title: Coactivation of SF-1-mediated transcription of steroidogenic enzymes by Ubc9 and PIAS1.
abstract: 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.
authors.affiliation: Department of Internal Medicine, School of Medicine, Keio University, Shinjujku-ku, Tokyo 160-8582, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Suda
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Shibata
authors.forename: Isao
authors.initials: I
authors.lastname: Kurihara
authors.forename: Yayoi
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Sakiko
authors.initials: S
authors.lastname: Kobayashi
authors.forename: Kenichi
authors.initials: K
authors.lastname: Yokota
authors.forename: Ayano
authors.initials: A
authors.lastname: Murai-Takeda
authors.forename: Ken
authors.initials: K
authors.lastname: Nakagawa
authors.forename: Mototsugu
authors.initials: M
authors.lastname: Oya
authors.forename: Masaru
authors.initials: M
authors.lastname: Murai
authors.forename: William E
authors.initials: WE
authors.lastname: Rainey
authors.forename: Takao
authors.initials: T
authors.lastname: Saruta
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Itoh
mesh_terms: D000302:Adrenal Cortex / Q000201:enzymology*; D045744:Cell Line, Tumor; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000235:genetics / Q000378:metabolism; D019405:Cytochrome P-450 CYP11B2 / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics / Q000378:metabolism; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics / Q000378:metabolism; D054339:Steroidogenic Factor 1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: C515057:NR5A1 protein, human; C106529:PIAS1 protein, human; D050828:Protein Inhibitors of Activated STAT; D025841:Small Ubiquitin-Related Modifier Proteins; D054339:Steroidogenic Factor 1; D013254:Steroid 17-alpha-Hydroxylase; D019405:Cytochrome P-450 CYP11B2; D013252:Steroid 11-beta-Hydroxylase; D002786:Cholesterol Side-Chain Cleavage Enzyme; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9