Molecular Mediators and Regulators of Glucocorticoid Actions

糖皮质激素作用的分子介质和调节剂

基本信息

批准号：
7734762
负责人：
Tomoshige Kino
金额：
$ 28.88万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/7734762
关键词：
Acetyltransferase Acute Adenosine Allergic Amino Acids Animals Apoptosis Aspartic Acid Autoimmune Process BHLH Protein Binding Biological Assay Brain Cells Characteristics Chimeric Proteins Circadian Rhythms Clinical Endocrinology Co-Immunoprecipitations Complex Condition Cyclin-Dependent Kinase 5 DNA Defect Development Disease Energy Metabolism Fatty Liver Gene Targeting Genes Genetic Transcription Genomics Glucocorticoid Receptor Glucocorticoids Glucose Goals Growth Hand Histidine Histone Acetylation Histones Homeostasis Indium Inflammatory Intervention Journals Life Ligands Liver Lymphoproliferative Disorders Lysine Manuscripts Mediating Mediator of activation protein Metabolic Metabolic Diseases Metabolism Methods Mineralocorticoid Receptor Mitolactol Moderate Exercise Molecular Morphogenesis Mutation N-terminal Neuraxis Neurodegenerative Disorders Neurons Oncogene Proteins Organ Pathologic Pathway interactions Patients Peripheral Phase Phenotype Phosphorylation Phosphotransferases Physiological Play Protein Binding Protein Kinase Proteins Publishing RNA Rattus Reaction Receptor Gene Regulation Reporting Resistance Resources Role Screening procedure Serine Serum Shock Signal Pathway Small Interfering RNA Starvation Steroid Receptors Steroids Stress Structure Syndrome System Testing Threonine Time Tissues Undifferentiated Work Yeasts chromatin immunoprecipitation concept fatty acid metabolism glucocorticoid-induced orphan receptor growth arrest specific transcript 5 histone acetyltransferase human BIRC3 protein human MAPK14 protein hypothalamic-pituitary-adrenal axis inhibitor/antagonist leukemia mRNA Expression mutant novel prevent promoter protein expression receptor transcription factor transcriptomics uptake yeast two hybrid system

项目摘要

Glucocorticoids, the end-products of the hypothalamic-pituitary-adrenal (HPA) axis, have a broad array of life-sustaining functions, and play an important role in 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). Using panels of assay methods, we have characterized details of molecular defects of GRD401H and found that 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 published in this year a landmark review manuscript in the Journal of Clinical Endocrinology & Metabolism on the familial/sporadic glucocorticoid resistance syndrome and molecular defects of the pathologic mutant GRs reported to date. 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 on more than 90 percent of the endogenous glucocorticoid-responsive genes tested. CDK5 phosphorylated GR at multiple serines, including those located at 203, 211 and 226 in its N-terminal domain. 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 find more intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screening assays using the GR DBD, and found that SET/TAF-Ibeta and the noncoding (nc) Gas5 interacted with this portion of the GR. The former molecule is a part of the SET-CAN oncogene product, as well as a component of the inhibitor of acetyltransferases (INHAT) complex. We found that SET/TAF-Ibeta acts as a negative regulator of GR transcriptional activity and that ligand-activated GR stimulates transcription by displacing the INHAT complex from histones via physical interaction through its DBD. In contrast to Set/TAF-Ibeta, the Set-Can fusion protein bound GREs regardless of ligand availability and strongly suppressed GR-induced transcriptional activity/histone acetylation, possibly participating in the development of glucocorticoid insensitivity in acute undifferentiated leukemia with Set-Can translocation. Gas5 also interacted with the GR DBD. 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 as a single sense RNA in a yeast two-hybrid screening and in a RNA-protein co-immunoprecipitation assay. Gas5 RNA prevented the association of the GR with its regulatory DNA elements in a chromatin immunoprecipitation assay and suppressed its transcriptional activity on a glucocorticoid-responsive endogenous promoter. Serum starvation-induced Gas5 suppressed glucocorticoid-mediated cellular inhibitor of apoptosis 2 (cIAP2) mRNA expression, while Gas5 siRNA completely abolished this suppression. Furthermore, serum starvation-induced Gas5 prevented apoptosis of growth-arrested cells through cIAP2 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 an RNA mock 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. Glucocorticoids play central roles in the regulation of basal and stress-related energy metabolism. Adenosine 5 monophosphate-activated protein kinase (AMPK), on the other hand, is the master regulator of energy homeostasis, sensing energy depletion inside the body and stimulating pathways that increase fuel uptake and save on peripheral supplies. We hypothesized that these major regulatory systems communicate with each other to maintain proper metabolic activity in the body. We found that AMPK regulates the actions of glucocorticoids at the cellular level by targeting their intracellular glucocorticoid receptor (GR) and altering its ability to modify transcription of target genes in a tissue- and promoter-specific fashion. AMPK does this by phosphorylating serine 211 of the GR indirectly through phosphorylation and subsequent activation of p38 mitogen-activated protein kinase (MAPK). At the animal level, activation of AMPK in rats completely reversed glucocorticoid-induced hepatic steatosis and strongly suppressed glucocorticoid-mediated stimulation of glucose and fatty acid metabolism. Transcriptomic analysis of liver tissue from these rats using microarrays suggested mutual regulation between the AMPK and glucocorticoid signaling pathways directed mostly from the former to moderate the activities of the latter. Thus, the AMPK-mediated energy control system governs also glucocorticoid action at target tissues. Our results also indicate that activation of AMPK could be a promising target for the development of pharmacologic interventions to metabolic disorders caused by excess amounts of glucocorticoids. Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion as a result of the control exerted by central components of the HPA axis, 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, accessed longitudinally by mRNA expression of an endogenous responsive gene, 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.

糖皮质激素是下丘脑-垂体-肾上腺 (HPA) 轴的终产物，具有广泛的生命维持功能，在多种炎症/自身免疫/过敏性和淋巴增殖性疾病的治疗中发挥着重要作用。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。使用一系列检测方法，我们表征了 GRD401H 分子缺陷的细节，发现这种突变受体的转录活性比野生型受体强 2-3 倍，而患者表现出糖皮质激素抵抗和超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。今年，我们在《临床内分泌与代谢杂志》上发表了一篇具有里程碑意义的综述手稿，内容涉及迄今为止报道的家族性/散发性糖皮质激素抵抗综合征和病理突变 GR 的分子缺陷。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 对所测试的 90% 以上的内源性糖皮质激素反应基因的转录活性。 CDK5 在多个丝氨酸处磷酸化 GR，包括位于其 N 端结构域中 203、211 和 226 的丝氨酸。由于除了 GR 之外，糖皮质激素还利用盐皮质激素受体 (MR) 作为大脑中的功能受体，因此我们检查了 CDK5 对 MR 的影响，发现该激酶磷酸化 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相似。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 SET/TAF-Ibeta 和非编码 (nc) Gas5 与 GR 的这一部分相互作用。前一种分子是 SET-CAN 癌基因产物的一部分，也是乙酰转移酶抑制剂 (INHAT) 复合物的组成部分。我们发现 SET/TAF-Ibeta 作为 GR 转录活性的负调节因子，并且配体激活的 GR 通过其 DBD 的物理相互作用从组蛋白中取代 INHAT 复合物来刺激转录。与 Set/TAF-Ibeta 相比，Set-Can 融合蛋白无论配体可用性如何都能结合 GRE，并强烈抑制 GR 诱导的转录活性/组蛋白乙酰化，可能参与 Set-Can 易位的急性未分化白血病中糖皮质激素不敏感性的发展。 Gas5 还与 GR DBD 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，在酵母双杂交筛选和 RNA-蛋白质共免疫沉淀测定中，Gas5 作为单义 RNA 在其 DBD 处与 GR 特异性结合。在染色质免疫沉淀测定中，Gas5 RNA 阻止 GR 与其调节性 DNA 元件结合，并抑制其在糖皮质激素反应性内源启动子上的转录活性。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，而 Gas5 siRNA 完全消除了这种抑制。此外，血清饥饿诱导的 Gas5 通过 cIAP2 蛋白表达阻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 的生长停滞相关辅抑制因子，含有 RNA 模拟 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。糖皮质激素在基础能量代谢和应激相关能量代谢的调节中发挥核心作用。另一方面，腺苷 5 单磷酸激活蛋白激酶 (AMPK) 是能量稳态的主要调节器，感知体内能量消耗并刺激增加燃料摄取并节省外周供应的途径。我们假设这些主要调节系统相互沟通以维持体内适当的代谢活动。我们发现 AMPK 通过靶向细胞内糖皮质激素受体 (GR) 并改变其以组织和启动子特异性方式修改靶基因转录的能力，在细胞水平上调节糖皮质激素的作用。 AMPK 通过磷酸化并随后激活 p38 丝裂原激活蛋白激酶 (MAPK)，间接磷酸化 GR 的丝氨酸 211，从而实现这一点。在动物水平上，大鼠中 AMPK 的激活完全逆转了糖皮质激素诱导的肝脂肪变性，并强烈抑制了糖皮质激素介导的葡萄糖和脂肪酸代谢刺激。使用微阵列对这些大鼠的肝组织进行转录组分析表明，AMPK 和糖皮质激素信号通路之间存在相互调节，主要是前者调节后者的活性。因此，AMPK 介导的能量控制系统也控制糖皮质激素在靶组织中的作用。我们的结果还表明，AMPK 的激活可能是开发针对过量糖皮质激素引起的代谢紊乱的药物干预措施的有希望的目标。由于 HPA 轴中央组件的控制，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 的反式激活活性（通过内源响应基因的 mRNA 表达纵向访问）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。