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到60多年了，但涉及到它们的调节机制仍然存在争议。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，后期或成长期是持续存在刺激的结果。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(ERα和ERβ)中的一种的配体相互作用。然而，在缺乏ERα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。最近，我们通过雌激素非依赖性机制在子宫中发现了几个早期受雌激素调控的下游靶基因。初步研究表明，雌二醇-17β(E_2)能迅速激活野生型和ERα(-/-)小鼠子宫中ERK1/ERK2(MAP激酶)的磷酸化。此外，我们观察到，在注射E2前30分钟给予ERK1/ERK2激活的选择性抑制剂(SL327)完全取消了包括子宫湿重和晚期子宫营养(生长)反应在内的早期反应基因的表达。相反，在注射E_2后6h注射该抑制剂并不改变迟发反应。总而言之，这些结果使我们能够分别研究早期和晚期的雌激素反应，并确定这两个阶段之间的关系。我们的假设是，虽然雌激素早期反应是ER非依赖性的，但晚期反应是ER依赖的，这两个阶段之间的合作是由自然雌激素或异种雌激素在子宫中介导的雌激素反应的完整补充所必需的。我们的具体目标是确定：1.子宫内雌激素作用的早期反应与晚期生长反应相协调。2.雌激素受体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，抑制早期基因在体内和体外子宫细胞中确定雌激素反应的直接后果。4.雌激素依赖的早期反应基因以一种协调的方式调节ER依赖的活动。这一结果将有助于我们确定子宫内两相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.