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1.  FBXW7α attenuates inflammatory signalling by downregulating C/EBPδ and its target gene Tlr4 
Nature communications  2013;4:1662.
Toll-like receptor 4 (TLR4) plays a pivotal role in innate immune responses, and the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ, Cebpd) is a TLR4-induced gene. Here, we identify a positive feedback loop in which C/EBPδ activates Tlr4 gene expression in macrophages and tumour cells. In addition, we discovered a negative feedback loop whereby the tumour suppressor FBXW7α (FBW7, Cdc4), whose gene expression is inhibited by C/EBPδ, targets C/EBPδ for degradation when C/EBPδ is phosphorylated by GSK-3β. Consequently, FBXW7α suppresses Tlr4 expression and responses to the ligand lipopolysaccharide (LPS). FBXW7α depletion alone is sufficient to augment pro-inflammatory signalling in vivo. Moreover, as inflammatory pathways are known to modulate tumour biology, Cebpd null mammary tumours, which have reduced metastatic potential, show altered expression of inflammation-associated genes. Together, these findings reveal a role for C/EBPδ upstream of TLR4 signalling and uncover a function for FBXW7α as an attenuator of inflammatory signalling.
PMCID: PMC3625980  PMID: 23575666
2.  CCAAT/enhancer binding protein-δ expression by dendritic cells regulates CNS autoimmune inflammatory disease 
The Journal of Neuroscience  2011;31(48):17612-17621.
CCAAT enhancer binding protein-delta (C/EBPδ) is a transcription factor that regulates inflammatory processes mediating bystander neuronal injury and CNS autoimmune inflammatory disease. The mechanism of C/EBPδ’s involvement in these processes remains to be determined. Here we examined the cellular source(s) and mechanisms by which C/EBPδ may be involved in an animal model of multiple sclerosis. Mice deficient in C/EBPδ expression exhibited less severe clinical disease than wild type littermates in response to induction of experimental autoimmune encephalomyelitis (EAE) by vaccination with a myelin oligodendrocyte glycoprotein (MOG) fragment. This reduction in EAE severity was associated with a significant alteration in the complement of major CNS T-helper (Th) cell subtypes throughout disease, manifest as reduced ratios of Th17 cells to regulatory T-cells (Tregs). Studies in bone marrow chimeric mice indicated that C/EBPδ expression by peripherally derived immune cells mediates C/EBPδ involvement in EAE. Follow up in vitro and in vivo examination of dendritic cell (DC) mediated Th-cell development suggests C/EBPδ suppresses DC expression of interleukin-10 (IL-10) and favours Th17 over Treg development. In vitro and in vivo blockade of IL-10 signalling reduced the effect of reduced DC C/EBPδ expression on Th17:Treg ratios. These findings identify C/EBPδ as an important DC transcription factor in CNS autoimmune inflammatory disease by virtue of its capacity to alter the Th17:Treg balance in an IL-10 dependent fashion.
PMCID: PMC3334319  PMID: 22131422
3.  Identification and characterization of CCAAT/Enhancer Binding proteindelta (C/EBPdelta) target genes in G0 growth arrested mammary epithelial cells 
CCAAT/Enhancer Binding Proteinδ (C/EBPδ) is a member of the highly conserved C/EBP family of leucine zipper (bZIP) proteins. C/EBPδ is highly expressed in G0 growth arrested mammary epithelial cells (MECs) and "loss of function" alterations in C/EBPδ have been associated with impaired contact inhibition, increased genomic instability and increased cell migration. Reduced C/EBPδ expression has also been reported in breast cancer and acute myeloid leukemia (AML). C/EBPδ functions as a transcriptional activator, however, only a limited number of C/EBPδ target genes have been reported. As a result, the role of C/EBPδ in growth control and the potential mechanisms by which "loss of function" alterations in C/EBPδ contribute to tumorigenesis are poorly understood. The goals of the present study were to identify C/EBPδ target genes using Chromatin Immunoprecipitation coupled with a CpG Island (HCG12K) Array gene chip ("ChIP-chip") assay and to assess the expression and potential functional roles of C/EBPδ target genes in growth control.
ChIP-chip assays identified ~100 C/EBPδ target gene loci which were classified by gene ontology (GO) into cell adhesion, cell cycle regulation, apoptosis, signal transduction, intermediary metabolism, gene transcription, DNA repair and solute transport categories. Conventional ChIP assays validated the ChIP-chip results and demonstrated that 14/14 C/EBPδ target loci were bound by C/EBPδ in G0 growth arrested MCF-12A MECs. Gene-specific RT-PCR analysis also demonstrated C/EBPδ-inducible expression of 14/14 C/EBPδ target genes in G0 growth arrested MCF-12A MECs. Finally, expression of endogenous C/EBPδ and selected C/EBPδ target genes was also demonstrated in contact-inhibited G0 growth arrested nontransformed human MCF-10A MECs and in mouse HC11 MECs. The results demonstrate consistent activation and downstream function of C/EBPδ in growth arrested human and murine MECs.
C/EBPδ target genes were identified by a global gene array approach and classified into functional categories that are consistent with biological contexts in which C/EBPδ is induced, such as contact-mediated G0 growth arrest, apoptosis, metabolism and inflammation. The identification and validation of C/EBPδ target genes provides new insights into the mechanistic role of C/EBPδ in mammary epithelial cell biology and sheds new light on the potential impact of "loss of function" alterations in C/EBPδ in tumorigenesis.
PMCID: PMC2576343  PMID: 18828910
4.  The CCAAT/enhancer binding protein (C/EBP) δ is differently regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-β peptide 
The transcription factors CCAAT/enhancer binding proteins (C/EBP) α, β and δ have been shown to be expressed in brain and to be involved in regulation of inflammatory genes in concert with nuclear factor κB (NF-κB). In general, C/EBPα is down-regulated, whereas both C/EBPβ and δ are up-regulated in response to inflammatory stimuli. In Alzheimer's disease (AD) one of the hallmarks is chronic neuroinflammation mediated by astrocytes and microglial cells, most likely induced by the formation of amyloid-β (Aβ) deposits. The inflammatory response in AD has been ascribed both beneficial and detrimental roles. It is therefore important to delineate the inflammatory mediators and signaling pathways affected by Aβ deposits with the aim of defining new therapeutic targets.
Here we have investigated the effects of Aβ on expression of C/EBP family members with a focus on C/EBPδ in rat primary astro-microglial cultures and in a transgenic mouse model with high levels of fibrillar Aβ deposits (tg-ArcSwe) by western blot analysis. Effects on DNA binding activity were analyzed by electrophoretic mobility shift assay. Cross-talk between C/EBPδ and NF-κB was investigated by analyzing binding to a κB site using a biotin streptavidin-agarose pull-down assay.
We show that exposure to fibril-enriched, but not oligomer-enriched, preparations of Aβ inhibit up-regulation of C/EBPδ expression in interleukin-1β-activated glial cultures. Furthermore, we observed that, in aged transgenic mice, C/EBPα was significantly down-regulated and C/EBPβ was significantly up-regulated. C/EBPδ, on the other hand, was selectively down-regulated in the forebrain, a part of the brain showing high levels of fibrillar Aβ deposits. In contrast, no difference in expression levels of C/EBPδ between wild type and transgenic mice was detected in the relatively spared hindbrain. Finally, we show that interleukin-1β-induced C/EBPδ DNA binding activity to both C/EBP and κB sites is abolished after exposure to Aβ.
These data suggest that both expression and function of C/EBPδ are dysregulated in Alzheimer's disease. C/EBPδ seems to be differently regulated in response to different conformations of Aβ. We propose that Aβ induces an imbalance between NF-κB and C/EBP transcription factors that may result in abnormal responses to inflammatory stimuli.
PMCID: PMC3096570  PMID: 21492414
5.  The Many Faces of C/EBPδ and their Relevance for Inflammation and Cancer 
The CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ) is a transcription factor that modulates many biological processes including cell differentiation, motility, growth arrest, proliferation, and cell death. The diversity of C/EBPδ's functions depends in part on the cell type and cellular context and can have opposing outcomes. For example, C/EBPδ promotes inflammatory signaling, but it can also inhibit pro-inflammatory pathways, and in a mouse model of mammary tumorigenesis, C/EBPδ reduces tumor incidence but promotes tumor metastasis. This review highlights the multifaceted nature of C/EBPδ's functions, with an emphasis on pathways that are relevant for cancer and inflammation, and illustrates how C/EBPδ emerged from the shadow of its family members as a fascinating “jack of all trades.” Our current knowledge on C/EBPδ indicates that, rather than being essential for a specific cellular process, C/EBPδ helps to interpret a variety of cues in a cell-type and context-dependent manner, to adjust cellular functions to specific situations. Therefore, insights into the roles and mechanisms of C/EBPδ signaling can lead to a better understanding of how the integration of different signaling pathways dictates normal and pathological cell functions and physiology.
PMCID: PMC3805898  PMID: 24155666
C/EBP; transcription factor; cell signaling; inflammation; cancer.
6.  CCAAT/enhancer-binding protein δ (C/EBPδ) aggravates inflammation and bacterial dissemination during pneumococcal meningitis 
The prognosis of bacterial meningitis largely depends on the severity of the inflammatory response. The transcription factor CAAT/enhancer-binding protein δ (C/EBPδ) plays a key role in the regulation of the inflammatory response during bacterial infections. Consequently, we assessed the role of C/EBPδ during experimental meningitis.
Wild-type and C/EBPδ-deficient mice (C/EBPδ−/−) were intracisternally infected with Streptococcus pneumoniae and sacrificed after 6 or 30 h, or followed in a survival study.
In comparison to wild-type mice, C/EBPδ−/− mice showed decreased bacterial loads at the primary site of infection and decreased bacterial dissemination to lung and spleen 30 h after inoculation. Expression levels of the inflammatory mediators IL-10 and KC were lower in C/EBPδ−/− brain homogenates, whereas IL-6, TNF-α, IL-1β, and MIP-2 levels were not significantly different between the two genotypes. Moreover, C/EBPδ−/− mice demonstrated an attenuated systemic response as reflected by lower IL-10, IL-6, KC, and MIP-2 plasma levels. No differences in clinical symptoms or in survival were observed between wild-type and C/EBPδ−/− mice.
C/EBPδ in the brain drives the inflammatory response and contributes to bacterial dissemination during pneumococcal meningitis. C/EBPδ does, however, not affect clinical parameters of the disease and does not confer a survival benefit.
PMCID: PMC4430878  PMID: 25958220
Experimental meningitis; C/EBPδ; Infection; Streptococcus pneumoniae
7.  Molecular Mechanism Underlying Persistent Induction of LCN2 by Lipopolysaccharide in Kidney Fibroblasts 
PLoS ONE  2012;7(4):e34633.
The neutrophil gelatinase-associated lipocalin 2 (LCN2) is a critical inflammatory mediator persistently induced during endotoxemia, contributing to tubular damage and kidney failure. The intracellular process responsible for persistent induction of LCN2 by bacterial endotoxin Lipopolysaccharide (LPS) is not well understood. Using primary kidney fibroblasts, we observed that LPS-induced LCN2 expression requires a coupled circuit involving an early transient phase of AP-1 path and a late persistent phase of C/EBPδ path, both of which are dependent upon the interleukin 1 receptor associated kinase 1 (IRAK-1). Using immunoprecipitation analysis we observed transient binding of AP-1 to the promoters of both TNFα and C/ebpδ. On the other hand, we only observed persistent binding of C/EBPδ to its own promoter but not on TNFα. Blockage of new protein synthesis using cyclohexamide significantly reduced the expression of C/EBPδ as well as LCN2. By chromatin immunoprecipitation analyses, we demonstrated that LPS recruited C/EBPδ to the Lcn2 promoter in WT, but not IRAK-1 deficient fibroblasts. A differential equation-based computational model captured the dynamic circuit leading to the persistent induction of LCN2. In vivo, we observed elevated levels of LCN2 in kidneys harvested from LPS-injected WT mice as compared to IRAK-1 deficient mice. Taken together, this study has identified an integrated intracellular network involved in the persistent induction of LCN2 by LPS.
PMCID: PMC3326042  PMID: 22514649
8.  Modelling Negative Feedback Networks for Activating Transcription Factor 3 Predicts a Dominant Role for miRNAs in Immediate Early Gene Regulation 
PLoS Computational Biology  2014;10(5):e1003597.
Activating transcription factor 3 (Atf3) is rapidly and transiently upregulated in numerous systems, and is associated with various disease states. Atf3 is required for negative feedback regulation of other genes, but is itself subject to negative feedback regulation possibly by autorepression. In cardiomyocytes, Atf3 and Egr1 mRNAs are upregulated via ERK1/2 signalling and Atf3 suppresses Egr1 expression. We previously developed a mathematical model for the Atf3-Egr1 system. Here, we adjusted and extended the model to explore mechanisms of Atf3 feedback regulation. Introduction of an autorepressive loop for Atf3 tuned down its expression and inhibition of Egr1 was lost, demonstrating that negative feedback regulation of Atf3 by Atf3 itself is implausible in this context. Experimentally, signals downstream from ERK1/2 suppress Atf3 expression. Mathematical modelling indicated that this cannot occur by phosphorylation of pre-existing inhibitory transcriptional regulators because the time delay is too short. De novo synthesis of an inhibitory transcription factor (ITF) with a high affinity for the Atf3 promoter could suppress Atf3 expression, but (as with the Atf3 autorepression loop) inhibition of Egr1 was lost. Developing the model to include newly-synthesised miRNAs very efficiently terminated Atf3 protein expression and, with a 4-fold increase in the rate of degradation of mRNA from the mRNA/miRNA complex, profiles for Atf3 mRNA, Atf3 protein and Egr1 mRNA approximated to the experimental data. Combining the ITF model with that of the miRNA did not improve the profiles suggesting that miRNAs are likely to play a dominant role in switching off Atf3 expression post-induction.
Author Summary
Activating transcription factor 3 (Atf3) is an important regulatory transcription factor which is associated with inflammation, restraint of the immune response and cancer. In this work, we develop a series of mathematical models to understand how Atf3 may be regulated. Informed with data from the literature and our own experiments, we show that self-regulation of Atf3 does not allow for variation between experimentally observed Atf3 mRNA and Atf3 protein expression profiles. A fast-acting signal via phosphorylated RSK is also shown to be implausible for similar reasons. Extending our mathematical model further, we postulate for the first time, that the observed dynamical variation in Atf3 mRNA and protein can be described by microRNAs downstream of RSKs. The further inclusion of an inhibitory transcription factor for Atf3 expression has little effect on these findings.
PMCID: PMC4014390  PMID: 24811474
9.  RFX Transcription Factor DAF-19 Regulates 5-HT and Innate Immune Responses to Pathogenic Bacteria in Caenorhabditis elegans 
PLoS Genetics  2013;9(3):e1003324.
In Caenorhabditis elegans the Toll-interleukin receptor domain adaptor protein TIR-1 via a conserved mitogen-activated protein kinase (MAPK) signaling cascade induces innate immunity and upregulates serotonin (5-HT) biosynthesis gene tph-1 in a pair of ADF chemosensory neurons in response to infection. Here, we identify transcription factors downstream of the TIR-1 signaling pathway. We show that common transcription factors control the innate immunity and 5-HT biosynthesis. We demonstrate that a cysteine to tyrosine substitution in an ARM motif of the HEAT/Arm repeat region of the TIR-1 protein confers TIR-1 hyperactivation, leading to constitutive tph-1 upregulation in the ADF neurons, increased expression of intestinal antimicrobial genes, and enhanced resistance to killing by the human opportunistic pathogen Pseudomonas aeruginosa PA14. A forward genetic screen for suppressors of the hyperactive TIR-1 led to the identification of DAF-19, an ortholog of regulatory factor X (RFX) transcription factors that are required for human adaptive immunity. We show that DAF-19 concerts with ATF-7, a member of the activating transcription factor (ATF)/cAMP response element-binding B (CREB) family of transcription factors, to regulate tph-1 and antimicrobial genes, reminiscent of RFX-CREB interaction in human immune cells. daf-19 mutants display heightened susceptibility to killing by PA14. Remarkably, whereas the TIR-1-MAPK-DAF-19/ATF-7 pathway in the intestinal immunity is regulated by DKF-2/protein kinase D, we found that the regulation of tph-1 expression is independent of DKF-2 but requires UNC-43/Ca2+/calmodulin-dependent protein kinase (CaMK) II. Our results suggest that pathogenic cues trigger a common core-signaling pathway via tissue-specific mechanisms and demonstrate a novel role for RFX factors in neuronal and innate immune responses to infection.
Author Summary
Toll-interleukin receptor (TIR)–domain adaptor proteins are keys to activate signaling cascades inducing transcriptional responses to internal and external pathogenic signals in evolutionary disparate organisms. Despite lacking a homolog of the mammalian innate immunity transcriptional regulator nuclear factor-kappaB (NF-κB), the nematode Caenorhabditis elegans responds to infections by activating TIR-1 signaling targets in the innate immune system and in neurons. Through a genetic screen for factors required for TIR-1 signaling to upregulate the serotonin biosynthesis gene tph-1, we identified DAF-19, an ortholog of regulatory factor X (RFX) transcription factors that were initially discovered in human immune cells. We show that DAF-19 concerts with ATF-7, a member of the activating transcription factor (ATF)/cAMP response element-binding B (CREB) family of transcription factors, to upregulate tph-1 in the ADF chemosensory neurons and antimicrobial genes in the intestine in response to bacterial infection, reminiscent of RFX-CREB interaction in human immune cells. daf-19 mutants display heightened susceptibility to killing by the human pathogen Pseudomonas aeruginosa PA14. Our studies suggest that RFX transcriptional regulation, which is essential for human adaptive immunity, has an ancient role in controlling serotonin biosynthesis and innate immunity.
PMCID: PMC3591283  PMID: 23505381
10.  The phosphoproteome of toll-like receptor-activated macrophages 
First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control.
Toll-like receptors (TLR) are a family of pattern recognition receptors that enable innate immune cells to sense infectious danger. Recognition of microbial structures, like lipopolysaccharide (LPS) of Gram-negative bacteria by TLR4, causes within hours substantial re-programming of macrophage gene expression, including up-regulation of chemokines driving inflammation, anti-microbial effector molecules and cytokines directing adaptive immune responses. TLR signalling is initiated by the adapter protein Myd88 and leads to the activation of kinase cascades that result in activation of the MAPK and NFkB pathways. Phosphorylation has an essential role in these early steps of TLR signalling, and in addition regulates critical transcription factors (TFs). Although TLR signalling has been extensively studied, a comprehensive analysis of phosphorylation events in TLR-activated macrophages is lacking. It is therefore unknown whether the canonical MAPK and NFkB pathways comprise the main phosphorylation events and which other molecular functions and processes are regulated by phosphorylation after stimulation with LPS.
Recent progress in mass spectrometry-based proteomics has opened the possibility to quantitatively investigate global changes in protein abundance and post-translational modifications. Stable isotope labelling with amino acids in cell culture (SILAC) allows highly accurate quantification, and has proved especially useful for direct comparison of phosphopeptide abundance in time-course or treatment analyses.
Here, we adapted SILAC to primary mouse macrophages, and performed a global, quantitative and kinetic analysis of the macrophage phosphoproteome after LPS stimulation. Bioinformatic analyses were used to identify kinases, pathways and biological processes enriched in the LPS-regulated phosphoproteome. To connect TF phosphorylation with transcription, we generated a parallel dataset of nascent RNA and used in silico promoter analysis to identify transcriptional regulators with binding site enrichment among the LPS-regulated gene set.
After establishing SILAC conditions for efficient labelling of primary bone marrow-derived macrophages in two independent experiments 1850 phosphoproteins with a total of 6956 phosphorylation sites were reproducibly identified. Phosphoproteins were detected from all cellular compartments, with a clear enrichment for nuclear and cytoskeleton-associated proteins. LPS caused major regulation of a large fraction of phosphopeptides, with 24% of all sites up-regulated and 9% down-regulated after stimulation (Figure 3A and B). These changes were highly dynamic, as the majority of the regulated phosphopeptides were up-regulated or down-regulated transiently or in a delayed manner (Figure 3C). Overall, the extent of changes in the phosphoproteome was comparable to the transcriptional re-programming, underscoring the importance of phosphorylation cascades in TLR signalling. Our parallel transcriptome data also showed that widespread phosphorylation precedes massive transcriptional changes.
To obtain footprints of kinase activation in response to TLR ligation, we searched phosphopeptide sequences for known linear sequence motifs of 33 kinases and identified kinase motifs enriched among LPS-regulated phosphorylation sites (compared to non-regulated phosphorylation sites) (Table I). Motif ERK/MAPK was highly enriched, in accordance with the essential role of the MAPK module in TLR signalling. Other kinases with motif enrichment have also recently been linked to TLR signalling (e.g. PKD; AKT and its targets GSK3 and mTOR). However, the DNA damage-actviated kinases ATM/ATR and the cell cycle-associated kinases AURORA and CHK1/2 have not been associated with the macrophage response to TLR activation yet. These finding shed new light on older data on the effect of TLR on macrophage proliferation in response to macrophage colony stimulating factor. Of interest, in follow-up experiments using pharmacological inhibitors of the kinases with motif enrichment, we observed that inhibition of ATM kinase activity caused increased LPS-induced expression of several cytokines and chemokines, suggesting that this pathway regulates inflammatory responses.
In further bioinformatic analyses, the Gene Ontology and signalling pathway annotations of phosphoproteins were used to identify signalling pathways and cellular processes targeted by TLR4-controlled phosphorylation (Table II). Among the expected hits, based on the known TLR pathways, were TLR signalling, MAPK and AKT as well as mTOR signalling. Of interest, the annotation terms ‘Rho GTPase cycle' and ‘cytoskeleton' were significantly enriched among LPS-regulated phosphoproteins, indicating a more prominent role for cytoskeletal proteins in the transduction of TLR signals or in the biological response to it.
We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.
In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.
Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high-resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4-induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up-regulation and 9% down-regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS-regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.
PMCID: PMC2913394  PMID: 20531401
macrophage; nascent RNA; phosphoproteome; SILAC; toll-like receptors
11.  The Transcription Factor C/EBP delta Has Anti-Apoptotic and Anti-Inflammatory Roles in Pancreatic Beta Cells 
PLoS ONE  2012;7(2):e31062.
In the course of Type 1 diabetes pro-inflammatory cytokines (e.g., IL-1β, IFN-γ and TNF-α) produced by islet-infiltrating immune cells modify expression of key gene networks in β-cells, leading to local inflammation and β-cell apoptosis. Most known cytokine-induced transcription factors have pro-apoptotic effects, and little is known regarding “protective” transcription factors. To this end, we presently evaluated the role of the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) on β-cell apoptosis and production of inflammatory mediators in the rat insulinoma INS-1E cells, in purified primary rat β-cells and in human islets. C/EBPδ is expressed and up-regulated in response to the cytokines IL-1β and IFN-γ in rat β-cells and human islets. Small interfering RNA-mediated C/EBPδ silencing exacerbated IL-1β+IFN-γ-induced caspase 9 and 3 cleavage and apoptosis in these cells. C/EBPδ deficiency increased the up-regulation of the transcription factor CHOP in response to cytokines, enhancing expression of the pro-apoptotic Bcl-2 family member BIM. Interfering with C/EBPδ and CHOP or C/EBPδ and BIM in double knockdown approaches abrogated the exacerbating effects of C/EBPδ deficiency on cytokine-induced β-cell apoptosis, while C/EBPδ overexpression inhibited BIM expression and partially protected β-cells against IL-1β+IFN-γ-induced apoptosis. Furthermore, C/EBPδ silencing boosted cytokine-induced production of the chemokines CXCL1, 9, 10 and CCL20 in β-cells by hampering IRF-1 up-regulation and increasing STAT1 activation in response to cytokines. These observations identify a novel function of C/EBPδ as a modulatory transcription factor that inhibits the pro-apoptotic and pro-inflammatory gene networks activated by cytokines in pancreatic β-cells.
PMCID: PMC3275575  PMID: 22347430
12.  C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells 
In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by up-regulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes. We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to pro-inflammatory conditions. Human aortic SMCs were treated with interferon-γ (IFN-γ) for up to 24 hrs. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFN-γ dose-dependently induced Nox activity and expression, nuclear translocation and up-regulation of C/EBPα, C/EBPβ and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFN-γ-induced up-regulation of Nox activity, gene and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4 and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFN-γ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress.
PMCID: PMC4124029  PMID: 24797079
NADPH oxidase; C/EBP; atherosclerosis; oxidative stress; inflammation
13.  Identification of a Src Tyrosine Kinase/SIAH2 E3 Ubiquitin Ligase Pathway That Regulates C/EBPδ Expression and Contributes to Transformation of Breast Tumor Cells 
Molecular and Cellular Biology  2012;32(2):320-332.
The transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ, CEBPD) is a tumor suppressor that is downregulated during breast cancer progression but may also promote metastasis. Here, we have investigated the mechanism(s) regulating C/EBPδ expression and its role in human breast cancer cells. We describe a novel pathway by which the tyrosine kinase Src downregulates C/EBPδ through the SIAH2 E3 ubiquitin ligase. Src phosphorylates SIAH2 in vitro and leads to tyrosine phosphorylation and activation of SIAH2 in breast tumor cell lines. SIAH2 interacts with C/EBPδ, but not C/EBPβ, and promotes its polyubiquitination and proteasomal degradation. Src/SIAH2-mediated inhibition of C/EBPδ expression supports elevated cyclin D1 levels, phosphorylation of retinoblastoma protein (Rb), motility, invasive properties, and survival of transformed cells. Pharmacological inhibition of Src family kinases by SKI-606 (bosutinib) induces C/EBPδ expression in an SIAH2-dependent manner, which is necessary for “therapeutic” responses to SKI-606 in vitro. Ectopic expression of degradation-resistant mutants of C/EBPδ, which do not interact with SIAH2 and/or cannot be polyubiquitinated, prevents full transformation of MCF-10A cells by activated Src (Src truncated at amino acid 531 [Src-531]) in vitro. These data reveal that C/EBPδ expression can be regulated at the protein level by oncogenic Src kinase signals through SIAH2, thus contributing to breast epithelial cell transformation.
PMCID: PMC3255785  PMID: 22037769
14.  Myc interacts with Max and Miz1 to repress C/EBPδ promoter activity and gene expression 
Molecular Cancer  2010;9:92.
"Loss of function" alterations in CCAAT/Enhancer Binding Proteinδ (C/EBPδ) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPδ gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPδ exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing.
We previously reported that C/EBPδ expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPα, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPδ expression.
Myc represses C/EBPδ promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPδ promoter in proliferating cells, when C/EBPδ expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPδ promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPδ promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPδ promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPδ promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPδ expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPδ promoter activity.
Myc represses C/EBPδ expression by associating with the C/EBPδ proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPδ promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPδ, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.
PMCID: PMC2879254  PMID: 20426839
15.  ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes 
Toll-like receptors (TLRs) are sentinels of the host defense system, which recognize a large number of microbial pathogens. The host defense system may be inefficient or inflammatory diseases may develop if microbial recognition by TLRs and subsequent TLR-triggered cytokine production are deregulated. Activating transcription factor 4 (ATF4), a member of the ATF/CREB transcription factor family, is an important factor that participates in several pathophysiological processes. In this report, we found that ATF4 is also involved in the TLR-mediated innate immune response, which participates in TLR4 signal transduction and mediates the secretion of a variety of cytokines. We observed that ATF4 is activated and translocates to the nucleus following lipopolysaccharide (LPS) stimulation via the TLR4-MyD88-dependent pathway. Additionally, a cytokine array assay showed that some key inflammatory cytokines, such as IL-6, IL-8 and RANTES, are positively regulated by ATF4. We also demonstrate that c-Jun directly binds to ATF4, thereby promoting the secretion of inflammatory cytokines. Taken together, these results indicate that ATF4 acts as a positive regulator in TLR4-triggered cytokine production.
PMCID: PMC4003182  PMID: 23241898
ATF4; cytokine; MyD88; TLR4 signaling pathway; TRIF
16.  A Temporal Gate for Viral Enhancers to Co-opt Toll-Like-Receptor Transcriptional Activation Pathways upon Acute Infection 
PLoS Pathogens  2015;11(4):e1004737.
Viral engagement with macrophages activates Toll-Like-Receptors (TLRs) and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV) have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in macrophages. In a series of pharmacologic, siRNA and genetic loss-of-function experiments we determined that signalling mediated by the TLR-adaptor protein MyD88 plays a vital role for governing the inflammatory activation of the CMV enhancer in macrophages. Downstream TLR-regulated transcription factor binding motif disruption for NFκB, AP1 and CREB/ATF in the CMV enhancer demonstrated the requirement of these inflammatory signal-regulated elements in driving viral gene expression and growth in cells as well as in primary infection of neonatal mice. Thus, this study shows that the prototypical CMV enhancer, in a restricted time-gated manner, co-opts through DNA regulatory mimicry elements, innate-immune transcription factors to drive viral expression and replication in the face of on-going pro-inflammatory antiviral responses in vitro and in vivo and; suggests an unexpected role for inflammation in promoting acute infection and has important future implications for regulating latency.
Author Summary
Here we discover how inflammatory signalling may unintentionally promote infection, as a result of viruses evolving DNA sequences, known as enhancers, which act as a bait to prey on the infected cell transcription factors induced by inflammation. The major inflammatory transcription factors activated are part of the TLR-signalling pathway. We find the prototypical viral enhancer of cytomegalovirus can be paradoxically boosted by activation of inflammatory “anti-viral” TLR-signalling independent of viral structural proteins. This leads to an increase in viral gene expression and replication in cell-culture and upon infection of mice. We identify an axis of inflammatory transcription factors, acting downstream of TLR-signalling but upstream of interferon inhibition. Mechanistically, the central TLR-adapter protein MyD88 is shown to play a critical role in promoting viral enhancer activity in the first 6h of infection. The co-option of TLR-signalling exceeds the usage of NFκB, and we identify IRF3 and 5 as newly found viral-enhancer interacting inflammatory transcription factors. Taken together this study reveals how virus enhancers, employ a path of least resistance by directly harnessing within a short temporal window, the activation of anti-viral signalling in macrophages to drive viral gene expression and replication to an extent that has not been recognised before.
PMCID: PMC4391941  PMID: 25856589
17.  Phosphorylation of the Conserved Transcription Factor ATF-7 by PMK-1 p38 MAPK Regulates Innate Immunity in Caenorhabditis elegans 
PLoS Genetics  2010;6(4):e1000892.
Innate immunity in Caenorhabditis elegans requires a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway that regulates the basal and pathogen-induced expression of immune effectors. The mechanisms by which PMK-1 p38 MAPK regulates the transcriptional activation of the C. elegans immune response have not been identified. Furthermore, in mammalian systems the genetic analysis of physiological targets of p38 MAPK in immunity has been limited. Here, we show that C. elegans ATF-7, a member of the conserved cyclic AMP–responsive element binding (CREB)/activating transcription factor (ATF) family of basic-region leucine zipper (bZIP) transcription factors and an ortholog of mammalian ATF2/ATF7, has a pivotal role in the regulation of PMK-1–mediated innate immunity. Genetic analysis of loss-of-function alleles and a gain-of-function allele of atf-7, combined with expression analysis of PMK-1–regulated genes and biochemical characterization of the interaction between ATF-7 and PMK-1, suggest that ATF-7 functions as a repressor of PMK-1–regulated genes that undergoes a switch to an activator upon phosphorylation by PMK-1. Whereas loss-of-function mutations in atf-7 can restore basal expression of PMK-1–regulated genes observed in the pmk-1 null mutant, the induction of PMK-1–regulated genes by pathogenic Pseudomonas aeruginosa PA14 is abrogated. The switching modes of ATF-7 activity, from repressor to activator in response to activated PMK-1 p38 MAPK, are reminiscent of the mechanism of regulation mediated by the corresponding ancestral Sko1p and Hog1p proteins in the yeast response to osmotic stress. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans, and suggest that ATF2/ATF7 may function in a similar manner in the regulation of mammalian innate immunity.
Author Summary
We have investigated mechanisms of how the soil nematode Caenorhabditis elegans interacts with pathogenic bacteria. Previously, we have established that a conserved PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates immunity in C. elegans, establishing the conservation of key innate immune signaling pathways of mammals in the immune response of C. elegans. Whereas multiple proteins have been identified as potential targets of p38 MAPK in immunity, the identification of physiological substrates of p38 MAPK in mammalian organisms has been challenging. Here, using a forward genetic approach to identify downstream regulators of the C. elegans innate immune response, we have characterized the transcription factor ATF-7, a conserved member of the basic-region leucine zipper (bZIP) transcription factor family orthologous to mammalian ATF2. We find that ATF-7 functions as a transcriptional regulator of PMK-1 MAPK–mediated innate immunity, functioning as a repressor of immune gene expression that undergoes a switch to an activator upon activation by PMK-1. Our data point to the regulation of the ATF2/ATF7/CREB5 family of transcriptional regulators by p38 MAPK as an ancient conserved mechanism for the control of innate immunity in metazoans and suggests a mechanism by which the protean effects of p38 MAPK on the mammalian innate immune response may be mediated.
PMCID: PMC2848548  PMID: 20369020
18.  Requirement for nuclear factor kappa B signalling in the interleukin-1-induced expression of the CCAAT/enhancer binding protein-δ gene in hepatocytes 
Elevated circulating levels of acute phase proteins (APP) are associated with inflammation and inflammatory disorders such as cardiovascular disease. APP are mainly synthesised by hepatocytes and their transcription is induced by pro-inflammatory cytokines such as interleukin-1 (IL-1). The molecular mechanisms underlying the IL-1-induced expression of key transcription factors implicated in the regulation of APP are poorly understood. We have investigated this aspect using the CCAAT/enhancer binding protein-δ (C/EBPδ) as a model gene. IL-1 induced the expression of C/EBPδ mRNA and protein in the human hepatoma Hep3B cell line, a widely employed model system for studies on cytokine signalling in relation to the expression of APP. The IL-1-mediated induction of C/EBPδ expression was attenuated in the presence of pharmacological inhibitors against c-Jun N-terminal kinase (JNK) (curcumin and SP600125), casein kinase 2 (CK2) (apigenin) and nuclear factor-κB (NF-κB) (NF-κB activation inhibitor). RNA interference assays showed significant attenuation of the IL-1-induced expression of C/EBPδ following knockdown of the p50 and p65 subunits of NF-κB. IL-1 induced NF-κB DNA binding and activation by this transcription factor and this was attenuated by curcumin and apigenin. Taken together, these results suggest a potentially crucial role for NF-κB in the IL-1-induced expression of C/EBPδ, and thereby downstream APP genes regulated by this transcription factor.
PMCID: PMC2827769  PMID: 19800021
Acute phase proteins; CCAAT/enhancer binding proteins; Hepatocytes; Interleukin-1; Cell signalling
19.  The Innate Immune Database (IIDB) 
BMC Immunology  2008;9:7.
As part of a National Institute of Allergy and Infectious Diseases funded collaborative project, we have performed over 150 microarray experiments measuring the response of C57/BL6 mouse bone marrow macrophages to toll-like receptor stimuli. These microarray expression profiles are available freely from our project web site . Here, we report the development of a database of computationally predicted transcription factor binding sites and related genomic features for a set of over 2000 murine immune genes of interest. Our database, which includes microarray co-expression clusters and a host of web-based query, analysis and visualization facilities, is available freely via the internet. It provides a broad resource to the research community, and a stepping stone towards the delineation of the network of transcriptional regulatory interactions underlying the integrated response of macrophages to pathogens.
We constructed a database indexed on genes and annotations of the immediate surrounding genomic regions. To facilitate both gene-specific and systems biology oriented research, our database provides the means to analyze individual genes or an entire genomic locus. Although our focus to-date has been on mammalian toll-like receptor signaling pathways, our database structure is not limited to this subject, and is intended to be broadly applicable to immunology. By focusing on selected immune-active genes, we were able to perform computationally intensive expression and sequence analyses that would currently be prohibitive if applied to the entire genome. Using six complementary computational algorithms and methodologies, we identified transcription factor binding sites based on the Position Weight Matrices available in TRANSFAC. For one example transcription factor (ATF3) for which experimental data is available, over 50% of our predicted binding sites coincide with genome-wide chromatin immnuopreciptation (ChIP-chip) results. Our database can be interrogated via a web interface. Genomic annotations and binding site predictions can be automatically viewed with a customized version of the Argo genome browser.
We present the Innate Immune Database (IIDB) as a community resource for immunologists interested in gene regulatory systems underlying innate responses to pathogens. The database website can be freely accessed at .
PMCID: PMC2268913  PMID: 18321385
20.  An anti-inflammatory role for C/EBPδ in human brain pericytes 
Scientific Reports  2015;5:12132.
Neuroinflammation contributes to the pathogenesis of several neurological disorders and pericytes are implicated in brain inflammatory processes. Cellular inflammatory responses are orchestrated by transcription factors but information on transcriptional control in pericytes is lacking. Because the transcription factor CCAAT/enhancer binding protein delta (C/EBPδ) is induced in a number of inflammatory brain disorders, we sought to investigate its role in regulating pericyte immune responses. Our results reveal that C/EBPδ is induced in a concentration- and time-dependent fashion in human brain pericytes by interleukin-1β (IL-1β). To investigate the function of the induced C/EBPδ in pericytes we used siRNA to knockdown IL-1β-induced C/EBPδ expression. C/EBPδ knockdown enhanced IL-1β-induced production of intracellular adhesion molecule-1 (ICAM-1), interleukin-8, monocyte chemoattractant protein-1 (MCP-1) and IL-1β, whilst attenuating cyclooxygenase-2 and superoxide dismutase-2 gene expression. Altered ICAM-1 and MCP-1 protein expression were confirmed by cytometric bead array and immunocytochemistry. Our results show that knock-down of C/EBPδ expression in pericytes following immune stimulation increased chemokine and adhesion molecule expression, thus modifying the human brain pericyte inflammatory response. The induction of C/EBPδ following immune stimulation may act to limit infiltration of peripheral immune cells, thereby preventing further inflammatory responses in the brain.
PMCID: PMC4499812  PMID: 26166618
21.  Linking Transcriptional Changes over Time in Stimulated Dendritic Cells to Identify Gene Networks Activated during the Innate Immune Response 
PLoS Computational Biology  2013;9(11):e1003323.
The innate immune response is primarily mediated by the Toll-like receptors functioning through the MyD88-dependent and TRIF-dependent pathways. Despite being widely studied, it is not yet completely understood and systems-level analyses have been lacking. In this study, we identified a high-probability network of genes activated during the innate immune response using a novel approach to analyze time-course gene expression profiles of activated immune cells in combination with a large gene regulatory and protein-protein interaction network. We classified the immune response into three consecutive time-dependent stages and identified the most probable paths between genes showing a significant change in expression at each stage. The resultant network contained several novel and known regulators of the innate immune response, many of which did not show any observable change in expression at the sampled time points. The response network shows the dominance of genes from specific functional classes during different stages of the immune response. It also suggests a role for the protein phosphatase 2a catalytic subunit α in the regulation of the immunoproteasome during the late phase of the response. In order to clarify the differences between the MyD88-dependent and TRIF-dependent pathways in the innate immune response, time-course gene expression profiles from MyD88-knockout and TRIF-knockout dendritic cells were analyzed. Their response networks suggest the dominance of the MyD88-dependent pathway in the innate immune response, and an association of the circadian regulators and immunoproteasomal degradation with the TRIF-dependent pathway. The response network presented here provides the most probable associations between genes expressed in the early and the late phases of the innate immune response, while taking into account the intermediate regulators. We propose that the method described here can also be used in the identification of time-dependent gene sub-networks in other biological systems.
Author Summary
The innate immune response is the first level of protection in organisms against invading pathogens. It consists of a large number of proteins functioning in signaling cascades triggered by the binding of fragments from microbes to specific cellular receptors. Disruptions in these pathways can lead to numerous diseases. As such, the innate immune system has been the subject of a large number of studies. However, due to its complexity and the interplay of a large number of pathways, it is not yet completely understood. In this study, we measured transcriptional changes in activated immune cells and used this information in the context of a large network of protein-protein and protein-DNA interactions to identify a smaller network of response genes. We did this by identifying the most probable network paths connecting genes showing large changes in their expression patterns at successive stages of the response. Analysis of this activated gene network revealed the associations between various temporal regulators of the innate immune response. We also identified response networks for immune cells lacking important mediators, MyD88 and TRIF, to clarify the distinct functional modules affected by their associated pathways in the innate immune response.
PMCID: PMC3820512  PMID: 24244133
22.  TLR-signaling Networks 
Journal of Dental Research  2011;90(4):417-427.
Toll-like receptors play a critical role in innate immunity by detecting invading pathogens. The ability of TLRs to engage different intracellular signaling molecules and cross-talk with other regulatory pathways is an important factor in shaping the type, magnitude, and duration of the inflammatory response. The present review will cover the fundamental signaling pathways utilized by TLRs and how these pathways regulate the innate immune response to pathogens. Abbreviations: TLR, Toll-like receptor; PRR, pattern recognition receptor; PAMP, pathogen-associated molecular pattern; LPS, lipopolysaccharide; APC, antigen-presenting cell; IL, interleukin; TIR, Toll/IL-1R homology; MyD88, myeloid differentiation factor 88; IFN, interferon; TRIF, TIR-domain-containing adapter-inducing interferon-β; IRAK, IL-1R-associated kinase; TAK1, TGF-β-activated kinase; TAB1, TAK1-binding protein; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B-cells; MAPK, mitogen-activated protein kinase; NLR, NOD-like receptors; LRR, leucine-rich repeats; DC, dendritic cell; PI3K, phosphoinositide 3-kinases; GSK3, glycogen synthase kinase-3; mTOR, mammalian target of rapamycin; DAF, decay-accelerating factor; IKK, IκB kinase; IRF, interferon regulatory factors; TBK1, TANK-binding kinase 1; CARD, caspase activation and recruitment domain; PYD, pyrin N-terminal homology domain; ATF, activating transcription factor; and PTEN, phosphatase and tensin homolog.
PMCID: PMC3075579  PMID: 20940366
TLR; GSK3; PI3K; dendritic cell; complement; inflammation
23.  Inhibition of the Type I Interferon Antiviral Response During Arenavirus Infection 
Viruses  2010;2(11):2443-2480.
Arenaviruses merit interest both as tractable experimental model systems to study acute and persistent viral infections, and as clinically-important human pathogens. Several arenaviruses cause hemorrhagic fever (HF) disease in humans. In addition, evidence indicates that the globally-distributed prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is a human pathogen of clinical significance in congenital infections, and also poses a great danger to immunosuppressed individuals. Arenavirus persistence and pathogenesis are facilitated by their ability to overcome the host innate immune response. Mammalian hosts have developed both membrane toll-like receptors (TLR) and cytoplasmic pattern recognition receptors (PRRs) that recognize specific pathogen-associated molecular patterns (PAMPs), resulting in activation of the transcription factors IRF3 or IRF7, or both, which together with NF-κB and ATF-2/c-JUN induce production of type I interferon (IFN-I). IFN-I plays a key role in host anti-microbial defense by mediating direct antiviral effects via up-regulation of IFN-I stimulated genes (ISGs), activating dendritic cells (DCs) and natural killer (NK) cells, and promoting the induction of adaptive responses. Accordingly, viruses have developed a plethora of strategies to disrupt the IFN-I mediated antiviral defenses of the host, and the viral gene products responsible for these disruptions are often major virulence determinants. IRF3- and IRF7-dependent induction of host innate immune responses is frequently targeted by viruses. Thus, the arenavirus nucleoprotein (NP) was shown to inhibit the IFN-I response by interfering with the activation of IRF3. This NP anti-IFN activity, together with alterations in the number and function of DCs observed in mice chronically infected with LCMV, likely play an important role in LCMV persistence in its murine host. In this review we will discuss current knowledge about the cellular and molecular mechanisms by which arenaviruses can subvert the host innate immune response and their implications for understanding HF arenaviral disease as well as arenavirus persistence in their natural hosts.
PMCID: PMC3185579  PMID: 21994626
arenavirus; type I interferon; hemorrhagic fever; innate immunity; lymphocytic choriomeningitis virus; nucleoprotein; dendritic cells
24.  Reciprocal regulation of p63 by C/EBP delta in human keratinocytes 
Genetic experiments have clarified that p63 is a key transcription factor governing the establishment and maintenance of multilayered epithelia. Key to our understanding of p63 strategy is the identification of target genes. We perfomed an RNAi screening in keratinocytes for p63, followed by profiling analysis.
C/EBPδ, member of a family with known roles in differentiation pathways, emerged as a gene repressed by p63. We validated C/EBPδ as a primary target of ΔNp63α by RT-PCR and ChIP location analysis in HaCaT and primary cells. C/EBPδ is differentially expressed in stratification of human skin and it is up-regulated upon differentiation of HaCaT and primary keratinocytes. It is bound to and activates the ΔNp63 promoter. Overexpression of C/EBPδ leads to alteration in the normal profile of p63 isoforms, with the emergence of ΔNp63β and γ, and of the TA isoforms, with different kinetics. In addition, there are changes in the expression of most p63 targets. Inactivation of C/EBPδ leads to gene expression modifications, in part due to the concomitant repression of ΔNp63α. Finally, C/EBPδ is found on the p63 targets in vivo by ChIP analysis, indicating that coregulation is direct.
Our data highlight a coherent cross-talk between these two transcription factors in keratinocytes and a large sharing of common transcriptional targets.
PMCID: PMC2148061  PMID: 17903252
25.  Differential Role for c-Rel and C/EBPβ/δ in TLR- Mediated Induction of Proinflammatory Cytokines 
Toll-like receptor (TLR) stimulation triggers a signaling pathway via MyD88 and IRAK-4 that is essential for proinflammatory cytokine induction. Although NF-κB has been shown to be one of the key transcriptional regulators of these cytokines, evidence suggests that other factors may also be important. Here we showed that MyD88-deficient macrophages have defective c-Rel activation, which has been linked to IL-12 p40 induction, but not IL-6 or TNFα. We also investigated other transcription factors and showed that C/EBPβ and C/EBPδ expression was limited in MyD88- or IRAK-4-deficient macrophages treated with LPS. Importantly, the absence of both C/EBPβ and C/EBPδ resulted in the impaired induction of proinflammatory cytokines stimulated by several TLR ligands. Our results identify c-Rel and C/EBPβ/δ as important transcription factors in a MyD88-dependent pathway that regulate the induction of proinflammatory cytokines.
PMCID: PMC2805849  PMID: 19454718
Lipopolysaccharide; Signal Transduction; Inflammation

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