Biomedical subjects

Allan R Brasier

Publications and source records attributed to Allan R Brasier.

At least 19 recordsLinked to original sources

PubMed2006

Angiotensin II induces IL-6 expression and the Jak-STAT3 pathway in aortic adventitia of LDL receptor-deficient mice.

Angiotensin II (A-II), the major effector peptide of the renin angiotensin system potently accelerates progression of atherosclerosis. To investigate its effects on vascular inflammatory mechanisms, we elucidated vascular cytokine expression during early lesion development in A-II-infused atherosclerosis-prone LDLR-/- mice. Male LDLR-/- mice were placed on a "Western" high-fat diet for 4 weeks, followed by sham or A-II infusion for 7 weeks. Equal blood pressures and elevations in serum lipids were seen in both groups. Mice were sacrificed when significant A-II-induced plaque development was first detectable, aortae were explanted and culture media assayed for secreted cytokines. Nine cytokines were significantly induced with interleukin-6 (IL-6) being the most highly secreted. Local IL-6 production was confirmed by in situ mRNA hybridization and immunostaining, where the most abundant IL-6 was found in the aortic adventitia, with lesser production by the medial and intimal layers. Immunofluorescence colocalization showed IL-6 expression by fibroblasts and activated macrophages. Activation of downstream IL-6 signaling mediated by the Jak-STAT3 pathway was demonstrated by inducible phospho-Tyr705-STAT3 formation in the adventitia and endothelium (of IL-6+/+ mice only). These findings define cytokine profiles in the A-II infusion model and demonstrate that IL-6, produced by activated macrophages and fibroblasts in the adventitia, induces the Jak-STAT3 pathway during early A-II-induced atherosclerosis.

Angiotensin II↗

PubMed2006

Retinoic acid-inducible gene I mediates early antiviral response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells.

Respiratory syncytial virus (RSV) is one of the most common viral pathogens causing severe lower respiratory tract infections in infants and young children. Infected host cells detect and respond to RNA viruses using different mechanisms in a cell-type-specific manner, including retinoic acid-inducible gene I (RIG-I)-dependent and Toll-like receptor (TLR)-dependent pathways. Because the relative contributions of these two pathways in the recognition of RSV infection are unknown, we examined their roles in this study. We found that RIG-I helicase binds RSV transcripts within 12 h of infection. Short interfering RNA (siRNA)-mediated RIG-I "knockdown" significantly inhibited early nuclear factor-kappaB (NF-kappaB) and interferon response factor 3 (IRF3) activation 9 h postinfection (p.i.). Consistent with this finding, RSV-induced beta interferon (IFN-beta), interferon-inducible protein 10 (IP-10), chemokine ligand 5 (CCL-5), and IFN-stimulated gene 15 (ISG15) expression levels were decreased in RIG-I-silenced cells during the early phase of infection but not at later times (18 h p.i.). In contrast, siRNA-mediated TLR3 knockdown did not affect RSV-induced NF-kappaB binding but did inhibit IFN-beta, IP-10, CCL-5, and ISG15 expression at late times of infection. Further studies revealed that TLR3 knockdown significantly reduced NF-kappaB/RelA transcription by its ability to block the activating phosphorylation of NF-kappaB/RelA at serine residue 276. We further found that TLR3 induction following RSV infection was regulated by RIG-I-dependent IFN-beta secreted from infected airway epithelial cells and was mediated by both IFN response-stimulated element (ISRE) and signal transducer and activator of transcription (STAT) sites in its proximal promoter. Together these findings indicate distinct temporal roles of RIG-I and TLR3 in mediating RSV-induced innate immune responses, which are coupled to distinct pathways controlling NF-kappaB activation.

Cell Line↗

PubMed2006

RhoA mediates angiotensin II-induced phospho-Ser536 nuclear factor kappaB/RelA subunit exchange on the interleukin-6 promoter in VSMCs.

The vasoconstrictor angiotensin II (Ang II) accelerates atherosclerosis by inducing vascular gene expression programs, producing monocyte recruitment, and vascular remodeling. In vascular smooth muscle cells (VSMCs), Ang II signaling activates interleukin (IL)-6 expression, a cytokine producing acute-phase inflammation, mediated by the transcription factor nuclear factor kappaB (NF-kappaB). The classical NF-kappaB activation pathway involves cytoplasmic-to-nuclear translocation of the potent RelA transactivating subunit; however, because nuclear RelA is present in VSMCs, the mechanism by which NF-kappaB activity is controlled is incompletely understood. In this study, we focus on early activation steps controlling RelA activation. Although Ang II only weakly induces approximately 1.5-fold RelA nuclear translocation, RelA is nevertheless required because short interfering RNA-mediated RelA knockdown inhibits inducible IL-6 expression. We find instead that Ang II stimulation rapidly induces RelA phosphorylation at serine residue 536, a critical regulatory site in its transactivating domain. Chromatin immunoprecipitation assays indicate no significant changes in total RelA binding to the native IL-6 promoter, but an apparent increase in fractional binding of phospho-Ser536 RelA. Inactivation of RhoA by treatment with Clostridium botulinum exoenzyme C3 exotoxin or expression of dominant negative RhoA blocks Ang II-inducible RelA Ser536 phosphorylation and IL-6 expression. Finally, enhanced phospho-Ser536 RelA formation in the aortae of rats chronically infused with Ang II was observed. Together, these data indicate a novel mechanism for Ang II-induced NF-kappaB activation in VSMCs, mediated by RhoA-induced phospho-Ser536 RelA formation, IL-6 expression, and vascular inflammation.

Angiotensin II↗

PubMed2006

Ikkepsilon regulates viral-induced interferon regulatory factor-3 activation via a redox-sensitive pathway.

Respiratory syncytial virus (RSV)-induced chemokine gene expression occurs through the activation of a subset of transcription factors, including Interferon Regulatory Factor (IRF)-3. In this study, we have investigated the signaling pathway leading to RSV-induced IRF-3 activation and whether it is mediated by intracellular reactive oxygen species (ROS) generation. Our results show that RSV infection induces expression and catalytic activity of IKKepsilon, a noncanonical IKK-like kinase. Expression of a kinase-inactive IKKepsilon blocks RSV-induced IRF-3 serine phosphorylation, nuclear translocation and DNA-binding, leading to inhibition of RANTES gene transcription, mRNA expression and protein synthesis. Treatment of alveolar epithelial cells with antioxidants or with NAD(P)H oxidase inhibitors abrogates RSV-induced chemokine secretion, IRF-3 phosphorylation and IKKepsilon induction, indicating that ROS generation plays a fundamental role in the signaling pathway leading to IRF-3 activation, therefore, identifying a novel molecular target for the development of strategies aimed to modify the inflammatory response associated with RSV infection of the lung.

Cell Line, Tumor↗

PubMed2006

Functional analysis of the nuclear proteome of human A549 alveolar epithelial cells by HPLC-high resolution 2-D gel electrophoresis.

The airway epithelial cell plays a central role in coordinating airway inflammatory responses, where significant changes in the proteome occur in response to infectious stimuli. To further understand the spectrum of proteins and the biological processes they control, we have initially determined the nuclear proteome of human type II-like alveolar epithelial cells (A549) using a sequential method of organellar enrichment followed by HPLC prefractionation prior to 2-DE-based protein identification using MALDI-TOF MS. This approach yielded 719 high-confidence identifications, 433 mapping to unique gene identifiers. Expert classification showed that these proteins controlled chromatin remodeling, protein refolding, cytoskeletal structure, membrane function, metabolic processes, mitochondrial function, RNA binding, protein synthesis, signaling, and transcription factor activities. The proteins were mapped to gene ontology classifications, where metabolism and catalytic activity functions were significantly enriched, representing 43 and 32% of the protein set, respectively. Pathways analysis indicated a protein network affecting tumor necrosis factor-nuclear factor-kappaB signaling pathway interacting with intermediate cytoskeletal filaments. Forty-five proteins of unknown function were subjected to domain analysis and inferred to have additional nuclear functions controlling purine nucleotide metabolism and protein-protein interactions. This database represents the most comprehensive data set of mammalian nuclear proteins and will serve as a foundation for further discovery.

Cell Line, Transformed↗

PubMed2006

Identification of an NF-kappaB-dependent gene network in cells infected by mammalian reovirus.

Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.

Animals↗

PubMed2006

An alternative splice product of IkappaB kinase (IKKgamma), IKKgamma-delta, differentially mediates cytokine and human T-cell leukemia virus type 1 tax-induced NF-kappaB activation.

NF-kappaB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IkappaB kinase (IKK) "signalsome". The core IKK consists of two catalytic serine kinases, IKKalpha and IKKbeta, and a noncatalytic subunit, IKKgamma. IKKgamma is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKgamma mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKgamma-delta. Using a specific reverse transcription-PCR assay, we find that IKKgamma-delta is widely expressed in cultured human cells and normal human tissues. Because IKKgamma-Delta protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-kappaB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKgamma-delta has strong homo- and heterotypic association with wild-type (WT) IKKgamma and, like IKKgamma WT, associates with the IKKbeta kinase. Similarly, IKKgamma-delta mediates IKK kinase activity and downstream NF-kappaB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-kappaB-inducing kinase-IKKalpha signaling pathway. Surprisingly, however, in contrast to IKKgamma WT, IKKgamma-delta is not able to mediate HTLV-1 Tax-induced NF-kappaB-dependent transcription, even though IKKgamma-delta binds and colocalizes with Tax. These observations suggest that IKKgamma-delta is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKgamma-delta expression, therefore, may affect signal transduction cascades coupling to IKK.

Alternative Splicing↗

PubMed2006

Diabetes-induced activation of canonical and noncanonical nuclear factor-kappaB pathways in renal cortex.

Evidence of diabetes-induced nuclear factor-kappaB (NF-kappaB) activation has been provided with DNA binding assays or nuclear localization with immunohistochemistry, but few studies have explored mechanisms involved. We examined effects of diabetes on proteins comprising NF-kappaB canonical and noncanonical activation pathways in the renal cortex of diabetic mice. Plasma concentrations of NF-kappaB-regulated cytokines were increased after 1 month of hyperglycemia, but most returned to control levels or lower by 3 months, when the same cytokines were increased significantly in renal cortex. Cytosolic content of NF-kappaB canonical pathway proteins did not differ between experimental groups after 3 months of diabetes, while NF-kappaB noncanonical pathway proteins were affected, including increased phosphorylation of inhibitor of kappaB kinase-alpha and several fold increases in NF-kappaB-inducing kinase and RelB, which were predominantly located in tubular epithelial cells. Nuclear content of all NF-kappaB pathway proteins was decreased by diabetes, with the largest change in RelB and p50 (approximately twofold decrease). Despite this decrease, measurable increases in protein binding to DNA in diabetic versus control nuclear extracts were observed with electrophoretic mobility shift assay. These results provide evidence for chronic NF-kappaB activation in the renal cortex of db/db mice and suggest a novel, diabetes-linked mechanism involving both canonical and noncanonical NF-kappaB pathway proteins.

Animals↗

PubMed2005

Stochastic regulation in early immune response.

Living cells may be considered noisy or stochastic biochemical reactors. In eukaryotic cells, in which the number of protein or mRNA molecules is relatively large, the stochastic effects originate primarily in regulation of gene activity. Transcriptional activity of a gene can be initiated by transactivator molecules binding to the specific regulatory site(s) in the target gene. The stochasticity of activator binding and dissociation is amplified by transcription and translation, since target gene activation results in a burst of mRNAs molecules, and each copy of mRNA then serves as a template for numerous protein molecules. In this article, we reformulate our model of the NF-kappaB regulatory module to analyze a single cell regulation. Ordinary differential equations, used for description of fast reaction channels of processes involving a large number of molecules, are combined with a stochastic switch to account for the activity of the genes involved. The stochasticity in gene transcription causes simulated cells to exhibit large variability. Moreover, none of them behaves like an average cell. Although the average mRNA and protein levels remain constant before tumor necrosis factor (TNF) stimulation, and stabilize after a prolonged TNF stimulation, in any single cell these levels oscillate stochastically in the absence of TNF and keep oscillating under the prolonged TNF stimulation. However, in a short period of approximately 90 min, most cells are synchronized by the TNF signal, and exhibit similar kinetics. We hypothesize that this synchronization is crucial for proper activation of early genes controlling inflammation. Our theoretical predictions of single cell kinetics are supported by recent experimental studies of oscillations in NF-kappaB signaling made on single cells.

Animals↗

PubMed2005

A TNF-induced gene expression program under oscillatory NF-kappaB control.

BACKGROUND: The cytokine tumor necrosis factor (TNF) initiates tissue inflammation, a process mediated by the NF-kappaB transcription factor. In response to TNF, latent cytoplasmic NF-kappaB is activated, enters the nucleus, and induces expression of inflammatory and anti-apoptotic gene expression programs. Recently it has been shown that NF-kappaB displays two distinct activation modes, monophasic and oscillatory, depending on stimulus duration. Characterization of temporal expression patterns for the NF-kappaB network and determination of those genes under monophasic- or oscillatory control has not been experimentally addressed. RESULTS: To identify the kinetics of NF-kappaB-dependent gene expression and determine whether these two types of NF-kappaB translocation modes control distinct gene programs, a detailed kinetic analysis of a validated microarray data set was performed on 74 unique NF-kappaB-dependent genes in response to TNF. Hierarchical clustering identified distinct expression profiles termed the "Early", "Middle", "Late" response groups, peaking 1, 3, and 6 h after stimulation, respectively. These expression patterns were validated by Quantitative Real Time PCR (Q-RT-PCR) and NF-kappaB binding was demonstrated by chromatin immunoprecipitation (ChIP) assays. Each response group was mapped to its molecular function; this analysis indicated that the Early group encodes cytokines or negative regulators of the IKK-NF-kappaB pathway, and the Late group encodes cell surface receptors, adhesion molecules and signal adapters. That similar coordinated sequential cascades of gene expression were also seen in response to stimulation by the cytokine IL-1, and expression patterns observed in MRC-5 fibroblasts indicated that the epithelial NF-kappaB program is relatively stimulus- and cell type-independent. Bioinformatic analysis of the Early and Late gene promoters indicates that although both groups contain similar patterns of NF-kappaB-binding sites, only the Early gene promoters contain NF-kappaB-binding sites located in phylogenetically conserved domains. Stimulation protocols designed to produce either monophasic or oscillatory NF-kappaB activation modes showed that the oscillatory mode is required only for expression of the Late genes. CONCLUSION: This analysis provides important insights into the TNF-regulated genetic response program in epithelial cells, where NF-kappaB controls sequential expression patterns of functionally distinct genes that depend on its oscillatory activation mode.

Cell Adhesion↗

PubMed2005

Transcriptional stochasticity in gene expression.

Due to the small number of copies of molecular species involved, such as DNA, mRNA and regulatory proteins, gene expression is a stochastic phenomenon. In eukaryotic cells, the stochastic effects primarily originate in regulation of gene activity. Transcription can be initiated by a single transcription factor binding to a specific regulatory site in the target gene. Stochasticity of transcription factor binding and dissociation is then amplified by transcription and translation, since target gene activation results in a burst of mRNA molecules, and each mRNA copy serves as a template for translating numerous protein molecules. In the present paper, we explore a mathematical approach to stochastic modeling. In this approach, the ordinary differential equations with a stochastic component for mRNA and protein levels in a single cells yield a system of first-order partial differential equations (PDEs) for two-dimensional probability density functions (pdf). We consider the following examples: Regulation of a single auto-repressing gene, and regulation of a system of two mutual repressors and of an activator-repressor system. The resulting PDEs are approximated by a system of many ordinary equations, which are then numerically solved.

Animals↗

PubMed2005

Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

Binding Sites↗

PubMed2005

STAT3 NH2-terminal acetylation is activated by the hepatic acute-phase response and required for IL-6 induction of angiotensinogen.

BACKGROUND & AIMS: The signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors mediating acute-phase response (APR) of the human angiotensinogen (hAGT) gene in hepatocytes. The mechanisms of how STAT3 activates target genes are unknown. Here we analyzed the biochemistry of STAT3 activation by interleukin (IL)-6 in hepatocellular carcinoma HepG2 and Balb/C mice. METHODS: Immunoprecipitation-Western assays and Matrix Assisted Laser Desorption-Time of Flight mass spectrometry determined sites of STAT3 acetylation by the 300-kilodalton target of E1A (p300) co-activator. The subcellular localization of acetylation-deficient STAT3 molecules were studied by microscopic imaging, effects on DNA binding measured by gel shift and chromatin immunoprecipitation (ChIP) assays, and gene transactivation by Northern blot and reporter assays. RESULTS: Two Lys residues at amino acids 49 and 87 in the STAT3 NH2 terminus are acetylated by p300. Lys-to-Arg point mutations (STAT3 K49R/K87R) had no effect on inducible DNA binding, but blocked p300-mediated acetyl(Ac)-STAT3 formation and abrogated IL-6-induced hAGT activation. Although STAT3 K49R/K87R rapidly translocated into the nucleus, it did not bind p300 and had delayed cytoplasmic redistribution. ChIP assays show IL-6-inducible acetylated STAT3 and p300 binding to the native hAGT promoter. Activation of the APR in mice induces nuclear Tyr phosphorylated and acetylated STAT3 in hepatic nuclei. We also observed that STAT3 interacts with histone deacetylases (HDACs), specifically HDAC 1, that down-regulate IL-6-induced hAGT transactivation. CONCLUSIONS: IL-6-induced target gene activation requires p300-mediated STAT3 acetylation, and HDACs are involved in the termination of STAT3 action. These studies indicate the acetylation-deacetylation reaction as a novel signaling mechanism controlling the IL-6-STAT3 pathway in the hepatic APR.

Acetylation↗

PubMed2005

Respiratory syncytial virus influences NF-kappaB-dependent gene expression through a novel pathway involving MAP3K14/NIK expression and nuclear complex formation with NF-kappaB2.

A member of the Paramyxoviridae family of RNA viruses, respiratory syncytial virus (RSV), is a leading cause of epidemic respiratory tract infection in children. In children, RSV primarily replicates in the airway mucosa, a process that alters epithelial cell chemokine expression, thereby inducing airway inflammation. We investigated the role of the mitogen-activated protein kinase kinase kinase 14/NF-kappaB-inducing kinase (NIK) in the activation of NF-kappaB-dependent genes in alveolus-like A549 cells. RSV infection induces a time dependent increase of NIK mRNA and protein expression that peaks 12 to 24 h after viral exposure. Immunoprecipitation kinase assays indicate that NIK kinase activity is activated even more rapidly (within 6 h of RSV adsorption) associated with an endogenous approximately 50-kDa NF-kappaB2 substrate. Because NIK associates with IKKalpha to mediate processing of the 100-kDa NF-kappaB2 precursor into its 52-kDa DNA binding isoform ("p52"), the effects of RSV on NIK complex formation with IKKalpha and NF-kappaB2 were determined by coimmunoprecipitation assay. We find that NIK, IKKalpha, and both 100 kDa- and 52-kDa NF-kappaB2 isoforms strongly complex 15 h after exposure to RSV at times subsequent to NIK kinase activation. Western immunoblot and microaffinity DNA pull-down assays showed a parallel increase in nuclear translocation and DNA binding of the NF-kappaB2-Rel B complex. Interestingly, we make the novel observations that NIK also transiently translocates into the nucleus complexed with 52-kDa NF-kappaB2. Small interfering RNA-mediated NIK "knock-down" blocked RSV-inducible 52-kDa NF-kappaB2 processing and interfered with the early activation of a subset of NF-kappaB-dependent genes, indicating the importance of this activation pathway in the genomic NF-kappaB response to RSV. Together, these data indicate that RSV infection rapidly activates the noncanonical NF-kappaB activation pathway prior to the more potent canonical pathway activation. This appears to be through a novel mechanism involving induction of NIK kinase activity, expression, and nuclear translocation of a ternary complex with IKKalpha and processed NF-kappaB2.

Active Transport, Cell Nucleus↗

PubMed2005

Respiratory syncytial virus-inducible BCL-3 expression antagonizes the STAT/IRF and NF-kappaB signaling pathways by inducing histone deacetylase 1 recruitment to the interleukin-8 promoter.

Respiratory syncytial virus (RSV) is a paramyxovirus that produces airway inflammation, in part by inducing interleukin-8 (IL-8) expression, a CXC-type chemokine, via the NF-kappaB/RelA and STAT/IRF signaling pathways. In RSV-infected A549 cells, IL-8 transcription attenuates after 24 h in spite of ongoing viral replication and persistence of nuclear RelA, suggesting a mechanism for transcriptional attenuation. RSV infection induces B-cell lymphoma protein -3 (Bcl-3) expression 6 to 12 h after viral infection, at times when IL-8 transcription is inhibited. By contrast, 293 cells, deficient in inducible Bcl-3 expression, show no attenuation of IL-8 transcription. We therefore examined Bcl-3's role in terminating virus-inducible IL-8 transcription. Transient expression of Bcl-3 potently inhibited virus-inducible IL-8 transcription by disrupting both the NF-kappaB and STAT/IRF pathways. Although previously Bcl-3 was thought to capture 50-kDa NF-kappaB1 isoforms in the cytoplasm, immunoprecipitation (IP) and electrophoretic mobility shift assays indicate that nuclear Bcl-3 associates with NF-kappaB1 without affecting DNA binding. Additionally, Bcl-3 potently inhibited the STAT/IRF pathway. Nondenaturing co-IP assays indicate that nuclear Bcl-3 associates with STAT-1 and histone deacetylase 1 (HDAC-1), increasing HDAC-1 recruitment to the IL-8 promoter. Treatment with the HDAC inhibitor trichostatin A blocks attenuation of IL-8 transcription. A nuclear targeting-deficient Bcl-3 is unable to enhance HDAC-1-mediated chemokine repression. Finally, small inhibitory RNA-mediated Bcl-3 "knockdown" resulted in enhanced RSV-induced chemokine expression in A549 cells. These data indicate that Bcl-3 is a virus-inducible inhibitor of chemokine transcription by interfering with the NF-kappaB and STAT/IRF signaling pathways by complexing with them and recruiting HDAC-1 to attenuate target promoter activity.

Carrier Proteins↗

PubMed2005

Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.

The chromatin immunoprecipitation (ChIP) assay has recently been exploited as a powerful and versatile technique for probing protein-DNA interactions within the chromatin environment. In this method, intact cells are fixed with a reversible DNA-protein cross-linking agent (formaldehyde), and associated DNA is enriched by immunoprecipitating a target DNA binding protein. The bound DNA in the immune complexes is then used to identify that specific DNA binding protein's endogenous genomic targets. Nuclear factor kappaB (NF-kappaB) is a highly inducible transcription factor that controls genetic networks important for pathogen- or cytokine-induced inflammation, immune response, and cellular survival. In our studies of the genetic network under control of the inducible NF-kappaB transcription factor, we found that the conventional ChIP technique using a single formaldehyde cross-linking step did not reproducibly cross-link it to DNA. As a result, we have developed a novel ChIP assay using a two-step cross-linking procedure, incorporating N-hydroxysuccinimide (NHS)-ester-mediated protein-protein cross-linking prior to conventional DNA-protein cross-linking. We demonstrate that this technique is highly efficient, cross-linking virtually all NF-kappaB/Rel A into covalent complexes, resulting in quantitative and robust identification of inducible NF-kappaB family binding to a variety of validated NF-kappaB-dependent genomic targets. To demonstrate the general utility of this two-step cross-linking procedure, we performed enhanced capture of cytokine-inducible signal transducer and activator of transcription-3 (STAT3) binding to one of its known target genes. Our method represents a significant improvement in the efficiency of ChIP analysis in the study of endogenous targets for rare transcription factors.

Binding Sites↗

PubMed2004

Stochastic effects of multiple regulators on expression profiles in eukaryotes.

The stochastic nature of gene regulation still remains not fully understood. In eukaryotes, the stochastic effects are primarily attributable to the binary nature of genes, which are considered either switched "on" or "off" due to the action of the transcription factors binding to the promoter. In the time period when the gene is activated, bursts of mRNA transcript are produced. In the present paper, we investigate regulation of gene expression at the single cell level. We propose a mechanism of gene regulation, which is able to explain the observed distinct transcription profiles assuming the number of co-regulatory activities, without attempting to identify the specific proteins involved. The model is motivated by our experiments on NF-kappaB-dependent genes in HeLa cells. Our experimental data shows that NF-kappaB-dependent genes can be stratified into three characteristic groups according to their expression profiles: early, intermediate and late having maximum of expression at about 1, 3 and 6 h, respectively, from the beginning of TNF stimulation. We provide a tractable analytical approach, not only in the terms of expected expression profiles and their moments, which corresponds to the measurements on the cell population, but also in the terms of single cell behavior. Comparison between these two modes of description reveals that single cells behave qualitatively different from the cell population. This analysis provides insights useful for understanding of microarray experiments.

Animals↗

PubMed2004

Interleukin (IL)-1 beta in tracheal aspirates from premature infants induces airway epithelial cell IL-8 expression via an NF-kappa B dependent pathway.

Tracheal aspirate IL-8 concentration and airway epithelial cell IL-8 expression are each increased in premature infants undergoing mechanical ventilation. We sought to determine the cytokines responsible for IL-8 expression in this context. Tracheal aspirates were collected from 18 mechanically ventilated premature infants. IL-8 protein abundance was high in tracheal aspirates from ventilated premature infants (mean, 5806 +/- 4923 pg/mL). IL-1 alpha (mean, 20 +/- 6 pg/mL), IL-1 beta (mean 67 +/- 46 pg/mL), and tumor necrosis factor (TNF)-alpha (mean, 8 +/- 2 pg/mL) were also found. Incubation of tracheal aspirates with 16HBE14o- human bronchial epithelial cells increased IL-8 protein in both cell lysates and supernatants, as well as transcription from the IL-8 promoter. Aspirates also induced nuclear factor (NF)-kappa B activation. Mutation of the IL-8 promoter NF-kappa B site abolished aspirate-induced IL-8 transcription. Endotoxin concentrations in the tracheal aspirates were negligible and incapable of inducing IL-8 promoter activity. Finally, incubation of tracheal aspirates with a neutralizing antibody against IL-1 beta reduced epithelial cell IL-8 production, whereas neutralizing antibodies against IL-1 alpha and TNF-alpha had no effect. We conclude that airway fluid from mechanically ventilated premature infants contains soluble factors capable of inducing airway epithelial cell IL-8 expression via a NF-kappa B-dependent pathway, and that IL-1 beta plays a specific role in this process.

Cells, Cultured↗