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）的神经系统成本和收益。这项研究的数据引用了HT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。尽管经历绝经的女性的平均年龄为51岁（根据Kaplan-Meier生存分析），但WHI研究中的一些女性平均绝经后11-12年。当对绝经期和绝经后妇女分别进行检查时，HT的影响有显著差异。人们认为，在循环雌激素几乎耗尽一段时间后，再次暴露会导致有害影响，这一观点得到了时间假说的支持4。这一假说表明，在雌激素戒断期间有一个关键的时间窗口，决定了雌激素的重新引入将如何影响身体。HT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。）。来自我们实验室的数据揭示了？的配体非依赖性转录作用。具体来说，在雌激素的情况下，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子。因此，这一建议的目的是i）阐明具体的调控行动调制ER？在雌激素戒断期间，以及ii）确定调节成分如何通过ER影响焦虑调节。为了研究这些过程，目标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？和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将a）鉴定与ER相关的共调节因子？然后B）确定在配体不存在的情况下AVP反式激活所需的特异性辅助调节因子。为了研究这些问题，一组体外和体内实验将采用细胞培养和整体动物方法。将进行二维SDS-PAGE和质谱分析以鉴定SUMO化和共调节关联。确认ER的SUMO化？并确定这一过程如何影响AVP的反式激活，突变受体将被制造并使用报告基因分析进行分析。SUMO化对ER的影响将使用行为焦虑测试评估介导的焦虑行为。为了确认协同调节相互作用并确定AVP反式激活的特异性协同调节因子，将使用生物发光共振能量转移（BRET 2）、siRNA敲低和报告基因测定。总的来说，这个提议将揭示雌激素戒断过程中发生的分子机制，这可能会改变大脑对HT的感受性。

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.