Molecular Mediators and Regulators of Glucocorticoid Act

糖皮质激素作用的分子介质和调节剂

• 批准号: 7332773
• 负责人: Tomoshige Kino
• 金额: --
• 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/7332773
• 关键词: Molecular; Mediators; Regulators; Glucocorticoid; Act

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 responsiveness to glucocorticoids may develop into pathologic states and influence their course. We investigated pathophysiologic mechanism of one of such conditions, familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations in the glucocorticoid receptor (GR) gene. We examined molecular defects of known pathologic mutants GRR477H and GRG679S. GRR477H completely lost its transcriptional activity due to its inability to bind the glucocorticoid response elements (GREs), while GR679S demonstrated reduced transcriptional activity with attenuated ligand-binding activity. We recently found two new heterozygotic mutations that replace aspartic acid by histidine at amino acid 401 and phenylalanine by leucine at amino acid 737 (GRD401H and F737L). In preliminary experiments, GRD401H demonstrated 2-3 times stronger transcriptional activity than the wild type receptor, while GRF737L produced a right-shifted dexamethasone-titration curve of transcriptional activity. We will examine their molecular defects and clarify impact of the mutations found to patients? phenotypes. To examine biologic defects of pathologic GR mutant receptors in living cells, we examined their motility inside the nucleus by using the fluorescence recovery after photobleaching (FRAP) method. Motility of the ligand-bound wild type GR is regulated inside the nucleus through its multiple interactions with the chromatin-associated molecules, transcriptional intermediate proteins, target DNAs and the ubiquitine/proteasomal pathway. We found that all of the examined mutant receptors demonstrated increased motility, which was correlated with their ligand-dependent transcriptional activity. Thus, pathologic mutant GRs possess dynamic motility defects in the nucleus, possibly caused by their inability to properly interact with key partner nuclear molecules necessary for full activation of glucocorticoid-responsive genes. To find intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screenings using several GR domains as baits. We found in the screening using the N-terminal domain of GR as bait that the transforming growth factor (TGF) beta/bone morphogenetic protein-downstream Smad6 specifically interacted with GR. Smad6 suppressed GR transcriptional activity both at cellular and animal levels via attracting histone deacetylases and antagonizing histone acetylation induced by p160 type histone acetyltransferase coactivators. It appears that the anti-glucocorticoid actions of Smad6 may contribute to the neuroprotective, anti-catabolic and pro-wound healing properties of the TGF beta family of proteins. In another yeast two-hybrid screening using the GR DNA-binding domain, we found that SET/TAF-1beta and gas5 interacted with this portion of the GR. The former molecule is known as a part of the SET-CAN oncogene product, as well as a component of the inhibitor of acetyltransferases (INHAT) complex that binds lysine residues of histones and protects them from acetylation by histone acetyltransferases. We found that SET/TAF-1beta acts as a negative regulator of GR transcriptional activity and ligand-activated GR stimulates transcription by displacing the INHAT complex from histones via physical interaction through the DBD. In contrast to Set/TAF-I beta, the Set-Can fusion protein constitutively bound GREs and suppressed GR-induced transcriptional activity/histone acetylation regardless of ligand availability. Thus, Set/TAF-I beta acts as a dynamic regulator of GR-induced transcriptional activity, suppressing baseline transcription by preventing histone acetylation, while facilitating a switch from INHAT-corepressor to HAT-coactivator complexes on GR-bound promoters in response to ligand. Pathologic fusion of Set to Can might disrupt this physiologic switch, causing glucocorticoid insensitivity in acute undifferentiated leukemia with Set-Can translocation. The other interacting molecule gas5 that was found in the same screening is non protein-coding RNA. It is highly accumulated in growth-arrested cells, but its physiologic roles are not known. We found that gas5 bound GR at its DNA-binding domain, prevented its association 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. In addition to GR, gas5 suppressed the transcriptional activities of the progesterone and estrogen receptors, but not of other transcription factors. Thus, gas5 is a growth arrest-related co-repressor of steroid receptors in resting cells, restricting the expression of steroid-responsive genes, whose expression might be nonadaptive in growth factor-deprived cells. Glucocorticoids play an essential role in the homeostasis of the central nervous system and influence diverse functions of neuronal cells. In 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 nervous system, and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with this domain of the GR through its activators p35/p25. CDK5 phosphorylated GR at serines 203 and 211 located in its N-terminal domain and suppressed the transcriptional activity of this receptor on a glucocorticoid-responsive promoter by attenuating attraction of transcriptional cofactors to DNA. 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. Brx, a Rho type guanine nucleotide exchange factor, activates Rho family small G proteins by converting them from inactive GDP-bound to active GTP-bound form. This protein has a nuclear receptor-interacting domain in its C-terminal portion. We found that Brx enhanced GR transcriptional activity by activating and closely attracting small G proteins to GRE-bound GR. Brx mediated lysophosphatidic acid (LPA)-induced potentiation of GR transactivation. LPA is produced from activated platelets in patients with dysmetabolic syndrome who may have glucocorticoid hypersensitivity. Thus, Brx may contribute to theincreased glucocorticoid sensitivity seen in these patients, mediating LPA-induced signal to GR. Circulating glucocorticoid concentrations fluctuate in a circadian pattern coordinated with the animal?s rest-activity cycle, such that peak concentrations are reached around the time of onset of the active period and lowest concentrations in association with inactivity. 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. In preliminary experiments, we found that the CLOCK/BMAL1 expression potentiated GR-induced transcriptional activity by directly acetylating the GR. Thus, it appears that the circadian rhythm regulates glucocorticoid action at the level of target tissues through the interaction between GR and CLOCK/BMAL1.

糖皮质激素具有广泛的维持生命的功能，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，组织对糖皮质激素反应性的改变可能发展为病理状态并影响其病程。我们研究了其中一种情况的病理生理机制，家族性/散发性糖皮质激素抵抗综合征，由糖皮质激素受体（GR）基因突变引起。我们检测了已知病理突变体GRR477H和GRG679S的分子缺陷。GRR477H由于无法结合糖皮质激素反应元件（GREs）而完全丧失转录活性，而GR679S则表现出转录活性降低，配体结合活性减弱。我们最近发现了两个新的杂合突变（GRD401H和F737L），在401号氨基酸上用组氨酸取代天冬氨酸，在737号氨基酸上用亮氨酸取代苯丙氨酸。在初步实验中，GRD401H的转录活性比野生型受体强2-3倍，GRF737L的转录活性呈右移地塞米松滴定曲线。我们将检查它们的分子缺陷，并阐明发现的突变对患者的影响。表型。为了检测活细胞中病理性GR突变受体的生物学缺陷，我们采用光漂后荧光恢复（FRAP）法检测了它们在细胞核内的运动。配体结合野生型GR通过与染色质相关分子、转录中间蛋白、靶dna和泛素/蛋白酶体途径的多重相互作用，在细胞核内调节其运动。我们发现所有检测的突变受体表现出增加的运动性，这与它们的配体依赖性转录活性相关。因此，病理性突变的gr在细胞核中具有动态运动缺陷，这可能是由于它们无法与糖皮质激素应答基因充分激活所必需的关键核分子适当相互作用所致。为了寻找可能影响组织对糖皮质激素敏感性的细胞内分子，我们使用几个GR结构域作为诱饵进行了酵母双杂交筛选。我们以GR的n端结构域为诱饵进行筛选，发现转化生长因子(TGF) β /骨形态发生蛋白下游Smad6与GR特异性相互作用。Smad6通过吸引组蛋白去乙酰化酶和拮抗p160型组蛋白乙酰转移酶共激活因子诱导的组蛋白乙酰化，在细胞和动物水平上抑制GR的转录活性。看来Smad6的抗糖皮质激素作用可能有助于TGF β蛋白家族的神经保护、抗分解代谢和促进伤口愈合特性。在另一项酵母双杂交筛选中，使用GR dna结合域，我们发现SET/ taf -1 β和gas5与GR的这一部分相互作用。前者分子是SET- can癌基因产物的一部分，也是乙酰转移酶抑制剂（INHAT）复合物的一个组成部分，该复合物结合组蛋白的赖氨酸残基并保护它们不被组蛋白乙酰转移酶乙酰化。我们发现SET/ taf -1 β作为GR转录活性的负调节因子，配体激活的GR通过DBD的物理相互作用取代组蛋白上的INHAT复合物来刺激转录。与Set/ taf - 1 β相比，Set- can融合蛋白组成性结合GREs并抑制gr诱导的转录活性/组蛋白乙酰化，而不考虑配体的可用性。因此，Set/ taf - 1 β作为gr诱导的转录活性的动态调节剂，通过阻止组蛋白乙酰化来抑制基线转录，同时促进gr结合启动子上的inhat -辅抑制因子到hat -辅激活因子复合物的转换，以响应配体。Set-Can的病理融合可能会破坏这种生理开关，导致急性未分化白血病伴Set-Can易位的糖皮质激素不敏感。在同一筛选中发现的另一种相互作用分子是非蛋白质编码RNA。它在生长受阻的细胞中高度积累，但其生理作用尚不清楚。我们发现gas5在GR的dna结合区域结合GR，阻止其与GREs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 mRNA的表达，并阻止生长受阻细胞的凋亡。除GR外，gas5对孕酮和雌激素受体的转录活性有抑制作用，但对其他转录因子无抑制作用。因此，gas5是静止细胞中类固醇受体的生长抑制相关的共抑制因子，限制类固醇反应基因的表达，而这些基因的表达在生长因子剥夺的细胞中可能是不适应的。糖皮质激素在中枢神经系统的稳态中起着至关重要的作用，并影响神经元细胞的多种功能。在利用GR配体结合域的细胞诱捕酵母双杂交筛选中，我们发现在神经系统的形态发生和功能中起重要作用的细胞周期蛋白依赖性激酶5 (CDK5)，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的该结构域相互作用。CDK5磷酸化位于其n端区域的GR丝氨酸203和211，并通过减弱转录辅助因子对DNA的吸引力来抑制该受体在糖皮质激素应答启动子上的转录活性。在使用大鼠皮质神经元细胞的微阵列分析中，CDK5抑制剂罗斯科维汀对超过90%的内源性糖皮质激素应答基因的GR转录活性进行了差异调节。因此，CDK5通过GR在神经元细胞中发挥其部分生物活性，以靶启动子依赖的方式动态调节GR的转录活性。Brx是一种Rho型鸟嘌呤核苷酸交换因子，通过将Rho家族小G蛋白从无活性的gdp结合形式转化为活性的gtp结合形式来激活Rho家族小G蛋白。该蛋白在其c端部分有一个核受体相互作用域。我们发现Brx通过激活并紧密吸引小G蛋白到GR结合的GR中来增强GR的转录活性。Brx介导的溶血磷脂酸（LPA）诱导的GR转激活增强。LPA是由代谢异常综合征患者的活化血小板产生的，这些患者可能有糖皮质激素过敏。因此，Brx可能有助于这些患者糖皮质激素敏感性的增加，介导lpa诱导的GR信号。循环糖皮质激素浓度与动物的昼夜节律模式相协调。S休息-活动周期，因此，在活跃期开始时浓度达到峰值，而与不活动相关的浓度最低。昼夜节律钟由基本的螺旋-环-螺旋转录因子clock及其异二聚体伙伴BMAL1组成，控制着中枢神经系统和外周组织的昼夜节律。在初步实验中，我们发现CLOCK/BMAL1的表达通过直接乙酰化GR来增强GR诱导的转录活性。因此，昼夜节律似乎通过GR和CLOCK/BMAL1的相互作用在靶组织水平上调节糖皮质激素的作用。