Oakley Robert H, Busillo John M, Cidlowski John A
From the Signal Transduction Laboratory, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709.
From the Signal Transduction Laboratory, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709
J Biol Chem. 2017 Apr 7;292(14):5825-5844. doi: 10.1074/jbc.M116.758888. Epub 2017 Feb 21.
Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules are widely used in the clinic. The molecular factors that govern tissue specificity of glucocorticoids, however, 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 two-hybrid assay. The MyoD family inhibitor domain-containing protein (MDFIC) was identified as a binding partner for GR. MDFIC associated with GR in the cytoplasm of cells, and treatment with glucocorticoids resulted in the dissociation of the GR-MDFIC complex. To investigate the function of the GR-MDFIC interaction, we performed a genome-wide microarray in intact and MDFIC-deficient A549 cells that were treated with glucocorticoids. A large cohort of genes was differentially regulated by GR depending on the presence or absence of MDFIC. These gene changes were strongly associated with inflammation, and glucocorticoid regulation of the inflammatory response was altered in MDFIC-deficient cells. At a molecular level, the interaction of MDFIC with GR altered the phosphorylation status of the receptor. We demonstrate in COS-1 cells that changes in receptor phosphorylation underlie the ability of MDFIC to regulate the transcriptional activity of GR. Finally, we show that GR directly represses the MDFIC gene, revealing a negative feedback loop by which glucocorticoids limit MDFIC activity. These findings identify a new binding partner for cytoplasmic GR that modulates the receptor transcriptome and contributes to the tissue-specific actions of glucocorticoids.
糖皮质激素是调节许多生理过程的主要应激激素，这些分子的合成衍生物在临床上被广泛使用。然而，人们对控制糖皮质激素组织特异性的分子因素了解甚少。糖皮质激素的作用是由糖皮质激素受体（GR）介导的。为了发现与GR相互作用并调节其功能的新蛋白质，我们进行了酵母双杂交试验。含MyoD家族抑制结构域蛋白（MDFIC）被鉴定为GR的结合伴侣。MDFIC在细胞胞质中与GR结合，用糖皮质激素处理导致GR-MDFIC复合物解离。为了研究GR-MDFIC相互作用的功能，我们在用糖皮质激素处理的完整和MDFIC缺陷的A549细胞中进行了全基因组微阵列分析。大量基因根据MDFIC的存在与否受到GR的差异调节。这些基因变化与炎症密切相关，并且在MDFIC缺陷的细胞中糖皮质激素对炎症反应的调节发生了改变。在分子水平上，MDFIC与GR的相互作用改变了受体的磷酸化状态。我们在COS-1细胞中证明，受体磷酸化的变化是MDFIC调节GR转录活性能力的基础。最后，我们表明GR直接抑制MDFIC基因，揭示了一种负反馈环，通过该负反馈环糖皮质激素限制MDFIC活性。这些发现确定了一种用于胞质GR的新结合伴侣，其调节受体转录组并有助于糖皮质激素的组织特异性作用。
