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) 轴的最终产物，具有广泛的生命维持功能，对于治疗多种炎症/自身免疫/过敏性和淋巴增殖性疾病至关重要。因此，组织对糖皮质激素敏感性的变化可能会形成病理状态并影响其病程。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，该综合征是由糖皮质激素受体（GR）基因突变引起的。我们在一名哥伦比亚患者中发现了一种杂合突变，该突变将第 401 位氨基酸 (GRD401H) 处的天冬氨酸替换为组氨酸。这种突变型受体的转录活性比野生型受体强 2-3 倍，而患者则表现出糖皮质激素抵抗和过敏状态的混合表型特征。因此，这种突变受体引起糖皮质激素活性的组织特异性改变。我们还发现了另一个具有普遍糖皮质激素耐药性的病例，该病例具有杂合突变，在第 714 位氨基酸处用谷氨酸取代了精氨酸（GR R714Q）。该患者是迄今为止报告的所有携带 GR 基因病理突变病例中最年轻的患者。 GR R714Q 表现出糖皮质激素响应启动子上的转录活性降低、对地塞米松的亲和力降低以及激活功能 (AF) 2 的形成缺陷。对 GR R714Q 配体结合域的计算机结构分析表明，该突变破坏了氢由LBD的α-heices、7、8、9和10形成的键网络改变了它们的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）的稳态中发挥着重要作用，并影响神经元细胞的多种功能。我们之前报道过细胞周期蛋白依赖性激酶 5 (CDK5) 在 CNS 的形态发生和功能中发挥重要作用，其异常激活与神经退行性疾病的发展相关，通过其激活剂 p35/p25 与 GR 相互作用并进行差异调节GR 的转录活性。 CDK5 在多个丝氨酸处磷酸化 GR，包括位于 203、211 和 226 的丝氨酸。由于糖皮质激素除 GR 外还使用盐皮质激素受体 (MR) 作为大脑中的功能受体，因此我们检查了 CDK5 对 MR 的影响，发现这激酶磷酸化 MR 的丝氨酸 129 和 158 以及苏氨酸 250 并调节 MR 诱导的转录活性与GR类似。为了进一步研究 CDK5 介导的 GR 和 MR 调节的重要性，我们重点关注脑源性神经营养因子 (BDNF)。 MR特异性配体醛固酮和GR特异性地塞米松分别增加和抑制大鼠皮质神经元细胞中BDNF mRNA/蛋白的表达，而CDK5则增强它们对BDNF表达的影响。由于 BDNF 在神经活性、突触可塑性、记忆巩固和情绪变化中发挥着关键作用，我们认为 CDK5 的异常激活可能通过皮质类固醇受体/BDNF 调节这些神经元活动，进一步促进神经退行性疾病的发展，并且可能，情绪障碍。 为了找到更多可能影响组织对糖皮质激素敏感性的细胞内分子，我们使用 GR DBD 进行了酵母双杂交筛选测定，发现非编码 (nc) Gas5 与 GR 的这一部分相互作用。这种 ncRNA 在生长停滞的细胞中积累，但其生理作用尚不清楚。我们发现 Gas5 在其 DBD 处与 GR​​ 特异性结合，并响应糖皮质激素而与 GR 一起易位到细胞核中。 Gas5 RNA 阻止 GR 与其调控 DNA 元件的结合并抑制其转录活性。血清饥饿诱导的 Gas5 抑制糖皮质激素介导的细胞凋亡抑制剂 2 (cIAP2) mRNA 表达，并通过抑制该蛋白表达来防止生长停滞细胞的凋亡。 Gas5在其分子内双螺旋结构的第3部分有一个类GRE序列，通过该序列，该ncRNA通过模仿DNA GRE与GR DBD竞争性相互作用。因此，Gas5 是 GR 的生长停滞相关共抑制因子，含有诱饵 RNA GRE，限制类固醇反应基因的表达。这是一个新颖的概念，表明 ncRNA 和基因组 DNA 之间对类固醇受体 DBD 的竞争。这项工作还表明，Gas5 可能有助于细胞对饥饿的适应性反应，防止细胞凋亡并节省能源。 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。我们发现 CLOCK/BMAL1 以 HAT 活性依赖性方式抑制 GR 诱导的转录活性。在血清休克同步细胞中，GR 反式激活活性以昼夜节律方式自发波动，与 CLOCK/BMAL1 mRNA 表达反相。 CLOCK 和 GR 在物理上相互作用，并且 CLOCK 通过乙酰化位于其铰链区的一簇赖氨酸残基来抑制 GR 与启动子 GRE 的结合。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。此外，这些结果表明，外周靶组织昼夜节律通过调节糖皮质激素的普遍存在和多样化的作用，间接影响体内每个器官/组织的功能。作为我们对昼夜节律研究的延伸，我们目前正在分析一个家庭的遗传原因，其中 3 名受影响的个体表现出季节性睡眠延长/缩短。 人类糖皮质激素受体 (GR) 基因通过交替使用特定的外显子 9beta 和 9alpha 分别产生 C 末端 GRbeta 和 GRalpha 亚型。与介导大多数已知糖皮质激素作用的经典受体 GRalpha 相比，GRbeta 的功能在很大程度上尚未被探索。因此，我们通过开发稳定表达 EGFP-GRbeta 或 EGFP 的 HeLa 细胞，探索了 GRbeta 对内源基因的转录活性。微阵列分析表明，GRbeta 具有内在的基因特异性转录活性，负向或正向调节大量基因的 mRNA 表达。大多数 GRbeta 反应基因与 GRalpha 调节的基因不同，而 GRbeta 和 GRalpha 在基因亚群中相互调节彼此的转录活性。我们的结果表明，除了先前报道的对 GRalpha 诱导的 GRE 驱动启动子反式激活的显性负面影响外，GRbeta 还具有内在的、独立于 GRalpha 的基因特异性转录活性。