Xenoestrogens: Genomic and Non-genomic Mechanisms

描述(申请人提供)：双酚A(BPA)和辛基酚(OP)在环境中含量丰富，与雌激素受体(ER)结合，并作为部分雌激素激动剂。尽管关于它们对生殖道的多重影响的证据越来越多，但人们对它们在神经内分泌轴中的作用范围知之甚少，特别是对雌激素敏感的脑下垂体催乳素。我们先前报道，这些化合物可诱导催乳素(PRL)基因的表达和释放，促进催乳素(PRL)的增殖，改变垂体ERa和ERbeta的表达，并影响女性生殖道的生长和形态。虽然这些效应需要几小时到几天的时间才能发生，并涉及基因转录和蛋白质合成，但我们最近发现，低纳摩尔浓度的雌激素/异种雌激素可以非常迅速地(在10分钟内)诱导MAP K系统的激活，并刺激培养的垂体前叶细胞释放PRL。我们的主要目标是比较雌激素/异种雌激素在脑垂体中的基因组和非基因组作用，并确定细胞内钙、一氧化氮(NO)释放、MAPK激活和核ER反式激活之间的相互作用，这些作用导致激素释放增加、基因激活和催乳细胞增殖。具体目标1将测试雌激素通过膜ER快速刺激PRL释放的假设，该ER与钙依赖的胞吐作用有关，也可能涉及一氧化氮(NO)的释放。游离和结合的雌激素/异种雌激素将被用来确定：a)在没有和存在ER拮抗剂的情况下，迅速刺激分散的垂体前叶细胞或GH3催乳素细胞释放PRL，b)钙、NO或MAPK阻滞剂逆转这一效应，c)细胞内钙浓度的变化或雌激素对NO释放的反应，d)在多巴胺存在的情况下雌激素增加PRL释放的能力，以及e)PRL对雌激素的分泌反应是否受到受体脱敏。特殊目的2将检验这一假设，即短时间暴露于雌激素/异种雌激素足以启动MAPK和ER信号通路的顺序激活，导致选定的靶基因的诱导和改变的细胞增殖/凋亡。在存在或不存在MAPK和ER抑制剂的情况下，乳酸菌将受到游离和结合雌激素的脉冲追逐。在此后的不同时间，我们将确定：a)诱导PRL和血管内皮生长因子(VEGF)基因表达，b)ER亚型的表达改变细胞对雌激素的敏感性，以及c)增加催乳素的增殖。特定目标3将使用基因阵列方法来测试这一假设，即雌激素和双酚A反应上调/下调的垂体基因在雌激素敏感型Fischer 344(F344)和雌激素不敏感型Spraogue Dawley(SD)大鼠之间存在差异。这些研究的结果应该为评估脑下垂体对内分泌干扰物侮辱的脆弱性提供急需的实验基础。

DESCRIPTION (provided by applicant): Bisphenol A (BPA) and octylphenol (OP) are abundant in the environment, bind to the estrogen receptor (ER) and act as partial estrogen agonists. In spite of accumulating evidence on their multiple effects on the reproductive tract, little is known about their spectrum of actions within the neuroendocrine axis, especially on the estrogen-sensitive pituitary lactotroph. We previously reported that these compounds induced lactotroph proliferation, increased prolactin (PRL) gene expression and release, altered pituitary ERalpha and ERbeta expression and affected the growth and morphology of the female reproductive tract. Whereas these effects take hours to days to occur and involve gene transcription and protein synthesis, we recently found that low nanomolar concentrations of estrogens/xenoestrogens induced very rapid (within 10 min) activation of the MAP kinase (MAPK) system and stimulated PRL release from cultured anterior pituitary cells. Our main objective is to compare genomic vs. non-genomic actions of estrogens/ xenoestrogens in the pituitary and determine interactions between intracellular calcium, nitric oxide (NO) release, MAPK activation and nuclear ER transactivation which result in increased hormone release, gene activation and lactotroph cell proliferation. Specific aim 1 will test the hypothesis that estrogens rapidly stimulate PRL release via a membrane ER that is linked to calcium-dependent exocytosis and may also involve nitric oxide (NO) release. Free and conjugated estrogens/xenoestrogens will be used to determine: a) rapid stimulation of PRL release from dispersed anterior pituitary cells or GH3 lactotrophs in the absence and presence of ER antagonists, b) reversal of this effect by blockers of calcium, NO or MAPK, c) changes in intracellular calcium concentrations or NO release in response to estrogens, d) the ability of estrogens to increase PRL release in the presence of dopamine, and e) whether the PRL secretory response to estrogens is subjected to receptor desensitization. Specific aim 2 will test the hypothesis that a short exposure to estrogens/xenoestrogens is sufficient to initiate sequential activation of the MAPK and ER signaling pathways, resulting in the induction of selected target genes and altered cell proliferation/apoptosis. Lactotrophs will be pulse-chased with free and conjugated estrogens in the presence or absence of MAPK and ER inhibitors. At various times thereafter we will determine: a) induction PRL and vascular endothelial growth factor (VEGF) gene expression, b) expression of ER isoforms that alter cellular sensitivity to estrogens, and c) increased lactotroph proliferation. Specific aim 3 will use a gene array approach to test the hypothesis that the profile of pituitary genes that are upregulated/downregulated in response to estradiol and BPA differs between the estrogensensitive Fischer 344 (F344) and the estrogen-insensitive Sprague Dawley (SD) rat. The results of these studies should provide much needed experimental foundation for assessing the vulnerability of the pituitary gland to insults by endocrine disruptors.