MDFIC alters the GR transcriptome in A549 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE86115 | GEO | 2017/04/07
SECONDARY ACCESSION(S): PRJNA340285
REPOSITORIES: GEO
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Project description:Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules and are widely used in the clinic. The cellular response to glucocorticoids is remarkably diverse; however, the molecular factors that govern tissue specificity are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast 2 hybrid assay using as bait the hinge region of GR. The MyoD family inhibitor domain-containing (MDFIC) protein was identified as a binding partner for GR. Knockdown of MDFIC in A549 cells alters the GR transcriptome. To further investigate the functional effect of MDFIC on the GR transcriptome, we performed a genome-wide microarray in COS-1 cells that were transfected with GR alone or GR and MDFIC. The transfected cells were treated with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. Overexpression of MDFIC was found to expand the GR transcriptome.
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Project description:Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules and are widely used in the clinic. The cellular response to glucocorticoids is remarkably diverse; however, the molecular factors that govern tissue specificity are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast 2 hybrid assay using as bait the hinge region of GR. The MyoD family inhibitor domain-containing (MDFIC) protein was identified as a binding partner for GR. Knockdown of MDFIC in A549 cells alters the GR transcriptome. Overexpression of MDFIC with GR in COS-1 cells also modulates the GR transcriptome by expanding the number of genes regulated in response to glucocorticoid treatment. Our findings in A549 cells suggest that MDFIC alters the gene regulatory profile of GR by modulating receptor phosphorylation at several residues, including S211. To further investigate the molecular link between MDFIC-mediated effects on GR phosphorylation at S211 and alterations in the GR transcriptome, we performed a genome-wide microarray in COS-1 cells that were transfected with the GR phosphorylation mutant S211A or S211A and MDFIC. The transfected cells were treated with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. The ability of MDFIC to expand the GR transcriptome was attenuated in cells expressing S211A mutant.
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Project description:Glucocorticoids are primary stress hormones and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by severe side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced by alternative translation initiation; however, the role individual isoforms play in controlling tissue-specific responses to glucocorticoids in vivo is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. The GR-C3 knockin mice die at birth due to respiratory distress but can be completely rescued by antenatal glucocorticoid administration. To evaluate the GR-C3 transcriptome, we prepared mouse embryonic fibroblasts (MEFs) from E12.5 wild-type (WT) and GR-C3 knockin embryos and treated the cells with vehicle or the synthetic glucocorticoid Dexamethasone (Dex) for 6 hours. The GR-C3 isoform was found to have a markedly different gene-regulatory profile than GR in WT MEFs.
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Project description:The goal of this project is to investigate the role of glucocorticoids and their receptor, glucocorticoid receptor (GR) , in intestinal stress response and tissue homeostasis.We generated a mouse model with specific deletion of GR in intestinal epithelium (GR iKO mice) and examined colonic transcriptomes of adrenalectomized (ADX) Flox control and GR iKO mice in response to dexamethasone (DEX) treatment by microarray analysis.
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Project description:The functions of human testicular peritubular cells (HTPCs), which form a small compartment located between the seminiferous epithelium and the interstitials areas of the testis, go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR/NR3C1) by these cells indicates that they are a target of glucocorticoids (GCs), yet detailed insights into consequences of GC actions are unknown. To address this point, we studied cultured HTPCs, which serve as a window into the human testis. We used a cytokine profiler assay to screen for changes in secreted cytokines upon dexamethasone (Dex) treatment and employed qPCR and ELISA studies to verify the results. Dex, used in a therapeutic concentration, decreased proinflammatory factors, including IL6, IL8, MCP1 and CXCL1 within 24 - 48 h. An siRNA-mediated knockdown of GR reduced these actions. Exposure to Dex resulted in reduced GR levels, implying that exposure to Dex could also reduce anti-inflammatory actions of Dex. To evaluate the influence of Dex on HTPCs further, we performed a proteomic analysis of cellular components and secreted proteins. Strong responses were detectable after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were ECM- and basement membrane components (e.g. FBLN2, COL1A2, COL3A1), as well as PTX3 and StAR. These results reveal that Dex via GR exerts profound actions in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may result in men upon treatment with Dex for medical reasons. -Proteome Data Subset 24 h-
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Project description:The functions of human testicular peritubular cells (HTPCs), which form a small compartment located between the seminiferous epithelium and the interstitials areas of the testis, go beyond intratesticular sperm transport and include immunological roles. The expression of the glucocorticoid receptor (GR/NR3C1) by these cells indicates that they are a target of glucocorticoids (GCs), yet detailed insights into consequences of GC actions are unknown. To address this point, we studied cultured HTPCs, which serve as a window into the human testis. We used a cytokine profiler assay to screen for changes in secreted cytokines upon dexamethasone (Dex) treatment and employed qPCR and ELISA studies to verify the results. Dex, used in a therapeutic concentration, decreased proinflammatory factors, including IL6, IL8, MCP1 and CXCL1 within 24 - 48 h. An siRNA-mediated knockdown of GR reduced these actions. Exposure to Dex resulted in reduced GR levels, implying that exposure to Dex could also reduce anti-inflammatory actions of Dex. To evaluate the influence of Dex on HTPCs further, we performed a proteomic analysis of cellular components and secreted proteins. Strong responses were detectable after 24 h (31 significantly altered cellular proteins) and more pronounced ones after 72 h (30 less abundant and 42 more abundant cellular proteins). Dex also altered the composition of the secretome (33 proteins decreased, 13 increased) after 72 h. Among the regulated proteins were ECM- and basement membrane components (e.g. FBLN2, COL1A2, COL3A1), as well as PTX3 and StAR. These results reveal that Dex via GR exerts profound actions in HTPCs. If transferrable to the human testis, changes specifically in ECM and the immunological state of the testis may result in men upon treatment with Dex for medical reasons. -Proteome Data Subset 72 h-
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