Local L-type Calcium Channel Signaling in Anterior Pituitary Gonadotropes

垂体前叶促性腺激素中的局部 L 型钙通道信号传导

• 批准号: 8838361
• 负责人: An Dang
• 金额: $ 3.38万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8838361
• 关键词: 1,2-diacylglycerol; Actins; Anterior Pituitary Gland; Binding; Blood Circulation; Calcium; Calcium Signaling; Cell membrane; Cell physiology; Cells; Cellular Structures; Clinical; Complex; Cytoskeleton; Data; Defect; Diglycerides; Electrophysiology (science); Elements; Endoplasmic Reticulum; Ensure; Event; F Factor; Female; Fertility; Fertility Agents; Fluorescence Microscopy; Follicle Stimulating Hormone; Frequencies; Future; Gene Expression; Generations; Genetic Transcription; Goals; Gonadotrope Cell; Gonadotropin Hormone Releasing Hormone; Gonadotropin-Releasing Hormone Receptor; Gonadotropins; Hydrogen Peroxide; Hypothalamic structure; Image; Imaging Techniques; Immunoblotting; Infertility; Inositol; L-Type Calcium Channels; Lead; Location; Luteinizing Hormone; MAPK8 gene; Measures; Mediating; Mission; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Molecular; National Institute of Child Health and Human Development; Neuropeptides; Ovulation; Pathway interactions; Persons; Phospholipase C; Phosphorylation; Physiologic pulse; Pituitary Gland; Production; Protein Kinase C; Proteins; Reactive Oxygen Species; Receptor Activation; Reproduction; Reproductive Physiology; Reproductive Process; Research; Resolution; Role; Signal Transduction; Signaling Molecule; Structure; Testing; Therapeutic; Western Blotting; Woman; Work; actin 2; base; human GNRH2 protein; hypothalamic pituitary gonadal axis; innovation; novel; public health relevance; receptor; receptor expression; reproductive; research study; response; signal processing; spatiotemporal; tripolyphosphate

 DESCRIPTION (provided by applicant): Mammalian reproduction requires intercommunication within the hypothalamic-pituitary-gonadal (HPG) axis. Defects within the HPG axis lead to abnormal reproductive cycles and infertility. For ovulation to occur, gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates gonadotrope cells in the anterior pituitary gland to acutely release luteinizing hormone (LH), known as the "LH surge". This LH secretion regulated by GnRH is mandatory for reproduction and involves specific interactions and signaling events. Therefore, understanding the molecular mechanisms underlying GnRH-induced LH secretion by gonadotropes has important implications for clinical manipulation of female reproduction. Calcium (Ca2+) is one of most versatile and universal signaling molecules that controls cellular processes, and activates important factors for fertility including key signal transduction molecules, mitogen-activated protein kinases (MAPK), that ultimately leads to LH transcription in gonadotrope cells. Previous research has provided indirect evidence that specific MAPK pathways, ERK and JNK, are activated by either a localized Ca2+ release through L-type Ca2+ channels, or by global Ca2+ signals released from intracellular stores in the endoplasmic reticulum, respectively. Importantly, the phosphorylation (i.e., activation) of ERK is the key signal required for enhanced LH synthesis and the LH surge. My goal is to investigate novel mechanistic molecular components and structures necessary for transducing GnRH receptor activation to these localized Ca2+ microdomains and ultimately activation of ERK. Specifically, I hypothesize that GnRH leads to ERK activation via highly localized Ca2+ microdomains produced by L-type Ca2+ channels, which are regulated by reactive oxygen species (ROS) and actin dynamics. Using a unique Ca2+ imaging technique combining electrophysiology and total internal reflection fluorescence (TIRF) microscopy, I will record Ca2+ influx through L-type Ca2+ channels with high spatiotemporal resolution. I will also use protein immunoblotting to measure ERK activation as phosphorylated ERK (pERK). In Specific Aim 1, I will test the hypothesis that reactive oxygen species (ROS) modulate local L-type Ca2+ channel function and ERK activity. In Specific Aim 2, I will test the hypothesis that that reactive oxygen species (ROS) modulate local L-type Ca2+ channel function and ERK activity. In each Specific Aim, I include preliminary data as well as proposed experimental plans for each mechanism that influences localized Ca2+ activity. This proposal attempts to better understand specific molecular mechanisms critical for reproduction, and therefore directly supports the NICHD mission statement "to ensure that every person is born healthy and wanted, [and] that women suffer no harmful effects from reproductive processes."

 描述（由申请人提供）：哺乳动物生殖需要下丘脑-垂体-性腺（HPG）轴内的相互交流。HPG轴内的缺陷导致异常生殖周期和不育。为了发生排卵，来自下丘脑的促性腺激素释放激素（GnRH）刺激垂体前叶中的促性腺激素细胞急性释放促黄体生成激素（LH），称为“LH峰”。这种由GnRH调节的LH分泌对于生殖是强制性的，并且涉及特定的相互作用和信号事件。因此，了解促性腺激素释放激素诱导LH分泌的分子机制对女性生殖的临床操作具有重要意义。钙（Ca 2+）是控制细胞过程的最通用和普遍的信号分子之一，并激活生育的重要因素，包括关键信号转导分子，促分裂原活化蛋白激酶（MAPK），其最终导致促性腺细胞中LH的转录。先前的研究提供了间接证据，即特定的MAPK途径，ERK和JNK，分别通过L型Ca 2+通道的局部Ca 2+释放或通过内质网中细胞内储存释放的全局Ca 2+信号来激活。重要的是，磷酸化（即，ERK的激活）是增强LH合成和LH激增所需的关键信号。我的目标是研究将GnRH受体激活转导到这些局部Ca 2+微区并最终激活ERK所需的新机制分子组分和结构。具体而言，我假设GnRH导致ERK激活通过高度本地化的Ca 2+微域产生的L型Ca 2+通道，这是由活性氧（ROS）和肌动蛋白动力学调节。使用一种独特的钙离子成像技术结合电生理学和全内反射荧光（TIRF）显微镜，我将记录钙离子流入通过L型钙离子通道具有高时空分辨率。我还将使用蛋白质免疫印迹来测量磷酸化ERK（pERK）的ERK活化。在具体目标1中，我将检验活性氧（ROS）调节局部L型Ca 2+通道功能和ERK活性的假设。在具体目标2中，我将检验活性氧（ROS）调节局部L型Ca 2+通道功能和ERK活性的假设。在每一个具体的目标，我包括初步的数据，以及为每一个机制，影响本地化的Ca 2+活动提出的实验计划。该提案试图更好地理解生殖关键的特定分子机制，因此直接支持NICHD的使命声明“确保每个人出生时都是健康的和需要的，[和]妇女不受生殖过程的有害影响。"