fig. 1a). ...
The increased number of elderly people worldwide poses a major medical and socio-economic challenge: the search of strategies to combat the consequences of the aging process. Oxidative stress and inflammation have been pointed out as the leading causes of brain aging, which in turn alters the functionality of brain. In this context, decline in adult neurogenesis (AN), due to modifications in the neural progenitor stem cells (NSCs) and their microenvironment, is among the aging alterations contributing to cognitive decline. Therefore, the consumption or administration of antioxidant and anti-inflammatory molecules, such as dietary polyphenols, is under study as a potential beneficial strategy for preventing brain aging alterations including AN decline. Polyphenols, through their antioxidant and anti-inflammatory properties, modulate several cascades and effectors involved in the regulation of AN (e.g., SIRT1, Wnt, NF-κB and Nrf2, among others). This work summarizes the latest discoveries regarding the mechanisms whereby polyphenols preserve AN and counteract the cognitive decline present in aging.
... As a result, overexpression of miR-130b markedly enhanced the expression of IFN- in cells with IBDV infection or poly(I·C) treatment, while IFN-␣ expression was not affected ( Fig. 2A and B). Further, we tested the expression of two important transcriptional regulators of type I IFN expression: IRF3, also known as IRF7 in chickens (42,43), and p65, a member of the NF-B family with transcription activity (44,45). Consistently, the expression of NF-B and IRF3 was also upregulated in cells with miR-130b overexpression ( Fig. 2C and D). ...
MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression post transcriptionally through silencing or degrading its targets, playing important roles in immune response. However, the role of miRNAs in host response against infectious bursal disease virus (IBDV) infection is not clear. In this study, we show that the expression of a series of miRNAs was significantly altered in DF-1 cells after infectious bursal disease virus (IBDV) infection. We found that gga-miR-130b inhibited IBDV replication via targeting the specific sequence of IBDV segment A and enhanced the expression of IFN-β by targeting Suppressors of Cytokine Signaling 5(SOCS5) in host cells. These findings indicate that gga-miR-130b-3p plays a crucial role in host defense against IBDV infection.
Importance This work shows that gga-miR-130b suppresses IBDV replication via directly targeting the viral genome and cellular SOCS5, the negative regulator for type I interferon expression, revealing the mechanism underlying gga-miR-130-induced inhibition of IBDV replication. This information would be of great help to the understandings of how host cells combat pathogenic infection by self-encoded small RNA and further our knowledge of the role of MicroRNAs in cell response against viral infection.
... The transcriptional activation domain of NF-κB is in the p65 subunit [142,150,151]. This p65 subunit is also modulated by posttranslational modifications such as phosphorylation at serines (276, 311, 529 and 536) and acetylation at lysines 310 [110,151], 122, 123, 218 and 221 [149,152,153]. The over activation of this NF-κB signaling pathway is one of the transcriptional signs of aging process [141,154]. ...
Background:
Classically the oxidative stress and more recently inflammatory processes have been identified as the major causes of brain aging. Oxidative stress and inflammation affect each other, but there is more information about the effects of oxidative stress on aging than regarding the contribution of inflammation on it.
Methods:
In the intense research for methods to delay or mitigate the effects of aging, are interesting polyphenols, natural molecules synthesized by plants (e.g. resveratrol). Their antioxidant and anti-inflammatory properties make them useful molecules in the prevention of aging.
Results:
The antiaging effects of polyphenols could be due to several related mechanisms, among which are the prevention of oxidative stress, SIRT1 activation and inflammaging modulation, via regulation of some signaling pathways, such as NF-κB.
Conclusion:
In this review, we describe the positive effects of polyphenols on the prevention of the changes that occur during aging in the brain and their consequences on cognition, emphasizing the possible modulation of inflammaging by polyphenols through a SIRT1-mediated mechanism.
... M denotes molecular weight marker lane cell growth and development, and apoptosis [28]. ROS in cells are known to activate NF-кB leading to transactivation of targets involved in protection against ROS [29] and under high oxidative stress, activation of NF-кB enhances cell death [30]. The results from our study show that SIRT1 protects cells from oxidative stress in part by repressing the expression of NF-кB suggesting that possibly NF-кB enhances cell death on treatment with diquat or rotenone. ...
Background:
Sirtuins (SIRTs) are NAD+ dependent lysine deacetylases which are conserved from bacteria to humans and have been associated with longevity and lifespan extension. SIRT1, the best studied mammalian SIRT is involved in many physiological and pathological processes and changes in SIRT1 have been implicated in neurodegenerative disorders, with SIRT1 having a suggested protective role in Parkinson’s disease. In this study, we determined the effect of SIRT1 on cell survival and α-synuclein aggregate formation in SH-SY5Y cells following oxidative stress.
Results:
Over-expression of SIRT1 protected SH-SY5Y cells from toxin induced cell death and the protection conferred by SIRT1 was partially independent of its deacetylase activity, which was associated with the repression of NF-кB and cPARP expression. SIRT1 reduced the formation of α-synuclein aggregates but showed minimal co-localisation with α-synuclein. In post-mortem brain tissue obtained from patients with Parkinson’s disease, Parkinson’s disease with dementia, dementia with Lewy bodies and Alzheimer’s disease, the activity of SIRT1 was observed to be down-regulated.
Conclusions:
These findings suggests a negative effect of oxidative stress in neurodegenerative disorders and possibly explain the reduced activity of SIRT1 in neurodegenerative disorders. Our study shows that SIRT1 is a pro-survival protein that is downregulated under cellular stress.
... Previous in vitro and in vivo studies have reported that nuclear factor-κB (NF-κB) activity may be upregulated in dysplastic nevi and lesions of human melanoma compared with human nevi or melanocytes in normal skin (7)(8)(9). NF-κB is a transcription factor that is associated with the activation of several cell processes, including cell growth and apoptosis (10). This factor is activated in various cell types in response to numerous stimuli, including mitogens, inflammatory cytokines and extracellular stress. ...
Neokestose has superior prebiotic effects compared with the commercial fructooligosaccharides (FOS). In addition, the branched structure of neokestose, a type of neo‑FOS, confers improved chemical stability compared with conventional FOS; therefore, the investigation of the branched structure by the present study may be of high biomedical value. The present study aimed to determine whether neokestose may suppress growth of the A2058 melanoma cell line. The cells were initially treated with neokestose; subsequently, in vitro cytotoxicity was assessed using MTT, and cell cycle progression and apoptosis were detected using flow cytometry. The protein expression levels of cyclin D1, phosphorylated (p)‑inhibitor of κB (IκB) and nuclear factor‑κB (NF‑κB) were determined using western blotting. Treatment with neokestose led to a dose‑dependent inhibition of cell viability. Flow cytometry data indicated that neokestose increased the sub‑G1 cell population, and induced early and late apoptosis. Western blot analysis revealed that neokestose treatment reduced the expression levels of p‑IκB and cyclin D1. These findings suggest that neokestose treatment may induce suppression of A2058 melanoma cell viability via inhibition of the NF‑κB pathway. The present findings support the requirement for further investigation into the potential use of neokestose as an additional or chemopreventive therapeutic agent for the treatment of melanoma.
Ethnopharmacological relevance:
Ginseng (Panax ginseng C. A. Mey) is a common traditional Chinese medicine used for anti-inflammation, treating colitis, type 2 diabetes, diarrhea, and recovering hepatobiliary function. Ginsenosides, the main active components isolated from ginseng, possess liver and gallbladder diseases therapeutic potential.
Aims of the study:
Cholestatic liver injury (CLI) is a liver disease induced by intrahepatic accumulation of toxic bile acids and currently lacks clinically effective drugs. Our previous study found that ginsenosides alleviated CLI by activating sirtuin 1 (SIRT1), but the effective ingredients and the underlying mechanism have not been clarified. This study aimed to identify an effective ingredient with the most significant activation effect on SIRT1 from the five major monomer saponins of ginsenosides: Rb1, Rd, Rg1, 20s-Rg3, and Rc further explore its protective effects on CLI, and elaborate its underlying mechanism.
Materials and methods:
Discovery Studio 3.0 was used to conduct molecular docking between monomer saponins and SIRT1, and further detect the influence of monomer saponins on SIRT1 activity in vitro. Finally, it was determined that Rg1 had the most significant stimulative effect on SIRT1, and the hepatoprotective activity of Rg1 in CLI was explored in vivo. Wild-type mice were intragastrically α-naphthylisothiocyanate (ANIT) to establish an experimental model of intrahepatic cholestasis and Rg1 intervention, and then liver injury and cholestasis related indexes were detected. In addition, Liver-specific SIRT1 gene knockout (SIRT1-/-) mice were administered with ANIT and/or Rg1 to further investigate the mechanism of action of Rg1.
Results:
The results of molecular docking and in vitro experiments showed that all the five ginsenoside monomers could bind to the active site of SIRT1 and promote SIRT1 activity in HepG2 cells. Among them, Rg1 exhibited the most significant stimulation of SIRT1 activity in cholestasis. Besides, it could ameliorate ANIT-induced inflammation and oxidative stress in HepG2 cells. Therefore, we investigated the hepatoprotective effect and mechanism of Rg1 on CLI. Results showed that Rg1 reversed the ANIT-induced increase in biochemical parameters, improved liver pathological injury, and decreased liver lipid accumulation, reactive oxygen species and pro-inflammatory factor levels. Mechanistically, Rg1 induced SIRT1 expression, followed by promoted the activity of Nrf2 and suppressed the activation of NF-κB. Interestingly, the hepatoprotective effect of Rg1 was blocked in SIRT1-/- mice.
Conclusion:
Rg1 mitigated ANIT-induced CLI via upregulating SIRT1 expression, and our results suggested that Rg1 is a candidate compound for treating CLI.
Cadmium (Cd) exposure is known to cause gut inflammation. In this study, we investigated the protective effects of cordycepin, a natural compound with pharmacological properties, against gut inflammation induced by Cd exposure. Using zebrafish larvae and colon cell line models, we examined the impact of cordycepin on Cd-induced toxicity and inflammation. Zebrafish larvae were exposed to Cd (2µg/mL) and treated with different concentrations of cordycepin (12.5, 25 and 50µg/mL). Cordycepin treatment significantly reduced Cd-induced embryotoxicity in zebrafish larvae. It also alleviated Cd-induced oxidative stress by reducing reactive oxygen species (ROS), lipid peroxidation and apoptosis. Furthermore, cordycepin treatment normalized the levels of liver-related biomarkers affected due to Cd exposure. Additionally, cordycepin (50µg/mL) demonstrated a significant reduction in Cd bioaccumulation and downregulated the expression of inflammatory genes in both zebrafish larval gut and colon cell lines. These findings suggest that cordycepin could be an effective agent against Cd-induced gut inflammation.
Colorectal cancer is the cancer of the colon, located at the lower part of the digestive system. Although the role of STAT3 in cancer is known, its role particularly in colon cancer is largely unknown. STAT3 is a cytoplasmic transcription factor that involves extracellular signaling to the nucleus regulating fundamental functions, like cell proliferation, apoptosis, differentiation, and angiogenesis. STAT3 is a key regulator; abrogated activation leads to several diseases, including cancer. Aberrant interleukin (IL)-6-mediated JAK/STAT3 signaling pathway is closely related to the advancement of several human solid tumors including colorectal cancer. With other upstream regulators, IL-6/JAK signaling can activate STAT3, and its role appears to be critical in various types of cancer. STAT3 has been traditionally recognized as an oncogene; more recently the dual role of STAT3 in cancer, either tumor inductive or suppressive, has been appreciated. This chapter describes the potential role of STAT3 in colon cancer based on in vitro, in vivo, and patient studies. Furthermore, we will discuss the mechanism of action and roles of the IL-6/JAK/STAT3 pathway in colorectal cancer and exploit current therapeutic strategies, to treat colorectal cancer. Understanding the complexity of STAT3 function in colorectal cancer has the potential to elucidate important molecular aspects of colorectal cancer with significant therapeutic implications.
Obesity may create a mitogenic microenvironment that influences tumor initiation and progression. The obesity-associated adipokine, leptin regulates energy metabolism and has been implicated in cancer development. It has been shown that some cell types other than adipocytes can express leptin and leptin receptors in tumor microenvironments. It has been shown that peroxisome proliferator-activated receptors (PPAR) agonists can affect leptin levels and vice versa leptin can affect PPARs. Activation of PPARs affects the expression of several genes involved in aspects of lipid metabolism. In addition, PPARs regulate cancer cell progression through their action on the tumor cell proliferation, metabolism, and cellular environment. Some studies have shown an association between obesity and several types of cancer, including breast cancer. There is some evidence that suggests that there is crosstalk between PPARs and leptin during the development of breast cancer. Through a systematic review of previous studies, we have reviewed the published relevant articles regarding leptin signaling in breast cancer and its crosstalk with peroxisome proliferator-activated receptors α and γ.
The IκB kinase (IKK)-NF-κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double-strand breaks elicit two subsequent phases of NF-κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA-sequencing reveals that the first-phase controls anti-apoptotic gene expression, while the second drives expression of senescence-associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM-PARP1-TRAF6-IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re-expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF-κB family member p65/RelA, in part mediated by GSK3β, results in transcriptional silencing of NFKBIA and IKK-independent, constitutive activation of NF-κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF-κB activation with important implications for genotoxic cancer treatment.
Ganoderic Acid A (GA) has many pharmacological effects such as anti-tumor, antibacterial, anti-inflammatory, and immunosuppressive effects. However, the protective effect of GA on liver injury has not been reported. This study aimed to investigate the action of GA on insufficient methionine and choline combined with high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats. NAFLD model was established by insufficient methionine and choline combined with high fat feeding to rats. The levels of Acetyl-CoA carboxylase, fatty acid synthase, sterol regulatory element binding protein, liver X receptors, AMP-activated protein kinase, peroxisome proliferator-activated receptor α, PPARg coactivator 1α and NF-κB pathway in the liver were detected by western blot. The results of this study demonstrated that the expression of GA can not only significantly decrease the live weight and liver weight per body weight of HFD mice, but also restore the alanine aminotransferase, aspartate aminotransferase, total bilirubin levels, triglyceride and cholesterol in serum. In addition, the expression of GA increased the levels of high-density lipoprotein cholesterol in serum, ameliorated pathological changes and decreased NAS score of mice’s liver. In conclusion, the treatment with GA could improve NAFLD in rats by regulating the levels of signaling events involved in free fatty acid production, lipid oxidation and liver inflammation.
The alteration in cellular metabolism whereby cancer cell meets the demand of bioenergetics, biosynthesis, and redox status to support their uncontrolled cell proliferation, growth, tumor progression, and metastasis is considered as a prominent hallmark of cancer. Warburg effect is the most commonly noticed consequence of these metabolic reprogramming which aggravate cancer cell to opt for glycolytic pathway over more efficient oxidative phosphorylation even under normoxic condition to generate lactate, as well as intermediates for lipid, nucleotide, amino acids synthesis, which are essential to maintain tumorigenesis and cancer progression. In order to develop efficient chemotherapeutic drug, various enzymes and proteins involved or associated with glycolytic pathways such as PMK2, LDHA and signaling pathways such as PKI3-Akt-mTOR are being targeted to inhibit various stages of cancer progression. In that direction, phytochemicals that are bioactive compounds obtained from plant sources have displayed promising results in hampering the growth of various cancer cell lines. Compounds of flavonoid class such as quercetin and fisetin along with other polyphenols and non-flavonoids such as resveratrol, isothiocyanates, and curcumin have displayed remarkable inhibitory effect on cancer cell metabolism. Overall, this chapter will highlight the effect of different phytochemicals on the metabolic pathways of cancer cells to inhibit various stages of cancer progression.
NF-κB is a transcription factor that regulates the expression of genes by directly binding to DNA. It’s involved in immune system, cell survival, cell proliferation, learning and memory processes. The SUMO protein, which is involved in post-translational modification, regulates the function of exogenous proteins and there are four homologs in mammals. Another protein involved in post-translational modification is ubiquitin. It is involved in many biological pathways and leads the marked proteins to destruction by directing them to the proteosome system. In this review NF-κB, SUSMO and ubiquitin proteins and their relationship to each other will be discussed.
Keywords: NF-κB, SUMO, Sumolation, Ubiquitin, Ubiquitination, Ubiquitin-Proteosome system.
Atherosclerosis (AS) is a chronic inflammation, which is a major cause of morbidity and mortality in the world. Accumulative evidences have demonstrated that miRNAs exert crucial roles in the development of AS. However, the effects of miR-145 and its underlying molecular mechanism remain incompletely clear. The aim of the present study is to explore the function of miR-145 in the occurrence and development of AS through investigating its role in inflammatory reactions. High-fat diet (HFD)-treated ApoE-/- mice were used as an in vivo model of atherosclerosis (AS). OxLDL-induced macrophages was employed as cell models of atherosclerosis. RT-PCR was used to evaluate the transfected efficiency of miR-145 mimic and inhibitor. RT-PCR and ELISA were performed to detect the expression of miR-145, and inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), C-C motif chemokine ligand 2 (CCL-2), CCL-4 and CCL-7. Western blotting was used to evaluate the protein expression of nuclear factor κB (NF-κB) and its related proteins such as phosphorylated-signal transducer and activator of transcription 3 (p-STAT3), p-IκBα and acetylated p65 (ac-p65). Hematoxylin and eosin (H&E) staining were conducted to examine atherosclerotic lesion. Immunohistochemistry was carried out to detect the expression of α-smooth muscle Actin (α-SMA) and CD68. Luciferase reporter assay were carried out to examine the effect of miR-145 on the transcriptional activity of NF-κB. Our results showed that over-expression of miR-145 promoted the expression of IL-1β, TNF-α, CCL-2, CCL-4 and CCL-7 through promotion of NF-κB, p-IκBα, p-STAT3 and ac-p65 expression in vivo and in vitro. Besides, down-regulation of miR-145 expression relieved the aortic sinus lesion, increased the number of VSMCs and decreased the number of macrophages. In conclusion, our study demonstrated that miR-145 accelerated the inflammatory reaction through activation of NF-κB signaling in AS.
Nuclear factor κB (NF-κB) is one of the transcription factors involved in the progression of pancreatic cancer. Pancreatic cancer (PC) is a deadly cancer in today’s world, and treatment approaches become critical due to poor prognosis and chemoresistance of the cancer. The NF-κB signaling pathway is known to induce cell proliferation and metastasis, including migration and angiogenesis. Furthermore, the NF-κB pathway is also involved in the prevention of apoptosis in PC. In this present chapter, we discuss the role of NF-κB in the progression of PC, resistance to chemo drugs such as gemcitabine, and the influence of downstream targets on the metastatic characteristics of cancer. Therefore, targeting the NF-κB signaling pathway stands as a rational approach for future treatment of pancreatic cancer.
Glycine modulates inflammatory processes mediated by macrophages and adipocytes through decreasing the secretion of TNF-α, IL-6, and leptin, while increasing adiponectin. These effects have been associated with the inactivation of NF-κB in response to TNF-α, across an increase of its inhibitor IκB-α in adipocytes. However, glycine upstream mainly influences the IκB kinase (IKK) complex, a multi-protein kinase complex considered a critical point in regulation of the NF-κB pathway; whether that is responsible for the TNF-α-induced phosphorylation of IkB has not been explored. Additionally, although previous studies have described glycine interactions with specific receptors (GlyR) in different immune system cell types, it is currently unknown whether adipocytes present GlyR. In this research, participation of the IKK-α/β complex in the inhibition of the TNF-α/NF-κB pathway by glycine was evaluated and associated with the synthesis and secretion of inflammatory cytokines in 3T3-L1 adipocytes. Furthermore, we also explored GlyR expression, its localization on the plasmatic membrane, intracellular calcium concentrations [Ca2+]iand strychnine antagonist action over the GlyR in these cells. Glycine decreased the IKK-α/β complex and the phosphorylation of NF-κB, diminishing the expression and secretion of IL-6 and TNF-α, but increasing that of adiponectin. GlyR expression and its fluorescence in the plasma membrane were increased in the presence of glycine. In addition, glycine decreased [Ca2+]i; whereas strychnine + glycine treatment inhibited the activation of NF-κB observed with glycine. In conclusion, the reduction of TNF-α and IL-6 and suppression of the TNF-α/NF-κB pathway by glycine may be explained in part by inhibition of the IKK-α/β complex, with a possible participation of GlyR in 3T3-L1 adipocytes.
Whole-organism chemical screening can circumvent bottlenecks that impede drug discovery. However, in vivo screens have not attained throughput capacities possible with in vitro assays. We therefore developed a method enabling in vivo high-throughput screening (HTS) in zebrafish, termed automated reporter quantification in vivo (ARQiv). In this study, ARQiv was combined with robotics to fully actualize whole-organism HTS (ARQiv-HTS). In a primary screen, this platform quantified cell-specific fluorescent reporters in textgreater500,000 transgenic zebrafish larvae to identify FDA-approved (Federal Drug Administration) drugs that increased the number of insulin-producing $beta$ cells in the pancreas. 24 drugs were confirmed as inducers of endocrine differentiation and/or stimulators of $beta$-cell proliferation. Further, we discovered novel roles for NF-$kappa$B signaling in regulating endocrine differentiation and for serotonergic signaling in selectively stimulating $beta$-cell proliferation. These studies demonstrate the power of ARQiv-HTS for drug discovery and provide unique insights into signaling pathways controlling $beta$-cell mass, potential therapeutic targets for treating diabetes.
Incontinentia pigmenti is a rare neuroectodermal dysplasia caused by a defect in the IKBKG gene (formerly known as NEMO). There are 27.6 new cases per year worldwide; 65% to 75% are sporadic mutations, and 25% to 35% are familial. It is usually lethal in males, but females survive because of X-inactivation mosaicism. The disorder is typically identified by unique skin findings, a series of four stages that emerge throughout the first year of life. The central nervous system manifestations in the eye and in the brain cause the most disability. Defects of hair, nails, and teeth occur, and there can be other systemic involvement. Surveillance protocols for medical management have been established by the Incontinentia Pigmenti International Foundation. This article will summarize the existing knowledge of this condition and detail the protocols to help manage the care of the infant or child who presents with incontinentia pigmenti.
Background:
With global increase in elderly population, modern societies must find strategies to reduce the consequences of aging process; thereby decreasing the incidence of age-related neurodegenerative diseases. Oxidative stress and recently inflammation, have been pointed out as the leading causes of brain aging. Thereby, the consumption or administration of antioxidant and anti-inflammatory molecules, such as polyphenols, is a beneficial strategy recommended for preventing brain aging and several brain age-related diseases.
Methods and results:
Several studies suggest that long term consumption of dietary polyphenols offers protection against development of neurodegenerative diseases. These beneficial effects are in part due to their antioxidant and anti-inflammatory properties, together with their positive role in the modulation of processes involved in the physiopathology of several neurodegenerative diseases (e.g., epigenetic factors, amyloid deposition, cholinesterase inhibition, autophagy, and neurotrophic factors, among others). Altogether, these molecules open the door to the research of new neuroprotective strategies. This review summarizes the latest discoveries in how polyphenols can exert positive effects on brain health in aging, emphasizing those effects on the diseases that most commonly affect the brain during aging: Parkinson's Disease (PD), Alzheimer's disease (AD), dementia and depression. Moreover, within are addressed the epigenetic effects of polyphenols as possible mediators in their positive effects on brain health, and the future challenges of research in this topic Conclusion: In brief, this review presents a report of state-of the art knowledge regarding the positive influences of polyphenols on the most common brain age-related diseases as well as in healthy brain aging.
Processing of the NF-κB2 precursor p100 to the mature p52 subunit is regulated via a unique pathway. NF-κB-inducing kinase (NIK) induces IκB kinase α (IKKα)-mediated phosphorylation of specific serine residues in the C-terminal domain of p100, leading to recruitment of the SCFβ-TrCP ubiquitin ligase. We identified a single lysine residue, K855, that serves as the ubiquitin-anchoring residue required for signal-induced processing of p100. In a reconstituted system containing purified components, p100-K855R could not be ubiquitinated. In a crude extract and cells, only residual, signal-independent ubiquitination and processing were retained. Importantly, K855 is located in a site homologous to K22 that serves as an ubiquitination site in IκBα. This suggests a common recognition mechanism for the two molecules. In contrast, p105, the p100 homologue, lacks a similar Lys residue. We also demonstrate that the NEDD8 pathway is essential for the SCFβ-TrCP activity. In a reconstituted system, efficient ubiquitination of p100 required all three components of the pathway – E1, the UBC12 E2 and NEDD8. Experiments in reconstituted systems and in cells demonstrated that SCFβ-TrCP, which contains a mutant Cul-1 that cannot be NEDDylated, cannot stimulate ubiquitination and processing. Similarly, dominant negative UBC12 inhibits, in a reversible manner, both ubiquitination and processing of p100.
Antiapoptotic activity of NF-κB in tumors contributes to acquisition of resistance to chemotherapy. Degradation of IκB is
a seminal step in activation of NF-κB. The IκB kinases, IKK1 and IKK2, have been implicated in both IκB degradation and subsequent
modifications of NFκB. Using mouse embryo fibroblasts (MEFs) devoid of both IKK1 and IKK2 genes (IKK1/2−/−), we document a novel IκB degradation mechanism. We show that this degradation induced by a chemotherapeutic agent, doxorubicin
(DoxR), does not require the classical serine 32 and 36 phosphorylation or the PEST domain of IκBα. Degradation of IκBα is
partially blocked by phosphatidylinositol 3-kinase inhibitor LY294002 and is mediated by the proteasome. Free NF-κB generated
by DoxR-induced IκB degradation in IKK1/2−/− cells is able to activate chromatin based NF-κB reporter gene and expression of the endogenous target gene, IκBα. These results
also imply that modification of NF-κB by IKK1 or IKK2 either prior or subsequent to its release from IκB is not essential
for NF-κB-mediated gene expression at least in response to DNA damage. In addition, DoxR-induced cell death in IKK1/2−/− MEFs is enhanced by simultaneous inhibition of NF-κB activation by blocking the proteasome activity. These results reveal
an additional pathway of activating NF-κB during the course of anticancer therapy and provide a mechanistic basis for the
observation that proteasome inhibitors could be used as adjuvants in chemotherapy.
Glycogen synthase kinase-3 (GSK-3)- and - are closely related
protein-serine kinases, which act as inhibitory components of Wnt signalling
during embryonic development and cell proliferation in adult tissues1, 2. Insight into the physiological function of GSK-3 has emerged
from genetic analysis in Drosophila3, 4, Dictyostelium
5 and yeast6, 7. Here we show that disruption
of the murine GSK-3 gene results in embryonic lethality caused
by severe liver degeneration during mid-gestation, a phenotype consistent
with excessive tumour necrosis factor (TNF) toxicity, as observed in mice
lacking genes involved in the activation of the transcription factor activation
NF-B. GSK-3-deficient embryos were rescued by inhibition of TNF
using an anti-TNF- antibody. Fibroblasts from GSK-3-deficient
embryos were hypersensitive to TNF- and showed reduced NF-B
function. Lithium treatment (which inhibits GSK-3; refs
8, 9) sensitized wild-type fibroblasts
to TNF and inhibited transactivation of NF-B. The early steps leading
to NF-B activation (degradation of I-B and translocation of
NF-B to the nucleus) were unaffected by the loss of GSK-3, indicating
that NF-B is regulated by GSK-3 at the level of the transcriptional
complex. Thus, GSK-3 facilitates NF-B function.
The IKKβ and NEMO/IKKγ subunits of the NF-κB-activating signalsome complex are known to be essential for activating NF-κB
by inflammatory and other stress-like stimuli. However, the IKKα subunit is believed to be dispensable for the latter responses
and instead functions as an in vivomediator of other novel NF-κB-dependent and -independent functions. In contrast to this generally accepted view of IKKα's
physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKβ and NEMO/IKKγ, IKKα is also
a global regulator of tumor necrosis factor α- and IL-1-responsive IKK signalsome-dependent target genes including many known
NF-κB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth
factor, Ptx3, β2-microglobulin, IL-1α, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B,
c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of
NF-κB-dependent target genes were preferentially dependent on IKKα or IKKβ. Constitutive expression of a trans-dominant IκBα
superrepressor (IκBαSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-κB.
A subset of NF-κB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was
also required to regulate basal levels of activated NF-κB in established MEFs. Overall, a sizable number of novel NF-κB/IKK-dependent
genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-β and -δ,
osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin,
interferon (α and β) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins
B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-κB/IKK-induced gene, suggesting that IKK-mediated
NF-κB activation can coordinately illicit negative effects on STAT signaling.
NF-κB dimers, inhibitor IκB proteins, and NF-κB·IκB complexes exhibit distinct patterns in partitioning between nuclear and
cytoplasmic cellular compartments. IκB-dependent modulation of NF-κB subcellular localization represents one of the more poorly
understood processes in the NF-κB signaling pathway. In this study, we have combined in vitro biochemical and cell-based methods to elucidate differences in NF-κB regulation exhibited by the inhibitors IκBβ and IκBα.
We show that although both IκBα and IκBβ bind to NF-κB with similar global architecture and stability, significant differences
exist that contribute to their unique functional roles. IκBβ derives its high affinity toward NF-κB dimers by binding to both
NF-κB subunit nuclear localization signals. In contrast, IκBα contacts only one NF-κB NLS and employs its carboxyl-terminal
proline, glutamic acid, serine, and threonine-rich region for high affinity NF-κB binding. We show that the presence of one
free NLS in the NF-κB·IκBα complex renders it a dynamic nucleocytoplasmic complex, whereas NF-κB·IκBβ complexes are localized
to the cytoplasm of resting cells.
Activation of the transcription factor NF-kappaB by inflammatory cytokines involves the successive action of NF-kappaB-inducing kinase (NIK) and two IkappaB kinases, IKK-alpha and IKK-beta. Here we show that NIK preferentially phosphorylates IKK-alpha over IKK-beta, leading to the activation of IKK-alpha kinase activity. This phosphorylation of IKK-alpha occurs specifically on Ser-176 in the activation loop between kinase subdomains VII and VIII. A mutant form of IKK-alpha containing alanine at residue 176 cannot be phosphorylated or activated by NIK and acts as a dominant negative inhibitor of interleukin 1- and tumor necrosis factor-induced NF-kappaB activation. Conversely, a mutant form of IKK-alpha containing glutamic acid at residue 176 is constitutively active. Thus, the phosphorylation of IKK-alpha on Ser-176 by NIK may be required for cytokine-mediated NF-kappaB activation.
Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-κB. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-κB-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact KB sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-κB-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-κB in the nucleus. We showed that the p65 subunit of NF-κB was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-κB after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the IκBα inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-κB. This delay was due neither to a defect in IκBα mRNA production nor to a nuclear retention of IκBα but was rather due to a persistent proteasome-mediated degradation of IκBα. A prolongation of IκB kinase activity could explain, at least partially, the delayed IκBα cytoplasmic reappearance observed in presence of TNF plus TSA.
It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-κB to initiate transformation, at least partially through the ability of NF-κB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-κB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-κB as well as stimulation of the NF-κB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-κB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-κB nuclear translocation via IκB degradation. Inhibition of IκB kinase (IKK), using an IKKβ dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-κB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-κB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-κB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-κB, Akt stimulates NF-κB predominantly by upregulating of the transactivation potential of p65.
The nuclear form of the NF-kappa B transcription factor binds to DNA as a heterodimer of a 50 kDa (p50) and 65 kDa (p65) polypeptide. The two polypeptides are encoded by different genes but share a long region of homology, the NRD motif, encompassing domains required for DNA binding and dimerization. In this study we have analysed the contribution of the two subunits to the strong transactivating potential of NF-kappa B. Transient expression of the p65 subunit alone resulted in a potent transactivation of a CAT reporter construct under the control of two NF-kappa B binding sites in monkey COS and mouse L cells. The strongly DNA binding p50 subunit showed only very weak, if any, induction of gene expression. Co-expression of p50 suppressed the transactivation by p65 presumably by competitive DNA binding of transcriptionally inactive p50 dimers (KBF1). Fusion of p65 sequences to DNA binding domain of the yeast GAL4 transcription factor allowed detection of the principal transactivation domain of p65 (TA1) in the C-terminal 30 amino acid sequence. TA1 is likely to adopt an amphipathic alpha-helical structure which clusters serine residues on the hydrophilic surface, a structural feature conserved between human, mouse and Xenopus p65. The unique C-terminal third of p65 contained at least one more activation domain, TA2, within a 90 amino acid sequence directly adjacent to TA1. In two mammalian cell lines, TA1 and TA2 acted separately, while in an insect cell line, the two domains were inactive after their separation. Our study suggests that the p50 subunit in NF-kappa B might only serve a helper function in DNA binding whereas the p65 subunit is responsible for initiating transcription. Homodimers of p50 seem to have the potential of down-regulating kappa B-specific gene expression.
Cell division protein FtsA, predicted to belong to the actin family, is present in different cell compartments depending on its phosphorylation state. The FtsA fraction isolated from the cytoplasm is phosphorylated and capable of binding ATP, while the membrane-bound form is unphosphorylated and does not bind ATP. A variant of the protein FtsA102, in which the nucleotide binding site was destroyed by mutagenesis of a highly conserved residue predicted to be needed for the binding, does not bind ATP. Another variant, FtsA104, cannot be phosphorylated because the predicted phosphorylatable residue has been replaced by a non-phosphorylatable one. This protein although unable to bind ATP in vitro, is able to rescue the reversible ftsA2, the irreversible ftsA3 and, almost with the same efficiency, the ftsA16 amber alleles. Consequently, phosphorylation and ATP binding may not be essential for the function of FtsA. Alternatively they may have a regulatory role on the action of FtsA in the septator.
CBP (CREB-binding protein) and p300 are versatile coactivators that link transcriptional activators to the basal transcriptional apparatus. In the present study, we identify CBP and p300 as coactivators of the nuclear factor-kappaB (NF-kappaB) component p65 (RelA). Consistent with their role as coactivators, both CBP and p300 potentiated p65-activated transcription of E-selectin and VCAM-1-CAT reporter constructs. The N- and C-terminal domains of both CBP/p300 functionally interact with a region of p65 containing the transcriptional activation domain as demonstrated by mammalian two-hybrid assays. Direct physical interactions of CBP/p300 with p65 were demonstrated by glutathione S-transferase fusion protein binding, and coimmunoprecipitation/Western blot studies. The adenovirus E1A 12S protein, which complexes with CBP and p300, inhibited p65-dependent gene expression. Reporter gene expression could be rescued from E1A inhibition by overexpression of CBP or p300. CBP and p300 act as coactivators of p65-driven gene activation and may play an important role in the cytokine-induced expression of various immune and inflammatory genes.
The activity of transcription factor NFkappaB is regulated by its subcellular localization. In most cell types, NFkappaB is sequestered in the cytoplasm due to binding of the inhibitory protein IkappaB alpha. Stimulation of cells with a wide variety of agents results in degradation of IkappaB alpha which allows translocation of NFkappaB to the nucleus. Degradation of IkappaB alpha is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IkappaB alpha kinase. p90rsk1 phosphorylates IkappaB alpha at Ser32 and it physically associates with IkappaB alpha in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, IkappaB alpha degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFkappaB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IkappaB alpha in response to mitogens.
Transcriptional activation of nuclear factor kappaB (NF-kappaB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. However, NF-kappaB activation induces rapid resynthesis of IkappaBalpha, which is responsible for post-induction repression of transcription. Newly synthesized IkappaBalpha translocates to the nucleus, where it dissociates NF-kappaB from DNA and transports NF-kappaB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-kappaB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-kappaB-dependent transcriptional activation mediated by interleukin 1beta or tumor necrosis factor alpha. This is a consequence of the inhibition of signal-induced degradation of IkappaBalpha. Although LMB treatment does not affect the signal transduction pathway leading to IkappaBalpha degradation, it blocks IkappaBalpha nuclear export. IkappaBalpha is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IkappaBalpha is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IkappaBalpha is therefore essential for maintaining a low level of IkappaBalpha in the nucleus and allowing NF-kappaB to be transcriptionally active upon cell stimulation.
The activity of the transcription factor NF-kappaB is thought to be regulated mainly through cytoplasmic retention by IkappaB molecules. Here we present evidence of a second mechanism of regulation acting on NF-kappaB after release from IkappaB. In endothelial cells this mechanism involves phosphorylation of the RelA subunit of NF-kappaB through a pathway involving activation of protein kinase Czeta (PKCzeta) and p21(ras). We show that transcriptional activity of RelA is dependent on phosphorylation of the N-terminal Rel homology domain but not the C-terminal transactivation domain. Inhibition of phosphorylation by dominant negative mutants of PKCzeta or p21(ras) results in loss of RelA transcriptional activity without interfering with DNA binding. Raf/MEK, small GTPases, phosphatidylinositol 3-kinase, and stress-activated protein kinase pathways are not involved in this mechanism of regulation.
IkappaB kinases (IKKs) IKK1 and IKK2 are two putative IkappaBalpha kinases involved in NF-kappaB activation. To examine the in vivo functions of IKK1, we generated IKK1-deficient mice. The mutant mice are perinatally lethal and exhibit a wide range of developmental defects. Newborn mutant mice have shiny, taut, and sticky skin without whiskers. Histological analysis shows thicker epidermis, which is unable to differentiate. Limbs and tail are wrapped inside the skin and do not extend properly out of the body trunk. Skeleton staining reveals a cleft secondary palate, split sternebra 6, and deformed incisors. NF-kappaB activation mediated by TNFalpha and IL-1 is diminished in IKK1-deficient mouse embryonic fibroblast (MEF) cells. The IKK complex in the absence of IKK1 is capable of phosphorylating IkappaBalpha and IkappaBbeta in vitro. Our results support a role for IKK1 in NF-kappaB activation and uncover its involvement in skin and skeleton development. We conclude further that the two related kinases IKK1 and IKK2 have distinct functions and can not be substituted for each other's functions.
The work of Reddy et al. (S. A. Reddy, J. A. Huang, and W. S. Liao, J. Biol. Chem. 272:29167-29173, 1997) reveals that phosphatidylinositol 3-kinase (PI3K) plays a role in transducing a signal from the occupied interleukin-1 (IL-1) receptor to nuclear factor kappaB (NF-kappaB), but the underlying mechanism remains to be determined. We have found that IL-1 stimulates interaction of the IL-1 receptor accessory protein with the p85 regulatory subunit of PI3K, leading to the activation of the p110 catalytic subunit. Specific PI3K inhibitors strongly inhibit both PI3K activation and NF-kappaB-dependent gene expression but have no effect on the IL-1-stimulated degradation of IkappaBalpha, the nuclear translocation of NF-kappaB, or the ability of NF-kappaB to bind to DNA. In contrast, PI3K inhibitors block the IL-1-stimulated phosphorylation of NF-kappaB itself, especially the p65/RelA subunit. Furthermore, by using a fusion protein containing the p65/RelA transactivation domain, we found that overexpression of the p110 catalytic subunit of PI3K induces p65/RelA-mediated transactivation and that the specific PI3K inhibitor LY294,002 represses this process. Additionally, the expression of a constitutively activated form of either p110 or the PI3K-activated protein kinase Akt also induces p65/RelA-mediated transactivation. Therefore, IL-1 stimulates the PI3K-dependent phosphorylation and transactivation of NF-kappaB, a process quite distinct from the liberation of NF-kappaB from its cytoplasmic inhibitor IkappaB.
Nuclear factor-κB (NF-κB) plays a role in the transcriptional regulation of genes involved in inflammation and cell survival.
In this report we demonstrate that NF-κB recruits a coactivator complex that has striking similarities to that recruited by
nuclear receptors. Inactivation of either cyclic AMP response element binding protein (CREB)-binding protein (CBP), members
of the p160 family of coactivators, or the CBP-associated factor (p/CAF) by nuclear antibody microinjection prevents NF-κB-dependent
transactivation. Like nuclear receptor-dependent gene expression, NF-κB-dependent gene expression requires specific LXXLL
motifs in one of the p160 family members, and enhancement of NF-κB activity requires the histone acetyltransferase (HAT) activity
of p/CAF but not that of CBP. This coactivator complex is differentially recruited by members of the Rel family. The p50 homodimer
fails to recruit coactivators, although the p50-p65 heterodimeric form of the transcription factor assembles the integrator
complex. These findings provide new mechanistic insights into how this family of dimeric transcription factors has a differential
effect on gene expression.
Recent investigations have elucidated the cytokine-induced NF-kappaB activation pathway. IkappaB kinase (IKK) phosphorylates inhibitors of NF-kappaB (IkappaBs). The phosphorylation targets them for rapid degradation through a ubiquitin-proteasome pathway, allowing the nuclear translocation of NF-kappaB. We have examined the possibility that IKK can phosphorylate the p65 NF-kappaB subunit as well as IkappaB in the cytokine-induced NF-kappaB activation. In the cytoplasm of HeLa cells, the p65 subunit was rapidly phosphorylated in response to TNF-alpha in a time dependent manner similar to IkappaB phosphorylation. In vitro phosphorylation with GST-fused p65 showed that a p65 phosphorylating activity was present in the cytoplasmic fraction and the target residue was Ser-536 in the carboxyl-terminal transactivation domain. The endogenous IKK complex, overexpressed IKKs, and recombinant IKKbeta efficiently phosphorylated the same Ser residue of p65 in vitro. The major phosphorylation site in vivo was also Ser-536. Furthermore, activation of IKKs by NF-kappaB-inducing kinase induced phosphorylation of p65 in vivo. Our finding, together with previous observations, suggests dual roles for IKK complex in the regulation of NF-kappaB.IkappaB complex.
Nuclear factor kappaB (NF-kappaB)/Rel transcription factors are key regulators of a variety of genes involved in immune and inflammatory responses, growth, differentiation, apoptosis, and development. In unstimulated cells, NF-kappaB/Rel proteins are sequestered in the cytoplasm by IkappaB inhibitor proteins. Many extracellular stimuli, such as tumor necrosis factor alpha (TNFalpha), cause rapid phosphorylation of IkappaB at N-terminal serine residues leading to ubiquitination and degradation of the inhibitor. Subsequently, NF-kappaB proteins translocate to the nucleus and activate gene expression through kappaB response elements. TNFalpha, as well as certain other stimuli, also induces the phosphorylation of the NF-kappaB proteins. Previously, we have shown that TNFalpha induces RelA/p65 phosphorylation at serine 529 and that this inducible phosphorylation increases NF-kappaB transcriptional activity on an exogenously supplied reporter (). In this report, we demonstrate that casein kinase II (CKII) interacts with p65 in vivo and can phosphorylate p65 at serine 529 in vitro. A CKII inhibitor (PD144795) inhibited TNFalpha-induced p65 phosphorylation in vivo. Furthermore, our results indicate that the association between IkappaBalpha and p65 inhibits p65 phosphorylation by CKII and that degradation of IkappaBalpha allows CKII to phosphorylate p65 to increase NF-kappaB transactivation potential. These data may explain the ability of CKII to modulate cell growth and demonstrate a mechanism whereby CKII can function in an inducible manner.
Induction of NF-kappaB-dependent transcription requires phosphorylation and subsequent degradation of I-kappaB, an inhibitor of NF-kappaB, followed by nuclear translocation and DNA binding of NF-kappaB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-kappaB activation in response to cytokines such as tumor necrosis factor alpha (TNFalpha). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-kappaBalpha in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k(-/-) animals die at approximately E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k(-/-) embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFalpha-induced apoptosis. In response to either TNFalpha or IL-1 induction, t2k(-/-) embryonic fibroblasts exhibit normal degradation of I-kappaB and kappaB-binding activity. However, NF-kappaB-directed transcription is dramatically reduced. These results demonstrate that, like I-kappaB kinase beta and the RelA subunit of NF-kappaB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-kappaB-directed transcription, apparently independent of I-kappaB degradation and NF-kappaB DNA binding.
The nf-kb2 gene encodes the cytoplasmic NF-κB inhibitory protein p100 from which the active p52 NF-κB subunit is derived by proteasome-mediated proteolysis. Ligands which stimulate p100 processing to p52 have not been defined. Here, ligation of CD40 on transfected 293 cells is shown to trigger p52 production by stimulating p100 ubiquitylation and subsequent proteasome-mediated proteolysis. CD40-mediated p52 accumulation is dependent on de novo protein synthesis and triggers p52 translocation into the nucleus to generate active NF-κB dimers. Endogenous CD40 ligation on primary murine splenic B cells also stimulates p100 processing, which results in the delayed nuclear translocation of p52–RelB dimers. In both 293 cells and primary splenic B cells, the ability of CD40 to trigger p100 processing requires functional NF-κB-inducing kinase (NIK). In contrast, NIK activity is not required for CD40 to stimulate the degradation of IκBα in either cell type. The regulation of p100 processing by CD40 is likely to be important for the transcriptional regulation of CD40 target genes in adaptive immune responses.
Surface-expressed BCR mediates the proliferation and expansion of antigen-specific B lymphocytes during a humoral immune response. Although several studies extensively characterize BCR proliferative signaling, the mechanisms linking these pathways to the cell cycle remain elusive. Using knockout mice, we show that c-Rel, a proto-oncogenic member of the NF-kB transcription factor family, is essential to BCR-mediated proliferation and cell cycle progression. Splenic B cells obtained from gene-targeted c-Rel knockout mice display a defective proliferation response to antigen receptor cross-linking, resulting in G1 arrest. At the molecular level, we see that BCR stimulation of resting c-Rel‐/‐ B cells fails to induce proper cyclin D3 and cyclin E expression, thereby negatively impacting G1 phase cyclin-dependent kinase (CDK) activity. c-Reldeficient B cells also exhibit incomplete phosphorylation of the Retinoblastoma protein (pRb) and poor expression of E2Fs, thus impeding the G1 to S phase transition. Down-regulation of the pRbrelated p130 protein during the G0 to G1 transition and removal of the CDK inhibitor p27 KIP1 in late G1 parallel that of wild-type cells, suggesting that Rel-deficient B cells can exit the G0 resting state and enter G1 phase normally. Finally, we demonstrate that restoration of proliferation can be achieved partially upon reintroduction of cyclin E using a protein transduction method to reconstitute primary B cells. Collectively, these studies emphasize the importance of c-Rel in lymphocyte proliferation and oncogenesis, and highlight a requirement for c-Rel in establishing an effective humoral immune response.
Excerpt
In 1986, Sen and Baltimore (1986) identified a nuclearfactor specific for the immunoglobulin κ light-chain geneenhancer of B lymphocytes. Nearly a decade and a halflater, the transcription factor NF-κB is recognized for itsroles in such vital cellular processes as immune and inflammatory responses, cellular adhesion, growth and development, and apoptosis (Baeuerle and Henkel 1994;Baeuerle and Baltimore 1996; Baldwin 1996; Ghosh etal. 1998). Regulation of NF-κB activity is governed byseveral factors including selective dimer formation, sequence-specific DNA binding, and interactions with IκBinhibitor proteins. Most recently, the field of NF-κB biology has benefited from a series of timely and well-executed structural studies. X-ray crystallographic analysesof NF-κB, both alone and in complex with various DNAtargets, have resulted in the elucidation of rules that govern NF-κB dimerization and DNA-binding selectivity.Furthermore, the crystal structure of NF-κB in complexwith the IκBα inhibitor protein has provided a wealth ofinformation regarding the regulation of NF-κB activity.Here, we review these structures in the context of NF-κBsignaling and transcriptional activation...
NF-κB-inducing kinase (NIK) is involved in the signal transduction pathway leading to the NF-κB activation. In this report, we demonstrate that the NIK-mediated NF-κB activation involves the transactivation (TA) domain of p65 subunit of NF-κB and the nuclear translocation of IKKα. By using luciferase assay, we found that both IKKα and IKKβ could activate NF-κB in synergy with NIK. Interestingly, although IKKβ stimulated the NIK-mediated IκB degradation, IKKα stimulated the action of NF-κB without enhancing the IκB degradation. By using heterologous transactivation system with Gal4 DNA-binding domain in fusion with various portions of p65 TA domain, we found that the transactivation domain 1 (TA1) of p65 serves as the direct target for the NIK–IKKα cascade and that the serine residue at 536 within p65 TA1 is indispensable for this action. Furthermore, we found that this action of NIK depends on the energy-dependent action of Ras-related protein (Ran) since the dominant negative mutant of Ran (RanQ69L) inhibited the transcriptional activity of p65 by preventing the nuclear import of IKKα.
IKKα is a component of the IκB kinase (IKK) complex that plays a key role in the activation of NF-κB. In Ikkα mutant mice and mice expressing a transdominant negative mutant of IκBα (cIκBαΔN), molars have abnormal cusps, indicating that Ikkα is involved in cusp formation through the NF-κB pathway. However, Ikkα mutant incisors also have an earlier phenotype where epithelium evaginates outward into the developing oral cavity rather than invaginating into the underlying mesenchyme. A similar evagination of epithelium was also observed in whisker development, suggesting that Ikkα contributes to the direction of epithelial growth during the early stages of development in many ectodermal appendages. Since cIκBαΔN mice have normal incisor epithelial invagination, Ikkα's role appears to be NF-κB independent. Changes in Notch1, Notch2, Wnt7b, and Shh expression found in incisor epithelium of Ikkα mutants suggest that this NF-κB-independent function is mediated by Notch/Wnt/Shh signaling pathways.
IκB kinase-α and -β (IKK-α and IKK-β), the catalytic subunits of the IKK complex, phosphorylate IκB proteins on specific serine residues, thus targeting IκB for degradation and activating the transcription factor NF-κB. To elucidate the in vivo function of IKK-β, we generated IKK-β-deficient mice. The homozygous mouse embryo dies at ∼14.5 days of gestation due to liver degeneration and apoptosis. IKK-β-deficient embryonic fibroblasts have both reduced basal NF-κB activity and impaired cytokine-induced NF-κB activation. Similarly, basal and cytokine-inducible kinase activities of the IKK complex are greatly reduced in IKK-β-deficient cells. These results indicate that IKK-β is crucial for liver development and regulation of NF-κB activity and that IKK-α can only partially compensate for the loss of IKK-β.
NF-κB is activated in response to proinflammatory stimuli, infections, and physical stress. While activation of NF-κB by many stimuli depends on the IκB kinase (IKK) complex, which phosphorylates IκBs at N-terminal sites, the mechanism of NF-κB activation by ultraviolet (UV) radiation remained enigmatic, as it is IKK independent. We now show that UV-induced NF-κB activation depends on phosphorylation of IκBα at a cluster of C-terminal sites that are recognized by CK2 (formerly casein kinase II). Furthermore, CK2 activity toward IκB is UV inducible through a mechanism that depends on activation of p38 MAP kinase. Inhibition of this pathway prevents UV-induced IκBα degradation and increases UV-induced cell death. Thus, the p38-CK2-NF-κB axis is an important component of the mammalian UV response.
Transcription factors within a family usually share the ability to recognize similar or identical consensus sites. For example, the five mammalian NF-κB/Rel proteins generate more than 12 dimers recognizing 9–11 nucleotide κB sites. Each dimer selectively regulates a few target promoters; however, several genes are redundantly induced by more than one dimer. Whether this property simply generates redundancy in target gene activation or underlies more complex regulatory mechanisms is an open issue. We show here that during dendritic cell maturation, rapidly activated dimers (e.g., p50/RelA) bound to a subset of target promoters are gradually replaced by slowly activated dimers (e.g., p52/RelB). Since the dimers have different transcriptional activity at each promoter, the dimer exchange allows fine tuning of the response over time. Further, due to the insensitivity of p52/RelB to the NF-κB inhibitors, the IκBs, dimer exchange contributes to sustained activation of selected NF-κB targets in spite of the resynthesis of IκBα.
Processing of the nfκb2 gene product p100 to generate p52 is an important step in NF-κB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-κB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nfκb2 knockout mice exhibit similar immune deficiencies.
Almost exactly ten years following the first publication on NF-κB (Sen and Baltimore 1986), researchers working on transcriptional regulation by NF-κB/Rel and IκB proteins gathered for the third time to discuss recent developments in the field (Madrid, July 8-10, 1996). The first meeting of its kind was a Howard Hughes workshop at the NIH in November 1992 and the second one a Banbury Conference held at Cold Spring Harbor in October 1993. This year's meeting was organized by R. Bravo (Bristol-Myers Squibb, Princeton) and P. S. Lazo (Universidad de Oviedo) and held at the Juan March Foundation in Madrid, Spain.
Activation of the transcription factor NF-κB by tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the NF-κB-inducing kinase (NIK). In a yeast two-hybrid screen for NIK-interacting proteins, we have identified a protein kinase previously known as CHUK. Overexpression of CHUK activates a NF-κB-dependent reporter gene. A catalytically inactive mutant of CHUK is a dominant-negative inhibitor of TNF-, IL-1-, TRAF-, and NIK-induced NF-κB activation. CHUK associates with the NF-κB inhibitory protein, IκB-α, in mammalian cells. CHUK specifically phosphorylates IκB-α on both serine 32 and serine 36, modifications that are required for targeted degradation of IκB-α via the ubiquitin-proteasome pathway. This phosphorylation of IκB-α is greatly enhanced by NIK costimulation. Thus, CHUK is a NIK-activated IκB-α kinase that links TNF- and IL-1-induced kinase cascades to NF-κB activation.
Phosphorylation of inhibitor of kappa B (IκB) proteins is an important step in the activation of the transcription nuclear
factor kappa B (NF-κB) and requires two IκB kinases, IKK1 (IKKα) and IKK2 (IKKβ). Mice that are devoid of the IKK2 gene had
extensive liver damage from apoptosis and died as embryos, but these mice could be rescued by the inactivation of the gene
encoding tumor necrosis factor receptor 1. Mouse embryonic fibroblast cells that were isolated from IKK2–/– embryos showed a marked reduction in tumor necrosis factor–α (TNF-α)– and interleukin-1α–induced NF-κB activity and an enhanced
apoptosis in response to TNF-α. IKK1 associated with NF-κB essential modulator (IKKγ/IKKAP1), another component of the IKK
complex. These results show that IKK2 is essential for mouse development and cannot be substituted with IKK1.
Phosphorylation of several serine residues especially in the transactivation (TA) domain of p65 NF-κB subunit has been suggested to be important for its transcriptional activity. However, the responsible phosphorylation site of p65 remains controversial. To investigate the biological significance of phosphorylation and to determine the critical phosphorylation sites of p65, we reconstituted murine embryonic fibroblasts (MEFs) from p65−/− mice with various serine to alanine (SA)-substituted mutants of p65. Unexpectedly, mutants in the TA domain, including S529A, S536A, and S529A/S536A, completely rescued the defect of p65−/− MEFs as assessed by tumor necrosis factor (TNF)- or interleukin-1 (IL-1)-induced IL-6 production and protection from TNF-induced cell death. On the other hand, S276A mutant had an impaired ability to rescue these responses. Moreover, TNF-induced phosphorylation of p65 was severely impaired in S276A mutant, indicating that S276 is the major phosphorylation site of p65 and its phosphorylation is essential for p65-dependent cellular responses.
The regulation of the transcription factor NF-κB activity occurs at several levels including controlled cytoplasmic-nuclear shuttling and modulation of its transcriptional activity. A critical component in NF-κB regulation is the IκB kinase (IKK) complex. This review is focused on recent progress as well as unanswered questions regarding the regulation and function of NF-κB and IKK.
The oligomeric IκB kinase (IKK) is composed of three polypeptides: IKKα and IKKβ, the catalytic subunits, and IKKγ, a regulatory
subunit. IKKα and IKKβ are similar in structure and thought to have similar function—phosphorylation of the IκB inhibitors
in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IκB and activation of nuclear factor
κB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKα-deficient mice.
IKKα was not required for activation of IKK and degradation of IκB by proinflammatory stimuli. Instead, loss of IKKα interfered
with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal
keratinocytes.
The histone proteins that constitute the nucleosomes of chromatin around which yards of DNA are wrapped undergo a series of
chemical modifications during gene activation and gene silencing. In a Perspective,
Berger
discusses new results (
Nakayama
et al.) that elucidate the sequence of reactions that modify histone H3 and how these modifications translate into gene silencing
in fission yeast.
Activation of the transcription factor nuclear factor κB (NF-κB) requires its release from inhibitor of NF-κB (IκB) proteins in the cytoplasm. Much work has focussed on the identification of pathways regulating this cytosolic rate-limiting step of NF-κB activation. However, there is increasing evidence for another complex level of NF-κB activation, which involves modulatory phosphorylations of the DNA-binding subunits. These phosphorylations can control several functions of NF-κB, including DNA binding and transactivation properties, as well as interactions between the transcription factor and regulatory proteins. Although their overall impact on NF-κB function has yet to be determined, modifications of this factor will very probably provide a mechanism to fine tune NF-κB function.
The transcription factor NF-B is a pivotal regulator of innate immune
responses, whose activity is rapidly induced by proinflammatory stimuli, most
notably the tumour-necrosis factor TNF and interleukin-1, viruses,
and components of bacterial cell walls1. In addition, NF-B
protects cells from the induction of programmed cell death by pro-apoptotic
stimuli such as TNF (refs 2,
3). Another important anti-apoptotic signal-transducing protein
is the protein kinase Akt (also known as protein kinase B), whose activity
is strongly stimulated by growth factors4. Ozes et al.5 have suggested that Akt is involved in the TNF-mediated activation
of NF-B5, implying that some of the anti-apoptotic activity
of Akt may be mediated through NF-B. However, we have failed to detect
any involvement of Akt in the signalling pathway through which TNF
leads to NF-B activation.
The candidate oncogene bcl-3 was discovered as a translocation into the immunoglobulin alpha-locus in some cases of B-cell chronic lymphocytic leukaemias. The protein Bcl-3 contains seven so-called ankyrin repeats. Similar repeat motifs are found in a number of diverse regulatory proteins but the motifs of Bcl-3 are most closely related to those found in I kappa B proteins in which the ankyrin repeat domain is thought to be directly involved in inhibition of NF-kappa B activity. No biological function has yet been described for Bcl-3, but it was noted recently that Bcl-3 interferes with DNA-binding of the p50 subunit of NF-kappa B in vitro. Here we demonstrate that Bcl-3 can aid kappa B site-dependent transcription in vivo by counteracting the inhibitory effects of p50/NF-kappa B homodimers. Bcl-3 may therefore aid activation of select NF-kappa B-regulated genes, including those of the human immunodeficiency virus.
Transcription factor NF-kappa B (p50/p65) is generally localized to the cytoplasm by its inhibitor I kappa B. Overproduced I kappa B, free from NF-kappa B, is rapidly degraded. Overexpression of p65 increases endogenous I kappa B protein in both carcinoma and lymphoid cells by two mechanisms: protein stabilization and increased transcription of I kappa B mRNA. In contrast, p65 delta, a naturally occurring splice variant, fails to markedly augment I kappa B protein levels. Both overexpressed p65 and coexpressed p50 are cytoplasmic, whereas p65 delta is partly nuclear, indicating that the I kappa B induced by p65 can maintain NF-kappa B in the cytoplasm. Thus, p65 and I kappa B are linked in an autoregulatory loop, ensuring that NF-kappa B is held in the cytoplasm until cells are specifically induced to translocate it to the nucleus.
The NK-kappa B transcription factor complex is sequestered in the cytoplasm by the inhibitory protein I kappa B-alpha (MAD-3). Various cellular stimuli relieve this inhibition by mechanisms largely unknown, leading to NF-kappa B nuclear localization and transactivation of its target genes. It is demonstrated here with human T lymphocytes and monocytes that different stimuli, including tumor necrosis factor alpha and phorbol 12-myristate 13-acetate, cause rapid degradation of I kappa B-alpha, with concomitant activation of NF-kappa B, followed by a dramatic increase in I kappa B-alpha mRNA and protein synthesis. Transfection studies reveal that the I kappa B-alpha mRNA and the encoded protein are potently induced by NF-kappa B and by homodimers of p65 and of c-Rel. We propose a model in which NF-kappa B and I kappa B-alpha mutually regulate each other in a cycle: saturating amounts of the inhibitory I kappa B-alpha protein are destroyed upon stimulation, allowing rapid activation of NF-kappa B. Subsequently, I kappa B-alpha mRNA and protein levels are quickly induced by the activated NF-kappa B. This resurgence of I kappa B-alpha protein acts to restore an equilibrium in which NF-kappa B is again inhibited.
The nuclear factor kappaB (NF-kappaB) transcription factor is responsive to specific cytokines and stress and is often activated in association with cell damage and growth arrest in eukaryotes. NF-kappaB is a heterodimeric protein, typically composed of 50- and 65-kilodalton subunits of the Rel family, of which RelA(p65) stimulates transcription of diverse genes. Specific cyclin-dependent kinases (CDKs) were found to regulate transcriptional activation by NF-kappaB through interactions with the coactivator p300. The transcriptional activation domain of RelA(p65) interacted with an amino-terminal region of p300 distinct from a carboxyl-terminal region of p300 required for binding to the cyclin E-Cdk2 complex. The CDK inhibitor p21 or a dominant negative Cdk2, which inhibited p300-associated cyclin E-Cdk2 activity, stimulated kappaB-dependent gene expression, which was also enhanced by expression of p300 in the presence of p21. The interaction of NF-kappaB and CDKs through the p300 and CBP coactivators provides a mechanism for the coordination of transcriptional activation with cell cycle progression.
Stimulation of cells with inducers of NF-kappaB such as LPS and IL-1 leads to the degradation of IkappaB-alpha and IkappaB-beta proteins and translocation of NF-kappaB to the nucleus. We now demonstrate that, besides the physical partitioning of inactive NF-kappaB to the cytosol, the transcriptional activity of NF-kappaB is regulated through phosphorylation of NF-kappaB p65 by protein kinase A (PKA). The catalytic subunit of PKA (PKAc) is maintained in an inactive state through association with IkappaB-alpha or IkappaB-beta in an NF-kappaB-IkappaB-PKAc complex. Signals that cause the degradation of IkappaB result in activation of PKAc in a cAMP-independent manner and the subsequent phosphorylation of p65. Therefore, this pathway represents a novel mechanism for the cAMP-independent activation of PKA and the regulation of NF-kappaB activity.
A transcriptional regulator in yeast, Gcn5p, activates transcription by targeted acetylation of specific lysine residues in the amino-terminal tails of histones. This targeted modification is restricted to nucleosomes assembled on the promoters of Gcn5p-responsive genes.
The transcriptional activity of NF-kappa B is stimulated upon phosphorylation of its p65 subunit on serine 276 by protein kinase A (PKA). The transcriptional coactivator CPB/p300 associates with NF-kappa B p65 through two sites, an N-terminal domain that interacts with the C-terminal region of unphosphorylated p65, and a second domain that only interacts with p65 phosphorylated on serine 276. Accessibility to both sites is blocked in unphosphorylated p65 through an intramolecular masking of the N terminus by the C-terminal region of p65. Phosphorylation by PKA both weakens the interaction between the N- and C-terminal regions of p65 and creates an additional site for interaction with CBP/p300. Therefore, PKA regulates the transcriptional activity of NF-kappa B by modulating its interaction with CBP/p300.
NF-kappaB, a ubiquitous, inducible transcription factor involved in immune, inflammatory, stress and developmental processes, is retained in a latent form in the cytoplasm of non-stimulated cells by inhibitory molecules, IkappaBs. Its activation is a paradigm for a signal-transduction cascade that integrates an inducible kinase and the ubiquitin-proteasome system to eliminate inhibitory regulators. Here we isolate the pIkappaBalpha-ubiquitin ligase (pIkappaBalpha-E3) that attaches ubiquitin, a small protein which marks other proteins for degradation by the proteasome system, to the phosphorylated NF-kappaB inhibitor pIkappaBalpha. Taking advantage of its high affinity to pIkappaBalpha, we isolate this ligase from HeLa cells by single-step immunoaffinity purification. Using nanoelectrospray mass spectrometry, we identify the specific component of the ligase that recognizes the pIkappaBalpha degradation motif as an F-box/WD-domain protein belonging to a recently distinguished family of beta-TrCP/Slimb proteins. This component, which we denote E3RSIkappaB (pIkappaBalpha-E3 receptor subunit), binds specifically to pIkappaBalpha and promotes its in vitro ubiquitination in the presence of two other ubiquitin-system enzymes, E1 and UBC5C, one of many known E2 enzymes. An F-box-deletion mutant of E3RS(IkappaB), which tightly binds pIkappaBalpha but does not support its ubiquitination, acts in vivo as a dominant-negative molecule, inhibiting the degradation of pIkappaBalpha and consequently NF-kappaB activation. E3RS(IkappaB) represents a family of receptor proteins that are core components of a class of ubiquitin ligases. When these receptor components recognize their specific ligand, which is a conserved, phosphorylation-based sequence motif, they target regulatory proteins containing this motif for proteasomal degradation.
Phosphorylation of inhibitor of kappa B (IkappaB) proteins is an important step in the activation of the transcription nuclear factor kappa B (NF-kappaB) and requires two IkappaB kinases, IKK1 (IKKalpha) and IKK2 (IKKbeta). Mice that are devoid of the IKK2 gene had extensive liver damage from apoptosis and died as embryos, but these mice could be rescued by the inactivation of the gene encoding tumor necrosis factor receptor 1. Mouse embryonic fibroblast cells that were isolated from IKK2-/- embryos showed a marked reduction in tumor necrosis factor-alpha (TNF-alpha)- and interleukin-1alpha-induced NF-kappaB activity and an enhanced apoptosis in response to TNF-alpha. IKK1 associated with NF-kappaB essential modulator (IKKgamma/IKKAP1), another component of the IKK complex. These results show that IKK2 is essential for mouse development and cannot be substituted with IKK1.
The oligomeric IkappaB kinase (IKK) is composed of three polypeptides: IKKalpha and IKKbeta, the catalytic subunits, and IKKgamma, a regulatory subunit. IKKalpha and IKKbeta are similar in structure and thought to have similar function-phosphorylation of the IkappaB inhibitors in response to proinflammatory stimuli. Such phosphorylation leads to degradation of IkappaB and activation of nuclear factor kappaB transcription factors. The physiological function of these protein kinases was explored by analysis of IKKalpha-deficient mice. IKKalpha was not required for activation of IKK and degradation of IkappaB by proinflammatory stimuli. Instead, loss of IKKalpha interfered with multiple morphogenetic events, including limb and skeletal patterning and proliferation and differentiation of epidermal keratinocytes.
IkappaB kinase-alpha and -beta (IKK-alpha and IKK-beta), the catalytic subunits of the IKK complex, phosphorylate IkappaB proteins on specific serine residues, thus targeting IkappaB for degradation and activating the transcription factor NF-kappaB. To elucidate the in vivo function of IKK-beta, we generated IKK-beta-deficient mice. The homozygous mouse embryo dies at approximately 14.5 days of gestation due to liver degeneration and apoptosis. IKK-beta-deficient embryonic fibroblasts have both reduced basal NF-kappaB activity and impaired cytokine-induced NF-kappaB activation. Similarly, basal and cytokine-inducible kinase activities of the IKK complex are greatly reduced in IKK-beta-deficient cells. These results indicate that IKK-beta is crucial for liver development and regulation of NF-kappaB activity and that IKK-alpha can only partially compensate for the loss of IKK-beta.