Project description:Here we have analyzed the role of interferon regulatory factor-2 binding protein-2 (IRF2BP2) in glucocorticoid and tumor necrosis factor alpha (TNF) signaling. We used ChIP-seq to analyze chromatin binding of IRF2BP2 in glucocorticoid (dexamethasone, dex) and vehicle treated HEK293 cells expressing GR (HEK293-GR). Furthermore, we used RNA-seq to analyze how silencing of IRF2BP2 modulates transcriptional responses to dex treatment in HEK293-GR cells, and dex, TNF and co-treatment (dex and TNF, DT) in A549 cells. Overall design: For ChIP-seq, HEK293-GR cells were grown on steroid-depleted medium for two days and treated with dex or vehicle for one hour. Cells were fixed with 1% (v/v) formaldehyde, chromatin was fragmented to ~200-400 bp by sonication, chromatin immunoprecipitation was done using anti-IRF2BP2 antibody and libraries were prepared using NEBNext Ultra II kit (New England Biolabs). Two biological replicates of both conditions were sequenced using Illumina HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany). For RNA-seq, HEK293-GR or A549 cells were cultured in steroid-depleted media and transfected with siRNA against IRF2BP2 (siIRF2BP2, 20nM, Dharmacon, ON-TARGETplus SMARTpool, L-007177-02-0005) or control siRNA (siNON, Dharmacon, non-targeting pool) using Lipofectamine RNAiMAX (Invitrogen). After three days, cells were treated with dexamethasone (dex, 100 nM), Tumor Necrosis Factor alpha (TNF, 10 ng/ml), or vehicle (EtOH, equal amount of ethanol) for six hours. RNA from three biological replicates was extracted using RNeasy Plus Mini kit (Qiagen) and mRNA isolated using NEBNext Poly(a) mRNA Magnetic Isolation Module (New England Biolabs). Libraries were prepared using NEBNext Ultra II kit (New England Biolabs) and sequenced with HiSeq 2000 at EMBL GeneCore (Heidelberg, Germany).
Project description:In the last decades in vitro studies highlighted the potential for crosstalk between Hypoxia-Inducible Factor-(HIF) and glucocorticoid-(GC) signalling pathways. However, how this interplay precisely occurs in vivo is still debated. Here, we use zebrafish larvae (Danio rerio) to elucidate how and to what degree hypoxic signalling affects the endogenous glucocorticoid pathway and vice versa, in vivo. Firstly, our results demonstrate that in the presence of upregulated HIF signalling, both glucocorticoid receptor (Gr) responsiveness and endogenous cortisol levels are repressed in 5 days post fertilisation larvae. In addition, despite HIF activity being low at normoxia, our data show that it already impedes both glucocorticoid activity and levels. Secondly, we further analysed the in vivo contribution of glucocorticoids to HIF activity. Interestingly, our results show that both glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) play a key role in enhancing it. Finally, we found indications that glucocorticoids promote HIF signalling via multiple routes. Cumulatively, our findings allowed us to suggest a model for how this crosstalk occurs in vivo.
Project description:p38 MAPK inhibition may have additive and synergistic anti-inflammatory effects when used with corticosteroids. We investigated crosstalk between p38 MAPK inhibitors and corticosteroids in bronchial epithelial cells to investigate synergistic effects on cytokine production and the molecular mechanisms involved. Effects of the p38 MAPK inhibitor BIRB-796 and dexamethasone alone and in combination on LPS, polyI:C or TNF? -induced IL-6, CXCL8 and RANTES were assessed in 16HBEs (human epithelial cell line) and on TNF?-induced IL-6 and CXCL8 in primary human epithelial cells from asthma patients and healthy controls. 16HBEs were used to assess effects of BIRB-796 alone and in combination with dexamethasone on glucocorticoid receptor (GR) activity by reporter gene assay, expression of GR target genes and nuclear localisation using Western blot. The effects of BIRB-796 on TNF? stimulated phosphorylation of p38 MAPK and GR at serine (S) 226 by Western blot. Epithelial levels of phosphorylated p38 MAPK and GR S226 were determined by immunohistochemistry in bronchial biopsies from asthma patients and healthy controls. BIRB-796 in combination with dexamethasone increased inhibition of cytokine production in a synergistic manner. Combination treatment significantly increased GR nuclear localisation compared to dexamethasone alone. BIRB-796 inhibited TNF?-induced p38 MAPK and GR S226 phosphorylation. Phosphorylated GR S226 and p38 MAPK levels were increased in bronchial epithelium of more severe asthma patients. Molecular crosstalk exists between p38 MAPK activation and GR function in human bronchial epithelial cells, which alters GR activity. Combining a p38 MAPK inhibitor and a corticosteroid may demonstrate therapeutic potential in severe asthma. KEY MESSAGES: • Combination of corticosteroid and p38 inhibitor in human bronchial epithelial cells • Combination increased cytokine inhibition synergistically and nuclear GR • p38 MAPK inhibition reduced TNF?-induced phosphorylation of GR at S226 but not S211 • Phosphorylated GRS226 and p38 is increased in bronchial epithelium in severe asthma • Combining a p38 inhibitor and a corticosteroid may be effective in asthma treatment.
Project description:Bone marrow is a reservoir for regulatory T (T(reg)) cells, but how T(reg) cells are regulated in that environment remains poorly understood. We show that expression of glucocorticoid (GC)-induced leucine zipper (GILZ) in bone marrow mesenchymal lineage cells or bone marrow-derived mesenchymal stem cells (BMSCs) increases the production of T(reg) cells via a mechanism involving the up-regulation of developmental endothelial locus-1 (Del-1), an endogenous leukocyte-endothelial adhesion inhibitor. We found that the expression of Del-1 is increased ∼4-fold in the bone tissues of GILZ transgenic (Tg) mice, and this increase is coupled with a significant increase in the production of IL-10 (2.80 vs. 0.83) and decrease in the production of IL-6 (0.80 vs. 2.33) and IL-12 (0.25 vs. 1.67). We also show that GILZ-expressing BMSCs present antigen in a way that favors T(reg) cells. These results indicate that GILZ plays a critical role mediating the crosstalk between BMSCs and T(reg) in the bone marrow microenvironment. These data, together with our previous findings that overexpression of GILZ in BMSCs antagonizes TNF-α-elicited inflammatory responses, suggest that GILZ plays important roles in bone-immune cell communication and BMSC immune suppressive functions.
Project description:When modeling cell signaling networks, a balance must be struck between mechanistic detail and ease of interpretation. In this paper we apply a fuzzy logic framework to the analysis of a large, systematic dataset describing the dynamics of cell signaling downstream of TNF, EGF, and insulin receptors in human colon carcinoma cells. Simulations based on fuzzy logic recapitulate most features of the data and generate several predictions involving pathway crosstalk and regulation. We uncover a relationship between MK2 and ERK pathways that might account for the previously identified pro-survival influence of MK2. We also find unexpected inhibition of IKK following EGF treatment, possibly due to down-regulation of autocrine signaling. More generally, fuzzy logic models are flexible, able to incorporate qualitative and noisy data, and powerful enough to produce quantitative predictions and new biological insights about the operation of signaling networks.
Project description:Glucocorticoids acting through the glucocorticoid receptor (GR) inhibit TNF-induced lethal inflammation. Here, we demonstrate that GR dimerization plays a role in reducing TNF sensitivity. In mutant mice unable to dimerize GR, we found that TNF failed to induce MAPK phosphatase 1 (MKP1). We assessed TNF sensitivity in Mkp1(-/-) mice and found increased inflammatory gene induction in livers, increased circulating cytokines, cell death in intestinal epithelium, severe intestinal inflammation, hypothermia, and death. Mkp1(-/-) mice had increased levels of phosphorylated JNK, which promotes apoptosis, in liver tissue. We further examined JNK-deficient mice for their response to TNF. Although Jnk1(-/-) mice showed no change in sensitivity to TNF, Jnk2(-/-) mice were significantly protected against TNF, identifying JNK2 as an essential player in inflammation induced by TNF. Furthermore, we found that loss of Jnk2 partially rescued the increased sensitivity of Mkp1(-/-) and mutant GR mice to TNF. Our data show that GR dimerization inhibits JNK2 through MKP1 and protects from TNF-induced apoptosis and lethal inflammation.
Project description:Muscle atrophy is a consequence of chronic diseases (e.g., diabetes) and glucocorticoid-induced insulin resistance that results from enhanced activity of the ubiquitin-proteasome pathway. The PI3K/Akt pathway inhibits the FOXO-mediated transcription of the muscle-specific E3 ligase atrogin-1/MAFbx (AT-1), whereas the MEK/ERK pathway increases Sp1 activity and ubiquitin (UbC) expression. The observations raise a question about how the transcription of these atrogenes is synchronized in atrophic muscle. We tested a signaling model in which FOXO3a mediates crosstalk between the PI3K/Akt and MEK/ERK pathways to coordinate AT-1 and UbC expression. In rat L6 myotubes, dexamethasone (> or = 24 h) reduced insulin receptor substrate (IRS)-1 protein and PI3K/Akt signaling and increased AT-1 mRNA. IRS-2 protein, MEK/ERK signaling, Sp1 phosphorylation, and UbC transcription were simultaneously increased. Knockdown of IRS-1 using small interfering RNA or adenovirus-mediated expression of constitutively activated FOXO3a increased IRS-2 protein, MEK/ERK signaling, and UbC expression. Changes in PI3K/Akt and MEK/ERK signaling were recapitulated in rat muscles undergoing atrophy due to streptozotocin-induced insulin deficiency and concurrently elevated glucocorticoid production. IRS-1 and Akt phosphorylation were decreased, whereas MEK/ERK signaling and expression of IRS-2, UbC and AT-1 were increased. We conclude that FOXO3a mediates a reciprocal communication between the IRS-1/PI3K/Akt and IRS-2/MEK/ERK pathways that coordinates AT-1 and ubiquitin expression during muscle atrophy.
Project description:Signal transduction networks coordinate transcriptional programs activated by diverse extracellular stimuli, such as growth factors and cytokines. Cells receive multiple stimuli simultaneously, and mapping how activation of the integrated signaling network affects gene expression is a challenge. We stimulated colon adenocarcinoma cells with various combinations of the cytokine tumor necrosis factor (TNF) and the growth factors insulin and epidermal growth factor (EGF) to investigate signal integration and transcriptional crosstalk. We quantitatively linked the proteomic and transcriptomic data sets by implementing a structured computational approach called tensor partial least squares regression. This statistical model accurately predicted transcriptional signatures from signaling arising from single and combined stimuli and also predicted time-dependent contributions of signaling events. Specifically, the model predicted that an early-phase, AKT-associated signal downstream of insulin repressed a set of transcripts induced by TNF. Through bioinformatics and cell-based experiments, we identified the AKT-repressed signal as glycogen synthase kinase 3 (GSK3)-catalyzed phosphorylation of Ser(37) on the long form of the transcription factor GATA6. Phosphorylation of GATA6 on Ser(37) promoted its degradation, thereby preventing GATA6 from repressing transcripts that are induced by TNF and attenuated by insulin. Our analysis showed that predictive tensor modeling of proteomic and transcriptomic data sets can uncover pathway crosstalk that produces specific patterns of gene expression in cells receiving multiple stimuli.
Project description:Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor ?B (NF-?B) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-?B p65 response to Tumor Necrosis Factor ? (TNF?) treatment. The degree of inhibition of the NF-?B response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-?B responses while being consistent with the observed population-level responses. In particular "all-or-nothing" encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and I?B? phosphorylation, while others involving transport were "analogue". In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-?B p65 subunit. The single cell responses exhibited "all-or-nothing" encoding. While most cells did not respond to TNF? stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKK? and IKK? subunits in whole cell lysates. However, a combination of "all-or-nothing" crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-?B responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.