Estrogen receptor beta protein:protein interactions following estrogen withdrawal

描述(由申请人提供)：妇女健康倡议(WHI)评估了绝经后妇女激素治疗(HT)的神经学成本和好处。这项研究的数据引用了羟色胺的负面影响，不同于流行病学和基本科学证据，这些证据表明雌激素具有神经保护和神经营养作用，从而增强了认知的某些方面。尽管绝经女性的平均年龄为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。当分别检查围绝经期和绝经期妇女时，羟色胺的影响显著不同。人们认为，在循环中的雌激素几乎耗尽一段时间后，再次暴露会造成有害影响，这一观点得到了时间假说的支持。这一假说表明，在雌激素停用期间有一段关键的时间窗口，这段时间决定了重新注入雌激素将如何影响身体。羟色胺的不同作用揭示了我们对雌激素基本作用的认识中缺失的一环，雌激素通过受体α和β(ER-？)传递。还有急诊室？)我们实验室的数据揭示了？的非配体转录行为。具体来说，在没有雌激素的情况下，ER？激活靶基因的转录，包括精氨酸加压素(AVP)，这是焦虑的关键调节因素。因此，本提案旨在阐明调控内质网的具体调控行为。在雌激素停用期间，以及ii)确定调节成分如何影响ER对焦虑的调节？为了研究这些过程，目标1是否将a)确定低雌激素如何影响转录调节因子SUMO-1与ER的结合？以及b)确定SUMO化如何改变AVP的配体非依赖性反式激活和焦虑行为。目标2：a)确定与内质网相关的共同调节因子吗？然后b)确定在缺乏配体的情况下AVP反式激活所需的特定辅助调节因子。为了研究这些问题，一系列的体外和体内实验将采用细胞培养和全动物方法学。二维SDS-PAGE和质谱学将被用来鉴定SUMO化和共调节关联。以确认内质网的SUMO化？并确定这一过程如何影响AVP的反式激活，将制造突变受体并使用报告基因分析进行分析。SUMO化对ER？介导的焦虑行为的影响将通过行为焦虑测试来评估。为了确认协同调节相互作用并确定AVP反式激活的特定协同调节因子，将利用生物发光共振能量转移(BRET2)、siRNA敲除和报告基因分析。总体而言，这项提议将揭示雌激素停用期间发生的分子机制，可能会改变大脑对羟色胺的接受度。

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.