Neuroendocrine Control Of The Stress Response

应激反应的神经内分泌控制

• 批准号: 8351109
• 负责人: Greti Aguilera
• 金额: $ 83.67万
• 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/8351109
• 关键词: Acute; Address; Adrenal Cortex; Adrenal Glands; Affect; Behavioral; Binding; Brain; CREB1 gene; Cell Nucleus; Cells; Chronic; Circadian Rhythms; Complex; Corticosterone; Corticotropin; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytochrome P450; Cytoplasm; Development; Diagnostic; Ependyma; Family; Forskolin; Foundations; Genes; Genetic Transcription; Glucocorticoids; Glutamates; Goals; Heterogeneous Nuclear RNA; Hormonal; Hormones; Hour; Hypothalamic structure; In Situ Hybridization; Infusion procedures; Injection of therapeutic agent; Investigation; Knockout Mice; Laboratories; Lead; Location; Measures; Medial Dorsal Nucleus; Mediating; Messenger RNA; Metabolic; Methylprednisolone; Minor; Molecular; Mus; Neurons; Neuropeptides; Neurosecretory Systems; Norepinephrine; Nuclear; Nuclear Export; Nuclear Translocation; Pathology; Pathway interactions; Pattern; Peptide Receptor; Peptides; Phase; Phosphorylation; Phosphotransferases; Physiologic pulse; Physiological; Pia Mater; Pituitary Gland; Plasma; Production; Prosencephalon; Protein Isoforms; Protein Kinase; Proteins; RNA; Rattus; Regulation; Reporting; Research; Rest; Role; Side; Signal Transduction; Staurosporine; Steroid biosynthesis; Steroids; Stress; Structure; Structure of nucleus infundibularis hypothalami; Testing; Therapeutic; Time; Transcriptional Activation; Transcriptional Regulation; Transducers; V1b vasopressin receptor; Vasopressins; Work; base; biological adaptation to stress; hypothalamic-pituitary-adrenal axis; in vivo; kinase inhibitor; knock-down; magnocellular; overexpression; paraventricular nucleus; pituitary adenylate cyclase activating polypeptide; prevent; promoter; research study; response; restraint; restraint stress; salt-inducible kinase; small hairpin RNA; steroidogenic acute regulatory protein; suprachiasmatic nucleus; supraoptic nucleus; tool; trafficking

Normal activity of the hypothalamic pituitary adrenal axis, leading to the secretion of glucocorticoids by the adrenal gland, is essential for normal metabolic activity and for survival during challenging situations. Studies under this project have defined the role of the hypothalamic peptides corticotrophin releasing hormone (CRH) and vasopressin (VP) in the regulation of pituitary ACTH, and contributed to elucidating the regulation of the expression of CRH and VP during stress, and the mechanisms of action, topographic distribution, regulation and physiological role of the receptors for these peptides in the pituitary gland and in the brain. 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, rapid but transient activation of CRH transcription is required to restore mRNA and peptide levels. Termination of the response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. Current work is aimed to elucidate the mechanisms controlling negative and positive transcriptional regulation of CRH, as well as the mechanisms responsible to the normal circadian and ultradian pattern of glucocorticoid secretion. This laboratory has reported that cAMP/phospho-CREB signaling is essential but not sufficient to activate CRH transcription. This finding led to the discovery that transcriptional activation of the CRH gene requires the CREB co-activator Transducer Of Regulated CREB activity (TORC). In resting conditions, the co-activator is found in a phosphorylated, inactive state in the cytoplasm. Its activation and nuclear translocation requires protein kinase A (PKA)- mediated inhibition protein kinases mediating TORC phosphorylation. Experiments examining the trafficking of TORC from the cytoplasm to the nucleus of CRH producing neurons, and the effects of over-expression and knock down of TORC on CRH transcription demonstrated that TORC is essential for CRH transcription, and that during stress the co-activator shifts to the nucleus and binds to the CRH promoter as part of a complex with CREB, paralleling the activation of CRH transcription. Future research is planned to examine the importance of the co-activator TORC during physiological regulation of CRH transcription in vivo. Significant effort was invested on the mechanisms regulating TORC activity. In basal conditions, transcription is low because TORC remains in the cytoplasm, inactivated by phosphorylation through Ser/Thr protein kinases of the AMP-dependent protein kinases (AMPK) family, including salt-inducible kinase (SIK). To determine which kinase is responsible for TORC phosphorylation in CRH neurons, we measured AMPK, SIK1 and SIK2 mRNA in the PVN of rats by in situ hybridization. In basal conditions, low levels of the 3 kinases were found in the dorsomedial PVN, consistent with location in CRH neurons. Restraint stress increased SIK1 mRNA levels, while SIK2 and AMPK mRNA showed only minor increases. Overexpression of either SIK1 or SIK2 in 4B cells reduced nuclear TORC2 levels and inhibited forskolin-stimulated CRH transcription. Conversely, the non-selective SIK inhibitor, staurosporine, increased nuclear TORC2 content and stimulated CRH transcription in 4Bcells, and primary neuronal cultures (heteronuclear RNA). Specific shRNA knock down of endogenous SIK2 but not SIK1 induced nuclear translocation of TORC2 and CRH transcription, suggesting that SIK2 mediates TORC inactivation in basal conditions, while induction of SIK1 limits transcriptional activation. Current research aims to test the hypothesis that while SIK2 mediates sequestration of TORC in basal conditions, induction of SIK1 during stimulation of the CRH neuron mediates phosphorylation and nuclear export of TORC, thus contributing to the termination of the transcriptional response. Since increasing evidence indicates that CREB-dependent transcriptional activation of a number of genes requires the co-activator, TORC, and because of the importance of CREB in many brain functions, the topographic distribution of TORC1, 2 and 3 mRNAs in specific regions of the rat forebrain were studied. In situ hybridization analysis showed that TORC1 is the most abundant isoform in most forebrain structures, followed by TORC2 and TORC3. Although high levels of TORC1 were widely distributed in the forebrain, TORC2 was found in discrete nuclei and TORC3 mostly in the ependyma, and pia mater. In the paraventricular nucleus of the hypothalamus, TORC1 and 2 mRNAs were abundant in the parvicellular and magnocellular neuroendocrine compartments, whereas TORC3 expression was low. All three isoform mRNAs were found elsewhere in the hypothalamus, with the most prominent expression of TORC1 in the ventromedial nucleus, TORC2 in the dorsomedial and arcuate nuclei, TORCs 1 and 2 in the supraoptic nucleus, and TORC2 in the suprachiasmatic nucleus. These differential distribution patterns are consistent with complex roles for all three TORC isoforms in diverse brain structures, and provide a foundation for further studies on the mechanisms of CREB/TORC signaling on brain function. While cyclic AMP/PKA-dependent pathways are essential for transcriptional activation of the CRH gene, the main direct regulators of the CRH neuron, norepinephrine and glutamate do not increase cyclic AMP production. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in central control of the HPA axis. Studies using PACAP knockout mice showed that these mice fail to increase CRH mRNA in response to restraint. Furthermore, PACAP can directly stimulate CRH hnRNA in primary cultures of hypothalamic neurons. The role of PACAP mediating cyclic AMP-signaling in the CRH neuron during stress is under current investigation. The activity of the HPA axis is characterized by circadian and ultradian pattern of glucocorticoid secretion with one secretory pulse per hour. Because of increasing evidence for the importance of pulsatility in regulating glucocorticoid-responsive gene transcription, considerable effort during the past year focused on the mechanisms determining pulsatile secretion at the adrenal level. Availability of active steroidogenic acute regulatory protein (StAR) and side chain cleavage cytochrome P450 (P450scc) are rate-limiting steps for steroidogenesis. The relationship between TORC activation and ACTH-induced steroidogenesis were studied in vivo, by examining the time-course of the effect of ACTH injection (4ng, iv) on the transcriptional activity of StAR and P450scc genes and nuclear accumulation of TORC2 in rat adrenal cortex. ACTH produced rapid (5min) and transient increases in plasma corticosterone. This was followed by increases in StAR and P450scc hnRNA levels by 15 min. Concomitantly, ACTH increased nuclear phospho-CREB, and nuclear accumulation of TORC2, with maximal levels at 5 min and returning to basal by 30 min. The decline of nuclear TORC2 paralleled increases in SIK1 expression. The direct temporal relationship between nuclear accumulation of TORC2 and the increase in transcription of steroidogenic proteins, implicates TORC2 in the physiological regulation of steroidogenesis in the adrenal cortex. The delayed induction of SIK1 suggests a role for SIK1 in the declining phase of steroidogenesis. Further studies in methylprednisolone suppressed rats, with reduced plasma corticosterone, and adrenal StAR and P450scc hnRNA, demonstrated that pulsatile but not constant ACTH infusion restored pulsatile steroid secretion as well as StAR and P450scc hnRNA levels, indicating that pulsatile ACTH release is critical for optimal adrenocortical function.

下丘脑垂体肾上腺轴的正常活动导致肾上腺分泌糖皮质激素，这对于正常代谢活动和在困难情况下的生存至关重要。该项目的研究明确了下丘脑肽促肾上腺皮质激素释放激素（CRH）和加压素（VP）在调节垂体ACTH中的作用，有助于阐明应激过程中CRH和VP表达的调节及其机制。这些肽受体在垂体和大脑中的作用、地形分布、调节和生理作用。 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，需要快速但短暂地激活 CRH 转录来恢复 mRNA 和肽水平。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。 目前的工作旨在阐明控制 CRH 负转录和正转录调节的机制，以及负责糖皮质激素分泌的正常昼夜节律和超昼夜模式的机制。 该实验室报告说，cAMP/磷酸-CREB ​​信号传导对于激活 CRH 转录至关重要，但不足以激活。这一发现导致发现 CRH 基因的转录激活需要 CREB ​​共激活因子调节 CREB ​​活性转导器 (TORC)。在静息条件下，共激活剂在细胞质中处于磷酸化、非活性状态。其激活和核转位需要蛋白激酶 A (PKA) 介导的抑制蛋白激酶介导的 TORC 磷酸化。实验检查了 TORC 从产生 CRH 的神经元的细胞质到细胞核的运输，以及 TORC 的过表达和敲低对 CRH 转录的影响，表明 TORC 对于 CRH 转录至关重要，并且在应激期间，共激活因子发生变化与 CRH 启动子结合，作为与 CREB ​​复合物的一部分，与 CRH 转录的激活并行。未来的研究计划探讨共激活子 TORC 在体内 CRH 转录生理调节过程中的重要性。 在监管 TORC 活动的机制上投入了大量精力。在基础条件下，转录水平较低，因为 TORC 保留在细胞质中，通过 AMP 依赖性蛋白激酶 (AMPK) 家族（包括盐诱导激酶 (SIK)）的 Ser/Thr 蛋白激酶磷酸化而失活。为了确定哪种激酶负责 CRH 神经元中的 TORC 磷酸化，我们通过原位杂交测量了大鼠 PVN 中的 AMPK、SIK1 和 SIK2 mRNA。在基础条件下，背内侧 PVN 中发现 3 种激酶水平较低，与 CRH 神经元中的位置一致。束缚应激增加了 SIK1 mRNA 水平，而 SIK2 和 AMPK mRNA 仅略有增加。 4B 细胞中 SIK1 或 SIK2 的过表达会降低核 TORC2 水平并抑制毛喉素刺激的 CRH 转录。相反，非选择性 SIK 抑制剂星形孢菌素可增加核 TORC2 含量并刺激 4B 细胞和原代神经元培养物（异核 RNA）中的 CRH 转录。内源性 SIK2（而非 SIK1）的特异性 shRNA 敲低诱导 TORC2 和 CRH 转录的核转位，表明 SIK2 在基础条件下介导 TORC 失活，而 SIK1 的诱导则限制转录激活。 目前的研究旨在检验以下假设：虽然 SIK2 在基础条件下介导 TORC 的隔离，但在刺激 CRH 神经元期间 SIK1 的诱导介导 TORC 的磷酸化和核输出，从而有助于转录反应的终止。 由于越来越多的证据表明，许多基因的 CREB ​​依赖性转录激活需要辅激活因子 TORC，并且由于 CREB ​​在许多大脑功能中的重要性，TORC1、2 和 3 mRNA 在大脑特定区域的拓扑分布研究了大鼠的前脑。原位杂交分析表明，TORC1 是大多数前脑结构中最丰富的亚型，其次是 TORC2 和 TORC3。虽然高水平的TORC1广泛分布在前脑中，但TORC2存在于离散的细胞核中，而TORC3主要存在于室管膜和软脑膜中。在下丘脑室旁核中，小细胞和大细胞神经内分泌室中 TORC1 和 2 mRNA 丰富，而 TORC3 表达较低。所有三种亚型 mRNA 均在下丘脑的其他部位发现，其中 TORC1 在腹内侧核中表达最显着，TORC2 在背内侧核和弓状核中表达，TORC 1 和 2 在视上核中表达，TORC2 在视交叉上核中表达。这些差异分布模式与所有三种TORC亚型在不同大脑结构中的复杂作用一致，并为进一步研究CREB/TORC信号传导对大脑功能的机制提供了基础。 虽然环 AMP/PKA 依赖性途径对于 CRH 基因的转录激活至关重要，但 CRH 神经元的主要直接调节因子、去甲肾上腺素和谷氨酸不会增加环 AMP 的产生。垂体腺苷酸环化酶激活多肽 (PACAP) 参与 HPA 轴的中枢控制。 使用 PACAP 基因敲除小鼠的研究表明，这些小鼠无法因限制而增加 CRH mRNA。此外，PACAP 可以直接刺激下丘脑神经元原代培养物中的 CRH hnRNA。目前正在研究 PACAP 在应激期间介导 CRH 神经元中循环 AMP 信号传导的作用。 HPA 轴的活动以糖皮质激素分泌的昼夜节律和超节律模式为特征，每小时一次分泌脉冲。由于越来越多的证据表明脉动在调节糖皮质激素反应性基因转录中的重要性，过去一年的大量努力集中在确定肾上腺水平脉动分泌的机制上。活性类固醇生成急性调节蛋白 (StAR) 和侧链裂解细胞色素 P450 (P450scc) 的可用性是类固醇生成的限速步骤。 通过检查 ACTH 注射（4ng，iv）对大鼠肾上腺皮质中 StAR 和 P450scc 基因转录活性以及 TORC2 核积累的影响的时间过程，研究了 TORC 激活与 ACTH 诱导的类固醇生成之间的关系。 。 ACTH 使血浆皮质酮快速（5 分钟）短暂增加。随后 StAR 和 P450scc hnRNA 水平增加 15 分钟。与此同时，ACTH 增加了核磷酸化 CREB ​​和 TORC2 的核积累，5 分钟时达到最大水平，30 分钟恢复到基础水平。核TORC2的下降与SIK1表达的增加平行。 TORC2 的核积累与类固醇生成蛋白转录增加之间的直接时间关系表明 TORC2 参与肾上腺皮质类固醇生成的生理调节。 SIK1 的延迟诱导表明 SIK1 在类固醇生成衰退阶段发挥作用。 对甲基泼尼松龙抑制大鼠血浆皮质酮、肾上腺 StAR 和 P450scc hnRNA 降低的进一步研究表明，脉冲式但不是恒定的 ACTH 输注可恢复脉冲式类固醇分泌以及 StAR 和 P450scc hnRNA 水平，表明脉冲式 ACTH 释放对于最佳肾上腺皮质至关重要功能。