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.
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.
The toll-like receptor (TLR) system constitutes a pylogenetically ancient, evolutionary conserved, archetypal pattern recognition system, which underpins pathogen recognition by and activation of the immune system. Toll-like receptor agonists have long been used as immunoadjuvants in anti cancer immunotherapy. However, TLRs are increasingly implicated in human disease pathogenesis and an expanding body of both clinical and experimental evidence suggests that the neoplastic process may subvert TLR signalling pathways to advance cancer progression. Recent discoveries in the TLR system open a multitude of potential therapeutic avenues. Extrapolation of such TLR system manipulations to a clinical oncological setting demands care to prevent potentially deleterious activation of TLR-mediated survival pathways. Thus, the TLR system is a double-edge sword, which needs to be carefully wielded in the setting of neoplastic disease.
toll-like receptors; toll-like receptor ligands; NF-κB; innate immunity; immunotherapy
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.
Lipopolysaccharide; Signal Transduction; Inflammation
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.
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.
Acute phase proteins; CCAAT/enhancer binding proteins; Hepatocytes; Interleukin-1; Cell signalling
The CCAAT/enhancer binding protein (CEBP) family of transcription factors includes five genes. In the ovary, both Cebpa and Cebpb are essential for granulosa cell function. In this study we have explored the role of the Cebpd gene in ovarian physiology by expression and functional studies. Here we report that Cebpd (C/EBPδ) is expressed in the mouse ovary in a highly restricted temporal and spatial pattern. In response to luteinizing hormone (LH/hCG), CEBPD expression is transiently induced in interstitial cells and in theca cells of follicles from the primary to pre-ovulatory stage, and overlaps in part with expression of the alpha-smooth muscle actin protein. Efficient down-regulation of CEBPD was dependent on a functional Cebpb gene. Proliferating human theca cells in culture also express Cebpd. Cells from patients with polycystic ovarian syndrome (PCOS) exhibited higher Cebpd expression levels. However, deletion of Cebpd in mice had no overt effect on ovarian physiology and reproductive function. Very little is known at present about the molecular mechanisms underlying theca/interstitial cell functions. The expression pattern of CEBPD reported here identifies a novel functional unit of mouse theca cells of primary through tertiary follicles responding to LH/hCG together with a subset of interstitial cells. This acute stimulation of CEBPD expression may be exploited to further characterize the hormonal regulation and function of theca and interstitial cells.
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.
IL-17, hallmark cytokine of the newly-described “Th17” population, signals through a novel subclass of receptors, yet surprisingly little is known about mechanisms of IL-17 receptor signaling. IL-17 activates target gene expression via NF-κB and two members of the C/EBP transcription factor family, C/EBPδ and C/EBPβ. It has been shown that TRAF6 and Act1 are upstream of NF-κB and C/EBPδ, but the signaling pathways regulating C/EBPβ remain entirely undefined. In this report, we demonstrate that IL-17 signaling leads to phosphorylation of two sites in the C/EBPβ “regulatory 2 domain” in a sequential, interdependent fashion. First rapid phosphorylation of Thr188 occurs within 15 minutes and is ERK-dependent. A second phosphorylation event targets Thr179, which requires prior Thr188 phosphorylation and GSK3β activity. We further show that the pathways leading to C/EBPβ phosphorylation are mediated by distinct subdomains within IL-17RA. Whereas phosphorylation of Thr188 is mediated by the previously-identified SEFIR/TILL domain, activation of Thr179 occurs through a motif located in the IL-17RA distal tail. Functionally, C/EBPβ phosphorylation mediates a negative signal, as blocking ERK and GSK3β upregulates IL-17-induced genes, and a C/EBPβ-Thr188 mutant enhances activation of a C/EBP-dependent reporter. Consistently, GSK3β overexpression inhibits IL-17 signaling to a C/EBP-dependent reporter, and GSK3β-deficient cells fail to induce phosphorylation at T179. Thus, IL-17 triggers dual phosphorylation of C/EBPβ, which down-modulates expression of inflammatory genes. This is the first detailed dissection of the IL-17-mediated C/EBP pathway and is also the first known example of a negative signal mediated by this unique receptor.
The Toll-like receptors (TLRs) and complement are key innate defense systems that are rapidly triggered upon infection. Although both systems have been investigated primarily as separate entities, an emerging body of evidence indicates extensive crosstalk between complement and TLR signaling pathways. Analysis of these data suggests that the complement–TLR interplay reinforces innate immunity or regulates excessive inflammation, through synergistic or antagonistic interactions, respectively. However, the facility of complement and TLRs for communication is exploited by certain pathogens as a means to modify the host response in ways that favor the persistence of the pathogens. Further elucidation of regulatory links between complement and TLRs is essential for understanding their complex roles in health and disease.
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.
arenavirus; type I interferon; hemorrhagic fever; innate immunity; lymphocytic choriomeningitis virus; nucleoprotein; dendritic cells
Microbe-associated molecular patterns are recognized by Toll-like receptors of the innate immune system. This recognition enables a rapid response to potential pathogens but does not clearly provide a way for the innate immune system to discriminate between virulent and avirulent microbes. We find that pulmonary infection of mice with type 3 translocation-competent Pseudomonas aeruginosa triggers a rapid inflammatory response, while infection with isogenic translocation-deficient mutants does not. Discrimination between translocon-positive and -negative bacteria requires caspase-1 activity in bone marrow-derived cells and interleukin-1 receptor signaling. Thus, the activation of caspase-1 by bacteria expressing type 3 secretion systems allows for rapid recognition of bacteria expressing conserved functions associated with virulence.
Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP response element-binding (ATF/CREB) family of transcription factors. It is an adaptive-response gene that participates in cellular processes to adapt to extra- and/or intracellular changes, where it transduces signals from various receptors to activate or repress gene expression. Advances made in understanding the immunobiology of Toll-like receptors have recently generated new momentum for the study of ATF3 in immunity. Moreover, the role of ATF3 in the regulation of the cell cycle and apoptosis has important implications for understanding susceptibility to and progression of several cancers.
ATF3; Transcriptional regulation; Immunity; TLRs; Oncogenesis
Toll-like receptors (TLRs) are pivotal components of the innate immune response, which is responsible for eradicating invading microorganisms through the induction of inflammatory molecules. These receptors are also involved in responding to harmful endogenous molecules and have crucial roles in the activation of the innate immune system and shaping the adaptive immune response. However, TLR signaling pathways must be tightly regulated because undue TLR stimulation may disrupt the fine balance between pro- and anti-inflammatory responses. Such disruptions may harm the host through the development of autoimmune and inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Several studies have investigated the regulatory pathways of TLRs that are essential for modulating proinflammatory responses. These studies reported several pathways and molecules that act individually or in combination to regulate immune responses. In this review, we have summarized recent advancements in the elucidation of the negative regulation of TLR signaling. Moreover, this review covers the modulation of TLR signaling at multiple levels, including adaptor complex destabilization, phosphorylation and ubiquitin-mediated degradation of signal proteins, manipulation of other receptors, and transcriptional regulation. Lastly, synthetic inhibitors have also been briefly discussed to highlight negative regulatory approaches in the treatment of inflammatory diseases.
inflammatory diseases; inhibitors; innate immune system; miRNA; Toll-like receptors
Toll-like receptors are a family of pattern-recognition receptors in mammals that can discriminate between chemically diverse classes of microbial products, including bacterial cell-wall components, and that can elicit pathogen-specific cellular immune responses. Toll-like receptors share characteristic features with the interleukin-1 and interleukin-18 cytokine receptors and these receptor classes activate similar signal-transduction pathways.
The innate immune system uses a variety of germline-encoded pattern-recognition receptors that recognize conserved microbial structures or pathogen-associated molecular patterns, such as those that occur in the bacterial cell-wall components peptidoglycan and lipopolysaccharide. Recent studies have highlighted the importance of Toll-like receptors (TLRs) as a family of pattern-recognition receptors in mammals that can discriminate between chemically diverse classes of microbial products. First identified on the basis of sequence similarity with the Drosophila protein Toll, TLRs are members of an ancient superfamily of proteins, which includes related proteins in invertebrates and plants. TLRs activate innate immune defense reactions, such as the release of inflammatory cytokines, but increasing evidence supports an additional critical role for TLRs in orchestrating the development of adaptive immune responses. The sequence similarity between the intracellular domains of the TLRs and the mammalian interleukin-1 and interleukin-18 cytokine receptors reflects the use of a common intracellular signal-transduction cascade triggered by these receptor classes. But more recent findings have demonstrated that there are in fact TLR-specific signaling pathways and cellular responses. Thus, TLRs function as sentinels of the mammalian immune system that can discriminate between diverse pathogen-associated molecular patterns and then elicit pathogen-specific cellular immune responses.
The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family and are activated by environmental stress. JNK is also activated by proinflammatory cytokines, such as TNF and IL-1, and Toll-like receptor ligands. This pathway, therefore, can act as a critical convergence point in immune system signaling for both adaptive and innate responses. Like other MAPKs, the JNKs are activated via the sequential activation of protein kinases that includes two dual-specificity MAP kinase kinases (MKK4 and MKK7) and multiple MAP kinase kinase kinases. MAPKs, including JNKs, can be deactivated by a specialized group of phosphatases, called MAP kinase phosphatases. JNK phosphorylates and regulates the activity of transcription factors other than c-Jun, including ATF2, Elk-1, p53 and c-Myc and non-transcription factors, such as members of the Bcl-2 family. The pathway plays a critical role in cell proliferation, apoptosis, angiogenesis and migration. In this review, an overview of the functions that are related to rheumatic diseases is presented. In addition, some diseases in which JNK participates will be highlighted.
Signal transduction; inflammation; autoimmunity; rheumatoid arthritis.
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.
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.
Interleukin-1R like receptors (ILRs) and Toll Like Receptors (TLRs) are key receptors of innate immunity, inflammation, and orientation of the adaptive response. They belong to a superfamily characterized by the presence of a conserved intracellular domain, the Toll/IL-1R (TIR) domain, which is involved in the activation of a signaling cascade leading to activation of transcription factors associated to inflammation. The activation of inflammatory responses and immunity by ILRs or TLRs signaling is potentially detrimental for the host in acute and chronic conditions and is tightly regulated at different levels by receptor antagonists, decoy receptors or signaling molecules, and miRNAs. Recent evidence suggests that the ILRs family member TIR8 (also known as SIGIRR) is a regulatory protein acting intracellularly to inhibit ILRs and TLRs signaling. In particular, current evidence suggests that TIR8/SIGIRR dampens TLRs-mediated activation and inhibits signaling receptor complexes of IL-1 family members associated with Th1 (IL-18), Th2 (IL-33), and Th17 (IL-1) differentiation. Studies with Tir8/Sigirr-deficient mice showed that the ability to dampen signaling from ILRs and TLRs family members makes TIR8/SIGIRR a key regulator of inflammation. Here, we summarize our current understanding of the structure and function of TIR8/SIGIRR, focusing on its role in different pathological conditions, ranging from infectious and sterile inflammation, to autoimmunity and cancer-related inflammation.
cytokine; interleukin-1; toll like receptors; inflammation; infection; inflammation-associated cancer
Receptor tyrosine kinases (RTKs) exhibit basal tyrosine phosphorylation and activity in the absence of ligand stimulation, which has been attributed to the “leaky” nature of tyrosine kinase autoinhibition and stochastic collisions of receptors in the membrane bilayer. This basal phosphorylation does not produce a signal of sufficient amplitude and intensity to manifest in a biological response and hence is considered to be a passive, futile process that does not have any biological function. This paradigm has now been challenged by a study showing that the basal phosphorylation of RTKs is a physiologically relevant process that is actively inhibited by the intracellular adaptor protein growth factor receptor-bound 2 (Grb2) and serves to “prime” receptors for a rapid response to ligand stimulation. Grb2 is conventionally known for playing positive roles in RTK signaling. The discovery of a negative regulatory role for Grb2 reveals that this adaptor acts as a double-edged sword in the regulation of RTK signaling.
The innate immune system plays important roles in a number of disparate processes. Foremost, innate immunity is a first responder to invasion by pathogens and triggers early defensive responses and recruits the adaptive immune system. The innate immune system also responds to endogenous damage signals that arise from tissue injury. Recently it has been found that innate immunity plays an important role in neuroprotection against ischemic stroke through the activation of the primary innate immune receptors, Toll-like receptors (TLRs). Using several large-scale transcriptomic data sets from mouse and mouse macrophage studies we identified targets predicted to be important in controlling innate immune processes initiated by TLR activation. Targets were identified as genes with high betweenness centrality, so-called bottlenecks, in networks inferred from statistical associations between gene expression patterns. A small set of putative bottlenecks were identified in each of the data sets investigated including interferon-stimulated genes (Ifit1, Ifi47, Tgtp and Oasl2) as well as genes uncharacterized in immune responses (Axud1 and Ppp1r15a). We further validated one of these targets, Ifit1, in mouse macrophages by showing that silencing it suppresses induction of predicted downstream genes by lipopolysaccharide (LPS)-mediated TLR4 activation through an unknown direct or indirect mechanism. Our study demonstrates the utility of network analysis for identification of interesting targets related to innate immune function, and highlights that Ifit1 can exert a positive regulatory effect on downstream genes.
The prevalence of obesity, an established epidemiologic risk factor for many chronic diseases including cancer, has been steadily increasing in the US over several decades. The mechanisms used to regulate energy balance and adiposity and the relationship of these factors to cancer are not completely understood. Here we have used knockout mice to examine the roles of the transcription factors CCAAT/enhancer-binding protein (C/EBP) β and C/EBPδ in regulating body composition and systemic levels of hormones such as insulin-like growth factor-1 (IGF-1), leptin and insulin that mediate energy balance. Dual-energy X-ray absorptiometry showed that C/EBPβ, either directly or indirectly, modulated body weight, fat content and bone density in both males and females, while the effect of C/EBPδ was minor and only affected adiposity and body weight in female animals. Levels of IGF-1, leptin and insulin in the serum were decreased in both male and female C/EBPβ−/− mice, and C/EBPβ was associated with their promoters in vivo. Moreover, colon adenocarcinoma cells displayed reduced tumorigenic potential when transplanted into C/EBPβ-deficient animals, especially males. Thus, C/EBPβ contributes to endocrine expression of IGF-1, leptin and insulin, which modulate energy balance and can contribute to cancer progression by creating a favorable environment for tumor cell proliferation and survival.
The transcription factor ATF3 inhibits lipid body formation in macrophages during atherosclerosis in part by dampening the expression of cholesterol 25-hydroxylase.
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. We demonstrate that macrophage lipid body formation can be induced by modified lipoproteins or by inflammatory Toll-like receptor agonists. We used an unbiased approach to study the overlap in these pathways to identify regulators that control foam cell formation and atherogenesis. An analysis method integrating epigenomic and transcriptomic datasets with a transcription factor (TF) binding site prediction algorithm suggested that the TF ATF3 may regulate macrophage foam cell formation. Indeed, we found that deletion of this TF results in increased lipid body accumulation, and that ATF3 directly regulates transcription of the gene encoding cholesterol 25-hydroxylase. We further showed that production of 25-hydroxycholesterol (25-HC) promotes macrophage foam cell formation. Finally, deletion of ATF3 in Apoe−/− mice led to in vivo increases in foam cell formation, aortic 25-HC levels, and disease progression. These results define a previously unknown role for ATF3 in controlling macrophage lipid metabolism and demonstrate that ATF3 is a key intersection point for lipid metabolic and inflammatory pathways in these cells.
Extensive research in recent years suggests that exposure to xenobiotic stimuli plays a critical role in autoimmunity induction and severity and that the resulting response would be exacerbated in individuals with an infection-aroused immune system. In this context, heavy metals constitute a prominent category of xenobiotic substances, known to alter divergent immune cell responses in accidentally and occupationally exposed individuals, thereby increasing the susceptibility to autoimmunity and cancer, especially when accompanied by inflammation-triggered persistent sensitization. This perception is learned from experimental models of infection and epidemiologic studies and clearly underscores the interplay of exposure to such immunomodulatory elements with pre- or postexposure infectious events. Further, the TH17 cell subset, known to be associated with a growing list of autoimmune manifestations, may be the “superstar” at the interface of xenobiotic exposure and autoimmunity. In this review, the most recently established links to this nomination are short-listed to create a framework to better understand new insights into TH17's contributions to autoimmunity.
Our objective was to delineate the potential role of adipogenesis in insulin resistance and type 2 diabetes. Obesity is characterized by an increase in adipose tissue mass resulting from enlargement of existing fat cells (hypertrophy) and/or from increased number of adipocytes (hyperplasia). The inability of the adipose tissue to recruit new fat cells may cause ectopic fat deposition and insulin resistance.
Research Methods and Procedures
We examined the expression of candidate genes involved in adipocyte proliferation and/or differentiation [CCAAT/enhancer-binding protein (C/EBP) α, C/EBPδ, GATA domain-binding protein 3 (GATA3), C/EBPβ, peroxisome proliferator-activated receptor (PPAR) γ2, signal transducer and activator of transcription 5A (STAT5A), Wnt-10b, tumor necrosis factor α, sterol regulatory element-binding protein 1c (SREBP1c), 11 beta-hydroxysteroid dehydrogenase, PPARG angiopoietin-related protein (PGAR), insulin-like growth factor 1, PPARγ coactivator 1α, PPARγ coactivator 1β, and PPARδ] in subcutaneous adipose tissue from 42 obese individuals with type 2 diabetes and 25 non-diabetic subjects matched for age and obesity.
Insulin sensitivity was measured by a 3-hour 80 mU/m2 per minute hyperinsulinemic glucose clamp (100 mg/dL). As expected, subjects with type 2 diabetes had lower glucose disposal (4.9 ± 1.9 vs. 7.5 ± 2.8 mg/min per kilogram fat-free mass; p < 0.001) and larger fat cells (0.90 ± 0.26 vs. 0.78 ± 0.17 μm; p = 0.04) as compared with obese control subjects. Three genes (SREBP1c, p < 0.01; STAT5A, p = 0.02; and PPARγ2, p = 0.02) had significantly lower expression in obese type 2 diabetics, whereas C/EBPβ only tended to be lower (p = 0.07).
This cross-sectional study supports the hypothesis that impaired expression of adipogenic genes may result in impaired adipogenesis, potentially leading to larger fat cells in subcutaneous adipose tissue and insulin resistance.
insulin resistance; gene expression; adipogenesis; fat cell size; type 2 diabetes