Molecular Mediators and Regulators of Glucocorticoid Act

糖皮质激素作用的分子介质和调节剂

• 批准号: 6993547
• 负责人: Tomoshige Kino
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6993547
• 关键词: HIV infections; corticosteroid receptors; gene induction /repression; genetically modified animals; glucocorticoids; host organism interaction; human immunodeficiency virus 1; human tissue; laboratory mouse; microarray technology; mutant; protein structure function; receptor expression; transcription factor; virus genetics; virus protein; virus replication

Glucocorticoids have a broad array of life-sustaining functions and play important roles in the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Changes of tissue responsiveness to glucocorticoids may produce pathologic states and influence both the course of a disease and its response to therapy. We investigated the pathophysiologic mechanisms of the familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations of the glucocorticoid receptor (GR) gene. We analyzed the functional defects of pathologic mutant receptors GRV571A, GRD641V, GRV729I, GRGRI559N and GRI747M by examining their nuclear shuttling, DNA-binding activity, interaction with p160 coactivators and transdominant negative activity on the wild type receptor, and compared observed mutants? defects with the clinical phenotypes of the patients. We also found a novel pathologic heterozygote mutation that replaces leucine at amino acid 773 of the GR with proline. This mutant demonstrated reduced transactivation activity due to its inability to form a ligand-dependent transactivation surface in its ligand-binding domain and behaved as a dominant negative inhibitor of the normal receptor, thereby producing clinical pathology at the heterozygotic condition. To discover intracellular molecules influencing tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screenings. GR specifically bound one of the orphan nuclear receptors, chicken ovalbumin-upstream promoter-transcription factor II (COUP-TFII). GR and COUP-TFII mutually affected each other's transcriptional activity on the promoters of several genes involved in intermediary metabolism. We also found that the G protein coupled receptor downstream mediator guanine nucleotide-binding protein (G protein) beta and the transforming growth factor beta/bone morphogenetic protein-induced SMAD6 repressor specifically interacted with the GR. Both Gbeta and SMAD6 proteins suppressed GR-induced transactivation of glucocorticoid-responsive promoters. Contrary to dogma, the former protein, with its partner molecule G gamma, co-migrated with the GR into the nucleus in response to glucocorticoids, and was attracted to glucocorticoid response elements (GREs) located in the promoter regions of glucocorticoid-responsive genes. These results indicate that the G protein beta/gamma complexes may suppress the transcriptional activity of the GR by interfering with the GR-associated transcriptosomes on GR-regulated promoters. In contrast, SMAD6 suppressed GR transcriptional activity by attracting histone deacetylases, functioning as a corepressor similarly to other classic molecules, like the nuclear receptor corepressors (NCoR) and the silencing mediator for retinoid and thyroid hormone receptors (SMRT). In addition to the above proteins, we found that the autoimmune regulator protein (AIRE), whose mutations cause the autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy (APECED) syndrome, interacted with the GR and enhanced its transcriptional activity. AIRE has four LXXLL signature motifs through which classic nuclear receptor coactivators interact with ligand-activated receptor molecules. Destruction of these motifs in AIRE disturbed its interaction with the GR and inactivated its enhancing effect on GR transactivation, indicating that AIRE may act as a tissue-specific (thymus) coactivator of GR by using such signature motifs. We are also working on Brx, a Rho type guanine nucleotide exchange factor, which activates the Rho family of small G proteins by converting them from an inactive GDP-bound to an active GTP-bound form. This protein has a nuclear receptor-interacting domain in its C-terminal portion. We have demonstrated that Brx enhanced GR transcriptional activity by activating and closely attracting small G proteins to the GR. Endogenous Brx is necessary for the action of glucocccorticoids in splenic lymphocytes and mediates lysophosphatidic acid (LPA)-induced potentiation of GR transactivation. We are investigating the roles of GRbeta and transcriptional coactivator TRAP220 in regulating tissue sensitivity to glucocorticoids. The former is a splicing variant of the classic receptor GRalpha, while the latter is a component of the huge coactivator protein complex DRIP/TRAP. We developed a stable cell line that over-expresses GFP-GRbeta and obtained TRAP220 KO cells from Dr. R. Roeder?s laboratory. We are examining the impact of their expression/depletion on glucocorticoid-induced transcriptional activity by using regular DNA microarray chips. To elucidate endogenous genes that attract GR to their promoter region, we have developed CpG microarray chips, which include the promoter regions of over 10,000 endogenous genes. We performed chromatin immunoprecipitation assays using antibodies against the GR and will treat the chips with the harvested DNA to discover endogenous promoters that attract GR. Patients with the acquired immunodeficiency syndrome (AIDS), caused by infection with the Human Immunodeficiency Virus type-1 (HIV-1), have several clinical manifestations compatible with increased tissue sensitivity to glucocorticoids. We focused on one of the HIV-1-encoding accessory proteins, Vpr, and demonstrated that this viral polypeptide acts as a coregulator of the GR. We further examined the mechanism of Vpr-induced coactivation of GR transactivation focusing on its interaction with host nuclear receptor coactivators p300/CBP, and addressed the biological significance of Vpr coactivator activity by testing it on the production/secretion of glucocorticoid-responsive interleukin-12 and other gene products. Our findings may explain several manifestations of AIDS, including immunosuppression related to a T-helper 1/T-helper 2 shift, myopathy, muscle weakness/atrophy and obesity-related insulin resistance with metabolic and cardiovascular manifestations.

糖皮质激素具有广泛的生命维持功能，在治疗多种炎症/自身免疫/过敏性和淋巴组织增生性疾病中发挥重要作用。组织对糖皮质激素反应性的改变可产生病理状态并影响疾病的进程及其对治疗的反应。我们研究了家族性/散发性糖皮质激素抵抗综合征的病理生理机制，这是由糖皮质激素受体（GR）基因突变引起的。我们分析了病理性突变受体GRV 571 A，GRD 641 V，GRV 729 I，GRGRI 559 N和GRI 747 M的功能缺陷，通过检查它们的核穿梭，DNA结合活性，与p160共激活因子的相互作用和对野生型受体的反式显性负活性，并比较观察到的突变体？患者临床表型的缺陷。我们还发现了一个新的病理性杂合子突变，取代亮氨酸在氨基酸773的GR与脯氨酸。这种突变体表现出降低的反式激活活性，由于其无法形成一个配体依赖性的反式激活表面在其配体结合结构域，并表现为一个显性负抑制剂的正常受体，从而产生临床病理学的杂合子条件。 为了发现影响组织对糖皮质激素敏感性的细胞内分子，我们进行了酵母双杂交筛选。GR特异性结合孤儿核受体之一，鸡卵清蛋白上游启动子转录因子II（COUP-TFII）。GR和COUP-TFII相互影响对方的转录活性的几个基因的启动子参与中间代谢。我们还发现，G蛋白偶联受体下游介体鸟嘌呤核苷酸结合蛋白（G蛋白）β和转化生长因子β/骨形态发生蛋白诱导的SMAD 6阻遏物特异性地与GR相互作用。G β和SMAD 6蛋白抑制GR诱导的糖皮质激素应答启动子的反式激活。与教条相反，前一种蛋白质及其伴侣分子G γ与GR共同迁移到细胞核中以响应糖皮质激素，并被吸引到位于糖皮质激素响应基因启动子区域的糖皮质激素响应元件（GRES）。这些结果表明，G蛋白β/γ复合物可以通过干扰GR调节启动子上的GR相关转录体来抑制GR的转录活性。相比之下，SMAD 6通过吸引组蛋白脱乙酰酶来抑制GR转录活性，其功能与其他经典分子类似，如核受体辅阻遏物（NCoR）和类维生素A和甲状腺激素受体（SMRT）的沉默介体。除了上述蛋白质，我们发现，自身免疫调节蛋白（AIRE），其突变导致自身免疫性多内分泌病念珠菌病-外胚层营养不良（APECED）综合征，与GR相互作用，并增强其转录活性。AIRE有四个LXXLL特征基序，经典的核受体辅激活剂通过这些基序与配体激活的受体分子相互作用。破坏这些图案在AIRE干扰其与GR的相互作用，并灭活其增强GR反式激活的作用，表明AIRE可能作为一个组织特异性（胸腺）的GR共激活剂通过使用这样的签名图案。我们还在研究Brx，这是一种Rho型鸟嘌呤核苷酸交换因子，它通过将小G蛋白从失活的GDP结合形式转化为活性的GDP结合形式来激活Rho家族。该蛋白在其C-末端部分具有核受体相互作用结构域。我们已经证明，Brx增强GR转录活性的激活和密切吸引小G蛋白的GR。内源性Brx是必要的脾淋巴细胞中的糖皮质激素的作用和介导的溶血磷脂酸（LPA）诱导的增强GR反式激活。 我们正在研究GR β和转录辅激活因子TRAP 220在调节组织对糖皮质激素敏感性中的作用。前者是经典受体GR α的剪接变体，而后者是巨大的共激活蛋白复合物DRIP/TRAP的组成部分。我们开发了过表达GFP-GR β的稳定细胞系，并从R.罗德？的实验室。我们正在研究他们的表达/消耗糖皮质激素诱导的转录活性的影响，通过使用常规的DNA微阵列芯片。为了阐明吸引GR到其启动子区域的内源性基因，我们开发了CpG微阵列芯片，其包括超过10，000个内源性基因的启动子区域。我们使用GR抗体进行染色质免疫沉淀试验，并将用收获的DNA处理芯片以发现吸引GR的内源性启动子。 由人类免疫缺陷病毒1型（HIV-1）感染引起的获得性免疫缺陷综合征（AIDS）患者具有与组织对糖皮质激素的敏感性增加相一致的几种临床表现。我们专注于HIV-1编码的辅助蛋白Vpr，并证明该病毒多肽作为GR的共调节因子。我们进一步研究了Vpr诱导的GR反式激活的共激活机制，重点是其与宿主核受体共激活因子p300/CBP的相互作用，并通过测试Vpr辅激活因子对糖皮质激素应答性白细胞介素-1的产生/分泌的影响，阐明了Vpr辅激活因子活性的生物学意义。12和其他基因产物。我们的研究结果可以解释艾滋病的几种表现，包括与辅助性T细胞1/辅助性T细胞2转移相关的免疫抑制，肌病，肌无力/萎缩和肥胖相关的胰岛素抵抗与代谢和心血管表现。