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: 7968640
• 负责人: Tomoshige Kino
• 金额: $ 46.5万
• 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/7968640
• 关键词: 3-Dimensional; AF2; Affect; Affinity; Aldosterone; Allergic; Amino Acids; Apoptosis; Arginine; Aspartic Acid; Autoimmune Process; BHLH Protein; Binding; Biological Assay; Brain; Brain-Derived Neurotrophic Factor; C-terminal; Cell Nucleus; Cells; Characteristics; Circadian Rhythms; Computers; Corticosteroid Receptors; Cyclin-Dependent Kinase 5; DNA; Development; Dexamethasone; Disease; Dominant-Negative Mutation; Elements; Emotional; Exons; Family; Genes; Genomics; Glucocorticoid Receptor; Glucocorticoids; Glutamic Acid; Goals; Growth; Hela Cells; Histidine; Homeostasis; Human; Hydrogen Bonding; Individual; Inflammatory; Life; Ligand Binding; Ligand Binding Domain; Ligands; Lymphoproliferative Disorders; Lysine; Mediating; Mediator of activation protein; Memory; Messenger RNA; Mineralocorticoid Receptor; Molecular; Mood Disorders; Morphogenesis; Mutation; Neuraxis; Neurodegenerative Disorders; Neurons; Organ; Pathologic; Patients; Peripheral; Phase; Phenotype; Phosphotransferases; Physiological; Play; Protein Isoforms; RNA; Rattus; Reaction; Receptor Gene; Regulation; Reporting; Research; Resistance; Resources; Role; Screening procedure; Serine; Serum; Shock; Sleep; Starvation; Steroid Receptors; Steroids; Structure; Surface; Synaptic plasticity; Syndrome; Threonine; Time; Tissues; Transactivation; Work; Yeasts; base; genetic analysis; glucocorticoid receptor alpha; glucocorticoid receptor beta; glucocorticoid-induced orphan receptor; growth arrest specific transcript 5; histone acetyltransferase; human BIRC3 protein; hypothalamic-pituitary-adrenal axis; mRNA Expression; mutant; novel; prevent; promoter; protein expression; receptor; response; transcription factor; yeast two hybrid system

Glucocorticoids, the end-products of the hypothalamic-pituitary-adrenal (HPA) axis, have a broad array of life-sustaining functions, and are essential for the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Thus, changes of tissue sensitivity to glucocorticoids may develop pathologic states and influence their disease course. We investigated the pathophysiologic mechanism of the familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations in the glucocorticoid receptor (GR) gene. We found in a Colombian patient a heterozygotic mutation that replaced aspartic acid to histidine at amino acid 401 (GRD401H). This mutant receptor demonstrated 2-3 times stronger transcriptional activity than the wild type receptor, while the patient showed a mixed phenotype characteristic to both glucocorticoid resistant and hypersentivitvity states. Thus, this mutant receptor causes tissue-specific alterations of glucocorticoid activity. We also found another case with generalized glucocorticoid resistance harboring a hetrozygotic mutation replacing arginine with glutamic acid at amino acid 714 (GR R714Q). The patient is the youngest among the all cases ever reported who carried pathologic mutations in the GR gene. GR R714Q demonstrated reduced transcriptional activity on the glucocorticoid-responsive promoter, reduced affinity to dexamethasone and defective formation of the activation function (AF) 2. Computer-based structural analysis on the ligand-binding domain of GR R714Q revealed that the mutation disrupts the hydrogen bond network formed by α-heices, 7, 8 9 and 10 of the LBD, alters their 3-dimensional arrangement, and eventually destroys the ligand-binding pocket and the AF2 surface. Glucocorticoids play an essential role in the homeostasis of the central nervous system (CNS) and influence diverse functions of neuronal cells. We previously reported that the cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of CNS, and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with GR through its activators p35/p25 and differentially regulated the transcriptional activity of the GR. CDK5 phosphorylated GR at multiple serines, including those located at 203, 211 and 226. Since glucocorticoids employ the mineralocorticoid receptor (MR) as a functional receptor in the brain in addition to GR, we examined the effects of CDK5 on MR and found that this kinase phosphorylated serines 129 and 158, and threonine 250 of MR and modulated MR-induced transcriptional activity similarly to GR. To further examine importance of CDK5-mediated regulation of GR and MR, we focused on the brain-derived neurotrophic factor (BDNF). MR-specific ligand aldosterone and GR-specific dexamethasone respectively increased and suppressed BDNF mRNA/protein expression in rat cortical neuronal cells, while CDK5 enhanced their effects on the BDNF expression. Since BDNF plays a critical roles in the neurobiability, synaptic plasticity, consolidation of memory and emotional changes, we suggest that aberrant activation of CDK5 may regulate these neuronal activity through corticosteroid receptors/BDNF, further contributing to the development of neurodegenerative disease, and possibly, mood disorders. To find more intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screening assays using the GR DBD, and found the noncoding (nc) Gas5 interacted with this portion of the GR. This ncRNA accumulates in growth-arrested cells, but its physiologic roles are not known as yet. We found that Gas5 bound specifically to GR at its DBD and translocated into the nucleus with GR in response to glucocorticoids. Gas5 RNA prevented the association of the GR with its regulatory DNA elements and suppressed its transcriptional activity. Serum starvation-induced Gas5 suppressed glucocorticoid-mediated cellular inhibitor of apoptosis 2 (cIAP2) mRNA expression and prevented apoptosis of growth-arrested cells through suppression of this protein expression. Gas5 has one GRE-like sequence in its 3 portion in its intra-molecular double helical structure, through which this ncRNA interacts competitively with the GR DBD by mimicking DNA GREs. Thus, Gas5 is a growth arrest-related co-repressor of the GR harboring a decoy RNA GRE, restricting the expression of steroid-responsive genes. This is a novel concept suggesting competition between ncRNA and genomic DNA for the DBD of steroid receptors. This work will also indicate that Gas5 may contribute to the cellular adaptive reaction to starvation, preventing apoptosis and saving energy resources. 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 in both the central nervous system and periphery. We found that CLOCK/BMAL1 repressed GR-induced transcriptional activity in a HAT activity-dependent fashion. In serum shock-synchronized cells, transactivational activity of GR fluctuated spontaneously in a circadian fashion, in reverse phase with CLOCK/BMAL1 mRNA expression. CLOCK and GR interacted with each other physically and CLOCK suppressed binding of GR to promoter GREs by acetylating a cluster of lysine residues located in its hinge region. 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. Further, these results suggest that a peripheral target tissue circadian rhythm indirectly influences functions of every organ/tissue inside the body through modulation of the ubiquitous and diverse actions of glucocorticoids. As an extension of our research on the circadian rhythm, we are currently analyzing the genetic cause(s) of a family in which 3 affected individuals demonstrate seasonal elongation/shortening of sleep. The human glucocorticoid receptor (GR) gene produces C-terminal GRbeta and GRalpha isoforms through alternative use of specific exons 9beta and 9alpha, respectively. In contrast to the classic receptor GRalpha, which mediates most of the known actions of glucocorticoids, the functions of GRbeta have been largely unexplored. We thus explored the transcriptional activity of GRbeta on endogenous genes by developing HeLa cells stably expressing EGFP-GRbeta or EGFP. Microarray analyses revealed that GRbeta had intrinsic gene-specific transcriptional activity, regulating mRNA expression of a large number of genes negatively or positively. Majority of GRbeta-responsive genes was distinct from those modulated by GRalpha, while GRbeta and GRalpha mutually modulated each others' transcriptional activity in a subpopulation of genes. Our results indicate that GRbeta has intrinsic, GRalpha-independent, gene-specific transcriptional activity, in addition to its previously reported dominant negative effect on GRalpha-induced transactivation of GRE-driven promoters.

糖皮质激素是下丘脑 - 垂体 - 肾上腺（HPA）轴的终产物，具有广泛的生命活性功能，对于几种炎症/自身免疫/过敏性和淋巴增生性疾病的治疗至关重要。因此，组织对糖皮质激素的敏感性的变化可能会发展出病理状态并影响其疾病病程。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，这是由糖皮质激素受体（GR）基因突变引起的。我们在哥伦比亚患者中发现了杂合子突变，该突变将天冬氨酸取代为氨基酸401（GRD401H）的组氨酸。该突变体受体比野生型受体表现出2-3倍的转录活性，而患者表现出与糖皮质激素耐药性和催眠病态状态的混合表型。因此，该突变受体会导致糖皮质激素活性的组织特异性改变。我们还发现了另一个病例，具有携带的糖皮质激素耐药性，该耐药性含有Hetrozygotic突变，用氨基酸714（GR R714Q）用谷氨酸代替精氨酸。患者是有史以来携带GR基因病理突变的所有病例中最年轻的。 GR R714Q表明在糖皮质激素反应性启动子上的转录活性降低，对地塞米松的亲和力降低，激活函数的形成不良，基于计算机的结构分析（AF）对GR R714Q的配体结合结构域进行了基于计算机的结构分析，该突变揭示了突变的hydogen键网络均受氢键网的形式。 3维排列，并最终破坏了配体结合口袋和AF2表面。 糖皮质激素在中枢神经系统（CNS）的稳态中起着至关重要的作用，并影响神经元细胞的各种功能。我们先前报道说，Cyclin依赖性激酶5（CDK5）在CNS的形态发生和功能中起重要作用，并且其异常激活与神经退行性疾病的发展有关，通过其激活剂P35/P25与GR相互作用，并差异地调节了GR的转录活性。 CDK5 phosphorylated GR at multiple serines, including those located at 203, 211 and 226. Since glucocorticoids employ the mineralocorticoid receptor (MR) as a functional receptor in the brain in addition to GR, we examined the effects of CDK5 on MR and found that this kinase phosphorylated serines 129 and 158, and threonine 250 of MR and modulated MR-induced transcriptional activity similarly to GR.为了进一步研究CDK5介导的GR和MR调节的重要性，我们专注于脑衍生的神经营养因子（BDNF）。 MR特异性配体醛固酮和GR特异性地塞米松分别增加并抑制了大鼠皮质神经元细胞中的BDNF mRNA/蛋白质表达，而CDK5则增强了其对BDNF表达的影响。由于BDNF在神经障碍，突触可塑性，记忆和情绪变化的巩固中起着至关重要的作用，因此我们建议CDK5的异常激活可以通过皮质类固醇受体/BDNF调节这些神经元活性，从而进一步有助于神经变性疾病的发展，并可能导致情绪失调。 为了找到更多的细胞内分子，可能会影响组织对糖皮质激素的敏感性，我们使用GR DBD进行了酵母两杂化筛选测定，并发现非编码（NC）GAS5与GR的这一部分相互作用。这种NCRNA积累在生长序列的细胞中，但其生理作用尚不称为。我们发现，气体在其DBD处专门与GR结合，并用GR转移到核中，以响应糖皮质激素。 GAS5 RNA阻止了GR与其调节性DNA元件的关联，并抑制了其转录活性。血清饥饿诱导的GAS5抑制凋亡2（CIAP2）mRNA表达的糖皮质激素介导的细胞抑制剂，并通过抑制该蛋白质表达来阻止生长降落细胞的凋亡。 GAS5在其3部分中具有一个类似于GRE的序列，其分子内双螺旋结构通过模仿DNA GRES与GR DBD竞争性相互作用。因此，GAS5是带有诱饵RNA GRE的GR的生长逮捕的共抑制剂，限制了类固醇反应性基因的表达。这是一个新的概念，表明NCRNA和基因组DNA之间的类固醇受体DBD竞争。这项工作还将表明GAS5可能有助于细胞自适应对饥饿的反应，防止凋亡和节省能源。 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是自我振荡的转录因子，它们在中枢神经系统和外围产生昼夜节律。我们发现时钟/BMAL1以帽子活动依赖性方式抑制了GR诱导的转录活性。在血清休克同步细胞中，GR的反式激活活性以昼夜节律自发波动，以时期为钟/bmal1 mRNA表达。时钟和GR彼此相互作用，并通过乙酰基于其铰链区域的一组赖氨酸残基来抑制GR与启动子GR的结合。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。此外，这些结果表明，外围靶组织昼夜节律通过调节糖皮质激素的无处不在和多样化的作用，间接影响体内每个器官/组织的功能。作为我们对昼夜节律的研究的扩展，我们目前正在分析一个家庭的遗传原因，其中3个受影响的个体表现出季节性伸长/睡眠的缩短。 人糖皮质激素受体（GR）基因分别通过替代使用特定外显子9Beta和9alpha产生C末端GRBETA和GRALPHA同工型。与介导大多数已知糖皮质激素作用的经典受体谷物相反，Grbeta的功能在很大程度上没有探索。因此，我们通过稳定地表达EGFP-GRBETA或EGFP的HELA细胞来探索Grbeta对内源基因的转录活性。微阵列分析表明，GRBETA具有固有的基因特异性转录活性，对大量基因的mRNA表达进行了负或积极的调节。大多数GRBETA响应基因与Gralpha调节的基因不同，而Grbeta和Gralpha在基因亚群中相互调节的转录活性。我们的结果表明，除了其先前报道的对Gralpha诱导的GRE驱动启动子反式激活的主要负面影响外，Grbeta具有固有的，gralpha独立的基因转录活性。