kappaB Motif Research Articles

AB0221 Estrogen modulation of endosome-associated toll-like receptors and ZAS3: An underlying mechanism of gender-BIAS in systemic lupus erythematosus?

BackgroundSystemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by significant gender bias. Previous studies have demonstrated the contributions of hormones, particularly estrogen (E2), to SLE pathogenesis. E2 has...

Dynamic Repositioning of Dorsal to Two Different κB Motifs Controls Its Autoregulation during Immune Response in Drosophila

Autoregulation is one of the mechanisms of imparting feedback control on gene expression. Positive autoregulatory feedback results in induction of a gene, and negative feedback leads to its suppression. Here, we report an interesting mechanism of autoregulation operating on Drosophila Rel gene dorsal that can activate as well as repress its expression. Using biochemical and genetic approaches, we show that upon immune challenge Dorsal regulates its activation as well as repression by dynamically binding to two different kappaB motifs, kappaB(I) (intronic kappaB) and kappaB(P) (promoter kappaB), present in the dorsal gene. Although the kappaB(I) motif functions as an enhancer, the kappaB(P) motif acts as a transcriptional repressor. Interestingly, Dorsal binding to these two motifs is dynamic; immediately upon immune challenge, Dorsal binds to the kappaB(I) leading to auto-activation, whereas at the terminal phase of the immune response, it is removed from the kappaB(I) and repositioned at the kappaB(P), resulting in its repression. Furthermore, we show that repression of Dorsal as well as its binding to the kappaB(P) depends on the transcription factor AP1. Depletion of AP1 by RNA interference resulted in constitutive expression of Dorsal. In conclusion, this study suggests that during acute phase response dorsal is regulated by following two subcircuits: (i) Dl-kappaB(I) for activation and (ii) Dl-AP1-kappaB(P) for repression. These two subcircuits are temporally delineated and bring about overall regulation of dorsal during immune response. These results suggest the presence of a previously unknown mechanism of Dorsal autoregulation in immune-challenged Drosophila.

NFκB Promotes Inflammation, Coagulation, and Fibrosis in the Aging Glomerulus

The peak prevalence of ESRD from glomerulosclerosis occurs at 70 to 79 years. To understand why old glomeruli are prone to failure, we analyzed the Fischer 344 rat model of aging under ad libitum-fed (rapid aging) and calorie-restricted (slowed aging) conditions. All glomerular cells contained genes whose expression changed "linearly" during adult life from 2 to 24 months: mesangial cells (e.g., MMP9), endothelial cells (e.g., ICAM and VCAM), parietal epithelial cells (e.g., ceruloplasmin), and podocytes (e.g., nephrin and prepronociceptin). Patterns of aging glomerular gene expression closely resembled atherosclerosis, including activation of endothelial cells, epithelial cells, and macrophages, as well as proinflammatory pathways related to cell adhesion, chemotaxis, blood coagulation, oxidoreductases, matrix metalloproteinases, and TGF-beta activation. We used a nonbiased data-mining approach to identify NFkappaB as the likely transcriptional regulator of these events. We confirmed NFkappaB activation by two independent methods: translocation of NFkappaB p50 to glomerular nuclei and ChIP assays demonstrating NFkappaB p50 binding to the kappaB motif of target genes in old versus young glomeruli. These data suggest that old glomeruli exhibit NFkappaB-associated up-regulation of a proinflammatory, procoagulable, and profibrotic phenotype compared with young glomeruli; these distinctions could explain their enhanced susceptibility to failure. Furthermore, these results provide a potential mechanistic explanation for the close relationship between ESRD and atherosclerotic organ failure as two parallel arms of age-associated NFkappaB-driven processes.

Nuclear delivery of NFκB-assisted DNA/polymer complexes: plasmid DNA quantitation by confocal laser scanning microscopy and evidence of nuclear polyplexes by FRET imaging

Quantification of a plasmid DNA (pDNA) and investigation of its polymer-associated state in the nucleus are crucial to evaluate the effectiveness of a gene-delivery system. This study was conducted with p3NF-luc-3NF, a pDNA-bearing optimized κB motif to favour NFκB-driven nuclear import. Here, a quantification of pDNA copies in the nucleus was performed by real-time confocal laser scanning microscopy in HeLa and C2C12 cells transfected with linear polyethylenimine or histidylated polylysine. Förster Resonance Energy Transfer (FRET) from the fluorescein-p3NF-luc-3NF donor to the co-localized rhodamine-polymer acceptor was carried out to investigate whether the pDNA was still condensed with the polymer in the nucleus. Upon 5 h of transfection, the nuclear amount of p3NF-luc3NF was ∼1500 copies in both cell lines whereas that of pTAL-luc, a 3NF-free counterpart pDNA, was less than 250. This quantity of p3NF-luc-3NF dropped dramatically to that of pTAL-luc in the presence of the BAY 11-7085, an inhibitor of NFκB activation. These data strongly support a nuclear import of p3NF-luc3NF mediated by NFκB. Moreover, FRET experiments clearly revealed that most of nuclear pDNA were still condensed with the polymer raising the question of their passage through the nuclear pore complex and their impact on the gene-expression efficiency.

Evidence for BAG3 modulation of HIV‐1 gene transcription

A family of co-chaperone proteins that share the Bcl-2-associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV-1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV-1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV-1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the kappaB motif of the HIV-1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF-kappaB with the kappaB DNA motif of the LTR. Results from binding and transcriptional assay identified the C-terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV-1 infection, and may provide a new avenue for the suppression of HIV-1 gene expression.

20‐Hydroxyecdysone indirectly regulates Hemolin gene expression in Hyalophora cecropia

Development and innate immune defence are two vital processes that have been demonstrated to use the same or similar molecules and signalling pathways in insects. Hemolin is a moth haemolymph protein belonging to the immunoglobulin superfamily. It is strongly induced upon bacterial infection. However, recent studies indicate a developmental regulation of hemolin. We show that the steroid hormone 20-hydroxyecdysone (20E) can activate the expression of Hyalophora cecropia Hemolin (HcHemolin) in the fat body of diapausing pupae. Using the protein synthesis inhibitor cycloheximide we demonstrate that Hemolin up-regulation by 20E requires ongoing protein synthesis. Moreover, 20E enhances transcription of the Hemolin gene in response to bacteria. Comparing the upstream regions of Manduca sexta Hemolin (MsHemolin) and HcHemolin, we identified four putative regulatory sites. Two are putative hormone response elements (HREs), one with an imperfect inverted repeat (HRE-IR) and one with a monomeric site (HRE-M). An additional monomeric hormone receptor site (MRE) is present only in HcHemolin. The third conserved motif is similar to the interferon (IFN) regulatory factor binding element (IRF-E) and IFN-stimulated response element (ISRE). The fourth conserved element is a kappaB motif situated between the Cap-site and the TATA-box. Finally, by electrophoresis mobility shift assay we demonstrate that the HRE-IR forms specific complexes with nuclear extract proteins of normal pupae that increase after 20E stimulation.

Interactions at a dioxin responsive element (DRE) and an overlapping kappaB site within the hs4 domain of the 3'alpha immunoglobulin heavy chain enhancer.

Our previous results describing the CH12.LX (AhR-expressing) and BCL-1 (AhR-deficient) B cell lines have supported an AhR/dioxin-responsive element (DRE)-mediated mechanism for TCDD-induced inhibition of micro heavy chain expression and thus of IgM secretion. Transcriptional regulation of the Ig heavy chain genes involves several regulatory elements including the 3'alpha Ig heavy chain enhancer, which is composed of four regulatory domains that span approximately 40 kb. One of these domains, hs4, contains a DRE-like site that overlaps a kappaB motif. We have previously demonstrated TCDD-inducible binding of both the AhR nuclear complex and NF-kappaB/Rel proteins to the DRE and kappaB motifs, respectively, as well as TCDD and LPS-induced transcriptional activity through the hs4 domain. The objective of the present study was to determine if the AhR nuclear complex and NF-kappaB/Rel proteins converge at these two overlapping cis-elements and act cooperatively to influence enhancer activity. To eliminate the potential influence of other transcription factors which bind to the hs4 domain, the approach was to construct a series of luciferase reporters containing a variable heavy chain (VH) promoter and a 42 bp fragment of the 1.4 kb hs4 regulatory domain, that included only the overlapping DRE and kappaB motif or mutations of these motifs for transient transfection experiments in CH12.LX and BCL-1 cells. In the CH12.LX cells, TCDD activated the hs4 fragment; however, co-treatment with LPS led to a marked and synergistic activation as previously observed with the wild type 1.4 kb hs4 domain. Mutation of either or both of the DRE and kappaB motifs diminished the effect of TCDD and LPS on the luciferase reporters possessing the 42 bp portion of hs4, and resembled the effect of these treatments on the promoter alone. In the BCL-1 cells, activity of the hs4 fragment was not induced by TCDD and/or LPS treatment. These results suggest that the AhR nuclear complex and NF-kappaB/Rel proteins converge at the DRE and kappaB motif to influence transcriptional activity of the hs4 enhancer fragment.

Transcriptional Regulation of Limulus Factor C: REPRESSION OF AN NFκB MOTIF MODULATES ITS RESPONSIVENESS TO BACTERIAL LIPOPOLYSACCHARIDE

Serine proteases play fundamental roles in invertebrate development, hemostasis, and innate immunity. This is exemplified by the limulus Factor C, which is a serine protease that binds a pathogen-associated molecule, lipopolysaccharide (LPS) to trigger a blood coagulation cascade. As a central molecule in the limulus innate immunity and hemostasis, Factor C gene expression has been detected in two major immune defense tissues, the amebocytes and hepatopancreas. Infection of the limulus with live Gram-negative bacteria induces a 2-3-fold increase in mRNA transcripts in both tissues. However, in vitro studies in Drosophila cell lines using Factor C promoter-reporter chimera DNA constructs, and site-directed mutagenesis of the promoter demonstrated that a proximal kappaB binding site, aided by an adjacent dorsal-like binding motif responds dramatically to LPS and dorsal transcription factor overexpression. Electrophoretic mobility shift assay further confirmed a strong interaction of the limulus kappaB motif with Rel proteins. However, deletion constructs of the Factor C promoter harboring different numbers of dorsal-like binding sites upstream of the kappaB motif as well as the electrophoretic mobility shift assay of these motifs with Rel proteins strongly suggest that the up-regulation of Factor C gene expression is attenuated during microbial challenge. The repression of the dramatic activation of this pathogen-responsive gene by LPS is probably effected via competition between the dorsal-like motifs over the proximal LPS-responsive kappaB unit, or through inhibition from the upstream repressive element(s), which accounts for the gene expression pattern observed in vivo. Our findings demonstrate that blood coagulation and innate immune response are integrated at the transcriptional level in this ancient organism, and that this LPS-responsive serine protease is controlled by an evolutionarily conserved NFkappaB pathway.

Developmental anomalies and neoplasia in animals and cells deficient in the large zinc finger protein KRC.

The large zinc finger protein KRC binds to the signal sequences of V(D)J recombination and the kappaB motif. Disruption of KRC expression in cell lines resulted in increased cell proliferation, anchorage independence of growth, and uncoupling of nuclear division and cell division. In this report, the function of KRC was studied in a RAG2-deficient blastocyst complementation animal model. KRC-deficient embryonic stem cells were generated by homologous recombination and were introduced into RAG2(-/-) blastocysts to generate KRC(-/-);RAG2(-/-) chimeric mice. The lymphoid compartments of chimeras examined at 5 weeks of age were developed, suggesting that KRC is not essential for V(D)J recombination development. However, by 6 months of age, there was a marked deficit in CD4(+)CD8(+) thymocytes in the chimeras, suggesting that KRC may be involved in T-lymphocyte survival. Additionally, one chimera developed anomalies, including postaxial polydactyly, hydronephrosis, and an extragonadal malignant teratoma. DNA analysis showed that the teratoma was derived from KRC(-/-) embryonic stem cells. The teratoma had compound tissue organization and was infiltrated with B lymphocytes. Subsequently, several immortalized KRC-deficient cell lines were established from the teratoma. In this study, growth anomalies and neoplasia were observed in animals and cells deficient in KRC, and other studies have shown allelic loss occurring at the chromosomal region of the human KRC counterpart in various tumors. We propose that KRC may be a previously unidentified tumor-suppresser gene.

An intron enhancer activates the immunoglobulin-related Hemolin gene in Hyalophora cecropia.

Hemolin is the only insect member of the immunoglobulin (Ig) superfamily reported to be up-regulated during an immune response. In diapausing pupae of Hyalophora cecropia the gene is expressed in fat body cells and in haemocytes. Like the mammalian Ig kappa light chain gene, the Hemolin gene harbours an enhancer including a kappaB motif in one of its introns. This motif binds the H. cecropia Rel factor Cif (Cecropia immunoresponsive factor). The Hemolin third intron also mediates transient reporter gene expression in immunoresponsive Drosophila mbn-2 cells. Co-transfections of Drosophila SL2 cells showed that the Drosophila Rel factor Dif (Dorsal-related immunity factor), transactivates reporter gene constructs through the intron. Moreover, a 4.8-fold synergistic activation was obtained when Dif is combined with the rat C/EBP (CCAAT/enhancer element-binding protein) and human HMGI (high mobility group protein I). This is the first report of an insect immune-related gene that is up-regulated by an enhancer activity conferred through an intron.

Interplay between cdk9 and NF-kappaB factors determines the level of HIV-1 gene transcription in astrocytic cells.

Basal transcription of the HIV-1 genome is controlled by a variety of ubiquitous and inducible regulatory factors, some with the ability to associate with the viral DNA sequences within the promoter spanning the long terminal repeat (LTR). In this report we demonstrate that activation of the HIV-1 promoter through the inducible DNA binding NF-kappaB transcription factors can be affected by cdk9 in human astrocytic cells. Our results show that ectopic expression of cdk9, but not its mutant variant which lacks the domain responsible for its kinase activity, augments transcription of the LTR. Moreover, we demonstrate that induction of the NF-kappaB pathway by PMA, or overexpression of its subunits including p50/p65 have a negative effect on the ability of cdk9 to stimulate viral gene transcription in these cells. Results from band-shift experiments demonstrated significant suppression of p50/p65 association to its DNA target motif by cdk9. Further, data from GST pull-down and combined immunoprecipitation/Western blot analysis of the protein extracts from cells expressing cdk9, p50 and p65 have revealed the interaction of cdk9 with both p50 and p65 in the absence of DNA containing the kappaB motif. All of these observations led us to conclude that the interaction of cdk9 with the NF-kappaB factors can determine the ability of NF-kappaB to modulate HIV-1 gene transcription.

ZAS: C2H2 zinc finger proteins involved in growth and development.

A ZAS gene encodes a large protein with two separate C2H2 zinc finger pairs that independently bind to specific DNA sequences, including the kappaB motif. Three paralogous mammalian genes, ZAS1, ZAS2, and ZAS3, and a related Drosophila gene, Schnurri, have been cloned and characterized. The ZAS genes encode transcriptional proteins that activate or repress the transcription of a variety of genes involved in growth, development, and metastasis. In addition, ZAS3 associates with a TNF receptor-associated factor to inhibit NF-kappaB- and JNK/ SAPK-mediated signaling of TNF-alpha. Genetic experiments show that ZAS3 deficiency leads to proliferation of cells and tumor formation in mice. The data suggest that ZAS3 is important in controlling cell growth, apoptosis, and inflammation. The potent vasoactive hormone endothelin and transcription factor AP2 gene families also each consist of three members. The ZAS, endothelin, and transcription factor AP2 genes form several linkage groups. Knowledge of the chromosomal locations of these genes provides valuable clues to the evolution of the vertebrate genome.

The κB transcriptional enhancer motif and signal sequences of V(D)J recombination are targets for the zinc finger protein HIVEP3/KRC: a site selection amplification binding study

BackgroundThe ZAS family is composed of proteins that regulate transcription via specific gene regulatory elements. The amino-DNA binding domain (ZAS-N) and the carboxyl-DNA binding domain (ZAS-C) of a representative family member, named κB DNA binding and recognition component (KRC), were expressed as fusion proteins and their target DNA sequences were elucidated by site selection amplification binding assays, followed by cloning and DNA sequencing. The fusion proteins-selected DNA sequences were analyzed by the MEME and MAST computer programs to obtain consensus motifs and DNA elements bound by the ZAS domains.ResultsBoth fusion proteins selected sequences that were similar to the κB motif or the canonical elements of the V(D)J recombination signal sequences (RSS) from a pool of degenerate oligonucleotides. Specifically, the ZAS-N domain selected sequences similar to the canonical RSS nonamer, while ZAS-C domain selected sequences similar to the canonical RSS heptamer. In addition, both KRC fusion proteins selected oligonucleoties with sequences identical to heptamer and nonamer sequences within endogenous RSS.ConclusionsThe RSS are cis-acting DNA motifs which are essential for V(D)J recombination of antigen receptor genes. Due to its specific binding affinity for RSS and κB-like transcription enhancer motifs, we hypothesize that KRC may be involved in the regulation of V(D)J recombination.

Benzo[a]pyrene Activates the Human p53 Gene through Induction of Nuclear Factor κB Activity

p53 is known to be recruited in response to DNA-damaging genotoxic stress and plays an important role in maintaining the integrity of the genome. In the present study, the effect of a potent lung cancer carcinogen, benzo[a]pyrene (B[a]P) on p53 expression was investigated. We showed that exposure of A549 and NIH 3T3 cells to B[a]P resulted in an increase in p53 mRNA levels and in p53 promoter activation, indicating that B[a]P-induced p53 expression is partly regulated at the transcriptional level. The p53 promoter region which extends from -58 to -43, overlapping the kappaB motif, is essential for both the p53 basal promoter activity and p53 promoter activation induced by B[a]P. Nuclear factor kappaB (NF-kappaB) proteins have been revealed to be activated in B[a]P-induced p53 expression. Activated NF-kappaB complexes were shown to contain predominantly p50 and p65 subunit components in A549 cells and p65 subunit in NIH 3T3 cells. In addition, the overexpression of IkappaBalpha completely inhibited NF-kappaB activation, p53 promoter transactivation and the stimulatory effect on p53 transcription induced by B[a]P. We therefore conclude that B[a]P transcriptionally activates the human p53 gene through the induction of NF-kappaB activity.

Role of p52 (NF-kappaB2) in LPS tolerance in a human B cell line.

Cells of the weakly CD14 positive human B cell line RPMI 8226, clone 1, will mobilize NF-kappaB (p50/p65 and p50/p50) proteins and produce TNF mRNA when stimulated with lipopolysaccharide (LPS). When such cells are precultured with a low amount of LPS (50-250 ng/ml) for 3 - 4 days followed by a secondary stimulation with a high dose of LPS (1 microg/ml) then the cytokine expression is strongly reduced, i. e. the cells have become tolerant. Western blot analysis of proteins of the NF-kappaB/rel family demonstrates cytoplasmic p50 and p65 for naive B cells plus a low level of p52. While with tolerance induction the pattern of p50 and p65 proteins remains essentially unchanged, the LPS tolerant 8226 cells show a dramatic increase of both p52 protein and its p100 precursor in the cytosol. This p52 is found strongly upregulated in Western blots of extracts from purified nuclei of tolerant cells. Also, gelshift analysis with the -605 kappaB motif of the human TNF 5'-region shows an additional high mobility complex in LPS tolerant cells -a complex that is supershifted with an anti-p52 antibody. Functional analysis with the -1064 TNF 5'-region in front of the luciferase reporter gene demonstrates that transactivation of the TNF promoter is strongly reduced in tolerant cells. Also, overexpression of p52 will suppress activity of TNF promoter reporter gene constructs. Taken together these data show that tolerance to LPS in the human RPMI 8226 B cell line involves upregulation of the p52 (NF-kappaB2) gene, which appears to be instrumental in the blockade of TNF gene expression.

Regulation by phosphorylation of the zinc finger protein KRC that binds the kappaB motif and V(D)J recombination signal sequences.

The DNA binding protein KRC (forkappaB binding andrecognitioncomponent of the V(D)J recombination signal sequence) belongs to a family of large zinc finger proteins that bind to the kappaB motif and contains two widely separated DNA binding structures. In addition to the kappaB motif, KRC fusion proteins bind to the signal sequences of V(D)J recombination to form highly ordered complexes. Here, we report that KRC may be regulated by post-translational modifications. Specific protein kinases present in the nucleus of pre-B cells phosphorylated a KRC fusion protein at tyrosine and serine residues. Such protein modifications increased DNA binding, thereby providing a mechanism by which KRC responds to signal transduction pathways. KRC is a substrate of epidermal growth factor receptor kinase and P34cdc2 kinase in vitro. Our results suggest that activation of the KRC family of transcription factors may provide a mechanism by which oncogenic tyrosine kinases regulate genes with kappaB-controlled gene regulatory elements.

KRC transcripts: identification of an unusual alternative splicing event.

Mouse KRC is a large zinc finger protein that binds to the kappaB motif of gene transcription and to the recognition signal sequences for the somatic recombination of the immunoglobulin and T-cell receptor gene segments. The mouse KRC gene is more than 70 kilobases (kb) in size, and contains at least seven exons, with the largest transcript being approximately 9.5 kb. Multiple differentially spliced transcripts of KRC were identified in thymus and brain, which would result in the production of multiple KRC protein isoforms with different N-termini and number of DNA binding domains. Alternative splicing events leading to the production of these multiple transcripts have been elucidated. Of particular interest are the exclusions in some transcripts of sequences from a gigantic exon of 5487 base pairs (bp), or from an exon of 176 bp. Both potentially deleted exons code for zinc finger motifs that are essential components of the N-terminal and C-terminal DNA binding domains, respectively. Another intriguing phenomenon found in some KRC transcripts is the skipping of a 459 bp fragment within the gigantic exon that would code for the N-terminal DNA binding domain. Bacterial fusion proteins derived from this fragment bind specifically to KRC target DNAs. Apparently, distinct alternative splicing events could eliminate the N-terminal DNA binding domain of KRC.

Expression of the leukocyte early activation antigen CD69 is regulated by the transcription factor AP-1.

The leukocyte Ag CD69, one of the earliest cell surface activation Ags, is up-regulated at the transcriptional level by proinflammatory stimuli involving the NF-kappaB/Rel family of transcription factors. However, promoter fragments lacking a critical kappaB motif respond to other stimuli such as phorbol esters and triggering Abs against TCR/CD3. Since the 5' promoter flanking region of the CD69 gene contains several putative binding sequences for transcription factor activating protein-1 (AP-1), we explored its role in the inducible expression of CD69. Stimuli that induce AP-1, but not NF-kappaB, such as pyrrolidine dithiocarbamate, augmented the cell surface expression of CD69 as well as its mRNA levels, and the promoter activity of the CD69 gene. This up-regulation is accompanied by an increased binding of jun and fos family members to a consensus AP-1 binding site of the proximal (-16) CD69 promoter region, which seems to be functionally responsive to different activation signals and is trans activated by c-jun expression vectors. Furthermore, cotransfection of a dominant negative version of c-jun, but not IkappaB, abolished the inducible transcriptional activity of the CD69 promoter. In conclusion, the inducible expression of the CD69 gene by mitogenic signals is regulated by the transcription factor AP-1.

Regulation of NFKB1 proteins by the candidate oncoprotein BCL-3: generation of NF-kappaB homodimers from the cytoplasmic pool of p50-p105 and nuclear translocation.

The candidate oncoprotein BCL-3 has been shown to function as a transcriptional co-activator for homodimers of NF-kappaB p50 and p50B. We expressed BCL-3 ectopically in pro-B cell lines and found that these cells exhibited a dramatic increase in nuclear kappaB motif binding activity of p50 homodimers containing BCL-3 in the complex. Co-transfection and in vitro reconstitution experiments revealed that the complex of p50 with its precursor p105 (p50-p105), which was shown to accumulate in the cytoplasm of the pro-B cell lines, is required for induction of DNA binding of p50 homodimers by BCL-3. However, we could see no in vivo or in vitro evidence of a BCL-3-induced increase in proteolytic processing. Instead, BCL-3-mediated reorganization of NFKB1 subunits was demonstrated in vitro. Immunofluorescence staining clearly demonstrated that the transition from cytoplasmic p50-p105 to nuclear p50 homodimers was induced by BCL-3 expression. Thus BCL-3 has versatile functions: cytoplasmic activation of p50 homodimers, their nuclear translocation and, as previously shown, modulation of the transcriptional machinery in the nucleus.

Synergy between Interferon-γ and Tumor Necrosis Factor-α in Transcriptional Activation Is Mediated by Cooperation between Signal Transducer and Activator of Transcription 1 and Nuclear Factor κB

Interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) cooperate to induce the expression of many gene products during inflammation. The present report demonstrates that a portion of this cooperativity is mediated by synergism between two distinct transcription factors: signal transducer and activator of transcription 1 (STAT1) and nuclear factor kappaB (NF-kappaB). IFNgamma and TNFalpha synergistically induce expression of mRNAs encoding interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1, Mig (monokine induced by gamma-interferon), and RANTES (regulated on activation normal T cell expressed and secreted) in normal but not STAT1-deficient mouse fibroblasts, indicating a requirement for STAT1. Transient transfection assays in fibroblasts using site-directed mutants of a 1.3-kilobase pair sequence of the IRF-1 gene promoter revealed that the synergy was dependent upon two sequence elements; a STAT binding element and a kappaB motif. Artificial constructs containing a single copy of both a STAT binding element and a kappaB motif linked to the herpes virus thymidine kinase promoter were able to mediate synergistic response to IFNgamma and TNFalpha; such response varied with both the relative spacing and the specific sequence of the regions between these two sites. Cooperatively responsive sequence constructs bound both STAT1alpha and NF-kappaB in nuclear extracts prepared from IFNgamma- and/or TNFalpha-stimulated fibroblasts, although binding of individual factors was not cooperative. Thus, the frequently observed synergy between IFNgamma and TNFalpha in promoting inflammatory response depends in part upon cooperation between STAT1alpha and NF-kappaB, which is most likely mediated by their independent interaction with one or more components of the basal transcription complex.