Environmental Toxins and Uterine Gene Expression

描述(由申请人提供)：子宫中的早期和晚期雌激素反应已经被认识了60多年，但其调控机制仍然存在争议。一种概念是，早期事件(S)发生在前6小时内，使子宫为以后(18-30小时)DNA合成、细胞增殖和蛋白质合成的增加做好准备。另一种观点认为，增长后期是刺激因素持续存在的结果。对这两个概念的讨论通常假设所有的反应都依赖于与两种雌激素受体亚型(ER1和ER2)中的一种的配体相互作用。然而，在缺乏ER1的小鼠中，或在雌激素拮抗剂ICI 182,780(ICI)抑制所有ER活性的小鼠中，注射雌激素或异种雌激素后基因表达增加，已表明这是过于简单化。在这方面，越来越多的证据表明，雌激素通过ER依赖和非依赖的方式调节子宫生物学中不同但相互依赖的信号通路。虽然我们长期的假设是雌激素的某些早期反应是ER非依赖性的，而晚期的反应是ER1依赖的，并且两个阶段之间的串扰对于子宫中的雌激素反应的完全补充是必要的。我们先前和初步的观察表明，在几个这样的早期基因中，Bip和Sik-SP是由雌二醇-172(E_2)和酮在小鼠子宫中通过ER非依赖性的I相反应诱导的。此外，我们最近发现，Bip在介导涉及ER1功能的E2或Kepone依赖的雌激素信号中至关重要。我们的初步研究还表明，E2可诱导GPR30和ERK1/2的磷酸化，而不涉及ER1。此外，我们观察到，在注射E2前30分钟给予ERK1/2激活的选择性抑制剂(SL327)可以消除早期的ER非依赖性基因，以及在早期和晚期反应阶段的子宫湿重。相反，在注射E2后6h注射该抑制剂并不能改变迟发效应。总而言之，这些结果使我们能够研究早期和晚期的雌激素反应，以确定各时相之间的分子关系。我们的具体目的是阐明：1.子宫中涉及SIK-SP、Bip和GPR30的E_2或Kepone的早期分子信号。2.Sik-SP和GPR30在E2或Kepone介导的子宫晚期雌激素反应中的功能意义研究应该会产生新的分子洞察力，涉及ER1介导的雌激素信号转导过程中的Bip、SIK-SP和GPR30。总体而言，这些结果将有助于我们确定子宫内两个阶段的雌激素反应之间的关系和分子相互作用。公共卫生相关性：我们的具体目标是阐明：1.子宫中涉及SIK-SP、Bip和GPR30的E2或Kepone的早期分子信号。2.Sik-SP和GPR30在E_2或Kepone介导的子宫晚期雌激素反应中的作用研究应该产生新的分子洞察力，涉及Bip，Sik-SP和GPR30对ER1介导的雌激素信号的调节。总体而言，这些结果将有助于我们确定子宫内两个阶段的雌激素反应之间的关系和分子串扰。

DESCRIPTION (provided by applicant): Early- and late-phase estrogenic responses in the uterus have been recognized for more than 60 years, yet mechanisms involved in their regulation remain controversial. One concept is that an early events(s), occurring within the first 6 h, prepares the uterus for later (18-30 h) increase in DNA synthesis, cell proliferation and protein synthesis. An alternate view is that the late growth phase is a result of the continuous presence of a stimulus. Discussion of either concept usually makes the assumption that all of the responses are dependent upon ligand interaction with one of the two estrogen receptor isoforms (ER1 and ER2). However, increased gene expression following injection of estrogen or xenoestrogen, in mice lacking ER1, or in which all ER-activity has been suppressed by an estrogen-antagonist, ICI 182,780 (ICI), has shown this to be an oversimplification. In this regard, accumulating evidence suggests that estrogen regulates diverse but interdependent signaling pathways in uterine biology via ER- dependent and -independent manners. While our long standing hypothesis is that estrogenic certain early responses are ER-independent, late responses are ER1- dependent, and a cross-talk between the two phases is necessary for a full complement of estrogenic responses in the uterus. Our previous and preliminary observations suggest that among several such early genes, Bip and Sik-SP are induced by estradiol- 172 (E2) and kepone in the mouse uterus via ER-independent manner as a phase-I response. Moreover, we recently showed that Bip is critically necessary in mediating E2- or kepone-dependent estrogen signaling that involves ER1 functions. Our preliminary studies also indicated that E2 induces GPR30 and ERK1/2 phoshorylation without involving ER1. Furthermore, we observed that the administration of a selective inhibitor of ERK1/2 activation (SL327), 30 min prior to E2 injection abrogates early ER- independent genes, as well as uterine wet weights during the early and late responsive phases. In contrast, injection of the inhibitor 6 h after E2 injection did not alter late effects. Collectively, these results positioned us to study early and late estrogenic responses to determine molecular relationship between the phases. Our specific aims are to elucidate: 1. Early molecular signaling by E2 or kepone involving Sik-SP, Bip and GPR30 in the uterus. 2. Functional significance of Sik-SP and GPR30 in E2- or kepone- mediated late estrogenic responses in the uterus. Studies should yield new molecular insights involving Bip, Sik-SP and GPR30 during ER1-mediated estrogen signaling. Overall, the results will help us to define relationship and a molecular cross-talk between the two-phase estrogenic responses in the uterus. PUBLIC HEALTH RELEVANCE: Our specific aims are to elucidate: 1. Early molecular signaling by E2 or kepone involving Sik-SP, Bip and GPR30 in the uterus. 2. Functional significance of Sik-SP and GPR30 in E2 or kepone mediated late estrogenic responses in the uterus. Studies should yield new molecular insights involving Bip, Sik-SP and GPR30 regulation of ER1-mediated estrogen signaling. Overall, the results will help us to define the relationship and a molecular cross-talk between the two-phase estrogenic responses in the uterus.