Member Of IκB Family Research Articles

A Helminth Glycan Induces APC Maturation via Alternative NF-κB Activation Independent of IκBα Degradation

Activation of APCs via TLRs leads to activation of NF-kappaB, a key transcription factor in cells of the immune system most often associated with induction of Th1-type and proinflammatory responses. The neoglycoconjugate lacto-N-fucopentaose III (12-25 molecules)-dextran (LNFPIII-Dex) activates dendritic cells (DCs) via TLR4, as does LPS. However, unlike LPS, LNFPIII-Dex-activated cells induce Th2-type CD4+ T cell responses. This observation led us to ask whether LNFPIII-activated APCs were differentially activating NF-kappaB, and if so, could this partly account for how DCs mature in response to these two different pathogen-associated molecular patterns (PAMPs). In this study, we show that LNFPIII-Dex stimulation of APCs induces rapid, but transient NF-kappaB translocation and activity in the nucleus, in comparison with the persistent activation induced by LPS. We then demonstrate that transient vs persistent NF-kappaB activation has important implications in the development of the APC phenotype, showing that the second wave of NF-kappaB translocation in response to LPS is required for production of the proinflammatory mediator NO. In contrast to LPS, LNFPIII-stimulated APCs that only transiently activate NF-kappaB do not induce degradation of the known IkappaB family members or production of NO. However, cells stimulated with LNFPIII rapidly accumulate p50, suggesting that an alternative p105 degradation-dependent mechanism is primarily responsible for NF-kappaB activation downstream of LNFPIII. Finally, we show that while NF-kappaB translocation in LNFPIII-stimulated APCs is transient, it is required for the development of the DC 2 phenotype, confirming a crucial and multifaceted role for NF-kappaB in innate immune responses.

FBI-1 Enhances Transcription of the Nuclear Factor-κB (NF-κB)-responsive E-selectin Gene by Nuclear Localization of the p65 Subunit of NF-κB

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-kappaB (NF-kappaB) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-kappaB in both in vivo and in vitro protein-protein interaction assays. FBI-1 enhanced NF-kappaB-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IkappaB alpha or IkappaB beta. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-kappaB-mediated transcription, and the repression was cooperative with IkappaB alpha or IkappaB beta. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-kappaB-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-kappaB movement into the nucleus and increased the stability of NF-kappaB in the nucleus, which enhanced NF-kappaB-mediated transcription of the E-selectin gene. FBI-1 also interacted with IkappaB alpha and IkappaB beta.

A Novel NF-κB Pathway Involving IKKβ and p65/RelA Ser-536 Phosphorylation Results in p53 Inhibition in the Absence of NF-κB Transcriptional Activity

Nuclear factor kappaB (NF-kappaB) plays an important role in regulating cellular transformation and apoptosis. The human T-cell lymphotropic virus type I protein, Tax, which is critical for viral transformation, modulates the transcription of several cellular genes through activation of NF-kappaB. We have demonstrated previously that Tax inhibits p53 activity through the p65/RelA subunit of NF-kappaB. We now present evidence that suggests that the upstream kinase IKKbeta plays an important role in Tax-induced p53 inhibition through phosphorylation of p65/RelA at Ser-536. First, mouse embryo fibroblast (MEF) IKKbeta-/-cells did not support Tax-mediated p53 inhibition, whereas MEFs lacking IKKalpha allowed Tax inhibition of p53. Second, transfection of IKKbeta wild type (WT), but not a kinase-dead mutant, into IKKbeta-/-cells rescued p53 inhibition by Tax. Third, the IKKbeta-specific inhibitor SC-514 decreased the ability of Tax to inhibit p53. Fourth, we show that phosphorylation of p65/RelA at Ser-536 is important for Tax inhibition of p53 using MEF p65/RelA-/-cells transfected with p65/RelA WT or mutant plasmids. Moreover, Tax induced p65/RelA Ser-536 phosphorylation in WT or IKKalpha-/- cells but failed to induce the phosphorylation of p65/RelA Ser-536 in IKKbeta-/-cells, suggesting a link between IKKbeta and p65/RelA phosphorylation. Consistent with this observation, blocking IKKbeta kinase activity by SC-514 decreases the phosphorylation of p65/RelA at Ser-536 in the presence of Tax in human T-cell lymphotropic virus type I-transformed cells. Finally, the ability of Tax to inhibit p53 is distinguished from the NF-kappaB transcription activation pathway. Our work, therefore, describes a novel Tax-NF-kappaB p65/RelA pathway that functions to inhibit p53 but does not require NF-kappaB transcription activity.

Per-Arnt-Sim domain-dependent association of cAMP-phosphodiesterase 8A1 with IκB proteins

Phosphodiesterase (PDE) 8A1 is a cAMP-specific PDE isozyme characterized by the presence of a Per-Arnt-Sim (PAS) domain. However, the function(s) of the PAS domain has remained unknown. In this study, using a lysate of HEK293 cells overexpressing recombinant human PDE8A1, we detected a physical association between PDE8A1 and endogenous IkappaBbeta by an antibody array technique. The association was specific for PDE8A1 and depended on the presence of the PAS domain. Subsequent coimmunoprecipitation experiments revealed that, in addition to IkappaBbeta, other IkappaB family members examined (p105, p100, and IkappaBalpha) also associated with PDE8A1. Furthermore, it was found that PDE8A1 competed with the p65/p50 NF-kappaB for IkappaBbeta binding. Taken together, these data indicate that PDE8A1, through its PAS domain, may bind with IkappaB proteins in a region containing their ankyrin repeats. Functionally, in vitro and in vivo experiments demonstrated that the association with IkappaB greatly enhanced the enzyme activity of PDE8A1. However, the PDE8A1-IkappaB association did not affect NF-kappaB activation. The biological role of the PDE8A1-IkappaB association remains to be elucidated.

Effects of TCDD upon IκB and IKK subunits localized in microsomes by proteomics

Biochemical studies have shown that microsomes represent an important subcellular fraction for determining 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) effects. Proteomic analysis by two-dimensional gel-mass spectrometry of liver microsomes was undertaken to gain new insight into the actions of TCDD in male and female rats. Proteomic analysis showed TCDD induced several xenobiotic metabolism enzymes as well as a protein at 90 kDa identified by mass spectrometry as IκB kinase β/IKK2. This observation led to the discovery of other NF-κB binding proteins and kinases in microsomes and effects by TCDD. Western blotting for IKK and IκB family members in microsomes showed a distinct pattern from cytosol. IKK1 and IKK2 were both present in microsomes and were catalytically active although, unlike cytosol, IKKγ/NEMO was not detectable. TCDD exposure produced an elevation in cytosolic and microsomal IKK activity of both genders. The NF-κB binding proteins IκBβ and IκBγ were prevalent in microsomes, while IκBα and IκBε proteins were absent. TCDD treatment produced hyperphosphorylation of microsomal IκBβ in both sexes with females being most sensitive. In cytosol, IκBα, IκBβ, and IκBε, but not IκBγ, were clearly observed but were not changed by TCDD. Overall, proteomic analysis indicated the presence of NF-κB pathway members in microsomes, selectively altered by dioxin, which may influence immune and inflammatory responses within the liver.

Regulation of the transcriptional activity of the nuclear factor-κB p65 subunit

Nuclear factor-κB (NF-κB) is well known for its role in inflammation, immune response, control of cell division and apoptosis. The function of NF-κB is primarily regulated by IκB family members, which ensure cytoplasmic localisation of the transcription factor in the resting state. Upon stimulus-induced IκB degradation, the NF-κB complexes move to the nucleus and activate NF-κB-dependent transcription. Over the years, a second regulatory mechanism, independent of IκB, has become generally accepted. Changes in NF-κB transcriptional activity have been assigned to phosphorylation of the p65 subunit by a large variety of kinases in response to different stimuli. Here, we give an overview of the kinases and signalling pathways mediating this process and comment on the players involved in tumour necrosis factor-induced regulation of NF-κB transcriptional activity. Additionally, we describe how other posttranslational modifications, such as acetylation and methylation of transcription factors or of the chromatin environment, may also affect NF-κB transcriptional activity.

Peptide-Induced Negative Selection of Thymocytes Activates Transcription of an NF-ΚB Inhibitor

Negative selection eliminates thymocytes bearing autoreactive T cell receptors (TCR) via an apoptotic mechanism. We have cloned an inhibitor of NF-ΚB, IΚBNS, which is rapidly expressed upon TCR-triggered but not dexamethasone- or γ irradiation-stimulated thymocyte death. The predicted protein contains seven ankyrin repeats and is homologous to IΚB family members. In class I and class II MHC-restricted TCR transgenic mice, transcription of IΚBNS is stimulated by peptides that trigger negative selection but not by those inducing positive selection (i.e., survival) or nonselecting peptides. IΚBNS blocks transcription from NF-ΚB reporters, alters NF-ΚB electrophoretic mobility shifts, and interacts with NF-ΚB proteins in thymic nuclear lysates following TCR stimulation. Retroviral transduction of IΚBNS in fetal thymic organ culture enhances TCR-triggered cell death consistent with its function in selection.

A distinct bipartite motif is required for the localization of inhibitory κB-like (IκBL) protein to nuclear speckles

The inhibitory κB (IκB)-like (IκBL) gene is located within the Class III region of the MHC on human chromosome 6. Previous analysis of the predicted amino acid sequence of the human IκBL protein revealed three putative functional domains; 2–3 ankyrin repeat sequences, which are similar to the second and third ankyrin repeats of the nuclear factor κB (NF-κB) protein; three PEST sequence motifs (a sequence that is rich in proline, serine, aspartic acid and threonine residues), which are also found in other IκB family members; and a C-terminal leucine zipper-like motif. In the present study we have identified a novel bipartite motif, which is required for nuclear localization of the IκBL protein. Analyses of IκBL-specific transcripts revealed the existence of a widely expressed spliced variant form of IκBL (IκBLsv1), which lacks the amino acid sequence GELEDEWQEVMGRFE (where single-letter amino-acid notation has been used). Interestingly, translation of IκBL mRNA in vivo was found to initiate predominantly from the second available methionine, thereby resulting in the disruption of the predicted N-terminal PEST sequence. Also, transient expression of T7 epitope-tagged IκBL and IκBLsv1 proteins in mammalian cells showed that both proteins were targeted to the nucleus, where they accumulate in nuclear speckles. To define the protein domains required for nuclear import and subnuclear localization, a complementary set of deletion mutants and enhanced green fluorescent protein—IκBL domain fusions were expressed in mammalian cells. Data from these experiments show that a combination of the ankyrin-repeat region and an adjacent arginine-rich sequence are necessary and sufficient for both nuclear import and speckle localization.

NF-κB-inducible BCL-3 Expression Is an Autoregulatory Loop Controlling Nuclear p50/NF-κB1 Residence

NF-kappa B is a transcription factor whose nuclear residence is controlled by I kappa B family members. In the NF-kappa B-I kappa B autoregulatory loop, activated (nuclear) Rel A.NF-kappa B1 induces the resynthesis of I kappa B alpha recapturing nuclear Rel A back into the cytoplasm within 1 h of stimulation. In contrast, NF-kappa B1 subunits redistribute more slowly into the cytoplasm (from 6 to 12 h). Here we examine the role of inducible cytoplasmic BCL-3 expression in terminating nuclear NF-kappa B1. Although BCL-3 is a nuclear protein in B lymphocytes, surprisingly, BCL-3 is primarily a cytoplasmic protein in HepG2 cells. Cytoplasmic BCL-3 abundance is induced 6-12 h after tumor necrosis factor-alpha stimulation where it complexes with NF-kappa B1 homodimers. Moreover, BCL-3 mRNA and protein expression are induced by NF-kappa B-activating agents. Two observations are interpreted to indicate that bcl-3 is transactivated by NF-kappa B/Rel A: 1) expression of a dominant negative NF-kappa B inhibitor blocks tumor necrosis factor-alpha-induced BCL-3 expression and 2) expression of constitutively active Rel A is sufficient to induce BCL-3 expression. In gene transfer studies, we identify two high affinity NF-kappa B-binding sites, kappa B1 (located at -872 to -861 nucleotides) and kappa B2 (-106 to -96 nucleotides), and although both bind with high affinity to Rel A, only kappa B2 is required for NF-kappa B-dependent induction of the native BCL-3 promoter. Down-regulation of BCL-3 induction results in prolonged, enhanced NF-kappa B1 binding and increased NF-kappa B-dependent transcription. Together, these data suggest the presence of an NF-kappa B-BCL-3 autoregulatory loop important in terminating NF-kappa B1 action and that individual NF-kappa B isoforms are actively terminated through coordinate induction of inhibitory I kappa B molecules to restore cellular homeostasis.

Postrepression activation of NF-kappaB requires the amino-terminal nuclear export signal specific to IkappaBalpha.

One of the most prominent NF-kappaB target genes in mammalian cells is the gene encoding one of its inhibitor proteins, IkappaBalpha. The increased synthesis of IkappaBalpha leads to postinduction repression of nuclear NF-kappaB activity. However, it is unknown why IkappaBalpha, among multiple IkappaB family members, is involved in this process and what significance this feedback regulation has beyond terminating NF-kappaB activity. Herein, we report an important IkappaBalpha-specific function dictated by its amino-terminal nuclear export sequence (N-NES). The IkappaBalpha N-NES is necessary for the postinduction export of nuclear NF-kappaB, which is a critical event in reestablishing a permissive condition for NF-kappaB to be rapidly reactivated. We show that although IkappaBalpha and another IkappaB member, IkappaBbeta, can enter the nucleus and repress NF-kappaB DNA-binding activity during the postinduction phase, only IkappaBalpha allows the efficient export of nuclear NF-kappaB. Moreover, swapping the N-terminal region of IkappaBbeta for the corresponding IkappaBalpha sequence is sufficient for the IkappaB chimera protein to export NF-kappaB similarly to IkappaBalpha during the postinduction state. Our findings provide a mechanistic explanation of why IkappaBalpha but not other IkappaB members is crucial for postrepression activation of NF-kappaB. We propose that this IkappaBalpha-specific function is important for certain physiological and pathological conditions where NF-kappaB needs to be rapidly reactivated.

An IκB Sink

The transcription factor NF-κB has critical functions in control of immune and inflammatory responses (see STKE Review by Rothwarf and Karin ). The required strict control of NF-κB transcriptional activity is mediated in part by inhibitory IκB proteins. When the IκB proteins are degraded, NF-κB activity is increased. IκB proteins appear to act in multiple ways to hold NF-κB activity in check. When it interacts with NF-κB, IκBα appears to hide the nuclear localization sequence of the NF-κB Rel subunit and also to inhibit DNA binding by NF-κB. IκBα also moves between the cytoplasm and nucleus through the actions of a nuclear localization sequence and a nuclear export sequence that interacts with the importin protein CRM1. Such nuclear export may also carry NF-κB-IκB complexes out of the nucleus and contribute to transcriptional control. Tam and Sen now report that the regulatory properties appear to differ in various IκB family members. In particular, they show that IκBβ and IκBϵ do not shuttle out of the nucleus by CRM1-dependent transport. The authors suggest that, rather than facilitating nuclear export of NF-κB, IκBβ and IκBϵ function primarily by sequestration of NF-κB in the cytoplasm. W. F. Tam, R. Sen, IκB family members function by different mechanisms. J. Biol. Chem. 276, 7701-7704 (2001). [Abstract] [Full Text]

IκB Family Members Function by Different Mechanisms

The IkappaB family of proteins regulates NF-kappaB-dependent transcription by inhibiting DNA binding and localizing these factors to the cell cytoplasm. IkappaBalpha does this by shifting the balance between nuclear import of Rel proteins and their export from the nucleus. Here we show that, unlike IkappaBalpha, IkappaBbeta and IkappaBepsilon appear to sequester p65 or c-Rel in the cytoplasm by inhibiting nuclear import. Furthermore, because IkappaBbeta does not undergo nucleocytoplasmic shuttling, it cannot remove nuclear proteins like IkappaBalpha does. We conclude that the mechanism of action differs among IkappaB family members.

Functional Characterization of a Purified, Recombinant NF-κB/IκB Complex

The NF-κB signal transduction pathway involves the interaction of several NF-κB and IκB family members that are activated by a diverse range of extracellular signals and that stimulate many different cellular responses. The biochemical regulation of this cascade can be studied by establishing a cell-free system using purified proteins. As a first step toward establishing an in vitro model incorporating multiple combinations of NF-κB and IκB proteins, we produced purified human p65 (RelA) and human IκBα proteins and tested their functionality. Full-length RelA and IκBα proteins were overproduced by coinfection of TN5-JE cells with two recombinant baculoviruses. RelA and IκBα formed a stable complex that could be purified to >95% homogeneity. Protein–protein interactions, protein–DNA binding, and protein phosphorylation were similar to the native proteins.

Effect of Staurosporine on Transcription Factor NF-κB in Human Keratinocytes

Activation of the transcription factor NF-κB is known to be important in the regulated expression of a large number of pro-inflammatory genes including interleukin-8 (IL-8). Previously, we showed that the protein kinase inhibitor staurosporine potentiates IL-1-stimulated IL-8 production in human keratinocytes. Moreover, recent studies by other investigators demonstrated that staurosporine treatment alone results in a concentration-dependent increase in IL-8 mRNA and protein production. Therefore, in order to understand the mechanism underlying this observation, the effect of staurosporine on the activation of NF-κB was investigated. Electrophoretic mobility shift assays using an oligonucleotide containing the NF-κB consensus motif demonstrated that staurosporine treatment resulted in the activation of NF-κB by 15 min post-treatment. The ability of staurosporine to activate NF-κB was investigated further, using luciferase reporters under the control of the HIV-LTR or IL-8 core promoter transfected into human U937 cells. Stimulation with staurosporine resulted in a concentration-dependent induction of luciferase activity. In contrast, the very selective protein kinase C inhibitor 3-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido-[1,2- a]indol-10-yl]-4-(1-methyl-3-indolyl)-1 H-pyrrole-2,5-dione hydrochloride (Ro32-0432) did not stimulate the activation of NF-κB, as measured in the luciferase reporter assay. The mechanism underlying NF-κB activation does not appear to involve the classical activation pathways in that staurosporine does not induce the disappearance of IκB family members. In conclusion, staurosporine appears to stimulate the activation of NF-κB in at least two cell types, and this effect appears to be independent of protein kinase C.