During pathogen infection, innate immunity is initiated via the recognition of microbial products by pattern recognition receptors and the subsequent activation of transcription factors that upregulate proinflammatory genes. By controlling the expression of cytokines, chemokines, anti-bacterial peptides and adhesion molecules, the transcription factor nuclear factor-kappa B (NF-κB) has a central function in this process. In a typical model of NF-κB activation, the recognition of pathogen associated molecules triggers the canonical NF-κB pathway that depends on the phosphorylation of Inhibitor of NF-κB (IκB) by the catalytic subunit IκB kinase β (IKKβ), its degradation and the nuclear translocation of NF-κB dimers.
Here, we performed an RNA interference (RNAi) screen on Shigella flexneri-induced NF-κB activation to identify new factors involved in the regulation of NF-κB following infection of epithelial cells by invasive bacteria. By targeting a subset of the human signaling proteome, we found that the catalytic subunit IKKα is also required for complete NF-κB activation during infection. Depletion of IKKα by RNAi strongly reduces the nuclear translocation of NF-κB p65 during S. flexneri infection as well as the expression of the proinflammatory chemokine interleukin-8. Similar to IKKβ, IKKα contributes to the phosphorylation of IκBα on serines 32 and 36, and to its degradation. Experiments performed with the synthetic Nod1 ligand L-Ala-D-γ-Glu-meso-diaminopimelic acid confirmed that IKKα is involved in NF-κB activation triggered downstream of Nod1-mediated peptidoglycan recognition.
Taken together, these results demonstrate the unexpected role of IKKα in the canonical NF-κB pathway triggered by peptidoglycan recognition during bacterial infection. In addition, they suggest that IKKα may be an important drug target for the development of treatments that aim at limiting inflammation in bacterial infection.
The prosurvival transcription factor NF-κB specifically binds promoter DNA to activate target gene expression. NF-κB is regulated through interactions with IκB inhibitor proteins. Active proteolysis of these IκB proteins is, in turn, under the control of the IκB kinase complex (IKK). Together, these three molecules form the NF-κB signaling module. Studies aimed at characterizing the molecular mechanisms of NF-κB, IκB, and IKK in terms of their three-dimensional structures have lead to a greater understanding of this vital transcription factor system.
Structural studies of the NF-κB transcription factor and its regulators I-κB and IKK provide insights into NF-κB dimerization, activation, and DNA binding.
Mutational activation of BRAF is a frequent event in human malignant melanomas suggesting that BRAF-dependent signaling is conducive to melanoma cell growth and survival. Previously published work reported that melanoma cells exhibit constitutive anti-apoptotic nuclear factor κB (NF-κB) transcription factor activation triggered by proteolysis of its inhibitor IκB. IκB degradation is dependent upon its phosphorylation by the IκB kinase (IKK) complex and subsequent ubiquitination facilitated by β-Trcp E3 ubiquitin ligase. Here, we report that melanocytes expressing a conditionally oncogenic form of BRAFV600E exhibit enhanced β-Trcp expression, increased IKK activity and a concomitant increase in the rate of IκBα degradation. Conversely, inhibition of BRAF signaling using either a broad-spectrum Raf inhibitor (BAY 43-9006) or by selective knock-down of BRAFV600E expression by RNA interference in human melanoma cells leads to decreased IKK activity and β-Trcp expression, stabilization of IκB, inhibition of NF-κB transcriptional activity and sensitization of these cells to apoptosis. Taken together, these data support a model in which mutational activation of BRAF in human melanomas contributes to constitutive induction of NF-κB activity and to increased survival of melanoma cells.
BRAF; melanoma; β-Trcp; NF-κB; IκB kinase
Inhibitors of kappa B (IκBs) -α, -β and -ε effect selective regulation of specific nuclear factor of kappa B (NF-κB) dimers according to cell lineage, differentiation state or stimulus, in a manner that is not yet precisely defined. Lymphocyte antigen receptor ligation leads to degradation of all three IκBs but activation only of subsets of NF-κB-dependent genes, including those regulated by c-Rel, such as anti-apoptotic CD40 and BAFF-R on B cells, and interleukin-2 (IL-2) in T cells. We report that pre-culture of a mouse T cell line with tumour necrosis factor-α (TNF) inhibits IL-2 gene expression at the level of transcription through suppressive effects on NF-κB, AP-1 and NFAT transcription factor expression and function. Selective upregulation of IκBε and suppressed nuclear translocation of c-Rel were very marked in TNF-treated, compared to control cells, whether activated via T cell receptor (TCR) pathway or TNF receptor. IκBε associated with newly synthesised c-Rel in activated cells and, in contrast to IκBα and -β, showed enhanced association with p65/c-Rel in TNF-treated cells relative to controls. Studies in IκBε-deficient mice revealed that basal nuclear expression and nuclear translocation of c-Rel at early time-points of receptor ligation were higher in IκBε−/− T and B cells, compared to wild-type. IκBε−/− mice exhibited increased lymph node cellularity and enhanced basal thymidine incorporation by lymphoid cells ex vivo. IκBε−/− T cell blasts were primed for IL-2 expression, relative to wild-type. IκBε−/− splenic B cells showed enhanced survival ex vivo, compared to wild-type, and survival correlated with basal expression of CD40 and induced expression of CD40 and BAFF-R. Enhanced basal nuclear translocation of c-Rel, and upregulation of BAFF-R and CD40 occurred despite increased IκBα expression in IκBε−/− B cells. The data imply that regulation of these c-Rel-dependent lymphoid responses is a non-redundant function of IκBε.
Constitutive activation of NF-κB is a frequent event in human cancers, playing important roles in cancer development and progression. In nontransformed cells, NF-κB activation is tightly controlled by IκBs. IκBs bind NF-κB in the cytoplasm, preventing it from translocating to the nucleus to modulate gene expression. Stimuli that activate NF-κB signaling trigger IκB degradation, enabling nuclear translocation of NF-κB. Among the genes regulated by NF-κB are those encoding the IκBs, providing a negative feedback loop that limits NF-κB activity. How transformed cells override this NF-κB/IκB negative feedback loop remains unclear. Here, we report in human glioma cell lines that microRNA-30e* (miR-30e*) directly targets the IκBα 3ι-UTR and suppresses IκBα expression. Overexpression of miR-30e* in human glioma cell lines led to hyperactivation of NF-κB and enhanced expression of NF-κB–regulated genes, which promoted glioma cell invasiveness in in vitro assays and in an orthotopic xenotransplantation model. These effects of miR-30e* were shown to be clinically relevant, as miR-30e* was found to be upregulated in primary human glioma cells and correlated with malignant progression and poor survival. Hence, miR-30e* provides an epigenetic mechanism that disrupts the NF-κB/IκBα loop and may represent a new therapeutic target and prognostic marker.
Two related kinases, IκB kinase α (IKKα) and IKKβ, phosphorylate the IκB proteins, leading to their degradation and the subsequent activation of gene expression by NF-κB. IKKβ has a much higher level of kinase activity for the IκB proteins than does IKKα and is more critical than IKKα in modulating tumor necrosis factor alpha activation of the NF-κB pathway. These results indicate an important role for IKKβ in activating the NF-κB pathway but leave open the question of the role of IKKα in regulating this pathway. In the current study, we demonstrate that IKKα directly phosphorylates IKKβ. Moreover, IKKα either directly or indirectly enhances IKKβ kinase activity for IκBα. Finally, transfection studies to analyze NF-κB-directed gene expression suggest that IKKα is upstream of IKKβ in activating the NF-κB pathway. These results indicate that IKKα, in addition to its previously described ability to phosphorylate IκBα, can increase the ability of IKKβ to phosphorylate IκBα.
A primary level of control for nuclear factor kappa B (NF-κB) is effected through its interactions with the inhibitor protein, inhibitor of kappa B (IκB). Several lines of evidence confirm the existence of multiple forms of IκB that appear to regulate NF-κB by distinct mechanisms. Therefore, we performed a comprehensive bioinformatics analysis to understand the evolutionary history and intrinsic functional diversity of IκB family members. Phylogenetic relationships were constructed to trace the evolution of the IκB family genes. Our phylogenetic analysis revealed 10 IκB subfamily members that clustered into 5 major clades. Since the ankyrin (ANK) domain appears to be more ancient than the Rel homology domain (RHD), our phylogenetic analysis suggests that some undefined ancestral set of ANK repeats acquired an RHD before any duplication and was later duplicated and then diverged into the different IκB subfamilies. Functional analysis identified several functionally divergent sites in the ANK repeat domains (ARDs) and revealed that this region has undergone strong purifying selection, suggesting its functional importance in IκB genes. Structural analysis showed that the major variations in the number of ANK repeats and high conformational changes in the finger loop ARD region contribute to the differing binding partner specificities, thereby leading to distinct IκB functions. In summary, our study has provided useful information about the phylogeny and structural and functional divergence of the IκB family. Additionally, we identified a number of amino acid sites that contribute to the predicted functional divergence of these proteins.
Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-κB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IκB, until an external stimulus triggers IκB degradation through an IκB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-κB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-κB-bound and -unbound IκB proteins that are critical for accurate computational predictions of steady-state IκB protein levels and basal NF-κB activity. Simulations reveal a homeostatic NF-κB signaling module in which differential degradation rates of free and bound pools of IκB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.
cross-regulation; homeostasis; mathematical modeling; robustness; signaling module
Inflammatory activation of NF-κB involves the stimulus-induced degradation of the NF-κB-bound inhibitor, IκB, via the IκB kinase (IKK). In response to UV irradiation, however, the mechanism and function of NF-κB activation remain unclear. Using a combined biochemical, genetic, and computational modeling approach, we delineate a dual requirement for constitutive IKK-dependent and IKK-independent IκB degradation pathways in conjunction with UV-induced translational inhibition. Interestingly, we find that the high homeostatic turnover of IκB in resting cells renders the NF-κB system remarkably resistant to metabolic stresses, but the two degradation pathways critically and differentially tune NF-κB responsiveness to UV. Indeed, in the context of low chronic inflammation that accelerates NF-κB-bound IκB degradation, UV irradiation results in dramatic NF-κB activation. Our work suggests that the human health relevance of NF-κB activation by UV lies with cellular homeostatic states that are associated with pathology rather than with healthy physiology.
Cytoplasmic IκB proteins are primary regulators that interact with NF-κB subunits in the cytoplasm of unstimulated cells. Upon stimulation, these IκB proteins are rapidly degraded, thus allowing NF-κB to translocate into the nucleus and activate the transcription of genes encoding various immune mediators. Subsequent to translocation, nuclear IκB proteins play an important role in the regulation of NF-κB transcriptional activity by acting either as activators or inhibitors. To date, molecular basis for the binding of IκBα, IκBβ and IκBζ along with their partners is known; however, the activation and inhibition mechanism of the remaining IκB (IκBNS, IκBε and Bcl-3) proteins remains elusive. Moreover, even though IκB proteins are structurally similar, it is difficult to determine the exact specificities of IκB proteins towards their respective binding partners. The three-dimensional structures of IκBNS, IκBζ and IκBε were modeled. Subsequently, we used an explicit solvent method to perform detailed molecular dynamic simulations of these proteins along with their known crystal structures (IκBα, IκBβ and Bcl-3) in order to investigate the flexibility of the ankyrin repeat domains (ARDs). Furthermore, the refined models of IκBNS, IκBε and Bcl-3 were used for multiple protein-protein docking studies for the identification of IκBNS-p50/p50, IκBε-p50/p65 and Bcl-3-p50/p50 complexes in order to study the structural basis of their activation and inhibition. The docking experiments revealed that IκBε masked the nuclear localization signal (NLS) of the p50/p65 subunits, thereby preventing its translocation into the nucleus. For the Bcl-3- and IκBNS-p50/p50 complexes, the results show that Bcl-3 mediated transcription through its transactivation domain (TAD) while IκBNS inhibited transcription due to its lack of a TAD, which is consistent with biochemical studies. Additionally, the numbers of identified flexible residues were equal in number among all IκB proteins, although they were not conserved. This could be the primary reason for their binding partner specificities.
Activation of the transcription factor NF-κB is controlled by the sequential phosphorylation, ubiquitination, and degradation of its inhibitory subunit, IκB. We recently purified a large multiprotein complex, the IκB kinase (IKK) signalsome, which contains two regulated IκB kinases, IKK1 and IKK2, that can each phosphorylate IκBα and IκBβ. The IKK signalsome contains several additional proteins presumably required for the regulation of the NFκB signal transduction cascade in vivo. In this report, we demonstrate reconstitution of IκB kinase activity in vitro by using purified recombinant IKK1 and IKK2. Recombinant IKK1 or IKK2 forms homo- or heterodimers, suggesting the possibility that similar IKK complexes exist in vivo. Indeed, in HeLa cells we identified two distinct IKK complexes, one containing IKK1-IKK2 heterodimers and the other containing IKK2 homodimers, which display differing levels of activation following tumor necrosis factor alpha stimulation. To better elucidate the nature of the IKK signalsome, we set out to identify IKK-associated proteins. To this end, we purified and cloned a novel component common to both complexes, named IKK-associated protein 1 (IKKAP1). In vitro, IKKAP1 associated specifically with IKK2 but not IKK1. Functional analyses revealed that binding to IKK2 requires sequences contained within the N-terminal domain of IKKAP1. Mutant versions of IKKAP1, which either lack the N-terminal IKK2-binding domain or contain only the IKK2-binding domain, disrupt the NF-κB signal transduction pathway. IKKAP1 therefore appears to mediate an essential step of the NF-κB signal transduction cascade. Heterogeneity of IKK complexes in vivo may provide a mechanism for differential regulation of NF-κB activation.
Curcumin metabolites are detectable in body fluids such as serum and urine. We have developed a novel assay that can detect metabolites in such body fluids by measuring their effect on the nuclear factor kappa B/inhibitor of kappa B (NF-κB/IκB) pathway.
Patients and Methods
Fifteen healthy individuals were enrolled in the study and randomly assigned to two groups: control group (five) and curcumin group (ten). The test group ingested 8 g of the curcuminoids (C3-Complex™) with 16 oz of bottled water. Blood and urine were collected at 0, 4, 8, and 24 h after ingestion. Degradation of the NF-κB/IκB complex was detected by the Genetic Expression and Measurement (GEM™) assay using HCT116 cells stably transfected with PGL3-IκB firefly luciferase.
Using our novel GEM assay, the five controls who had not taken curcumin were identified.
The GEM assay is a very sensitive and accurate non-invasive assay that could be utilized to detect metabolites in body fluids. It could also serve as a tool to determine participants’ compliance during clinical research studies.
Curcumin; clinical trial; urinalysis; GEM assay
Latently infected cell reservoirs represent the main barrier to HIV eradication. Combination antiretroviral therapy (cART) effectively blocks viral replication but cannot purge latent provirus. One approach to HIV eradication could include cART to block new infections plus an agent to activate latent provirus. NF-κB activation induces HIV expression, ending latency. Before activation, IκB proteins sequester NF-κB dimers in the cytoplasm. Three canonical IκBs, IκBα, IκBβ, and IκBε, exist, but the IκB proteins' role in HIV activation regulation is not fully understood. We studied the effects on HIV activation of targeting IκBs by single and pairwise small interfering RNA (siRNA) knockdown. After determining the relative abundance of the IκBs, the relative abundance of NF-κB subunits held by the IκBs, and the kinetics of IκB degradation and resynthesis following knockdown, we studied HIV activation by IκB knockdown, in comparison with those of known HIV activators, tumor necrosis factor alpha (TNF-α), tetradecanoyl phorbol acetate (TPA), and trichostatin A (TSA), in U1 monocytic and J-Lat 10.6 lymphocytic latently infected cells. We found that IκBα knockdown activated HIV in both U1 and J-Lat 10.6 cells, IκBβ knockdown did not activate HIV, and, surprisingly, IκBε knockdown produced the most HIV activation, comparable to TSA activation. Our data show that HIV reactivation can be triggered by targeting two different IκB proteins and that IκBε may be an effective target for HIV latency reactivation in T-cell and macrophage lineages. IκBε knockdown may offer attractive therapeutic advantages for HIV activation because it is not essential for mammalian growth and development and because new siRNA delivery strategies may target siRNAs to HIV latently infected cells.
Many actions of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) on gene expression are mediated by the transcription factor NF-κB. Activation of NF-κB by TNF and IL-1 is initiated by the phosphorylation of the inhibitory subunit, IκB, which targets IκB for degradation and leads to the release of active NF-κB. The nonsteroidal anti-inflammatory drug sodium salicylate (NaSal) interferes with TNF-induced NF-κB activation by inhibiting phosphorylation and subsequent degradation of the IκBα protein. Recent evidence indicated that NaSal activates the p38 mitogen-activated protein kinase (MAPK), raising the possibility that inhibition of NF-κB activation by NaSal is mediated by p38 MAPK. We now show that inhibition of TNF-induced IκBα phosphorylation and degradation by NaSal is prevented by treatment of cells with SB203580, a highly specific p38 MAPK inhibitor. Both p38 activation and inhibition of TNF-induced IκBα degradation were seen after only 30 s to 1 min of NaSal treatment. Induction of p38 MAPK activation and inhibition of TNF-induced IκBα degradation were demonstrated with pharmacologically achievable doses of NaSal. These findings provide evidence for a role of NaSal-induced p38 MAPK activation in the inhibition of TNF signaling and suggest a possible role for the p38 MAPK in the anti-inflammatory actions of salicylates. In addition, these results implicate the p38 MAPK as a possible negative regulator of TNF signaling that leads to NF-κB activation.
Recognition of pathogens by immune receptors leads to activation of macrophages, dendritic cells, and lymphocytes. Signals are communicated to enhance expression of target molecules such as cytokines and adhesion molecules, depending on activation of various inducible transcription factors, among which the family NF-κB transcription factors plays an evolutionarily conserved and critical role. Classical activation of NF-κB involves phosphorylation, polyubiquitination and subsequent degradation of the inhibitor molecules of NF-κB, referred to as IκB. Modification of IκBα, one of the mammalian IκB isoforms, with the small ubiquitin-like modifier (SUMO) results its protection from degradation.
Presentation of the hypothesis
SUMO-IκBα localizes in the nucleus. The nuclear SUMO-IκBα pool may be dynamic. SUMO-IκBα functions as synergy control factor.
Testing the hypothesis
Immunoprecipitation from cellular fractions, 35S methionine pulse-chase, and FRET assays should reveal the localization of SUMO-IκBα and the dynamics of the pool. Expression of SUMOylation defective IκBα in an IκBα -/- background should yield insights into the function of SUMO-IκBα.
Implication of the hypothesis
IκBα contains the required SUMOylation motif but IκBβ does not. The suggested study would provide evidence whether or not IκBα and IκBβ can substitute each other. In addition, the suggested assays would reveal a possible redundancy in controlling transcriptional activity of NF-κB.
SUMO; IκB; NF-κB; signaling
We previously showed that the signal transcription factor Nuclear Factor-kappaB (NF-κB) is aberrantly activated and that inhibition of NF-κB induces cell death and inhibits tumorigenesis in Head and Neck Squamous Cell Carcinomas (HNSCC). Thus, identification of specific kinases underlying the activation of NF-κB could provide targets for selective therapy. Inhibitor KappaB Kinase (IKK) is known to activate NF-κB by inducing N-terminal phosphorylation and degradation of its endogenous inhibitor, Inhibitor-κB (IκB). Casein Kinase II (CK2) was previously reported to be over expressed in HNSCC cells, and to be a C-terminal IκB kinase, but its relationship to NF-κB activation in HNSCC cells is unknown. In this study, we examined the contribution of IKK and CK2 in the regulation of NF-κB in HNSCC in vitro. NF-κB activation was specifically inhibited by kinase dead mutants of the IKK1 and IKK2 subunits or siRNA targeting the β subunit of CK2. CK2 and IKK kinase activity, as well as NF-κB transcriptional activity, were shown to be serum responsive, indicating that these kinases mediate aberrant activation of NF-κB in response to serum factor(s) in vitro. Recombinant CK2α was shown to phosphorylate rIKK2, as well as to promote immunoprecipitated IKK complex from HNSCC to phosphorylate the N-terminal S32/S36 of IκBα. We conclude that the aberrant NF-κB activity in HNSCC cells in response to serum is partially through a novel mechanism involving CK2 mediated activation of IKK2, making these kinases candidates for selective therapy to target the NF-κB pathway in HNSCC.
Casein Kinase 2; Inhibitor-KappaB Kinase; Nuclear Factor kappaB; squamous cell carcinoma; Serum
In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor κ B (IκB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IκB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IκB is directly transported into isolated lysosomes in a process that requires binding of IκB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IκB uptake by lysosomes. Ubiquitination and phosphorylation of IκB are not required for its targeting to lysosomes. The lysosomal degradation of IκB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IκB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IκB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IκB is increased. There are both short- and long-lived pools of IκB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IκB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IκB degradation in lysosomes. This selective pathway of lysosomal degradation of IκB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor κ B. In addition, the response of nuclear factor κ B to several stimuli increases when this lysosomal pathway of proteolysis is activated.
The transcription factor nuclear factor-κB (NF-κB) regulates expression of a variety of genes involved in immune responses, inflammation, proliferation, and programmed cell death (apoptosis). Here, we show that in rat neonatal ventricular cardiomyocytes, activation of NF-κB is involved in the hypertrophic response induced by myotrophin, a hypertrophic activator identified from spontaneously hypertensive rat heart and cardiomyopathic human hearts. Myotrophin treatment stimulated NF-κB nuclear translocation and transcriptional activity, accompanied by IκB-α phosphorylation and degradation. Consistently, myotrophin-induced NF-κB activation was enhanced by wild-type IκB kinase (IKK) β and abolished by the dominant-negative IKKβ or a general PKC inhibitor, calphostin C. Importantly, myotrophin-induced expression of two hypertrophic genes (atrial natriuretic factor [ANF] and c-myc) and also enhanced protein synthesis were partially inhibited by a potent NF-κB inhibitor, pyrrolidine dithio-carbamate (PDTC), and calphostin C. Expression of the dominant-negative form of IκB-α or IKKβ also partially inhibited the transcriptional activity of ANF induced by myotrophin. These findings suggest that the PKC–IKK–NF-κB pathway may play a critical role in mediating the myotrophin-induced hypertrophic response in cardiomyocytes.
myotrophin; NF-κB; PKC; cardiac hypertrophy; signal transduction
Klotho is an antiaging hormone present in the kidney that extends the lifespan, regulates kidney function, and modulates cellular responses to oxidative stress. We investigated whether Klotho levels and signaling modulate inflammation in diabetic kidneys.
RESEARCH DESIGN AND METHODS
Renal Klotho expression was determined by quantitative real-time PCR and immunoblot analysis. Primary mouse tubular epithelial cells were treated with methylglyoxalated albumin, and Klotho expression and inflammatory cytokines were measured. Nuclear factor (NF)-κB activation was assessed by treating human embryonic kidney (HEK) 293 and HK-2 cells with tumor necrosis factor (TNF)-α in the presence or absence of Klotho, followed by immunoblot analysis to evaluate inhibitor of κB (IκB)α degradation, IκB kinase (IKK) and p38 activation, RelA nuclear translocation, and phosphorylation. A chromatin immunoprecipitation assay was performed to analyze the effects of Klotho signaling on interleukin-8 and monocyte chemoattractant protein-1 promoter recruitment of RelA and RelA serine (Ser)536.
Renal Klotho mRNA and protein were significantly decreased in db/db mice, and a similar decline was observed in the primary cultures of mouse tubule epithelial cells treated with methylglyoxal-modified albumin. The exogenous addition of soluble Klotho or overexpression of membranous Klotho in tissue culture suppressed NF-κB activation and subsequent production of inflammatory cytokines in response to TNF-α stimulation. Klotho specifically inhibited RelA Ser536 phosphorylation as well as promoter DNA binding of this phosphorylated form of RelA without affecting IKK-mediated IκBα degradation, total RelA nuclear translocation, and total RelA DNA binding.
These findings suggest that Klotho serves as an anti-inflammatory modulator, negatively regulating the production of NF-κB–linked inflammatory proteins via a mechanism that involves phosphorylation of Ser536 in the transactivation domain of RelA.
Nuclear factor κB (NF-κB) subunits comprise a family of eukaryotic transcription factors that are critically involved in cell proliferation, inflammation, and apoptosis. Under basal conditions, NF-κB subunits are kept under inhibitory regulation by physical interaction with NF-κB inhibitors (IκB subunits) in the cytosol. Upon stimulation, IκB subunits become phosphorylated, ubiquitinated, and subsequently degraded, allowing NF-κB subunits to translocate to the nucleus and bind as dimers to κB responsive elements of target genes. Previously, we have shown that AEBP1 enhances macrophage inflammatory responsiveness by inducing the expression of various proinflammatory mediators. Herein, we provide evidence suggesting that AEBP1 manifests its proinflammatory function by up-regulating NF-κB activity via hampering IκBα, but not IκBβ, inhibitory function through protein–protein interaction mediated by the discoidin-like domain (DLD) of AEBP1. Such interaction renders IκBα susceptible to enhanced phosphorylation and degradation, subsequently leading to augmented NF-κB activity. Collectively, we propose a novel molecular mechanism whereby NF-κB activity is modulated by means of protein–protein interaction involving AEBP1 and IκBα. Moreover, our study provides a plausible mechanism explaining the differential regulatory functions exhibited by IκBα and IκBβ in various cell types. We speculate that AEBP1 may serve as a potential therapeutic target for the treatment of various chronic inflammatory diseases and cancer.
Transcription factor NF-κB controls the expression of multiple genes involved in immunity and inflammation. The initial activation and duration of NF-κB signaling is regulated by posttranslational modifications to IκB kinase, which earmarks inhibitors of NF-κB for degradation. Prior studies suggest that K63-linked ubiquitination of NEMO (NF-κB essential modulator), an IκB kinase regulatory subunit, is critical for NF-κB and MAPK signaling following engagement of Ag receptors. We now demonstrate that NF-κB and MAPK pathways are largely unaffected in primary cells from mice harboring a ubiquitination-defective form of NEMO, NEMO-KR. TLR- but not Ag receptor-induced cellular responses are impaired in NEMO-KR mice, which are more resistant to LPS-induced endotoxic shock than wild-type animals. Thus, one function of NEMO ubiquitination is to fine tune innate immune responses under TLR control.
NF-κB corresponds to an inducible eukaryotic transcription factor complex that is negatively regulated in resting cells by its physical assembly with a family of cytoplasmic ankyrin-rich inhibitors termed IκB. Stimulation of cells with various proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), induces nuclear NF-κB expression. TNF-α signaling involves the recruitment of at least three proteins (TRADD, RIP, and TRAF2) to the type 1 TNF-α receptor tail, leading to the sequential activation of the downstream NF-κB-inducing kinase (NIK) and IκB-specific kinases (IKKα and IKKβ). When activated, IKKα and IKKβ directly phosphorylate the two N-terminal regulatory serines within IκBα, triggering ubiquitination and rapid degradation of this inhibitor in the 26S proteasome. This process liberates the NF-κB complex, allowing it to translocate to the nucleus. In studies of NIK, we found that Thr-559 located within the activation loop of its kinase domain regulates NIK action. Alanine substitution of Thr-559 but not other serine or threonine residues within the activation loop abolishes its activity and its ability to phosphorylate and activate IKKα. Such a NIK-T559A mutant also dominantly interferes with TNF-α induction of NF-κB. We also found that ectopically expressed NIK both spontaneously forms oligomers and displays a high level of constitutive activity. Analysis of a series of NIK deletion mutants indicates that multiple subregions of the kinase participate in the formation of these NIK-NIK oligomers. NIK also physically assembles with downstream IKKα; however, this interaction is mediated through a discrete C-terminal domain within NIK located between amino acids 735 and 947. When expressed alone, this C-terminal NIK fragment functions as a potent inhibitor of TNF-α-mediated induction of NF-κB and alone is sufficient to disrupt the physical association of NIK and IKKα. Together, these findings provide new insights into the molecular basis for TNF-α signaling, suggesting an important role for heterotypic and possibly homotypic interactions of NIK in this response.
In the course of studies on bioactive metabolites from marine fungi, a new 10-membered lactone, named penicillinolide A (1) was isolated from the organic extract of Penicillium sp. SF-5292 as a potential anti-inflammatory compound. The structure of penicillinolide A (1) was mainly determined by analysis of NMR and MS data and Mosher’s method. Penicillinolide A (1) inhibited the production of NO and PGE2 due to inhibition of the expression of iNOS and COX-2. Penicillinolide A (1) also reduced TNF-α, IL-1β and IL-6 production, and these anti-inflammatory effects were shown to be correlated with the suppression of the phosphorylation and degradation of IκB-α, NF-κB nuclear translocation, and NF-κB DNA binding activity. In addition, using inhibitor tin protoporphyrin (SnPP), a competitive inhibitor of HO activity, it was verified that the inhibitory effects of compound 1 on the production of pro-inflammatory mediators and NF-κB DNA binding activity were partially associated with HO-1 expression through Nrf2 nuclear translocation.
Penicillium sp.; marine-derived fungi; 10-membered lactone; anti-inflammatory effect; heme oxygenase-1
The NF-κB signalling pathway regulates many different biological processes from the cellular level to the whole organism. The majority of these functions are completely dependent on the activation of the cytoplasmic IKK kinase complex that leads to IκB degradation and results in the nuclear translocation of specific NF-κB dimers, which, in general, act as transcription factors. Although this is a well-established mechanism of action, several publications have now demonstrated that some members of this pathway display additional functions in the nucleus as regulators of NF-κB-dependent and independent gene expression. In this review, we compiled and put in context most of the data concerning specific nuclear roles for IKK and IκB proteins.
NF-κB (nuclear factor -κB); IκB (inhibitor of NF-κB); IKK (IκB kinases); chromatin; gene transcription
The nuclear factor κ light chain enhancer of activated B cells (NFκB) is constitutively active in most cancers, controlling multiple cellular processes including proliferation, invasion and resistance to therapy. NFκB is primarily regulated through the association with inhibitory proteins that are known as inhibitors of NFκB (IκBs). Increased NFκB activity in tumor cells has been correlated with decrease stability of IκB proteins, in particular IκBα. In responso to a large number of stimuli, IκB proteins are degraded by the proteasome. Cytotoxic T lymphocytes (CTLs) recognize HLA-restricted antigenic peptides that are generated by proteasomal degradation in target cells. In the present study, we demonstrate the presence of naturally occurring IκBα -specific T cells in the peripheral blood of patients suffering from several unrelated tumor types, i.e., breast cancer, malignant melanoma and renal cell carcinoma, but not of healthy controls. Furthermore, we show that such IBα-specific T cells are granzyme B-releasing, cytotoxic cells. Hence, the increased proteasomal degradation of IκBα in cancer induces IκBα-specific CTLs.
CTL; NFκB; T cells; antigens; inhibitor of κB; proteasome