Environmental Toxins and Uterine Gene Expression

描述（由申请人提供）：子宫中的“早期”和“晚期”雌激素反应已被认识超过60年，但涉及其调节的机制仍有争议。 一个概念是，在前6小时内发生的早期事件为子宫后期（18-30小时）DNA合成、细胞增殖和蛋白质合成的增加做好准备。 另一种观点是，晚期或增长阶段是刺激持续存在的结果。 对这两个概念的讨论通常假设所有的反应都依赖于配体与两种雌激素受体亚型（ER α和ER β）之一的相互作用。 然而，在缺乏ER α或所有ER活性均被雌激素拮抗剂ICI 182，780（ICI）抑制的小鼠中，注射雌激素或异种雌激素后基因表达增加，表明这是一种过度简化。 最近，我们确定了几个下游靶基因调节早期雌激素在子宫中的ER非依赖性机制。 初步研究表明，雌二醇-17 β（E2）可迅速激活野生型和ER α（-/-）小鼠子宫内ERK 1/ERK 2（MAP激酶）的磷酸化。此外，我们观察到，E2注射前30分钟给予ERK 1/ERK 2活化的选择性抑制剂（SL 327）完全消除了早期反应基因表达，包括子宫湿重和晚期子宫营养（生长）反应。 相反，注射抑制剂6小时后，E2注射并没有改变迟发反应。 总的来说，这些结果使我们能够分别研究早期和晚期雌激素反应，并确定这两个阶段之间的关系。 我们的假设是，虽然雌激素的早期反应是ER独立的，晚期反应是ER依赖性的，和两个阶段之间的合作是必要的天然雌激素或异种雌激素介导的雌激素反应在子宫内的充分补充。 我们的具体目标是确定：1。早期反应与子宫内雌激素作用的晚期生长反应相协调。 2. ER介导的早期和晚期雌激素反应的分子相互作用。 3.通过腺病毒载体驱动的递送，在体内和体外确定子宫细胞雌激素反应中抑制早期基因的直接后果。 4.雌激素依赖性早期反应基因以协调的方式发挥作用，以调节ER依赖性活动。 这些结果将帮助我们确定子宫内两阶段雌激素反应之间的关系和分子串扰。

DESCRIPTION (provided by applicant): An "early" and a "late" phase estrogenic response in the uterus has been recognized for more than 60 years yet mechanisms involved in their regulation remain controversial. One concept is that an early event(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 or 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 (ERalpha and ERbeta). However, increased gene expression following injection of estrogen or xenoestrogen, in mice lacking ERalpha, or in which all ER-activity has been suppressed by an estrogen-antagonist, ICI 182,780 (ICI), has shown this to be an oversimplification. Recently we identified several downstream target genes regulated early by estrogens in the uterus by an ER-independent mechanism. Preliminary studies indicated that estradiol-17beta (E2) rapidly activates phosphorylation of ERK1/ERK2 (MAP kinases) in uteri of both wild-type and ERalpha (-/-) mice. Furthermore, we observed that the administration of a selective inhibitor of ERK1/ERK2 activation (SL327), 30 min prior to an E2 injection totally abrogates the early responsive gene expression including uterine wet weights and the late uterotrophic (growth) responses. In contrast, injection of the inhibitor 6 h after E2 injection did not alter the late responses. Collectively, these results positioned us to study early and late estrogenic responses separately and determine the relationship between these two phases. Our hypothesis is that while estrogenic early responses are ER-independent, late responses are ER-dependent, and cooperation between the two phases is necessary for a full complement of estrogenic responses mediated by natural estrogen or xenoestrogen in the uterus. Our specific aims are to determine: 1. Early responses coordinate with the late growth responses for estrogenic actions in the uterus. 2. Molecular interactions of early and late estrogenic responses mediated by ER. 3. Direct consequences of inhibition of early genes in defining the estrogenic responses in uterine cells both in vivo and in vitro by adenoviral vector-driven delivery. 4. Estrogen-dependent early responsive genes function in a coordinated fashion to regulate ER-dependent activities. The results will help us define the relationship and a molecular cross-talk between the two-phase estrogenic responses in the uterus.