Neuroendocrine Control Of The Stress Response

应激反应的神经内分泌控制

• 批准号: 8736818
• 负责人: Greti Aguilera
• 金额: $ 63.82万
• 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/8736818
• 关键词: Acute; Adrenal Glands; Adrenalectomy; Affect; Anterior Pituitary Gland; Area; Automobile Driving; Behavioral; Binding; Brain; CREB1 gene; Cell Line; Cell Nucleus; Cell membrane; Cells; Chronic; Collaborations; Corticosterone; Corticotropin; Corticotropin-Releasing Hormone; Cyclic AMP; Cyclic AMP Response Element; Cyclic AMP-Dependent Protein Kinases; DNA Methylation; Data; Development; Diagnostic; Down-Regulation; EGF gene; ERG gene; Enzymes; Epigenetic Process; Exhibits; Feedback; Forskolin; Foundations; Gene Expression Profile; Gene Targeting; Generations; Genes; Genetic Transcription; Glucocorticoid Receptor; Glucocorticoids; Goals; Hormones; Hour; Hypothalamic structure; Injection of therapeutic agent; Ion Channel; Japan; Lead; Ligands; MAP Kinase Gene; Mediating; Membrane; Messenger RNA; Metabolic; Minor; Mitogen-Activated Protein Kinases; Molecular; National Institute of Child Health and Human Development; Neurons; Neuropeptide Receptor; Neuropeptides; Neurosecretory Systems; Neurotransmitter Receptor; Nuclear; Nuclear Translocation; Pathology; Pathway interactions; Pattern; Peptide Receptor; Peptides; Periodicity; Phorbol Esters; Phosphotransferases; Physiologic pulse; Pituitary Gland; Plasma; Pro-Opiomelanocortin; Production; Protein Kinase C; Proteins; Rattus; Receptor Inhibition; Regulation; Relative (related person); Research; Role; Side; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Sodium Chloride; Steroid biosynthesis; Steroids; Stress; System; Therapeutic; Transcriptional Activation; Transducers; Trypsin; Universities; Up-Regulation; V1b vasopressin receptor; Vasopressins; Withdrawal; Work; analog; base; biological adaptation to stress; chromatin immunoprecipitation; gene repression; genome wide association study; hypothalamic-pituitary-adrenal axis; kinase inhibitor; melanocortin receptor; non-genomic; paraventricular nucleus; prevent; promoter; protein protein interaction; receptor; receptor binding; research study; steroidogenic acute regulatory protein; tool; transcription factor; transcriptome sequencing

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. Previous 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 understanding the regulation of CRH and VP expression during stress. Studies under this project also led to the characterization of the receptors for these peptides in the pituitary gland and in the brain, the identification of their topographic distribution and mechanisms of regulation and action of these receptors. Limitation of the stress response is essential to prevent pathology associated with chronic elevation of CRH and glucocorticoid production. Current work is aimed to elucidate the molecular mechanisms controlling activation and inactivation of HPA axis activity, as well as the mechanisms responsible for normal episodic patterns of glucocorticoid secretion. Regulation of CRH transcription depends on cyclic AMP/protein kinase A (PKA) signaling and binding of phospho-CREB to a cyclic AMP response element (CRE) at 270 in the CRH promoter. This CRE is essential for activation of the CRH promoter, and epigenetic DNA methylation at the internal CpG of this site reduces CREB binding to the promoter affecting CRH expression. However, phospho-CREB alone is not sufficient for driving CRH transcription and requires the CREB co-activator, Transducer Of Regulated CREB activity (TORC) and its recruitment by the CRH promoter. We have also provided evidence that activation/inactivation of TORC in the CRH neuron involves the TORC kinases salt induced kinases (SIK) 1 and SIK2. The overall evidence indicates that TORC is essential for activation of CRH transcription, and suggests that regulation of the SIK/TORC system by stress-activated signaling pathways acts as a sensitive switch mechanism for rapid activation and inactivation of CRH transcription. While positive regulation of CRH expression is important for HPA axis responsiveness, negative feedback by adrenal glucocorticoids is also essential for preventing deleterious effects of excessive corticotrophin releasing hormone (CRH) and glucocorticoid production. One target of glucocorticoid feedback is CRH transcription in the hypothalamic paraventricular nucleus (PVN). To elucidate the unanswered question of whether glucocorticoid receptors (GR) directly interact with the proximal CRH promoter, we examined the effects of glucocorticoids on GR recruitment by the hypothalamic CRH promoter using chromatin immunoprecipitation. In contrast to the marked GR recruitment by promoters of glucocorticoid dependent genes, such as Per 1, stress or corticosterone injection had no significant effect on GR binding to the proximal CRH promoter. Stress increased pCREB recruitment by the CRH promoter, irrespective of circulating glucocorticoids. In the hypothalamic cell line 4B, glucocorticoids had no effect on forskolin-induced nuclear accumulation of phospho-CREB or TORC2. These data suggest that transcriptional repression of CRH by glucocorticoids involves protein-protein interactions or/and modulation of afferent inputs to the PVN rather than direct interaction with the proximal promoter. In order to identify target genes for glucocorticoid feedback in the PVN region, in collaboration with Drs Keiichi Itoi (Sendai University, Japan) and David Klein, NICHD, we conducted genome wide analysis of the transcriptome by RNA-Seq in microdissected PVN region of rats subjected to changes in circulating glucocorticoids. Chronic glucocorticoid withdrawal (7-days adrenalectomy) or elevation (s.c. corticosterone pellets) induced upregulation or downregulation of a relative small number of genes compared with the large number (near 3,000) significantly induced one hour after an acute corticosterone injection increasing plasma levels in the stress range. A small number of genes were downregulated following acute injection. Unexpectedly, early response genes during stress such as Fos, Egr, and Nur77, increased after 1 corticosterone injection and returned to values significant below basal after 3h. CRH mRNA levels increased after acute corticosterone injection, despite exhibiting the expected changes following adrenalectomy and high corticosterone exposure. In addition, a number of ion channels, neuropeptide and neurotransmitter receptors were highly regulated by glucocorticoids suggesting that they are target genes for glucocorticoid feedback. These data uncover a number of genes as target of glucocorticoid regulation in the hypothalamic PVN area and provides a foundation for further studies on the mechanisms by which glucocorticoids regulate CRH expression in the PVN. Another important target of glucocorticoid feedback is the pituitary corticotroph where the steroid inhibits ACTH secretion and proopiomelanocortin (POMC) transcription. The rapid inhibition of ACTH secretion by glucocorticoids has suggested the involvement of non-genomic mechanisms. Rapid non-genomic effects of glucocorticoids at the pituitary level have been postulated to drive a pituitary-adrenal feedforward/feedback mechanism responsible for hourly (ultradian) rhythmicity of glucocorticoid secretion. Ongoing studies provide evidence that the classical glucocorticoid receptor (GR) could mediate rapid effects through a ligand-dependent association to membrane fractions. Perifusion experiments in trypsin-dispersed anterior pituitary cells shows that while inhibition of POMC transcription parallels nuclear translocation of the GR, inhibition of ACTH secretion is associated with membrane localization of GR. On the other hand, the fact that only high glucocorticoid concentrations, in the stress range, were able to inhibit ACTH secretion suggests that glucocorticoid feedback at the pituitary levels is not responsible for ultradian pulse generation. Current efforts in this area are aimed to identify the target proteins of GR in the plasma membrane. At the adrenal level, studies have been continued to uncover mechanisms determining pulsatile secretion at the adrenal level. We have shown that secretory pulses induced by ACTH are associated with episodes of transcription of genes encoding critical proteins for steroidogenesis including steroidogenic acute regulatory protein (StAR), side chain cleavage enzyme and melanocortin receptor associated protein (MRAP). Although a number of transcription factors have been implicated, the precise mechanisms regulating StAR transcription are unclear. We showed that increases in transcription are preceded by nuclear translocation of TORC, supporting role for the CREB co-activator in transcriptional initiation. However, studies using the ACTH-responsive clone of the adrenocortical cell line Y1, showed that suppression of TORC by siRNA had only minor effects of StAR transcription. Cyclic AMP analogs but not stimulation of protein kinase C by phorbol esters, or MAP kinase pathway by EGF mimicked ACTH stimulation of StAR transcription. However, simultaneous inhibition of both protein kinase A and MAP kinase were necessary to block the effect of ACTH, indicating that the effect of ACTH on StAR transcription requires cAMP-dependent activation of both PKA and MAPK pathways. Interestingly, the combination of PKA and MAP kinase inhibitors had no effect on TORC translocation to the nucleus. These data show that although cyclic AMP is essential, multiple cyclic AMP- mediated signaling pathways are required for full transcriptional activation of StAR. In addition, it appears that the CREB co-activator TORC is not essential for initiation of StAR transcription.

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。该项目前期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，有助于了解应激期间CRH和VP表达的调节。 该项目的研究还确定了垂体和大脑中这些肽的受体的特征，确定了它们的拓扑分布以及这些受体的调节和作用机制。 限制应激反应对于预防与 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 HPA 轴活性激活和失活的分子机制，以及负责糖皮质激素分泌正常发作模式的机制。 CRH 转录的调节取决于环 AMP/蛋白激酶 A (PKA) 信号传导以及磷酸-CREB ​​与 CRH 启动子中 270 处环 AMP 反应元件 (CRE) 的结合。该 CRE 对于 CRH 启动子的激活至关重要，并且该位点内部 CpG 处的表观遗传 DNA 甲基化会减少 CREB ​​与影响 CRH 表达的启动子的结合。然而，单独的磷酸化 CREB ​​不足以驱动 CRH 转录，需要 CREB ​​共激活子、调节 CREB ​​活性的转导子 (TORC) 及其由 CRH 启动子的募集。我们还提供了证据表明 CRH 神经元中 TORC 的激活/失活涉及 TORC 激酶盐诱导激酶 (SIK) 1 和 SIK2。总体证据表明TORC对于CRH转录的激活至关重要，并表明应激激活信号通路对SIK/TORC系统的调节是CRH转录快速激活和失活的敏感开关机制。 虽然 CRH 表达的正向调节对于 HPA 轴反应性很重要，但肾上腺糖皮质激素的负反馈对于防止过量促肾上腺皮质激素释放激素 (CRH) 和糖皮质激素产生的有害影响也至关重要。糖皮质激素反馈的目标之一是下丘脑室旁核 (PVN) 中的 CRH 转录。 为了阐明糖皮质激素受体 (GR) 是否直接与近端 CRH 启动子相互作用这一尚未解答的问题，我们使用染色质免疫沉淀检查了糖皮质激素对下丘脑 CRH 启动子募集 GR 的影响。 与糖皮质激素依赖性基因启动子（例如 Per 1）明显的 GR 募集相反，应激或皮质酮注射对 GR 与近端 CRH 启动子的结合没有显着影响。 无论循环中的糖皮质激素如何，应激都会增加 CRH 启动子对 pCREB ​​的募集。在下丘脑细胞系 4B 中，糖皮质激素对毛喉素诱导的磷酸-CREB ​​或 TORC2 的核积累没有影响。这些数据表明，糖皮质激素对 CRH 的转录抑制涉及蛋白质-蛋白质相互作用或/和 PVN 传入输入的调节，而不是与近端启动子的直接相互作用。 为了确定 PVN 区域糖皮质激素反馈的靶基因，我们与 Keiichi Itoi 博士（日本仙台大学）和 NICHD 的 David Klein 合作，通过 RNA-Seq 对显微解剖的大鼠 PVN 区域的转录组进行了全基因组分析。受到循环糖皮质激素变化的影响。 慢性糖皮质激素戒断（7 天肾上腺切除术）或升高（皮下皮质酮颗粒）会引起相对少量基因的上调或下调，而急性注射皮质酮一小时后显着诱导大量基因（近 3,000 个），从而增加血浆水平应力范围。 急性注射后少数基因下调。出乎意料的是，应激期间的早期反应基因（例如 Fos、Egr 和 Nur77）在注射 1 次皮质酮后增加，并在 3 小时后恢复到显着低于基础值。 尽管在肾上腺切除术和高皮质酮暴露后出现了预期的变化，但急性皮质酮注射后 CRH mRNA 水平有所增加。 此外，许多离子通道、神经肽和神经递质受体受到糖皮质激素的高度调节，表明它们是糖皮质激素反馈的靶基因。 这些数据揭示了下丘脑PVN区域糖皮质激素调节靶点的大量基因，为进一步研究糖皮质激素调节PVN中CRH表达的机制奠定了基础。 糖皮质激素反馈的另一个重要目标是垂体促肾上腺皮质激素，其中类固醇抑制 ACTH 分泌和阿片黑皮质素原 (POMC) 转录。糖皮质激素对 ACTH 分泌的快速抑制表明非基因组机制的参与。据推测，糖皮质激素在垂体水平上的快速非基因组效应可驱动垂体-肾上腺前馈/反馈机制，负责糖皮质激素每小时（超）节律性的分泌。正在进行的研究提供的证据表明，经典的糖皮质激素受体（GR）可以通过与膜组分的配体依赖性关联来介导快速效应。 胰蛋白酶分散的垂体前叶细胞的灌注实验表明，虽然 POMC 转录的抑制与 GR 的核转位平行，但 ACTH 分泌的抑制与 GR 的膜定位相关。 另一方面，只有在应激范围内高糖皮质激素浓度才能抑制 ACTH 分泌，这一事实表明垂体水平的糖皮质激素反馈并不负责超电脉冲的产生。 目前该领域的工作旨在鉴定质膜中 GR 的靶蛋白。 在肾上腺水平，研究仍在继续揭示决定肾上腺水平脉动分泌的机制。我们已经证明，ACTH 诱导的分泌脉冲与编码类固醇生成关键蛋白的基因转录事件相关，这些蛋白包括类固醇生成急性调节蛋白 (StAR)、侧链裂解酶和黑皮质素受体相关蛋白 (MRAP)。 尽管涉及许多转录因子，但调节 StAR 转录的精确机制尚不清楚。我们发现，TORC 的核易位先于转录增加，从而支持 CREB ​​共激活因子在转录起始中的作用。 然而，使用肾上腺皮质细胞系 Y1 的 ACTH 反应性克隆进行的研究表明，siRNA 对 TORC 的抑制仅对 StAR 转录产生轻微影响。 环AMP类似物但不通过佛波酯刺激蛋白激酶C，或通过EGF模拟ACTH刺激StAR转录的MAP激酶途径。然而，同时抑制蛋白激酶 A 和 MAP 激酶对于阻断 ACTH 的作用是必要的，这表明 ACTH 对 StAR 转录的影响需要 pKA 和 MAPK 途径的 cAMP 依赖性激活。有趣的是，PKA 和 MAP 激酶抑制剂的组合对 TORC 转位至细胞核没有影响。 这些数据表明，尽管环 AMP 是必需的，但 StAR 的完全转录激活需要多个环 AMP 介导的信号通路。此外，CREB ​​共激活因子 TORC 似乎对于 StAR 转录的起始并不是必需的。