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，具有广泛的维持生命的功能，对治疗多种炎症/自身免疫/过敏和淋巴细胞增生性疾病至关重要。因此，组织对糖皮质激素敏感性的改变可能发展成病理状态并影响其病程。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异合子突变，在401号氨基酸（GRD401H）处将天冬氨酸替换为组氨酸。该突变受体表现出比野生型受体强2-3倍的转录活性，而患者表现出糖皮质激素耐药和高敏感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广泛性糖皮质激素耐药病例，其中包含一个异卵突变，在714号氨基酸（GR R714Q）上精氨酸被谷氨酸取代。该患者是所有携带GR基因病理突变的病例中最年轻的一例。GR R714Q表现出糖皮质激素应答启动子的转录活性降低，对地塞米松的亲和力降低，激活功能（AF）形成缺陷2。对GR R714Q配体结合域的计算机结构分析表明，突变破坏了LBD的&#945；-heices, 7,8,9和10形成的氢键网络，改变了它们的三维排列，最终破坏了配体结合袋和AF2表面。