Neuroendocrine Control Of The Stress Response

应激反应的神经内分泌控制

• 批准号: 7968518
• 负责人: Greti Aguilera
• 金额: $ 61.73万
• 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/7968518
• 关键词: Acute; Address; Adrenal Glands; Affect; Amygdaloid structure; Antibodies; Area; Behavior; Behavioral; Binding; Biological Assay; Birth; CREB1 gene; Cell Line; Cell Nucleus; Cells; Chronic; Co-Immunoprecipitations; Confocal Microscopy; Control Groups; Corticosterone; Corticotropin; Corticotropin-Releasing Hormone; Cyclic AMP; Cytoplasm; DNA Methylation; DNA-Binding Proteins; Data; Development; Diagnostic; Dose; Elements; Epigenetic Process; Evaluation; Exposure to; Failure; Feedback; Female; Forskolin; Genetic Transcription; Glucocorticoids; Goals; Heterogeneous Nuclear RNA; Histones; Hormonal; Hormones; Hypersensitivity; Hypothalamic structure; Immigration; In Vitro; Indium; Knock-out; Laboratories; Laboratory Study; Lead; Life Stress; Longitudinal Studies; Luciferases; Maternal Deprivation; Mediating; Messenger RNA; Metabolic; Methylation; Minor; Molecular; Neonatal; Neurons; Neuropeptides; Neurosecretory Systems; Nuclear; Nuclear Protein; Nuclear Proteins; Nuclear Translocation; Pathology; Peptides; Phorbol Esters; Phosphorylation; Physiological; Pituitary Gland; Plasma; Production; Proteins; RNA Interference; Rattus; Regulation; Reporter Genes; Reporting; Research; Signal Transduction; Solid; Staining method; Stains; Steroid biosynthesis; Stress; System; Testing; Therapeutic; Time; Tissues; Transcript; Transcriptional Activation; Transcriptional Regulation; Transducers; V1b vasopressin receptor; Vasopressins; Weight; Western Blotting; base; biological adaptation to stress; chromatin immunoprecipitation; hormone regulation; hypothalamic-pituitary-adrenal axis; in vivo; indexing; killings; magnocellular; male; paraventricular nucleus; parvocellular; prevent; promoter; research study; response; restraint; restraint stress; sex; tool; transcription factor; transfection/expression vector

Summary: Studies of this laboratory have been pivotal for understanding the interaction between CRH and vasopressin (VP) in the regulation of pituitary ACTH, and the regulation of the expression of these peptides in the PVN during stress and other alterations of the hypothalamic pituitary adrenal (HPA) axis. Both peptides co-expressed in the same parvocellular neuron of the paraventricular nucleus (PVN) are differentially regulated during stress or exposure to glucocorticoids. CRH coordinates behavioral, autonomic and hormonal responses to stress and is the main regulator of ACTH secretion in acute and chronic conditions. Following CRH release, activation of CRH transcription is required to restore mRNA and peptide levels, but termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. This laboratory has made important contributions on the understanding of the mechanisms controlling negative and positive transcriptional regulation of CRH. CRH transcription is under positive control by cAMP/phospho-CREB signaling and negatively regulated by glucocorticoid feedback. Concerning the positive regulation, this laboratory has reported solid evidence that cAMP/phospho-CREB signaling, believed to mediate activation of the CRH promoter, is essential but not sufficient to activate CRH transcription. This finding strongly suggested that transcriptional activation requires a co-activator of CREB. In a number of systems it has been shown that CREB mediated transcription potentially involved in the regulation of CRH transcription is Transducer Of Regulated CREB activity (TORC) is required for CREB mediated transcription. The ability of TORC to regulate CRH transcription was examined in the hypothalamic cell line 4B transfected with a CRH promoter driven luciferase reporter gene. Studies were performed in the hypothalamic cell line, 4B, to examine the hypothesis that the CREB co-activator, TORC, is required for activation of CRH transcription. This cell line does not express endogenous CRH but it has been proven to provide a good system for studying CRH transcription using reporter gene assays. Quantitative real time PCR indicated that all 3 TORC subtypes are present in 4B cells with TORC 2 being the most abundant expressed (TORC 2, 5-fold>TORC 1, 15x>TORC 3). Western blot analysis of cytoplasmic and nuclear proteins revealed rapid and transient nuclear translocation of TORC 2 and 3, and to a minor extent TORC 1, by forskolin in a dose dependent manner. In contrast, the phorbol ester, PMA, had no effect on nuclear TORC levels and caused a delay in migration in the cytoplasm suggesting hyper-phosphorylation. In reporter gene assays, co-transfection of expression vectors for TORC 1 or 2 increased basal CRH promoter activity and potentiated the stimulatory effect of forskolin. The phorbol ester PMA had no significant effect of CRH promoter activity, with or without TORC over-expression. Silencing RNA knock out of each endogenous TORC subtype partially inhibited forskolin-stimulated CRH promoter activity, while simultaneous knockout of TORC 2 and 3 was sufficient to prevent it. Co-immunoprecipitation and chromatin immunoprecipitation experiments revealed association of CREB and TORC in the nucleus, and recruitment of TORC 2 by the CRH promoter, following 30min incubation with forskolin. The data demonstrates that TORC 2 is required for transcriptional activation of the CRH promoter in the hypothalamic cell line 4B, by acting as a CREB co-activator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB. The physiological relevance of these findings was studied in primary cultures of hypothalamic neurons in vitro and hypothalamic tissue from control and stressed rats. Western blot analysis of hypothalamic proteins and TORC 1, 2 and 3 antibodies revealed the presence of TORC 1 and 2 in cytoplasmic and nuclear fractions. Thirty min restraint stress caused a slight increase of TORC 2 in the nuclear fractions followed by a decrease to basal by 2h. There were no significant changes in TORC1 levels at either 30min or 2h of the stress in cytoplasmic of nuclear proteins. Similar results were observed in hypothalamic neuronal cultures following 20 min incubation with forskolin, with only TORC 2 translocating to the nucleus. Immunohistochemical studies using TORC2 antibody in rat hypothalamic tissue, revealed specific staining in the parvocellular and magnocellular areas of the paraventricular nucleus (PVN). Staining was mostly cytoplasmic in controls and 4h after restraint, at 30 min restraint, there was a significant increase in nuclear staining in the parvocellular but not magnocellular region. Double fluorescent immunostaining and confocal microscopy showed that 61 3.5% of CRH immunoreactive cells also stained for TORC2. The demonstration of the presence of TORC 2 in CRH neurons and its transient translocation to the nucleus during stress supports the involvement of this TORC subtype in CRH regulation. Current research is focused on the interactions of TORC with the CRH promoter and on the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo. During the past year considerable effort was placed in studying the long-term consequences of early life stress on the function of the HPA axis. It is well recognized that stress exposure during early development causes long-lasting alterations in behavior and HPA axis activity, including increased levels of CRH mRNA in the PVN. The aim of this study was to test the hypothesis that early life stress causes epigenetic changes in the CRH promoter leading to increased CRH transcription. Groups of 8-week old female and male rats, which had been subjected to maternal deprivation between days 2and 10 post-birth, were killed either in basal conditions, or following 30 or 60 min restraint stress for evaluation of ACTH and corticosterone, and CRH primary transcript or hnRNA levels (as an index of CRH transcription). Additional groups of control and MD were used for methylation analysis of the CRH promoter in the PVN and the amygdala. Adrenal weight, basal levels of plasma corticosterone and hypothalamic CRH hnRNA were higher in MD females but not in males. However, plasma corticosterone and CRH hnRNA responses to acute restraint stress were higher in MD of both sexes. DNA methylation analysis of the CRH promoter in the PVN and amygdala revealed a lower percent of methylation specifically in 2 CpGs located immediately preceding (1) and inside (2) the cAMP-responsive element (CRE) at -230 in the CRH promoter, in both sexes. This CRE has been shown to be an absolute requirement for activation of the CRH promoter. In contrast to the PVN, the percentage of methylation of CpGs 1 and 2 in the amygdala were identical in control in rats subjected to maternal deprivation. These findings demonstrate that HPA axis hypersensitivity caused by neonatal stress causes long-lasting enhanced CRH transcriptional activity in the PVN of both sexes. Hypomethylation of the -230 CRE in the CRH promoter is likely to serve as a mechanism for the increased transcriptional responses to stress observed in maternal deprivation in rats. Current efforts are directed to elucidate the functional consequences of hypomethylation of CpGs 1 and 2. Specifically studies will be performed to examine on the capacity of the CRH promoter CRE to bind the transcription factor, phospho-CREB, and the co-activator, TORC 2, as well as the association of acetylated histones and methyl DNA binding protein to the CRH promoter.

摘要：该实验室的研究对于理解 CRH 和加压素 (VP) 在垂体 ACTH 调节中的相互作用，以及在压力和下丘脑垂体肾上腺的其他变化期间这些肽在 PVN 中的表达调节至关重要。 HPA）轴。两种肽在室旁核（PVN）的同一小细胞神经元中共表达，在应激或暴露于糖皮质激素期间受到不同的调节。 CRH 协调对压力的行为、自主神经和激素反应，是急性和慢性疾病中 ACTH 分泌的主要调节因子。 CRH 释放后，需要激活 CRH 转录来恢复 mRNA 和肽水平，但终止反应对于预防与 CRH 和糖皮质激素产生慢性升高相关的病理学至关重要。该实验室在理解CRH负转录和正转录调控机制方面做出了重要贡献。 CRH 转录受到 cAMP/磷酸-CREB ​​信号传导的正控制，并受到糖皮质激素反馈的负调节。 关于正向调节，该实验室报告了确凿的证据，表明 cAMP/磷酸-CREB ​​信号传导（据信介导 CRH 启动子的激活）对于激活 CRH 转录至关重要，但不足以激活。这一发现强烈表明转录激活需要 CREB ​​的共激活因子。在许多系统中，已经表明CREB介导的转录可能参与CRH转录的调节，调节CREB活性的转导器(TORC)是CREB介导的转录所必需的。在用 CRH 启动子驱动的荧光素酶报告基因转染的下丘脑细胞系 4B 中检查了 TORC 调节 CRH 转录的能力。 在下丘脑细胞系 4B 中进行了研究，以检验 CREB ​​共激活因子 TORC 是激活 CRH 转录所必需的假设。该细胞系不表达内源 CRH，但已被证明为使用报告基因测定研究 CRH 转录提供了良好的系统。定量实时 PCR 表明所有 3 个 TORC 亚型均存在于 4B 细胞中，其中 TORC 2 表达最丰富（TORC 2，5 倍>TORC 1，15x>TORC 3）。细胞质和核蛋白的蛋白质印迹分析显示，福司可林以剂量依赖性方式使 TORC 2 和 3 以及较小程度的 TORC 1 快速且短暂地发生核转位。相比之下，佛波酯 PMA 对核 TORC 水平没有影响，并导致细胞质迁移延迟，表明过度磷酸化。在报告基因检测中，TORC 1 或 2 表达载体的共转染增加了基础 CRH 启动子活性并增强了毛喉素的刺激作用。无论有或没有TORC 过表达，佛波酯PMA 对CRH 启动子活性均无显着影响。 沉默RNA敲除每种内源TORC亚型会部分抑制毛喉素刺激的CRH启动子活性，而同时敲除TORC 2和3足以阻止这种情况。免疫共沉淀和染色质免疫沉淀实验揭示了与毛喉素孵育 30 分钟后，CREB ​​和 TORC 在细胞核中的关联，以及 CRH 启动子对 TORC 2 的募集。数据表明，TORC 2 作为 CREB ​​共激活剂，是下丘脑细胞系 4B 中 CRH 启动子转录激活所必需的。此外，PMA 处理期间 TORC 的细胞质滞留可能解释了尽管有效磷酸化 CREB，佛波酯仍无法激活 CRH 转录。 在体外原代培养的下丘脑神经元以及来自对照和应激大鼠的下丘脑组织中研究了这些发现的生理相关性。下丘脑蛋白以及 TORC 1、2 和 3 抗体的蛋白质印迹分析揭示了细胞质和核部分中存在 TORC 1 和 2。 30 分钟的约束应力导致核分数中 TORC 2 略有增加，随后 2 小时降至基础分数。应激30分钟和2小时后，核蛋白胞质中TORC1水平没有显着变化。与毛喉素孵育 20 分钟后，在下丘脑神经元培养物中观察到类似的结果，仅 TORC 2 易位至细胞核。在大鼠下丘脑组织中使用 TORC2 抗体进行的免疫组织化学研究揭示了室旁核 (PVN) 的小细胞和大细胞区域的特异性染色。对照中染色主要是细胞质，抑制后4小时，抑制30分钟时，细小细胞区域的核染色显着增加，而大细胞区域则没有。双荧光免疫染色和共聚焦显微镜显示，61±3.5%的CRH免疫反应性细胞也被TORC2染色。 CRH 神经元中 TORC 2 的存在及其在应激期间短暂易位至细胞核的证明支持了该 TORC 亚型参与 CRH 调节。目前的研究重点是TORC与CRH启动子的相互作用以及共激活子TORC在体内CRH转录的生理调节过程中的重要性。 在过去的一年里，人们投入了大量精力来研究早期生活压力对 HPA 轴功能的长期影响。 众所周知，早期发育过程中的压力暴露会导致行为和 HPA 轴活动的长期改变，包括 PVN 中 CRH mRNA 水平的增加。本研究的目的是检验以下假设：早期生活压力会导致 CRH 启动子发生表观遗传变化，从而导致 CRH 转录增加。 8周龄雌性和雄性大鼠组在出生后第2天至第10天期间被剥夺母性，在基础条件下或在30或60分钟的束缚应激后处死，以评估ACTH和皮质酮以及CRH初级转录物或 hnRNA 水平（作为 CRH 转录的指标）。 另外的对照组和MD组用于PVN和杏仁核中CRH启动子的甲基化分析。 MD 女性的肾上腺重量、血浆皮质酮基础水平和下丘脑 CRH hnRNA 较高，但男性则不然。然而，两性 MD 中血浆皮质酮和 CRH hnRNA 对急性束缚应激的反应均较高。对 PVN 和杏仁核中 CRH 启动子的 DNA 甲基化分析显示，甲基化百分比较低，特别是位于 CRH 启动子中 -230 处 cAMP 响应元件 (CRE) 之前 (1) 和内部 (2) 的 2 个 CpG，男女皆宜。该 CRE 已被证明是激活 CRH 启动子的绝对必要条件。与 PVN 相比，在母性剥夺的大鼠中，杏仁核中 CpGs 1 和 2 的甲基化百分比与对照组相同。这些发现表明，新生儿应激引起的 HPA 轴超敏反应会导致两性 PVN 中 CRH 转录活性的持久增强。 CRH 启动子中 -230 CRE 的低甲基化可能是在大鼠母性剥夺中观察到的应激转录反应增加的机制。 目前的努力旨在阐明 CpG 1 和 2 低甲基化的功能后果。将进行具体研究以检查 CRH 启动子 CRE 结合转录因子磷酸-CREB ​​和共激活剂 TORC 2 的能力，以及乙酰化组蛋白和甲基 DNA 结合蛋白与 CRH 启动子的关联。