Project description:An unresolved molecular paradox is how the glucocorticoid receptor (GR) activates some genes while potently repressing others. We carried out genome-wide localization and expression profiling experiments in primary bone marrow-derived mouse macrophages treated with Dexamethasone in the presence or absence of LPS. Unexpectedly, we find that the anti-inflammatory GR cistrome, which is principally composed of 'canonical' GREs colocalizing with NFkB and AP-1 co-enriched with the myeloid lineage factors C/EBP and Pu.1, is shaped by TLR4-directed chromatin dynamics, suggesting that context rather than sequence may be a critical determinant of function. Identification of GR, cJun, NFkB(p65) binding sites in primary bone-marrow derived macrophages unstimulated and LPS-stimulated (3hrs) that were untreated or pre-treated with Dexamethasone for 16 hrs
Project description:To identify cross-talk between the cytoplasmic kinase AMPK and the nuclear receptor, glucocorticoid receptor, during inflammatory responses, bone-marrow-derived macrophages lacking the catalytic domain of AMPK, AMPKa1, were exposed to vehicle, dexamethasone, a synthetic glucocorticoid, lipopolysaccharide, crushed muscle extract (damage-associated molecular patterns) or a combination of the inflammatory stimulus and dexamethasone.
Project description:We performed Brg1 (Smarac4) knock-down by siRNA in murine primary bone marrow-derived macrophages and profiled the effect on gene expression by RNAseq after lipopolysaccharide (LPS) or LPS plus dexamethasone (Dex) treatment. Brg1 knock-down cells are compared to cells treated with a control siRNA. We analysed the requirement of Brg1/Smarca4 for the expression of inflammatory and glucocorticoid receptor target genes.
Project description:To identify cross-talk between the transcription factors, glucocorticoid receptor and FOXO3 after activation of the glucocorticoid receptor with dexamethasone.Bone marrow derived macrophages lacking FOXO3 were exposed to vehicle, dexamethasone, a synthetic glucocorticoid.
Project description:An unresolved molecular paradox is how the glucocorticoid receptor (GR) activates some genes while potently repressing others. We carried out genome-wide localization and expression profiling experiments in primary bone marrow-derived mouse macrophages treated with Dexamethasone in the presence or absence of LPS. Unexpectedly, we find that the anti-inflammatory GR cistrome, which is principally composed of ‘canonical’ GREs colocalizing with NF-κB and AP-1 co-enriched with the myeloid lineage factors C/EBP and Pu.1, is shaped by TLR4-directed chromatin dynamics, suggesting that context rather than sequence may be a critical determinant of function.
Project description:An unresolved molecular paradox is how the glucocorticoid receptor (GR) activates some genes while potently repressing others. We carried out genome-wide localization and expression profiling experiments in primary bone marrow-derived mouse macrophages treated with Dexamethasone in the presence or absence of LPS. Unexpectedly, we find that the anti-inflammatory GR cistrome, which is principally composed of 'canonical' GREs colocalizing with NFkB and AP-1 co-enriched with the myeloid lineage factors C/EBP and Pu.1, is shaped by TLR4-directed chromatin dynamics, suggesting that context rather than sequence may be a critical determinant of function.
Project description:Macrophages are amongst the major targets of glucocorticoids (GC) as therapeutic anti-inflammatory agents. Here we show that GC treatment of mouse and human macrophages initiates a cascade of induced gene expression including many anti-inflammatory genes. Inducible binding of the glucocorticoid receptor (GR) was detected at candidate enhancers in the vicinity of induced genes in both species and this was strongly associated with canonical GR binding motifs. However, the sets of inducible genes, the candidate enhancers, and the GR motifs within them, were highly-divergent between the two species. Mouse bone marrow derived macrophages were generated from two male 10 week old C57BL/6 mice, treated with dexamethsone 100nM or vehicle and glucocorticoid receptor bound DNA extracted by chromatin immunoprecipitation
Project description:Glucocorticoids are widely used to treat inflammatory disorders. Prolonged use results in side effects including osteoporosis, diabetes and obesity. The selective glucocorticoid receptor (GR) modulator Compound A (CpdA) exhibits an inflammation-suppressive effect, largely in absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated using an unbiased proteomics approach. We found that the autophagy receptor p62 but not GR mediates the anti-inflammatory action of CpdA in macrophages. CpdA drives the upregulation of p62 by recruiting the NRF2 transcription factor to its promoter. Contrarily, the classic GR ligand dexamethasone recruits GR to p62 and other NRF2 controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA are able to induce autophagy, albeit in a cell-type and time-dependent manner. Suppression of LPS-induced IL-6 and MCP1 genes in bone marrow-derived macrophages by CpdA is hampered upon p62 silencing, confirming that p62 is essential for the anti-inflammatory capacity of CpdA. Together, these results demonstrate how off-target mechanisms of selective GR ligands may establish a more efficient anti-inflammatory therapy
Project description:Glucocorticoids are widely used to treat inflammatory disorders; however, prolonged use of glucocorticoids results in side effects including osteoporosis, diabetes and obesity. Compound A (CpdA), identified as a selective NR3C1/glucocorticoid receptor (nuclear receptor subfamily 3, group C, member 1) modulator, exhibits an inflammation-suppressive effect, largely in the absence of detrimental side effects. To understand the mechanistic differences between the classic glucocorticoid dexamethasone (DEX) and CpdA, we looked for proteins oppositely regulated in bone marrow-derived macrophages using an unbiased proteomics approach. We found that the autophagy receptor SQSTM1 but not NR3C1 mediates the anti-inflammatory action of CpdA. CpdA drives SQSTM1 upregulation by recruiting the NFE2L2 transcription factor to its promoter. In contrast, the classic NR3C1 ligand dexamethasone recruits NR3C1 to the Sqstm1 promoter and other NFE2L2-controlled gene promoters, resulting in gene downregulation. Both DEX and CpdA induce autophagy, with marked different autophagy characteristics and morphology. Suppression of LPS-induced Il6 and Ccl2 genes by CpdA in macrophages is hampered upon Sqstm1 silencing, confirming that SQSTM1 is essential for the anti-inflammatory capacity of CpdA, at least in this cell type. Together, these results demonstrate how off-target mechanisms of selective NR3C1 ligands may contribute to a more efficient anti-inflammatory therapy.