Molecular Mediators and Regulators of Glucocorticoid Actions

糖皮质激素作用的分子介质和调节剂

• 批准号: 8351165
• 负责人: Tomoshige Kino
• 金额: $ 160.15万
• 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/8351165
• 关键词: 16p11.2; 3-Dimensional; Acetylation; Achievement; Affect; Agonist; Allergic; Androgens; Anti-Inflammatory Agents; Anti-inflammatory; Attenuated; Autoimmune Process; BHLH Protein; Beds; Binding; Biopsy; Blood Pressure; Cells; Cholesterol; Circadian Rhythms; Code; Collaborations; Computers; Cultured Cells; DNA Sequence; Defect; Dexamethasone; Disease; Enzymes; Excretory function; Family; Family member; Fatigue; Fatty Acids; Fatty acid glycerol esters; Female; Force of Gravity; Genes; Genetic; Genetic Transcription; Genome; Glucocorticoid Receptor; Glucocorticoids; Glucose; Goals; Greece; Hepatic; Hour; Human; Hydrocortisone; Immune; Inflammatory; Life; Ligand Binding Domain; Ligands; Lipids; Liver; Lymphocyte; Lymphoproliferative Disorders; Lysine; Manuscripts; Measures; Mediating; Mediator of activation protein; Minnesota; Molecular; Moon; Movement; Mus; Mutation; Neuraxis; Nuclear Receptors; Obesity; Pathologic; Patients; Pattern; Periodicity; Peripheral; Phase; Phosphoenolpyruvate Carboxylase; Physiological; Play; Primary carcinoma of the liver cells; Production; Prostaglandins; Proteins; RXR; Rattus; Receptor Gene; Reporting; Resistance; Response Elements; Role; Sampling; Screening procedure; Sequence Analysis; Small Interfering RNA; Stress; Symptoms; Syndrome; System; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Transactivation; absorption; base; boys; comparative genomic hybridization; exome; glucocorticoid-induced orphan receptor; glucose metabolism; glucose-6-phosphatase; histone acetyltransferase; hormone resistance; human study; interest; lipid metabolism; mRNA Expression; macrophage; medical schools; microdeletion; nicotinamide phosphoribosyltransferase; overexpression; peripheral blood; proband; promoter; prototype; receptor; receptor binding; steroid hormone; subcutaneous; transcription factor

We investigated the pathophysiologic mechanism of familial/sporadic generalized glucocorticoid resistance syndrome, a prototype of glucocorticoid resistance caused by mutations in the glucocorticoid receptor (GR) gene. We found three new heterozygotic cases with mutations in the GR gene (GR V423A, GR V575G and GR H726R) in collaboration with Dr. Evangelia Charmandari, the Univ of Athens Medical School, Athens, Greece. We are currently analyzing their molecular defects. We have almost completed the computer-based 3-dimensional structural analysis for the ligand-binding domain of all pathologic GR mutations ever reported in addition to these newly identified mutations by using the algorism we established for GR R714Q in the last year. We also encountered a 7-year boy with mild resistance to glucocorticoids, androgens and thyroid hormones. Array-based comparative genomic hybridization analysis showed that this patient has 1.1 Mb size heterozygotic 16p11.2 microdeletion, while the siRNA-based screening and subsequent molecular analyses revealed that heterozygotic deletion of the ZNF764 gene by his microdeletion is responsive for his multi-hormone resistance, as this protein acts as a common coactivator for the glucocorticoid, androgen and thyroid hormone receptors. This is the first case demonstrating resistance to multiple steroid hormones with identification of the causative gene. We are now preparing a manuscript based on these results. The liver X receptors (LXRs), which belong to the nuclear receptor superfamily, mediate the biologic actions of various lipids, such as the cholesterol metabolites oxysterols, prostanoids and some fatty acids, by directly binding to these molecules. LXRs exist as two subtypes, LXRα and LXRβ, which display distinct patterns of tissue expression. Once LXRs bind their lipid ligands, they form a heterodimer with the retinoid X receptor (RXR), and stimulate the transcription of an array of genes involved in the absorption, efflux, transport, and excretion of cholesterol and other lipids. LXRs also regulate glucose metabolism by decreasing the expression of its rate-limiting enzymes G6Pase and PEPCK, and have anti-inflammatory activity by repressing a set of inflammatory genes in macrophages and other immune cells. Overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion, and activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats and suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells. Mechanistically, we found that LXRα/RXRα bound GREs and inhibited GR binding to these DNA sequences in a gene-specific fashion. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism. Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of the GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery. We previously reported that CLOCK/BMAL1 repressed GR-induced transcriptional activity by acetylating GR at several lysine residues located in its hinge region and by suppressing binding of GR to promoter GREs. These findings indicate that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids. We thus performed a human study in which we sampled peripheral blood in the morning and evening from normal subjects, and measured mRNA expression of known glucocorticoid-responsive genes and GR acetylation in circulating lymphocytes. We found that GR was acetylated higher in the morning than in the evening, positively correlating with mRNA expression of CLOCK and BMAL1, while circulating glucocorticoid-stimulated mRNA expression of glucocorticoid responsive genes were repressed by CLOCK/BMAL1 in a gene-specific fashion. These results indicate that the peripheral CLOCK system negatively regulates GR transcriptional activity through acetylation of GR not only in cultured cells but also in humans. In another clinically oriented study in which we measured mRNA expression of 190 GR action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsied from 25 obese subjects, we found that the morning/evening cortisol ratio, an indicator for the amplitude of circadian rhythmicity of circulating cortisol, negatively correlated with mRNA expression of the nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme for the production of intracellular NAD+. It is known that NAMPT/NAD+ is necessary for maintaining circadian rhythms of the CLOCK transcriptional system. Our results thus indicate that diurnal fluctuation of circulating glucocorticoids provides negative input to local CLOCK activity. In connection with our study on circadian rhythm, we found one interesting family with seasonal alteration of circadian rhythmicity in collaboration with Dr. F. Halberg, the Univ. of Minnesota. The proband, 61-year old female, has suffered from an annual cycle of severe fatigue including the inability to get out of bed, which lasts 2-3 months in the summer and the winter. She, however, is free from symptoms with high achievement during the unaffected months between these bad periods. The proposita demonstrated elongation of circadian rhythmicity (24.84 hours) in blood pressure and vigor rates during the affected periods, while she was in normal 24-hour rhythm in the unaffected months. The cycle of 24.84 hours is exactly the double of the tidal cycle, which is hypothesized to come about gravity-changes caused by movement of the moon around the earth. To identify genetic cause(s) of these manifestations, we have started whole exome analysis, sequencing the entire coding region of genome in 3 affected subjects and 5 unaffected members of the family.

我们研究了家族性/散发性全身性糖皮质激素抵抗综合征的病理生理机制，该综合征是由糖皮质激素受体（GR）基因突变引起的糖皮质激素抵抗的原型。我们与希腊雅典大学医学院的 Evangelia Charmandari 博士合作，发现了三个新的 GR 基因突变的杂合病例（GR V423A、GR V575G 和​​ GR H726R）。我们目前正在分析它们的分子缺陷。通过使用我们去年为 GR R714Q 建立的算法，除了这些新发现的突变外，我们几乎完成了对所有已报道的病理 GR 突变的配体结合域的基于计算机的 3 维结构分析。 我们还遇到了一名对糖皮质激素、雄激素和甲状腺激素有轻度抵抗力的7岁男孩。基于阵列的比较基因组杂交分析显示，该患者具有 1.1 Mb 大小的杂合 16p11.2 微缺失，而基于 siRNA 的筛选和随后的分子分析表明，ZNF764 基因的微缺失杂合缺失对他的多激素抵抗有反应，因为该蛋白质充当多激素抵抗的常见共激活剂。 糖皮质激素、雄激素和甲状腺激素受体。这是第一个通过鉴定致病基因证明对多种类固醇激素具有抗性的病例。我们现在正在根据这些结果准备一份手稿。 肝脏 X 受体 (LXR) 属于核受体超家族，通过直接与这些分子结合来介导各种脂质的生物学作用，例如胆固醇代谢物氧甾醇、前列腺素和一些脂肪酸。 LXR 有两种亚型：LXRα 和 LXRβ，它们显示出不同的组织表达模式。一旦 LXR 与其脂质配体结合，它们就会与类视黄醇 X 受体 (RXR) 形成异二聚体，并刺激涉及胆固醇和其他脂质的吸收、流出、运输和排泄的一系列基因的转录。 LXR 还通过降低限速酶 G6Pase 和 PEPCK 的表达来调节葡萄糖代谢，并通过抑制巨噬细胞和其他免疫细胞中的一组炎症基因来具有抗炎活性。 LXR/RXR 的过表达/配体 (GW3965) 激活以基因特异性方式抑制 GR 刺激的糖皮质激素反应元件 (GRE) 驱动的启动子的反式激活，GW3965 激活 LXR 可减弱地塞米松刺激的大鼠循环葡萄糖升高并抑制地塞米松诱导的 mRNA 表达 大鼠、小鼠和人肝癌 HepG2 细胞中肝葡萄糖 6 磷酸酶 (G6Pase) 的变化。从机制上讲，我们发现 LXRα/RXRα 结合 GRE，并以基因特异性方式抑制 GR 与这些 DNA 序列的结合。我们认为，通过直接减弱 GR 对葡萄糖和/或脂质代谢的转录活性，LXR 激动剂的施用可能有益于糖皮质激素治疗或应激相关的代谢异常状态。 糖皮质激素的循环水平以昼夜节律方式自然波动，并调节靶组织中 GR 的转录活性。基本的螺旋-环-螺旋蛋白 CLOCK（一种组蛋白乙酰转移酶 (HAT)）及其异二聚体伴侣 BMAL1 是自振荡转录因子，可在中枢神经系统和外周神经系统中产生昼夜节律。我们之前报道过，CLOCK/BMAL1 通过乙酰化 GR 铰链区的几个赖氨酸残基并抑制 GR 与启动子 GRE 的结合来抑制 GR 诱导的转录活性。这些发现表明，CLOCK/BMAL1 在靶组织中充当糖皮质激素作用的反相负调节剂，可能是通过拮抗昼夜波动的循环糖皮质激素的生物作用。因此，我们进行了一项人体研究，在早上和晚上从正常受试者身上采集外周血样本，并测量循环淋巴细胞中已知糖皮质激素反应基因的 mRNA 表达和 GR 乙酰化。我们发现GR在早上的乙酰化程度高于晚上，与CLOCK和BMAL1的mRNA表达呈正相关，而循环糖皮质激素刺激的糖皮质激素反应基因的mRNA表达被CLOCK/BMAL1以基因特异性方式抑制。这些结果表明，外周CLOCK系统不仅在培养细胞中而且在人类中通过GR乙酰化负向调节GR转录活性。在另一项临床导向的研究中，我们测量了 25 名肥胖受试者皮下脂肪活检中 190 个 GR 作用调节和糖皮质激素反应基因的 mRNA 表达，我们发现早晨/晚上皮质醇比率（循环皮质醇昼夜节律幅度的指标）与烟酰胺 mRNA 表达呈负相关 磷酸核糖基转移酶 (NAMPT)，是细胞内 NAD+ 产生的限速酶。众所周知，NAMPT/NAD+ 对于维持 CLOCK 转录系统的昼夜节律是必需的。因此，我们的结果表明循环糖皮质激素的昼夜波动为局部时钟活动提供了负输入。 在我们对昼夜节律的研究中，我们与大学的 F. Halberg 博士合作发现了一个有趣的昼夜节律随季节变化的家族。明尼苏达州。先证者是一名 61 岁女性，每年都会遭受严重疲劳的困扰，包括无法下床，这种情况在夏季和冬季持续 2-3 个月。然而，在这些糟糕时期之间未受影响的几个月里，她没有表现出高成就的症状。提案显示，受影响期间血压和活力率的昼夜节律（24.84 小时）延长，而在未受影响的月份，她处于正常的 24 小时节律。 24.84小时的周期恰好是潮汐周期的两倍，推测潮汐周期是由于月球绕地球运动引起的重力变化。为了确定这些表现的遗传原因，我们开始进行全外显子组分析，对 3 名受影响受试者和 5 名未受影响的家庭成员的基因组整个编码区域进行测序。