Estrogen receptor beta protein:protein interactions following estrogen withdrawal

描述（由申请人提供）：妇女健康倡议（WHI）评估了激素治疗（HT）对绝经后妇女的神经学成本和益处。这项研究的数据引用了激素的负面影响，与流行病学和基础科学证据不同，这些证据表明雌激素具有神经保护和神经营养作用，从而增强认知的某些方面。尽管经历更年期的女性平均年龄为51岁（根据Kaplan-Meier生存分析），但参与WHI研究的一些女性平均在绝经后11-12年。当围绝经期和绝经后妇女分开检查时，激素治疗的效果有显著差异。人们认为，在循环中的雌激素接近耗尽一段时间后，再次接触会造成有害影响，这一观点得到了时间假说的支持。这一假设表明，在雌激素停药期间存在一个关键的时间窗口，它决定了重新引入雌激素对身体的影响。激素的各种作用揭示了我们对雌激素基本作用的认识中缺失的一个环节，雌激素通过受体α和β (ER-?)传递。呃?)。我们实验室的数据揭示了与配体无关的转录作用。具体来说，在缺乏雌激素的情况下，ER？激活目标基因的转录，包括精氨酸抗利尿激素（AVP），一种焦虑的关键调节因子。因此，本提案旨在i)阐明调节ER的具体监管行动？ii)确定调节成分如何影响雌激素受体对焦虑的调节？为了研究这些过程，Aim 1将a)确定低雌激素性如何影响转录调节因子SUMO-1与ER的结合？b)确定SUMOylation如何改变AVP与配体无关的交易激活和焦虑行为。目标2将a)确定与ER相关的共调节因子？然后b)确定在没有配体的情况下AVP转激活所需的特定共调节因子。为了研究这些问题，一系列体外和体内实验将采用细胞培养和全动物方法。将进行二维SDS-PAGE和质谱分析来鉴定SUMOylation和协调节关联。确认ER的summoylation ？并确定这一过程如何影响AVP的转激活，突变受体将被制造并使用报告基因分析。SUMOylation对ER的影响？-介导的焦虑行为将使用行为焦虑测试进行评估。为了确认共调控相互作用并确定AVP转激活的特异性共调控因子，将使用生物发光共振能量转移（BRET2）、siRNA敲低和报告基因检测。总的来说，这一建议将揭示雌激素停药期间发生的分子机制，可能会改变大脑对激素的接受性。

DESCRIPTION (provided by applicant): The Women's Health Initiative (WHI) evaluated the neurological costs and benefits of hormone therapy (HT) for post-menopausal women. Data from this study, citing negative effects of HT, differ from epidemiological and basic scientific evidence that suggests that estrogens are neuroprotective and neurotrophic, thereby enhancing some aspects of cognition. Despite the mean age for women experiencing menopause being 51 years (according to the Kaplan-Meier survival analysis), some women involved in the WHI study were, on average, 11-12 years post-menopause. When peri- and postmenopausal women are examined separately, the effects of HT differ significantly. It is thought that after circulating estrogens are nearly deplete for some time, re-exposure causes detrimental effects, an idea supported by the timing hypothesis4. This hypothesis suggests that there is a critical window of time during estrogen withdrawal that determines how reintroduction of estrogens will affect the body. Varied effects of HT reveal a missing link in our knowledge of the basic actions of estrogens, transmitted through receptors alpha and beta (ER-?. and ER?). Data from our laboratory reveal ligand-independent transcriptional actions of ???. Specifically, in the absence of estrogens, ER? activates transcription of target genes, including arginine vasopressin (AVP), a critical regulator of anxiety. Hence, this proposal is designed i) to elucidate specific regulatory actions modulating ER? during periods of estrogen withdrawal and ii) to determine how regulatory components effect anxiety regulation by ER?. To investigate these processes, Aim 1 will a) identify how hypoestrogenicity affects the conjugation of a transcriptional regulator, SUMO-1, to ER? and b) determine how SUMOylation alters ligand-independent transactivation of AVP and anxiety behavior. Aim 2 will a) identify coregulators associated with ER? during periods of hypoestrogenicity and then b) determine specific coregulators that are required for transactivation of AVP in the absence of ligand. To examine these issues, a battery of in vitro and in vivo experiments will employ cell culture and whole animal methodology. Two-dimensional SDS-PAGE and mass spectrometry will be performed to identify SUMOylation and coregulatory associations. To confirm SUMOylation of ER? and determine how this process affects transactivation of AVP, mutant receptors will be made and analyzed using reporter gene assays. The effect of SUMOylation on ER?-mediated anxiety behaviors will be assessed using behavioral anxiety tests. To confirm coregulatory interactions and determine specific coregulators for AVP transactivation, bioluminescence resonance energy transfer (BRET2), siRNA knockdown and reporter gene assays will be utilized. Overall, this proposal will reveal molecular mechanisms occurring during estrogen withdrawal that may alter the brain's receptivity to HT. PUBLIC HEALTH RELEVANCE: Hormone replacement therapy is a highly controversial but critical topic that affects women's health. This proposal aims to identify mechanisms that are responsible for dichotomous age-dependent effects of hormone replacement therapy during and after menopause.