Ligand-independent signaling of estrogen receptor beta and the aging brain

雌激素受体β的配体独立信号传导和大脑老化

• 批准号: 9401430
• 负责人: Toni R. Pak
• 金额: $ 42.13万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9401430
• 关键词: Age; Aging; Alternative Splicing; Architecture; Automobile Driving; Biological; Biological feedback; Brain; Cells; ChIP-seq; Chromatin; Cis-Acting Sequence; Clinical; Cost Analysis; Costs and Benefits; Data; Detection; Disease; Estrogen Receptor beta; Estrogen Receptors; Estrogen Replacement Therapy; Estrogens; Event; Exons; Family; Female; Funding; Gene Targeting; Genes; Genetic Transcription; Goals; Health Benefit; Hippocampus (Brain); Hormone replacement therapy; Human; Kinetics; Knowledge; Length; Ligand Binding; Ligands; Link; Longevity; Mass Spectrum Analysis; Mediating; Menopause; Modeling; Modification; Molecular; Nuclear Receptors; Ovarian hormone; Pathologic; Pathway interactions; Pattern; Phosphorylation; Phosphorylation Site; Play; Post-Translational Protein Processing; Postmenopause; Predictive Factor; Process; Proteins; Proteomics; RNA Splicing; RNA-Binding Proteins; Rattus; Role; Signal Pathway; Signal Transduction; Signaling Protein; Site; Specificity; Testing; Therapeutic; Time; Tissues; Transactivation; Transcript; Transcriptional Activation; Treatment Efficacy; Variant; Woman; Work; age related; aged; aging brain; base; deprivation; driving force; experimental study; gene repression; hormone therapy; in vivo; interest; member; multiple reaction monitoring; mutant; novel; osteosarcoma; promoter; protein protein interaction; receptor; reproductive; theories; therapeutic development; transcription factor; treatment strategy

PROJECT SUMMARY. We propose that there is a biological switch in estrogen receptor action that occurs coincident with age and length of time after ovarian hormone depletion (i.e. menopause). Accumulating evidence suggests that ERb shifts from a predominantly ligand-activated transcription factor (major role during reproductive years) to a ligand-independent transcription factor post-menopause. Elucidating the molecular basis for this shift in signaling paradigms is critical for understanding clinical observations, which demonstrate a defined narrow window of time for therapeutic efficacy of hormone therapy in postmenopausal women. This application is a competitive renewal of R01 AG033605. Our previous aims for this project centered on factors that we predicted would differentially dictate a dominant ligand-dependent signaling pattern for ERb, as opposed to a ligand-independent signaling pattern. Through that work, we identified 3 key molecular factors that facilitated ligand-independent function of ERb in the aged brain: receptor phosphorylation, alternative RNA splicing, and coregulatory protein interactions. These mechanisms represented both direct changes to the receptor itself and indirect changes in protein:protein interactions. The experiments proposed in this renewal will be focused on the direct changes to the ERb protein itself, namely phosphorylation and alternative splicing. Aim 1 will focus on posttranslational phosphorylation of the receptor. Phosphorylation of ERb is a strong facilitator of ligand-independent activity, yet the extent of phosphorylated ERb present in the aged female brain, and the in vivo functional consequences of such a modification, represents a major gap in our current knowledge. We propose a comprehensive proteomics approach to a) quantify phosphor-ERb in the aged female brain, b) determine how site-specific phosphorylation alters ERb protein:protein interactions, and c) identify specific phospho-ERb target genes. Aim 2 will focus on alternative RNA splicing of the nascent ERb transcript. Our data and others have demonstrated the functional significance of ERb splice variants and we have shown that these alternative variants increase during aging. However, we lack a fundamental understanding of the molecular mechanisms regulating ERb splicing in the brain, or in any tissue type. We have now identified several putative splicing factors that regulate alternative splicing of ERb in the aged brain. This aim will test the direct effects of these splicing factors on ERb alternative splicing and also determine if E2 regulates global splicing events by modulating the expression of these splicing factors. Our preliminary data also showed that transcriptional kinetics play an important role in ERb splicing, with slower transcription favoring exon inclusion resulting in increased ERb2. This aim will further explore this relationship to test how transcriptional kinetics in the brain change in our aging female rat menopause model. Impact: Understanding the basic molecular signaling pathways of E2 in the aging brain will help drive therapeutic advances and inform treatment strategies for postmenopausal women.

项目总结。我们认为在雌激素受体的作用中存在一个生物开关。 与卵巢激素耗竭(即绝经)后的年龄和时间长短一致。积累 有证据表明，Erb从一个主要由配体激活的转录因子转变(在 生殖年限)到绝经后的非配基非依赖转录因子。阐明分子 这种信号模式转变的基础对于理解临床观察至关重要，这表明 绝经后妇女激素治疗疗效的明确狭窄时间窗。 本申请是R01 AG033605的竞争性续订。我们之前对这个项目的目标集中在 我们预测的因素将不同地决定ERb的主要配体依赖的信号模式，因为 与配体无关的信号模式相反。通过这项工作，我们确定了3个关键的分子因素 促进ERb在老年脑中的非配体依赖功能：受体磷酸化，替代RNA 剪接和共调控蛋白相互作用。这些机制代表了对 受体本身和蛋白质的间接变化：蛋白质相互作用。这项研究中提出的实验 更新将集中在ERb蛋白本身的直接变化上，即磷酸化和 另一种拼接。目标1将专注于该受体的翻译后磷酸化。磷酸化 Erb是一种强烈的配体非依赖性活性促进剂，但存在于 老化的女性大脑，以及这种修饰在体内的功能后果，代表着在 我们目前的知识。我们提出了一种全面的蛋白质组学方法来a)定量 老年女性大脑，b)确定位点特异性磷酸化如何改变Erb蛋白：蛋白质相互作用，以及 C)确定特定的磷酸化Erb靶基因。目标2将专注于新生Erb的替代RNA剪接 文字记录。我们的数据和其他数据已经证明了erb剪接变异体的功能意义，我们 已经表明，随着年龄的增长，这些可供选择的变种会增加。然而，我们缺乏一个基本的 了解调节ERb在大脑或任何组织类型中剪接的分子机制。我们 现在已经确定了几个可能的剪接因子，它们调节老年人大脑中Erb的选择性剪接。 这一目的将测试这些剪接因子对ERb选择性剪接的直接影响，并确定E2 通过调节这些剪接因子的表达来调节全局剪接事件。我们的初步数据 还表明转录动力学在erb剪接中起着重要作用，但转录速度较慢 有利于外显子包涵体导致ERB2增加。这一目标将进一步探索这种关系，以测试如何 我们的老年雌性大鼠更年期模型中脑转录动力学的变化。影响：了解 衰老大脑中E2的基本分子信号通路将有助于推动治疗进展和信息 绝经后妇女的治疗策略。