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: 7594207
• 负责人: Tomoshige Kino
• 金额: $ 22.23万
• 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/7594207
• 关键词: Acetyltransferase; Activity Cycles; Acute; Agonist; Allergic; Amino Acids; Androgen Receptor; Animals; Apoptosis; Aspartic Acid; Attenuated; Autoimmune Process; BHLH Protein; Binding; Cell Differentiation process; Cells; Characteristics; Chimeric Proteins; Cholesterol; Circadian Rhythms; Complex; Condition; Congenital adrenal hyperplasia; Cyclin-Dependent Kinase 5; DNA; DNA Binding Domain; Development; Dexamethasone; Disease; Estrogen Receptors; Fatty Acids; Fibroblast Growth Factor 2; GTP-Binding Proteins; Genes; Genetic Transcription; Genomics; Glucocorticoid Receptor; Glucocorticoids; Goals; Growth; Hand functions; Histidine; Histone Deacetylase; Histone Deacetylation; Histones; Homeostasis; Human; Immune; Inflammatory; Injury; Leucine; Life; Ligand Binding Domain; Ligands; Lipids; Liver; Lymphoproliferative Disorders; Mediating; Mediator of activation protein; Membrane; Metabolism; Mineralocorticoid Receptor; Mineralocorticoids; Molecular; Morphogenesis; Mouse Mammary Tumor Virus; Mutation; N-terminal; Natural regeneration; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neuronal Differentiation; Neurons; Nuclear; Nuclear Hormone Receptors; Nuclear Orphan Receptor; Nuclear Receptors; Nuclear Translocation; Oncogene Proteins; Orphan; Pathologic; Patients; Pattern; Peripheral; Phase; Phenotype; Phenylalanine; Physiological; Play; Population; Process; Progesterone Receptors; Property; Prostaglandins; Protein Binding; RNA; Rattus; Receptor Gene; Receptor Signaling; Regulation; Resistance; Rest; Retinoic Acid Receptor; Role; Screening procedure; Serine; Serum; Siblings; Starvation; Stem cells; Steroid Receptors; Steroids; Stress; Structure; Syndrome; System; Testing; Time; Tissues; Undifferentiated; Withdrawal; Yeasts; circadian pacemaker; concept; constitutive androstane receptor; dimer; glucocorticoid-induced orphan receptor; hepatoma cell; human BIRC3 protein; immune function; inhibitor/antagonist; leukemia; lipid metabolism; mRNA Expression; mutant; nerve stem cell; novel; precursor cell; prevent; promoter; receptor; response; roscovitine; sensor; yeast two hybrid system

Glucocorticoids 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 into pathologic states andor influence the disease course. We investigated the molecular pathophysiologic mechanism of the familial-sporadic glucocorticoid resistance syndrome, which is caused by mutations of the glucocorticoid receptor (GR) gene. We identified and examined a novel heterozygotic mutation (GRF737L) causing replacement of phenylalanine by leucine at amino acid 737. This mutant receptor has transdominant negative activity on the wild type GR and a delayed nuclear translocation. We found another novel heterozygotic mutation that leads to replacement of aspartic acid by histidine at 401 (GRD401H). This receptor demonstrated 2-3 times stronger transcriptional activity than the wild type receptor, while the patient showed a mixed phenotype characteristic of both glucocorticoid resistance and hypersentivity. Thus, this mutant receptor appears to cause tissue-specific alterations of glucocorticoid activity, an as yet undescribed state. We further found another novel heterozygotic mutation, GRV423A, in the DNA-binding domain (DBD), and yet another homozygotic mutation, GRM593A, in two siblings with congenital adrenal hyperplasia. We are characterizing the molecular properties of these mutant receptors. Glucocorticoids play an essential role in the homeostasis of the central nervous system (CNS) and influence diverse functions of neuronal cells. To find intracellular molecules that potentially influence tissue sensitivity to glucocorticoids in the CNS, we performed a cyto-trap yeast two-hybrid screening using the GR ligand-binding domain. We found that cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of the CNS, and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with this domain of the GR through its activators p35p25. CDK5 phosphorylated the GR at multiple serines, including those located at 203 and 211 in its N-terminal domain. In microarray analyses using rat cortical neuronal cells, the CDK5 inhibitor roscovitine differentially regulated the transcriptional activity of the GR on more than 90 percent of the endogenous glucocorticoid-responsive genes tested. Thus, CDK5 exerts some of its biologic activities in neuronal cells through the GR, dynamically modulating GR transcriptional activity in a target promoter-dependent fashion in the CNS. In another yeast two-hybrid screening using the GR DBD, we found that SET-TAF-Ibeta and 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 through histone deacetylation, possibly participating in the development of glucocorticoid insensitivity in acute undifferentiated leukemia with Set-Can translocation. The latter molecule, Gas5, a noncoding (nc) RNA, 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 GR and prevented the association of ligand-activated GR with GREs and suppressed its transcriptional activity. Serum starvation-induced Gas5 suppressed glucocorticoid-mediated cellular inhibitor of apoptosis 2 mRNA expression and prevented apoptosis of growth-arrested cells. Interestingly, Gas5 has one GRE in its 3 portion in its intra-molecular double helical structure, through which this ncRNA interacts with the GR DBD by mimicking DNA GREs. Thus, Gas5 is a growth arrest-related co-repressor of the GR harboring an 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. Circulating glucocorticoid concentrations fluctuate in a circadian pattern coordinated with the animals rest-activity cycle. The circadian clock, which consists of the basic helix-loop-helix transcription factors CLOCK and its hetero-dimer partner BMAL1, controls circadian rhythms both in the central nervous system and peripheral tissues. We found that the CLOCK-BMAL1 expression inhibited GR-induced transcriptional activity by directly acetylating the GR and negatively modulating GR-induced transcriptional activity in a circadian fashion. Thus, the circadian rhythm regulates not only glucocorticoid secretion but also action at the level of target tissues, in fact, in an opposite phase fashion. Glucocorticoids are crucial for the regulation of basal and stress-related intermediary metabolism and immune homeostasis, while the liver X receptor (LXR) is a sensor of bioactive lipids, such as the cholesterol metabolites oxysterols, prostanoids and fatty acids, which are essential for the regulation of lipid metabolism and immune function. We examined potential interactions between the glucocorticoid and LXR signaling systems. We found that agonist ligand-mediated activation of LXR strongly suppressed dexamethasone-stimulated GR-mediated transcriptional activity of the glucocorticoid-responsive mouse mammary tumor virus promoter, while activation of endogenous LXR completely attenuated dexamethasone-induced mRNA expression of endogenous glucocorticoid-responsive genes in human hepatoma cells. Thus, it appears that various biologically active lipids may influence the peripheral actions of glucocorticoids via LXR. CNS Stem cells are a well-defined population of precursor cells that differentiate into neurons and glial cells. Elucidation of neuronal differentiation mechanisms is essential for understanding both physiologic and pathologic neuronal development and regeneration after injury. nuclear hormone receptors and their transcriptional coregulators, on the other hand, function as an adaptive mechanism that modulates numerous cellular activities in response to changes of external circumstances by sensing their ligands. Thus, to identify key nuclear receptors and their transcriptional regulators in neural stem cell differentiation, we examined mRNA expression of a large spectrum of nuclear receptors and orphan nuclear receptors, including the glucocorticoid and mineralocorticoid (MR) receptors and many of their coregulators, during neural stem cell differentiation after withdrawal of fibroblast growth factor-2 (FGF2), a crucial determinant of this process. We found that 34 out of 47 nuclear receptorsorphan nuclear receptors examined were expressed at the basal state. Several receptors already known to play major roles in neuronal development were highly expressed at baseline, while the testicular receptors 2 and 4, liver X receptors and the constitutive androstane receptor were highly expressed in undifferentiated stem cells. GR, membrane G-protein-coupled progesterone receptor and androgen receptor, but not estrogen receptors, were also highly expressed in these cells. mRNA expression of the retinoic acid receptor-related orphan receptor gamma and MR were stimulated by over 40 and 30 fold, respectively. Most of the transcriptional coregulators examined were expressed basally and throughout differentiation without major changes. Only, mRNA expression of histone deacetylase-11 was highly stimulated.

糖皮质激素具有多种维持生命的功能，在多种炎症、自身免疫、过敏和淋巴细胞增生性疾病的治疗中发挥重要作用。因此，组织对糖皮质激素敏感性的改变可能发展为病理状态或影响病程。本研究探讨了由糖皮质激素受体（GR）基因突变引起的家族-散发性糖皮质激素耐药综合征的分子病理生理机制。我们鉴定并检测了一种新的杂合子突变（GRF737L），该突变导致氨基酸737处的苯丙氨酸被亮氨酸取代。该突变受体对野生型GR具有显性负活性和延迟核易位。我们发现了另一个新的杂合子突变，导致401位点的天冬氨酸被组氨酸取代（GRD401H）。该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抗性和超敏性的混合表型特征。因此，这种突变受体似乎引起糖皮质激素活性的组织特异性改变，这是一种尚未描述的状态。我们进一步在dna结合域（DBD）发现了另一个新的杂合子突变GRV423A，在患有先天性肾上腺增生的两个兄弟姐妹中发现了另一个纯合子突变GRM593A。我们正在描述这些突变受体的分子特性。