Search

Background: A disintegrin and metalloproteinase 17 (ADAM17) is a proteolytic cleaving protein with a crucial function in the inflammatory responses, especially sepsis. But the clear role of ADAM17 in sepsis and the underlying mechanism remained unknown. In this study, we aim to determine the clinical association of ADAM17 -172A > G (rs12692386) promoter polymorphism with sepsis and to further explore the effect and mechanism of the early growth response 1 (EGR1)/ADAM17 pathway in inflammatory process during sepsis., Methods: A total of 477 sepsis patients and 750 controls were enrolled in this study to determine the association of ADAM17 -172A > G polymorphism with sepsis. The transcription factor binding to the promoter region of ADAM17 gene was predicted by bioinformatics analysis and verified by Chromatin Immunoprecipitation (ChIP) and luciferase assays. Quantitative real-time PCR and Western blot were performed to detect EGR1 and ADAM17 expression. Cytokine production was detected by enzyme-linked immunosorbent assay. The effect of EGR1/ADAM17 pathway on sepsis-induced inflammatory responses was evaluated in EGR1-silenced cells and endotoxemia mouse model., Results: The frequencies of non-survivors among the sepsis patients with the -172AG/GG genotypes and G allele were distinctly higher than those among patients with the AA genotype (53.9% vs. 39.7%, OR = 1.779, 95% CI = 1.119-2.829, P = 0.0142) and A allele (30.9% vs. 22.2%, OR = 1.570, 95% CI = 1.095-2.251, P = 0.0136). The Kaplan-Meier survival analysis indicated that the 28-day survival in septic patients with -172AG/GG genotypes of this functional ADAM17 promoter polymorphism was much worse than in the AA genotype carriers (log-rank = 5.358, P = 0.021). The results of in vitro lipopolysaccharide-stimulated and luciferase assays indicated that the -172 A-to-G variation could functionally upregulate promoter activity and transcription of ADAM17 gene via enhancing the binding affinity of its promoter region with the EGR1. The ChIP assay identified the direct interaction. Further studies demonstrated that inhibition of EGR1 significantly decreased ADAM17 expression and the pro-inflammatory cytokine secretion in vitro, and improved the survival and inflammatory response of sepsis mouse model., Conclusions: These results provided evidence that the ADAM17 -172A > G polymorphism functionally promoted ADAM17 expression and enhanced sepsis-induced inflammatory responses via the EGR1/ADAM17 pathway, which ultimately conferred susceptibility to sepsis mortality and poor prognosis., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is known primarily for its function in DNA double-stranded break repair and nonhomologous end joining (NHEJ). However, DNA-PKcs also has a critical yet undefined role in immunity impacting both myeloid and lymphoid cell lineages spurring interest in targeting DNA-PKcs for therapeutic strategies in immune-related diseases. To gain insight into the function of DNA-PKcs within immune cells, we performed a quantitative phosphoproteomic screen in T cells to identify phosphorylation targets of DNA-PKcs. Our results indicate that DNA-PKcs phosphorylates the transcription factor Egr1 (early growth response protein 1) at serine 301. Expression of Egr1 is induced early upon T cell activation and dictates T cell response by modulating expression of cytokines and key costimulatory molecules such as IL (interleukin) 2, IL6, IFNγ, and NFκB. Inhibition of DNA-PKcs by treatment with a DNA-PKcs specific inhibitor NU7441 or shRNA knockdown increased proteasomal degradation of Egr1. Mutation of serine 301 to alanine via CRISPR-Cas9 reduced EGR1 protein expression and decreased Egr1-dependent transcription of IL2 in activated T cells. Our findings identify DNA-PKcs as a critical intermediary link between T cell activation and T cell fate and a novel phosphosite involved in regulating Egr1 activity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The transcription factor ZENK is an immediate early gene that has been employed as a surrogate marker to map neuronal activity in the brain. It has been used in a wide variety of species, however, commercially available antibodies have limited immunoreactivity in birds. To address this issue we generated a new mouse monoclonal antibody, 7B7-A3, raised against ZENK from the rock pigeon (Columba livia). We show that 7B7-A3 labels clZENK in both immunoblots and histological stainings with high sensitivity and selectivity for its target. Using a sound stimulation paradigm we demonstrate that 7B7-A3 can detect activity-dependent ZENK expression at key stations of the central auditory pathway of the pigeon. Finally, we compare staining efficiency across three avian species and confirm that 7B7-A3 is compatible with immunohistochemical detection of ZENK in the rock pigeon, zebra finch, and domestic chicken. Taken together, 7B7-A3 represents a useful tool for the avian neuroscience community to map functional activity in the brain.

Egr-1, an immediate-early gene product and master regulator was originally described as a phosphoprotein following its discovery in the 1980s. However specific residue(s) phosphorylated in Egr-1 remain elusive. Here we phosphorylated recombinant Egr-1 in vitro with ERK1 prior to mass spectrometry, which identified phosphorylation of Ser 12 and Ser 26 with the latter ∼12 times more abundant than Ser 12 . Phosphorylation of wild-type recombinant Egr-1 (as compared with Ser 26 >Ala 26 mutant Egr-1) revealed that Ser 26 accounts for the majority of phosphorylation of Egr-1 by ERK1. N-FGSFPH(pS)PTMDNYC-C was used as an antigen to generate mouse monoclonal antibodies (pS26 MAb). pS26 MAb recognised ERK1-phosphorylated Egr-1 but not Egr-1 bearing a point mutation at Ser 26 . pS26 MAb recognised inducible ∼75 kDa and 100 kDa species in nuclear extracts of cells exposed to FGF-2. Peptide blocking revealed both inducible species were phosphosite-specific. Immunoprecipitation of nuclear extracts of cells exposed to FGF-2 with pS26 MAb followed by SDS-PAGE and mass spectrometry identified Egr-1 sequences corresponding to the ∼75 kDa species but not ∼100 kDa species. This study identifies a specific amino acid phosphorylated in endogenous Egr-1., (Copyright © 2019. Published by Elsevier Inc.)

Beta-defensins 2 and 3 (BD2 and BD3) are inducible peptides present at the sites of infection, and they are well characterized for their antimicrobial activities and immune-regulatory functions. However, no study has thoroughly investigated their immunomodulatory effects on macrophage-mediated immune responses against Pseudomonas aeruginosa (PA). Here, we use THP-1 and RAW264.7 cell lines and demonstrate that BD2 and BD3 suppressed macrophage autophagy but enhanced the engulfment of PA and Zymosan bioparticles as well as the formation of phagolysosomes, using immunofluorescence staining and confocal microscopy. Plate count assay showed that macrophage-mediated phagocytosis and intracellular killing of PA were promoted by BD2 and BD3. Furthermore, microarray and real-time PCR showed that the expression of two genes, early growth response gene-1 (EGR1) and c-FOS, was attenuated by BD2 and BD3. Western blot revealed that BD2 and BD3 inhibited the expression and nuclear translocation of EGR1 and c-FOS. Knockdown of EGR1 and c-FOS by siRNA transfection suppressed macrophage autophagy before and after PA infection; while overexpression of these two transcription factors enhanced autophagy but reversed the role of BD2 and BD3 on macrophage-mediated PA eradication. Together, these results demonstrate a novel immune defense activity of BD2 and BD3, which promotes clearance of PA by inhibiting macrophage autophagy through downregulation of EGR1 and c-FOS.

Hyperactivation of proliferative and growth promoting pathways underlies the progression of vessel remodeling, leading to vascular dysfunction. An upregulation of early growth response protein 1 (Egr-1), a zinc finger transcription factor has been observed in several models of vascular diseases. In the vasculature, Egr-1 expression can be induced by multiple hormonal, metabolic and external stimuli, such as growth factors, cytokines, reactive oxygen species, hyperglycaemia and stretch-induced stress. The structure of the Egr-1 promoter allows both its auto-regulation and its binding with several regulatory transcription cofactors like the serum response factor and the cAMP response element binding protein. Pharmacological and genetic studies have revealed the involvement of several signaling pathways that contribute to the expression of Egr-1. Among them, the mitogen-activated protein kinase pathway has emerged as a predominant signaling cascade that regulates Egr-1 transcription in response to various stimuli. Moreover, targeted deletion of Egr-1 by DNAzymes, antisense oligonucleotides or RNA interference has also helped in defining the importance of Egr-1 in the pathophysiology of vascular diseases. Neointimal formation and expression of genes directly linked with proinflammatory processes have been demonstrated to be enhanced by Egr-1 expression and activity. This review provides an overview on the signaling components implicated in Egr-1 expression and discusses its potential involvement in vascular pathophysiology.

Formation of the central nervous system in ontogeny and function in adult mammals are controlled by universal ubiquitin-proteasome proteolytic system. The aim of this work was to study the dynamics of expression of immune proteasomes in comparison with the dynamics of ChLA and CLA proteasome and expression of the transcription factor Zif268 in the structures of the brain (cortex, hippocampus, and brainstem) in embryonic (E19, E21 days of embryonic development) and early postnatal (P1, P3, P4, P5, P7, P15 days of post-natal development) development in rats. ChLA and CLA in clarified homogenates of rat brain structures were determined by hydrolysis of fluorogenic commercial oligopeptides Suc-LLVY-AMC and Z-LLG-AMC, respectively. In the cortex and hippocampus of the brain was observed upregulation of immune subunits LMP7 during the active formation of biochemical mediatory structure and efferent neuronal projections at the period P7-P15. In the cerebral cortex during this period ChLA and CLA also are increased. In all structures of the brain the LMP2 immune subunits content was significantly increased at the period P7-P15. Contents of proteolytic constitutive subunit β1 in all structures decreased by P4 compare to P1 levels and was increased on P15 relative to the P1 levels. However, the level of expression of proteolytic constitutive subunit β5 increased in cortex, hippocampus and brainstem from E21 and reached maximum values on P3, P5 and P1, respectively with a sharp decrease to P7 in all studied structures. In all structures expression of LM P2 immune subunits and β1 constitutive subunits increased simultaneously with LMP7 immune subunits and sharply on P15. Also shown a positive correlation of increased expression regulator PA28 and constitutive β5 subunits in the hippocampus during the period P3-P5 and in the brainstem at the period P1-P5. The peculiarity of the studied brain regions during P7-P15 of rat early development is a correlation of expression of immune subunits LMP2 and LMP7 proteasome and ChLA with the expression of the transcription factor Zif268. Probably immune proteasome plays an important role in the regulation of key biochemical processes in the early ontogenesis of the central nervous system and are necessary for the emergence and realization of synaptic plasticity in the brain structures studied in rats.

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Obesity is associated with low-grade chronic inflammation, which contributes to cellular dysfunction promoting metabolic disease. Obesity during pregnancy leads to a proinflammatory milieu in the placenta; however, the underlying causes for obesity-induced placental inflammation remain unclear. Here, we examine the mechanisms by which saturated fatty acids and inflammatory cytokines induce inflammation in placental trophoblasts. We conducted global transcriptomic profiling in BeWo cells following palmitate and/or TNFα treatment and gene/protein expression analyses of MAPK pathways and characterized downstream transcription factors directly regulating inflammatory cytokines. Microarray analysis revealed increased expression of genes regulating inflammation, stress response, and immediate early response in cytotrophoblasts in response to palmitic acid (PA), TNFα, or a combination of both (PA + TNFα). Both gene ontology and gene set enrichment analysis revealed MAPK and EGR-1 signaling to be upregulated in BeWo cells, which was confirmed via immunoblotting. Importantly, activation of JNK signaling was necessary for increased proinflammatory cytokine (IL-6, TNFα, and IL-8) and EGR1 mRNA. Consistent with the requirement of JNK signaling, ChIP analysis confirmed the recruitment of c-Jun and other MAPK-responsive immediate early factors on the EGR1 promoter. Moreover, recruitment of EGR-1 on cytokine promoters (IL-6, TNFα, and IL-8) and an impaired proinflammatory response following knockdown of EGR-1 suggested it as a central component of the mechanism facilitating inflammatory gene expression. Finally, akin to in vitro findings, term placenta from obese women also had both increased JNK and p38 signaling and greater EGR-1 protein relative to lean women. Our results demonstrate that lipotoxic insults induce inflammation in placental cells via activation of JNK/EGR-1 signaling.

Appropriate innate immune responses are required to protect an organism against foreign pathogens, and the immune response must be tightly controlled. Here, we report a new microRNA (miRNA) identified from a small RNA library from the epididymis, termed miR-7578, that acts as a negative regulator of inflammatory responses. It was abundantly expressed in immune-related organs and induced by lipopolysaccharide in the lung and epididymis, as well as macrophages stimulated with diverse Toll-like receptor ligands, in an NF-κB-dependent manner. mmu-miR-7578 inhibited the release of pro-inflammatory cytokines, including TNFα and IL6, by regulating its target gene Egr1, which encodes a transcription factor that activates TNFα and NF-κB expression. Transgenic mice overexpressing mmu-miR-7578 displayed higher resistance to endotoxin shock and lower plasma levels of TNFα and IL6, indicating that this miRNA acted as a negative molecule of immune response. In sum, we report a previously uncharacterized LPS-responsive miRNA that controls inflammatory response in a feedback loop by fine-tuning a key transcription factor in vivo.

Based on previous findings that early growth response 1 (Egr-1) participates in leukocyte recruitment and cell proliferation in vitro, this study was designed to investigate its mode of action during arteriogenesis in vivo. In a model of peripheral arteriogenesis, Egr-1 was significantly upregulated in growing collaterals of wild-type (WT) mice, both on mRNA and protein level. Egr-1(-/-) mice demonstrated delayed arteriogenesis after femoral artery ligation. They further showed increased levels of monocytes and granulocytes in the circulation, but reduced levels in adductor muscles under baseline conditions. After femoral artery ligation, elevated numbers of macrophages were detected in the perivascular zone of collaterals in Egr-1(-/-) mice and mRNA of leukocyte recruitment mediators was upregulated. Other Egr family members (Egr-2 to -4) were significantly upregulated only in Egr-1(-/-) mice, suggesting a mechanism of counterbalancing Egr-1 deficiency. Moreover, splicing factor-1, downregulated in WT mice after femoral artery ligation in the process of increased vascular cell proliferation, was upregulated in Egr-1(-/-) mice. αSM-actin on the other hand, significantly downregulated in WT mice, showed no differential expression in Egr-1(-/-) mice. While cell cycle regulator cyclin E and cdc20 were upregulated in Egr-1(-/-) mice, cyclin D1 expression decreased below the detection limit in collaterals, and the proliferation marker ki67 was not differentially expressed. In conclusion, compensation for deficiency in Egr-1 function in leukocyte recruitment can presumably be mediated by other transcription factors; however, Egr-1 is indispensable for effective vascular cell cycle progression in arteriogenesis.

Interactions driven by the T cell antigen receptor (TCR) determine the lineage fate of CD4(+)CD8(+) thymocytes, but the molecular mechanisms that induce the lineage-determining transcription factors are unknown. Here we found that TCR-induced transcription factors Egr2 and Egr1 had higher and more-prolonged expression in precursors of the natural killer T (NKT) than in cells of conventional lineages. Chromatin immunoprecipitation followed by deep sequencing showed that Egr2 directly bound and activated the promoter of Zbtb16, which encodes the NKT lineage-specific transcription factor PLZF. Egr2 also bound the promoter of Il2rb, which encodes the interleukin 2 (IL-2) receptor β-chain, and controlled the responsiveness to IL-15, which signals the terminal differentiation of the NKT lineage. Thus, we propose that persistent higher expression of Egr2 specifies the early and late stages of NKT lineage differentiation, providing a discriminating mechanism that enables TCR signaling to 'instruct' a thymic lineage.

Modification/chlorination of high-density lipoprotein (HDL) by hypochlorous acid (HOCl), formed by the myeloperoxidase-H₂O₂-chloride system of activated phagocytes, converts an anti-atherogenic lipoprotein into a pro-inflammatory lipoprotein particle. Chlorinated HDL is present in human lesion material, binds to and is internalized by endothelial cells and impairs expression and activity of endothelial nitric oxide synthase. The present study aimed at clarifying whether exposure of endothelial cells to pro-inflammatory HOCl-HDL impacts on expression of heme oxygenase-1, a potential rescue pathway against endothelial dysfunction. Our findings revealed that HDL modified by HOCl, added as reagent or generated enzymatically, induced phosphorylation of p42/44 mitogen-activated protein kinase, expression of transcription factor early growth response-1 (Egr-1) and enhanced expression of heme oxygenase-1 in human endothelial cells. Upregulation of heme oxygenase-1 could be blocked by an inhibitor upstream of p42/44 mitogen-activated protein kinase and/or knockdown of Egr-1 by RNA-interference. Electrophoretic mobility shift assays demonstrated HOCl-HDL-mediated induction of the Egr-1 DNA binding activity. Immunocytochemical and immunoblotting experiments demonstrated HOCl-HDL-induced translocation of Egr-1 to the nucleus. The present study demonstrates a novel compensatory pathway against adverse effects of HOCl-HDL, providing cytoprotection in a number of pathological conditions including cardiovascular disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Influenza infection of the distal airways results in severe lung injury, a considerable portion of which is immunopathologic and attributable to the host responses. We have used a mouse model to specifically investigate the role of antiviral CD8(+) T cells in this injury, and have found that the critical effector molecule is TNF-alpha expressed by the T cells upon antigen recognition. Interestingly, the immunopathology which ensues is characterized by significant accumulation of host inflammatory cells, recruited by chemokines expressed by the target alveolar epithelial cells. In this study we analyzed the mechanisms involved in the induction of epithelial chemokine expression triggered by antigen-specific CD8(+) T cell recognition, and demonstrate that the early growth response-1 (Egr-1) transcription factor is rapidly induced in epithelial cells, both in vitro and ex vivo, and that this is a critical regulator of a host of inflammatory chemokines. Genetic deficiency of Egr-1 significantly abrogates both the chemokine expression and the immunopathologic injury associated with T cell recognition, and it directly regulates transcriptional activity of a model CXC chemokine, MIP-2. We further demonstrate that Egr-1 induction is triggered by TNF-alpha-dependent ERK activation, and inhibition of this pathway ablates Egr-1 expression. These findings suggest that Egr-1 may represent an important target in mitigating the immunopathology of severe influenza infection.

alphabeta and gammadelta T cells arise from a common thymocyte progenitor during development in the thymus. Emerging evidence suggests that the pre-T cell receptor (pre-TCR) and gammadelta T cell receptor (gammadeltaTCR) play instructional roles in specifying the alphabeta and gammadelta T-lineage fates, respectively. Nevertheless, the signaling pathways differentially engaged to specify fate and promote the development of these lineages remain poorly understood. Here, we show that differential activation of the extracellular signal-related kinase (ERK)-early growth response gene (Egr)-inhibitor of DNA binding 3 (Id3) pathway plays a defining role in this process. In particular, Id3 expression served to regulate adoption of the gammadelta fate. Moreover, Id3 was both necessary and sufficient to enable gammadelta-lineage cells to differentiate independently of Notch signaling and become competent IFNgamma-producing effectors. Taken together, these findings identify Id3 as a central player that controls both adoption of the gammadelta fate and its maturation in the thymus.

T-bet is a Th1-specific transcription factor that is directly involved in three important pathways for Th1 cell differentiation, namely TCR signaling, and the IFN-gamma-STAT1 and IL-12-STAT4 pathways. A recent study also showed that T-bet plays a vital role in innate immunity. However, the molecular mechanism responsible for transcriptional activation of T-bet during T cell development is not yet known. Here, we characterize the essential human T-bet promoter elements and show that binding of EGR1 to this promoter induces T-bet transcription. Notably, overexpression of EGR1 transactivates and, synergistically in concert with TCR signaling, induces T-bet expression in activated T cells. In contrast, depletion of EGR1 significantly decreases T-bet induction. Finally, we report a positive correlation between EGR1 and T-bet expression during T helper cell differentiation. Collectively, these findings provide molecular insight into T-bet transcription and suggest that EGR1 is an upstream regulator of T-bet induction.

Early growth response-1 (Egr-1) is expressed in human airways and found to modulate tumor necrosis factor, immunoglobulin E (IgE), airway responsiveness, and interleukin-13-induced inflammation in mice. We investigated the effects of Chinese-tagging single nucleotide polymorphisms (SNPs) of Egr-1 on asthma traits in 298 Chinese asthmatic children and 175 controls, and a replication community cohort of 191 controls. Tag SNP (-4071 A-->G) and three additional SNPs (-1427 C-->T, -151 C-->T and IVS1 -42 C-->T) were genotyped by restriction fragment length polymorphism (RFLP). Significant associations were found between plasma total IgE concentration and -4071 A-->G (p = 0.008) and IVS1 -42 C-->T (p = 0.027) in asthmatic patients. After Bonferroni correction, only -4071 A-->G showed significant association. Multivariate regression analysis confirmed this significant association with a standardized coefficient beta of 0.156 (95% CI: 0.046-0.317; p = 0.009) in asthmatics among the three SNPs with age and gender-adjusted. In -4071 A-->G, IgE(log) was significantly higher in patients with the GG genotype than the AA genotype (p = 0.009). In addition, -4071 A-->G was significantly associated with atopy (p = 0.016) and high total IgE concentration (p = 0.030) among asthmatics. Patients with the G allele had a 3.5-fold risk of having atopy and a 2.0-fold risk of having high total IgE concentration than those homozygous for the A allele. This is the first report to show significant association of Egr-1 polymorphisms with plasma total IgE and atopy in asthmatics. It may help to explore the pharmacogenetics of Egr-1 inhibitors.

The repertoire of TCR specificities is established by a selection process in the thymus, during which precursor survival and maturation is dictated by the nature of the TCR signals. The differences in signals that determine whether precursors will survive and mature or be induced to die remain poorly understood. Among the molecular effectors involved in executing the differentiation process initiated by TCR-ligand interactions is a family of Zn-finger transcription factors termed early growth response genes (Egr). Indeed, ablation of the Egr1 gene impairs ligand-induced maturation (positive selection) but not ligand-induced deletion (negative selection). The partial impairment of positive selection by Egr1 deficiency is not enhanced by simultaneous deletion of another Egr family member, Egr3. Accordingly, we asked whether this results from compensation by another family member, Egr2. In this manuscript, we demonstrate that deletion of Egr2 impairs positive selection of both CD4 and CD8 single-positive thymocytes. Interestingly, many of the genes involved in positive selection and T cell differentiation are up-regulated normally in the Egr2-deficient thymocytes. However, Bcl-2 up-regulation is not sustained during late stages of positive selection. This defect is at least partially responsible for the developmental blockade in Egr2-deficient thymocytes, as enforced expression of Bcl-2 rescues T cell development in Egr2(-/-) thymocytes. Taken together, these data suggest that Egr2 plays a central role in the up-regulation of the survival molecule Bcl-2 during positive selection.

Stem cell factor (SCF) is not only critical for mast cell development, but also an important mast cell functional regulator. However, roles of transcription factors involved in SCF-induced effects remain incompletely defined. Early growth response factor-1 (Egr-1) is a member of zinc-finger transcription factor family. Mouse bone marrow-derived mast cells (BMMC) were used to examine a role of Egr-1 in SCF-induced mast cell activation and growth. SCF induced a strong and rapid expression of Egr-1 mRNA as tested by real-time PCR analysis. SCF-induced Egr-1 nuclear translocation and DNA binding were demonstrated by electrophoretic mobility shift assay (EMSA) and immunofluorescence assay. To examine if Egr-1 is required for SCF-induced IL-13 expression, Egr-1-deficient BMMC were used. Levels of SCF-induced IL-13 mRNA and protein were reduced in Egr-1 deficient BMMC when compared with wild-type BMMC. Although Egr-1 is required for macrophage and lymphocyte development, SCF-induced mast cells growth was not affected by Egr-1 deficiency. Interestingly, SCF-induced Egr activation was blocked by a tyrosine kinase inhibitor PP2, suggesting a role of tyrosine phosphorylation in SCF-induced Egr-1 activation. Taken together, our results suggest that Egr-1 is required for SCF-induced IL-13 expression, but not mast cell growth.

The presence of multiple areas of hypoxia (low oxygen tension) is a hallmark feature of human and experimental tumours. Monocytes are continually recruited into tumours where they differentiate into tumour-associated macrophages (TAM) and often accumulate in hypoxic and/or necrotic areas. A number of recent studies have shown that macrophages respond to hypoxia by up-regulating transcription factors such as HIF-1alpha and HIF-2alpha, which in turn up-regulate the expression of a broad array of mitogenic, pro-invasive, pro-angiogenic and pro-metastatic genes. Here we show that primary human macrophages but not monocytes rapidly up-regulate HIF-1alpha and HIF-2alpha proteins upon exposure to hypoxia, and that these proteins then translocate to the nucleus. We also demonstrate differences in the temporal expression and responses to re-oxygenation for HIF-1alpha and HIF-2alpha in macrophages. Here we found that, compared to HIF-1alpha, HIF-2alpha expression was prolonged and persisted with re-oxygenation. ATF-4 and Egr-1 were also found to be hypoxia-responsive transcription factors in macrophages but not monocytes, but only early after exposure to hypoxia. Taken together, these findings indicate that a number of transcription factors work together in a tightly regulated fashion to control macrophage activities in ischaemic areas of diseased tissues.

Regulatory T cells (T(reg)) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and T(reg) development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, T(reg) development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4(+) cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3(+) and Foxp3(-) CD4(+) populations. In SAP-1-deficient CD4(+)CD25(+) T(reg) cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4(+)CD25(+) T(reg) cells to suppress CD4(+)CD25(-) cell proliferation in vitro. Finally the suppressive activity of CD4(+)CD25(+) T(reg) cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of T(reg) cells in the thymus are thus distinct from those required for "conventional" T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of T(reg) cells.

Upregulation of the activated Factor VII (FVIIa)/Tissue Factor complex, downregulation of natural anticoagulation pathways, and inhibition of fibrinolysis, are major contributors to coagulopathies associated with acute inflammation. Provision of FVIIa, and consequent downstream coagulation-related proteases, also stimulates further inflammatory changes, which can result in disseminated intravascular coagulation. Thus, the potential protective effects in vivo of a genetic-based reduction in FVII levels have been investigated in a murine model of acute inflammation, namely lipopolysaccharide (LPS)-induced lethal endotoxaemia. Mice with a total FVII deficiency do not survive the neonatal period. Therefore mice expressing low levels of FVII (FVII(tTA/tTA)), producing sufficient amounts of FVII for survival (approximately 5% of wild-type (WT) FVII), were employed to investigate in vivo pathways involved in the crosstalk between coagulation, inflammation, and survival, consequent to administration of a lethal dose of LPS. The FVII(tTA/tTA) mice presented with reduced mortality, coagulation, and inflammatory responses in comparison with similarly treated WT mice after administration of LPS. The attenuated inflammatory responses in FVII(tTA/tTA) mice were associated with downregulation of Egr-1 signalling. Administration, in vivo, of specific inhibitors of FXa and thrombin demonstrated that the inflammatory responses were unaltered in WT mice, but further reduced in FVII(tTA/tTA) mice. Therefore, a FVII deficiency enhances survival from lethal endotoxaemia both through attenuation of inflammatory responses that result directly from reduced FVIIa levels, and, indirectly, from downregulation of coagulation proteases downstream of the FVII-dependent cascade.

Carbon monoxide (CO) suppresses proinflammatory responses in macrophages reacting to LPS. We hypothesize that CO acts by inducing a molecule(s) that suppresses the inflammatory response to subsequent stress. Exposure of macrophages to CO alone in vitro produced a brief burst of mitochondrial-derived ROS, which led to expression of PPARgamma. PPARgamma expression proved essential for mediating the anti-inflammatory effects of CO. Blocking the CO-mediated increase in ROS generation prevented PPARgamma induction, and blocking PPARgamma prevented CO's anti-inflammatory effects. In a model of acute lung injury in mice, CO blocked expression of Egr-1, a central mediator of inflammation, and decreased tissue damage; inhibition of PPARgamma abrogated both effects. These data identify the mitochondrial oxidases as an (perhaps the) initial cellular target of CO and demonstrate that CO upregulates expression of PPARgamma via the mitochondria, which assures that a subsequent stress stimulus will lead to a cytoprotective as opposed to a proinflammatory phenotype.

The size of the thymus can be greatly influenced by changes in the small number of early progenitors in the thymus. However, it is not known whether thymic cellularity feeds back to regulate the recruitment, survival, and expansion of progenitors. The transcription factor early growth response gene 1 (Egr1) has been implicated in controlling proliferation and survival in many cell types. We have previously shown that mice deficient in Egr1 have increased thymic cellularity. We now show that Egr1 regulates a negative feedback signal that controls the entry of cells into the thymus. Egr1-deficient mice have higher percentages of early T lineage progenitors in the thymus, yet Egr1-deficient mice have normal numbers of myelolymphoid progenitors in the bone marrow, and Egr1-deficient thymocytes show normal rates of apoptosis and proliferation at all stages of development. Evidence from mixed bone marrow chimeras shows that the ability of Egr1 to control progenitor recruitment is mediated by bone marrow-derived cells, but is not cell autonomous. Furthermore, Egr1-deficient thymuses have increased P-selectin expression. The data suggest that Egr1 mediates a feedback mechanism whereby the number of resident double negative thymocytes controls the entry of new progenitors into the thymus by regulating P-selectin expression on thymic endothelial cells.

Early growth-response factor 1 (Egr-1) is a zinc-finger transcription factor that plays a regulatory role in the expression of many genes important for inflammation. Whether Egr-1 is involved in IgE-dependent mast-cell activation was investigated. We demonstrated that IgE and antigen (TNP) stimulation induced a rapid expression of Egr-1 mRNA in mouse bone marrow-derived mast cells (BMMCs). As early as 15 to 20 minutes after IgE + TNP stimulation, Egr-1 protein was detectable in the nucleus of BMMCs by immunofluorescence or electrophoretic mobility shift assay. To examine a role for Egr-1 in IgE-dependent cytokine production by mast cells, Egr-1-deficient (Egr-1-/-) BMMCs were developed from the bone marrow cells of Egr-1 knockout mice. Egr-1-/- BMMCs express similar levels of surface c-kit and IgE receptor as compared with those on Egr-1+/+ BMMCs. Importantly, IgE + TNP-induced TNF and IL-13 expression was significantly reduced at both mRNA and protein levels in Egr-1-/- BMMCs as compared with those in Egr-1+/+ BMMCs. Thus, our results suggest that de novo synthesis of Egr-1 represents a novel mechanism in FcepsilonRI signaling and is required for the full responsiveness of IgE-dependent TNF and IL-13 production by mast cells.