Analysis of Il36a induction by C/EBPβ via a half-CRE•C/EBP element in murine macrophages in dependence of its CpG methylation level.
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ABSTRACT:
SUBMITTER: Nerlich A
PROVIDER: S-EPMC8674125 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:DNA methylation of the cytosine in the CpG dinucleotide is typically associated with gene silencing. Genomic analyses have identified low CpG promoters that are both methylated and transcriptionally active, but the mechanism underlying the activation of these methylated promoters remains unclear. Here we show that CpG methylation of the CRE sequence (TGACGTCA) enhances the DNA binding of the C/EBP? transcription factor, a protein critical for activation of differentiation in various cell types. Transfection assays also show that C/EBP? activates the CRE sequence only when it is methylated. The biological significance of this observation was seen in differentiating primary keratinocyte cultures from newborn mice where certain methylated promoters are both bound by C/EBP? and activated upon differentiation. Experimental demethylation by either 5-azacytidine treatment or DNMT1 depletion diminished both C/EBP? binding and activation of the same methylated promoters upon differentiation suggesting that CpG methylation can localize C/EBP?. Transfection studies in cell cultures using methylated tissue-specific proximal promoters identified half-CRE (CGTCA) and half-C/EBP (CGCAA) sequences that need to be methylated for C/EBP? mediated activation. In primary dermal fibroblasts, C/EBP? activates a different set of methylated tissue-specific promoters upon differentiation into adipocytes. These data identify a new function for methyl CpGs: producing DNA binding sites at half-CRE and half-C/EBP sequences for C/EBP? that are needed to activate tissue-specific genes.
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Project description:The concept of macrophage polarization toward different phenotypes after CNS injury has been increasingly discussed. Here, we propose that CD200 treatment may help shift pro-inflammatory macrophages to an arginase 1 (Arg1)-, transglutaminase 2 (TGM2)-, and transforming growth factor beta 1 (TGF-β)-positive phenotype. Rat macrophages were stimulated by interferon γ and lipopolysaccharide (LPS) to induce pro-inflammatory phenotypes. Treatment with human CD200-Fc up-regulated expression levels of alternatively activated M2-like markers such as Arg1 and TGM2 but suppressed pro-inflammatory M1-like markers such as toll-like receptor 4, interleukin 1 beta (IL-1β), IL-6, and GM-CSF. Concomitantly, CD200-Fc enhanced (CCAAT/enhancer-binding protein) C/EBP-beta promoter activity, whereas NF-κB activity was suppressed. Treatment with CD200-Fc also up-regulated potentially beneficial TGF-β expression in macrophages. When C/EBP-beta signaling was suppressed with siRNA, the effect of CD200-Fc on Arg1, TGM2 and TGF-β up-regulation was canceled. Taken together, these data provide proof-of-principle that targeting CD200 signaling may be a novel therapeutic approach to shift macrophages toward M2-like polarization via modulating cAMP-response element binding protein-C/EBP-beta transcriptional activity. We showed that CD200 treatment decreased pro-inflammatory cytokines (IL-1β, IL-6, and GM-CSF) along with suppressed inflammatory NF-κB activity in pro-inflammatory Mφ. On the other hand, CD200 increased Arg1, TGM2, and TGF-β production through CREB-C/EBPβ signaling. We think that these findings provide proof-of-concept that CD200 signaling may play a key role in regulating macrophage polarization toward anti-inflammatory phenotypes.
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Project description:Macrophages and dendritic cells (DC) are distributed throughout the body and play important roles in pathogen detection and tissue homeostasis. In tissues, resident macrophages exhibit distinct phenotypes and activities, yet the transcriptional pathways that specify tissue-specific macrophages are largely unknown. We investigated the functions and origins of two peritoneal macrophage populations in mice: small and large peritoneal macrophages (SPM and LPM, respectively). SPM and LPM differ in their ability to phagocytose apoptotic cells, as well as in the production of cytokines in response to LPS. In steady-state conditions, SPM are sustained by circulating precursors, whereas LPM are maintained independently of hematopoiesis; however, both populations are replenished by bone marrow precursors following radiation injury. Transcription factor analysis revealed that SPM and LPM express abundant CCAAT/enhancer binding protein (C/EBP)-?. Cebpb(-/-) mice exhibit elevated numbers of SPM-like cells but lack functional LPM. Alveolar macrophages are also missing in Cebpb(-/-) mice, although macrophage populations in the spleen, kidney, skin, mesenteric lymph nodes, and liver are normal. Adoptive transfer of SPM into Cebpb(-/-) mice results in SPM differentiation into LPM, yet donor SPM do not generate LPM after transfer into C/EBP?-sufficient mice, suggesting that endogenous LPM inhibit differentiation by SPM. We conclude that C/EBP? plays an intrinsic, tissue-restricted role in the generation of resident macrophages.
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Project description:Our previous work has provided strong evidence that the proteasome is central to most of the genes induced in mouse macrophages in response to LPS stimulation. In the studies presented here, we evaluated the role of the macrophage proteasome in response to a second microbial product CpG DNA (unmethylated bacterial DNA). For these studies, we applied Affymetrix microarray analysis of RNA derived from murine macrophages stimulated with CpG DNA in the presence or absence of proteasome inhibitor, lactacystin. The results of these studies revealed that similar to LPS, most of those macrophage genes regulated by CpG DNA are also under the control of the proteasome at 4 h. In contrast to LPS stimulation, however, many of these genes were induced much later than 4 h, at 18 h, in response to CpG DNA. Lactacystin treatment of macrophages completely blocked the CpG DNA-induced gene expression of TNF-? and other genes involved in the production of inflammatory mediators. These data strongly support the conclusion that similar to LPS, the macrophage proteasome is a key regulator of CpG DNA-induced signaling pathways.
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Project description:Generally, knowledge of the mechanism regulating gene expression in primary spermatocytes is incomplete. We have used the lactate dehydrogenase gene (Ldhc) as a model to explore these mechanisms during spermatogenesis. Its 100-bp core promoter contained two essential elements common to many genes, a GC box and a CRE site. Here we report results that support a model in which transcription factor MYBL1 acts as a coactivator directing tissue-specific expression via the CRE cis element. We hypothesize that this is a common mechanism involving activation of multiple genes in the primary spermatocyte. MYBL1 is expressed predominantly as a tissue-specific transcription factor in spermatocytes and breast epithelial cells. Our finding that LDHC expression is lost in 21-day testes of MYBL1 mutant mice supports our hypothesis. In the GC1-spg germ cell line exogenous MYBL1 induces activity 4- to 8-fold, although extracts from these cells do not show MYBL1 binding activity for the Myb consensus sequences in the Ldhc promoter by EMSA. Rather, MYBL1 stimulates expression from a synthetic promoter containing only CRE elements, suggesting MYBL1 activates the promoter by interacting with protein that binds to a CRE element. Mutation of three Myb sites does not affect Ldhc promoter activity significantly (P > 0.05). CREB-binding protein (CBP) is a coactivator that interacts with CRE-binding protein CREB. We show that the transactivation domain (TAD) in MYBL1 interacts with the KIX domain in CBP, and the TAD domain and DNA binding domain in MYBL1 each interact with the CREB N-terminal domain. MYBL1 also stimulated expression from testis-specific genes Pgk2 (phosphoglycerate kinase 2) and Pdha2 (pyruvate dehydrogenase alpha 2) promoters, each of which contains CRE promoter elements and is expressed in primary spermatocytes. We propose that MYBL1 directs germ cell-specific activation via the CRE site of certain genes that are activated specifically in the primary spermatocyte, although other, more indirect effects of MYBL1 remain a possible explanation for our results.
| S-EPMC3290662 | biostudies-literature
Project description:Specific bacterial lipopolysaccharides (LPS), IFN-gamma, and unmethylated cytosine or guanosine-phosphorothioate containing DNAs (CpG) activate host immunity, influencing infectious responses. Macrophages detect, inactivate and destroy infectious particles, and synthetic CpG sequences invoke similar responses of the innate immune system. Previously, murine macrophage J774 cells treated with CpG induced the expression of nitric oxide synthase 2 (NOS2) and cyclo-oxygenase 2 (COX2) mRNA and protein. In this study murine J774 macrophages were exposed to vehicle, interferon gamma+lipopolysaccharide (IFN-g/LPS), non-CpG (SAK1), or two-CpG sequence-containing DNA (SAK2) for 0-18h and gene expression changes measured. A large number of immunostimulatory and inflammatory changes were observed. SAK2 was a stronger activator of TNFalpha- and chemokine expression-related changes than LPS/IFN-g. Up regulation included tumor necrosis factor receptor superfamily genes (TNFRSF's), IL-1 receptor signaling via stress-activated protein kinase (SAPK), NF-kappaB activation, hemopoietic maturation factors and sonic hedgehog/wingless integration site (SHH/Wnt) pathway genes. Genes of the TGF-beta pathway were down regulated. In contrast, LPS/IFN-g-treated cells showed increased levels for TGF-beta signaling genes, which may be linked to the observed up regulation of numerous collagens and down regulation of Wnt pathway genes. SAK1 produced distinct changes from LPS/IFN-g or SAK2. Therefore, J774 macrophages recognize LPS/IFN-g, non-CpG DNA or two-CpG DNA-containing sequences as immunologically distinct.
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Project description:The Cre/loxP system is a widely applied technology for site-specific genetic manipulation in mice. This system allows for deletion of the genes of interest in specific cells, tissues, and whole organism to generate a diversity of conditional knockout mouse strains. Additionally, the Cre/loxP system is useful for development of cell- and tissue-specific reporter mice for lineage tracing, and cell-specific conditional depletion models in mice. Recently, the Cre/loxP technique was extensively adopted to characterize the monocyte/macrophage biology in mouse models. Compared to other relatively homogenous immune cell types such as neutrophils, mast cells, and basophils, monocytes/macrophages represent a highly heterogeneous population which lack specific markers or transcriptional factors. Though great efforts have been made toward establishing macrophage-specific Cre driver mice in the past decade, all of the current available strains are not perfect with regard to their depletion efficiency and targeting specificity for endogenous macrophages. Here we overview the commonly used Cre driver mouse strains targeting macrophages and discuss their major applications and limitations.
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Project description:Periodontitis is characterized by the progressive destruction of tooth-supporting alveolar bone, which is mainly caused by chronic inflammation in response to persistent bacterial insult. It has recently become clear that the pathogenesis of periodontitis is associated with a high ratio of proinflammatory M1 (classically activated) macrophages to anti-inflammatory M2 (alternatively activated). To decrease the inflammatory activity, we locally delivered the C-C motif chemokine ligand 2 (CCL2) using controlled-release microparticles (MPs). CCL2 is known to promote chemotaxis of M0 or M2 phenotype macrophages to the inflamed site and induce M2 phenotype polarization locally. Our in vitro data showed that CCL2 increased the number of M2 phenotype macrophages, decreased TNF-? secretion, and enhanced chemotaxis of RAW264.7 cells toward CCL2 MPs. Moreover, we induced periodontal disease in 2 animal models through inoculation of Porphyromonas gingivalis and ligature around the murine molar. Micro-computed tomography analysis showed significant reduction of alveolar bone loss in the CCL2 MP treatment group when compared with a blank MP group and a no-treatment periodontitis group in both models. Immunohistologic analysis showed a significant increase in the M2 phenotype subset and a decrease in the M1 phenotype subset in the CCL2 MP group of the P. gingivalis-induced model. Also, in both models, tartrate-resistant acidic phosphatase staining showed significantly fewer numbers of osteoclasts in the CCL2 MP group in alveolar bone area. Moreover, quantitative polymerase chain reaction results showed a significant increase in IL-1RA (interleukin 1 receptor antagonist) mRNA expression and a decrease in RANKL (receptor activator of nuclear factor kappa-? ligand) mRNA expression in the CCL2 MP group in the ligature model. In summary, manipulation of endogenous M2 phenotype macrophages with CCL2 MPs decreased the M1 phenotype:M2 phenotype ratio and prevented alveolar bone loss in mouse periodontitis models. The delivery of CCL2 MPs provides a novel approach to treat periodontal disease.
| S-EPMC6761736 | biostudies-literature
Project description:Transcription factors C/EBP? and C/EBP? are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor ? (PPAR?) and C/EBP? and are required for adipogenesis in vivo However, the mechanism that controls the induction of C/EBP? and C/EBP? remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPAR? and C/EBP? during adipogenesis. Here, we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated glucocorticoid receptor to directly control the induction of C/EBP? and C/EBP? in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBP? and C/EBP? and leads to severe defects in adipogenesis. Adipogenesis defects in PAGR1-deficient cells can be rescued by the ectopic expression of C/EBP? or PPAR?. Finally, the deletion of Pagr1 in Myf5+ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBP? and C/EBP?, PAGR1 plays a critical role in adipogenesis.
| S-EPMC7431048 | biostudies-literature
Project description:ObjectiveTamoxifen is widely used for inducible Cre-LoxP systems but has several undesirable side effects for researchers investigating metabolism or energy balance, including weight loss, lipoatrophy, and drug incorporation into lipid stores. For this reason, we sought to determine whether a doxycycline-inducible system would be more advantageous for adipocyte-specific Cre mouse models, but serendipitously discovered widespread ectopic tetracycline response element Cre (TRE-Cre) recombinase activity.MethodsAdipocyte-specific tamoxifen- and doxycycline-inducible Cre mice were crossed to fluorescent Cre reporter mice and visualized by confocal microscopy to assess efficiency and background activity. TRE-Cre mice were crossed to stop-floxed diphtheria toxin mice to selectively ablate cells with background Cre activity.ResultsTamoxifen- and doxycycline-inducible systems performed similarly in adipose tissues, but ectopic Cre recombination was evident in numerous other cell types of the latter, most notably neurons. The source of ectopic Cre activity was isolated to the TRE-Cre transgene, driven by the pTet (tetO7) tetracycline-inducible promoter. Ablation of cells with ectopic recombination in mice led to stunted growth, diminished survival, and reduced brain mass.ConclusionsThese results indicate that tamoxifen- and doxycycline-inducible adipocyte-specific Cre mouse models are similarly efficient, but the TRE-Cre component of the latter is inherently leaky. TRE-Cre background activity is especially pronounced in the brain and peripheral nerve fibers, and selective ablation of these cells impairs mouse development and survival. Caution should be taken when pairing TRE-Cre with floxed alleles that have defined roles in neural function, and additional controls should be included when using this model system.
| S-EPMC9170755 | biostudies-literature