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/散发性糖皮质激素抗性综合征的病理生理机制，这是糖皮质激素受体（GR）基因突变引起的糖皮质激素耐药性的原型。我们在GR基因（GR V423A，GR V575G和GR H726R）中发现了三个新的杂合病例，并与希腊雅典医学院的大学合作。我们目前正在分析它们的分子缺陷。我们几乎完成了基于计算机的三维结构分析，用于除这些新鉴定的突变外，除了使用我们去年为GR R714Q建立的算法外，除了这些新鉴定的突变外，还完成了所有病理GR突变。 我们还遇到了一个7岁男孩，对糖皮质激素，雄激素和甲状腺激素具有轻度抗性。基于阵列的比较基因组杂交分析表明，该患者具有1.1 Mb大小的杂合16p11.2微骨骼，而基于siRNA的筛选和随后的分子分析表明，杂合性缺失的杂合性缺失是Znf764基因通过其微骨骼的响应，因为他的多构素耐药性，是一种蛋白质的作用，是一种蛋白质的作用，糖皮质激素，雄激素和甲状腺激素受体。这是第一种情况，表明对多种类固醇激素的抗性具有鉴定为病毒基因。我们现在正在根据这些结果准备手稿。 属于核受体超家族的肝脏X受体（LXR）介导了各种脂质的生物学作用，例如胆固醇代谢物氧甲醇，前列腺素和一些脂肪酸，通过直接与这些分子结合。 LXR作为两个亚型LXRα和LXRβ存在，它们表现出不同的组织表达模式。一旦LXR结合其脂质配体，它们就会形成与视乙他X受体（RXR）的异二聚体，并刺激参与吸收，外排，转运和排泄胆固醇和其他脂质的基因阵列的转录。 LXR还通过降低其限制酶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。 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大鼠，小鼠和人肝癌HEPG2细胞中肝葡萄糖-6-磷酸酶（G6Pase）的含量。从机械上讲，我们发现LXRα/RXRα结合的GR并以基因特异性方式抑制与这些DNA序列的结合。我们建议，通过直接衰减GR对葡萄糖和/或脂质代谢的转录活性，可以通过糖皮质激素治疗或与压力相关的非代谢状态有益。 循环水平的糖皮质激素水平以昼夜节律自然波动，并调节靶组织中GR的转录活性。基本的螺旋 - 环螺旋蛋白时钟，组蛋白乙酰转移酶（HAT）及其异二聚体伴侣BMAL1是在中枢神经系统和周围产生昼夜节律的自我振荡转录因子。我们先前报道说，时钟/BMAL1在其位于其铰链区域的几个赖氨酸残基上并抑制GR与启动子GR的结合在几个赖氨酸残基处抑制了GR诱导的转录活性。这些发现表明，时钟/BMAL1作为靶组织中糖皮质激素作用的反相负调节剂，可能是通过拮抗昼夜循环循环的糖皮质激素的生物学作用。因此，我们进行了一项人类研究，在早晨和晚上，我们从正常受试者中抽样外周血，并在循环淋巴细胞中测量了已知的糖皮质激素反应基因和GR乙酰化的mRNA表达。我们发现早晨的GR比晚上更高，与时钟和BMAL1的mRNA表达正相关，而循环糖皮质激素刺激的糖皮质激素反应基因的mRNA表达以基因特异性方式抑制了糖皮质激素反应基因。这些结果表明，外围时钟系统不仅在培养的细胞中，而且在人类中也通过GR的乙酰化来负调节GR转录活性。在另一项面向临床方向的研究中，我们测量了从25名肥胖受试者的皮下脂肪活检中的190 GR作用调节和糖皮质激素反应性基因的mRNA表达，我们发现早晨/晚上的皮质醇比，这是循环中循环的循环症状，循环症状相关的表现良好的指标磷酸贝糖基转移酶（NAMPT），这是用于生产细胞内NAD+的速率限制酶。众所周知，NAMPT/NAD+对于维持时钟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性提供了负输入。 关于我们关于昼夜节律的研究，我们发现了一个有趣的家庭，并与大学合作，与昼夜节律的季节性改变了。明尼苏达州。这位61岁的女性的概率遭受了严重疲劳的年周期，包括无法起床，夏季和冬季持续2-3个月。但是，在这些不良时期之间未受影响的几个月中，她没有症状很高。该提案表明在受影响的时期，血压和活力率的昼夜节律延伸（24.84小时），而她在未受影响的月份处于正常的24小时节奏。 24.84小时的循环正是潮汐周期的两倍，这是由于月球在地球周围的运动而引起的重力变化。为了确定这些表现形式的遗传原因，我们已经开始了整个外显子组分析，对3个受试者和5个不受影响的家族成员进行了整个基因组的编码区域。