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

项目摘要。我们认为雌激素受体的作用中有一个生物开关， 与年龄和卵巢激素耗竭（即绝经）后的时间长度一致。积累 有证据表明，ERb从一个主要的配体激活的转录因子（主要作用在 生殖年龄）与绝经后配体非依赖性转录因子的关系。阐明分子 信号模式的这种转变的基础对于理解临床观察是至关重要的，这表明 绝经后妇女激素治疗疗效的限定狭窄时间窗。 本申请是R01 AG033605的竞争性更新。我们之前对这个项目的目标是 我们预测的因素将差异决定ERb的主要配体依赖性信号模式， 与配体独立信号模式相反。通过这项工作，我们确定了3个关键的分子因子， 促进老年脑中ERb的配体非依赖性功能：受体磷酸化，替代RNA 剪接和共调节蛋白相互作用。这些机制既代表了直接的变化， 受体本身和蛋白质：蛋白质相互作用的间接变化。在此提出的实验 更新将集中在ERb蛋白本身的直接变化，即磷酸化和 选择性剪接目标1将集中在受体的翻译后磷酸化。磷酸化 ERb是配体非依赖性活性的强促进剂，然而，存在于细胞中的磷酸化ERb的程度是不确定的。 老年女性大脑，以及这种修饰的体内功能后果，代表了 我们目前的知识。我们提出了一个全面的蛋白质组学方法，a）定量磷酸化ERb在 老年女性脑，B）确定位点特异性磷酸化如何改变ER B蛋白：蛋白相互作用，和 c）鉴定特异性磷酸化ERb靶基因。目标2将集中在新生ERb的选择性RNA剪接 成绩单。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， 已经表明这些替代变体在老化过程中增加。然而，我们缺乏一个基本的 了解调节脑或任何组织类型中ERb剪接的分子机制。我们 现在已经确定了几个假定的剪接因子，调节老年大脑中ERb的选择性剪接。 这一目标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是否 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 也表明转录动力学在ERb剪接中起重要作用， 有利于外显子包含，导致ERb2增加。这一目标将进一步探讨这种关系，以测试如何 在我们的老龄雌性大鼠更年期模型中，脑变化的转录动力学。影响：理解 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， 绝经后妇女的治疗策略。

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.