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: 8553900
• 负责人: Tomoshige Kino
• 金额: $ 59.92万
• 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/8553900
• 关键词: 16p11.2; 3-Dimensional; 7 year old; 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; Fatigue; Fatty Acids; Fatty acid glycerol esters; Female; Force of Gravity; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Glucocorticoid Receptor; Glucocorticoids; Glucose; Goals; Greece; 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; Molecular Analysis; Moon; Movement; Mus; Mutation; Neuraxis; Nuclear Receptors; Obesity; Pathologic; Patients; Pattern; Periodicity; Peripheral; Phase; Phosphoenolpyruvate Carboxylase; Physiological; Play; Primary carcinoma of the liver cells; Prostaglandins; Proteins; Publishing; RXR; Rattus; Receptor Gene; Reporting; Resistance; Response Elements; Role; Sampling; Screening procedure; Serum; Small Interfering RNA; Stress; Symptoms; Syndrome; System; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Transactivation; Writing; absorption; base; boys; comparative genomic hybridization; exome; glucocorticoid-induced orphan receptor; glucose metabolism; histone acetyltransferase; hormone resistance; human study; interest; lipid metabolism; mRNA Expression; macrophage; medical schools; member; microdeletion; peripheral blood; proband; promoter; prototype; receptor; receptor binding; steroid hormone; subcutaneous; transcription factor

We have 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 have almost completed molecular analysis on the functional defects of the former two mutations and are now preparing manuscripts. We have 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. We are also writing a manuscript based on these findings. We encountered a 7-year old 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 responsible for his multi-hormone resistance, as this protein acts as a specific 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 have published these results in this fiscal year. 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. We found that activation of LXRs/RXRs repressed GR-stimulated transactivation of glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion: It attenuated dexamethasone-stimulated elevation of circulating glucose in rats and suppressed dexamethasone-induced mRNA expression of 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 GR on glucose and/or lipid metabolism. Based on these findings, we have published one manuscript in this fiscal year Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of 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 to 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. We published one manuscript based on these findings in this fiscal year. 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 levels of evening cortisol is much more important than those in the morning to regulate mRNA expression of glucocorticoid responsive genes. Ratios of morning/evening serum cortisol levels also have a unique effect. It appears that higher sensitivity of tissues to circulating glucocorticoids in the evening due to reduced GR acetylation by CLOCK underlies stronger impact of evening serum cortisol levels to glucocorticoid-regulated gene expression compared to its morning levels. The manuscript based on these findings is now under review. In connection with our study on circadian rhythms, 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 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 rhythms 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 just completed the whole exome analysis that reveals sequence of the entire coding region of genome in 3 affected subjects and 5 unaffected members. We will complete analysis on the obtained results soon.

我们已经研究了家族/零星广义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，与Univ的Evangelia Charmandari博士合作。希腊雅典雅典医学院的我们几乎完成了对前两个突变功能缺陷的分子分析，现在正在准备手稿。我们已经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报告的所有病理GR突变的配体结合域。我们还根据这些发现撰写手稿。 我们遇到了一个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。 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 responsible for his multi-hormone resistance, as this protein acts as a specific coactivator for the glucocorticoid,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。我们在这个财政年度发布了这些结果。 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。我们发现，LXRS/RXRS的激活以基因特异性的方式抑制了糖皮质激素反应元件（GRE）驱动子的转换：它减弱了右甲虫刺激的大鼠循环葡萄糖的升高，并抑制了dexamethashasone诱导的G6pase和人类的dexamethassone诱导的mRNA的表达，并抑制了人类的表达。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GR的转录活性，对LXR激动剂的给药可能对糖皮质激素治疗或与压力相关的差异代谢状态有益。根据这些发现，我们在这个财政年度发表了一份手稿 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。我们根据这些财政年度的这些发现出版了一份手稿。在另一项面向临床的研究中，我们在其中测量了从25名肥胖受试者进行的皮下脂肪活检中190 GR作用调节和糖皮质激素反应性基因的mRNA表达，我们发现夜间皮质醇的水平比早晨的夜间皮质醇水平重要得多，以调节糖皮质激素反应性基因的mRNA表达。早晨/晚上血清皮质醇水平的比率也具有独特的效果。看来，由于时钟降低了GR乙酰化的降低，组织对糖皮质醇水平对糖皮质激素调节的基因表达的影响更大，因此组织对糖皮质激素的循环敏感性更高。基于这些发现的手稿现在正在审查中。 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，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度周期，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，昼夜节律节奏（24.84小时）的血压和活力率延伸，而在未受影响的月份中，她处于正常的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。为了确定这些表现的遗传原因，我们刚刚完成了整个外显子组分析，该分析揭示了3个受试者和5名未受影响的成员中基因组整个编码区域的顺序。我们将尽快完成对获得结果的分析。