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Berlin Brandenburg

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  • 1
    Language: English
    In: The Journal of biological chemistry, 12 March 2010, Vol.285(11), pp.8084-93
    Description: The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.
    Keywords: Aldosterone -- Metabolism ; Ccaat-Binding Factor -- Metabolism ; Hydrocortisone -- Metabolism ; Kidney Tubules, Collecting -- Metabolism ; Receptors, Mineralocorticoid -- Metabolism
    ISSN: 00219258
    E-ISSN: 1083-351X
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  • 2
    Language: English
    In: Endocrinology, June 2011, Vol.152(6), pp.2266-77
    Description: Steroidogenic factor-1 (SF-1) is a nuclear orphan receptor, which is essential for adrenal development and regulation of steroidogenic enzyme expression. SF-1 is posttranslationally modified by small ubiquitin-related modifier-1 (SUMO-1), thus mostly resulting in attenuation of transcription. We investigated the role of sumoylation enzymes, Ubc9 and protein inhibitors of activated STAT1 (PIAS1), in SF-1-mediated transcription of steroidogenic enzyme genes in the adrenal cortex. Coimmunoprecipitation assays showed that both Ubc9 and PIAS1 interacted with SF-1. Transient transfection assays in adrenocortical H295R cells showed Ubc9 and PIAS1 potentiated SF-1-mediated transactivation of reporter constructs containing human CYP17, CYP11A1, and CYP11B1 but not CYP11B2 promoters. Reduction of endogenous Ubc9 and PIAS1 by introducing corresponding small interfering RNA significantly reduced endogenous CYP17, CYP11A1, and CYP11B1 mRNA levels, indicating that they normally function as coactivators of SF-1. Wild type and sumoylation-inactive mutants of Ubc9 and PIAS1 can similarly enhance the SF-1-mediated transactivation of the CYP17 gene, indicating that the coactivation potency of Ubc9 and PIAS1 is independent of sumoylation activity. Chromatin immunoprecipitation assays demonstrated that SF-1, Ubc9, and PIAS1 were recruited to an endogenous CYP17 gene promoter in the context of chromatin in vivo. Immunohistochemistry and Western blotting showed that SF-1, Ubc9, and PIAS1 were expressed in the nuclei of the human adrenal cortex. In cortisol-producing adenomas, the expression pattern of SF-1 and Ubc9 were markedly increased, whereas that of PIAS1 was decreased compared with adjacent normal adrenals. These results showed the physiological roles of Ubc9 and PIAS1 as SF-1 coactivators beyond sumoylation enzymes in adrenocortical steroidogenesis and suggested their possible pathophysiological roles in human cortisol-producing adenomas.
    Keywords: Gene Expression Regulation, Enzymologic ; Transcriptional Activation ; Adrenal Cortex -- Enzymology ; Protein Inhibitors of Activated Stat -- Metabolism ; Small Ubiquitin-Related Modifier Proteins -- Metabolism ; Steroidogenic Factor 1 -- Metabolism ; Ubiquitin-Conjugating Enzymes -- Metabolism
    ISSN: 00137227
    E-ISSN: 1945-7170
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  • 3
    Language: English
    In: The Journal of biological chemistry, 19 January 2007, Vol.282(3), pp.1998-2010
    Description: Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.
    Keywords: Transcriptional Activation ; Receptors, Mineralocorticoid -- Metabolism ; Ubiquitin-Conjugating Enzymes -- Chemistry
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 4
    Language: English
    In: The Journal of biological chemistry, 25 February 2005, Vol.280(8), pp.6721-30
    Description: Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.
    Keywords: Cytochrome P-450 Cyp11b2 -- Genetics ; DNA-Binding Proteins -- Physiology ; Proteins -- Metabolism ; Transcription Factors -- Physiology ; Ubiquitin-Conjugating Enzymes -- Metabolism
    ISSN: 0021-9258
    E-ISSN: 1083351X
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  • 5
    Language: English
    In: Journal of Steroid Biochemistry and Molecular Biology, 2003, Vol.85(2), pp.449-456
    Description: Hyperfunctioning adrenocortical adenomas produce excessive amounts of various corticosteroids due to dysregulated expression of steroidogenic enzymes. Since no genetic mutations in steroidogenic enzyme genes have been identified as yet, the dysregulated expression at the transcription level may be crucial. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) and steroidogenic factor-1 (SF-1) play key roles in the transcriptional regulation of steroidogenic P450 genes. Transfection studies showed that SF-1 activated and COUP-TFs repressed the transcription of bovine CYP17 gene promoter from the CRS2 element in a mutually exclusive manner in Y-1 cells. The results indicate that COUP-TFs negatively regulate the transcriptional activity of SF-1, a steroidogenic cell-specific activator of various steroidogenic P450 genes. Expression of both COUP-TFI and COUP-TFII was significantly decreased in the cortisol-producing adenomas, in which CYP17 was drastically overexpressed, indicating that decreased expression of COUP-TFs play a key role in overexpression of CYP17 in this type of tumors. We then screened for COUP-TFI- interacting proteins from a cortisol-producing adenoma cDNA library using a yeast two-hybrid system and identified a novel RING finger-containing protein which can function as a coregulator for COUP-TFI. Notably, COUP-TFI activated rather than repressed several target genes including the human CYP11B2 gene promoter, the results of which were opposite to those of the CYP17 promoter. The bifunctional activities of COUP-TFI may be derived from the promoter context and our newly identified COUP-TFI coregulator.
    Keywords: Coup-Tfs ; Corepressor ; Coactivator ; N-Cor ; Smrt ; Steroidogenesis ; Adrenal Cortex ; Cushing’s Syndrome ; Chemistry
    ISSN: 0960-0760
    E-ISSN: 1879-1220
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  • 6
    Language: English
    In: Endocrine Research, 01 January 2004, Vol.30(4), pp.617-621
    Description: Estrogen plays important roles in the pathophysiology of atherosclerosis and cardiovascular diseases mediated by estrogen receptor α (ERα). To elucidate the molecular mechanisms, we screened ERα-interacting proteins from a human heart cDNA library using a yeast two-hybrid system, and identified...
    Keywords: Erα ; Ubc9 ; Pias1 ; Fhl2 ; Coactivator ; Medicine ; Anatomy & Physiology
    ISSN: 0743-5800
    E-ISSN: 1532-4206
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  • 7
    Language: English
    In: Endocrine Research, 01 January 2004, Vol.30(4), pp.611-616
    Description: The ubiquitin-proteasome pathway regulates the turnover of many nuclear hormone receptors, such as the estrogen receptor. For estrogen receptor, proteasome inhibition decreases ligand-mediated transcription. We provide evidence that the mineralocorticoid receptor (MR) is degraded by the ubiquitin-proteasome...
    Keywords: Mineralcorticoid Receptor ; Ubiquitin ; Proteasome ; Aldosterone ; Medicine ; Anatomy & Physiology
    ISSN: 0743-5800
    E-ISSN: 1532-4206
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  • 8
    Language: English
    In: Endocrine Research, 01 January 2004, Vol.30(4), pp.795-801
    Description: Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) and steroidogenic factor-1 (SF-1) play key roles in the transcriptional regulation of steroidogenic P450 genes. Transfection studies showed that SF-1 activated bovine CYP17 promoter activity, whereas COUP-TFs repressed it...
    Keywords: Coup-Tfi ; Ubc9 ; Piasi ; Sumo ; Co-Activator ; Co-Repressor ; Medicine ; Anatomy & Physiology
    ISSN: 0743-5800
    E-ISSN: 1532-4206
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