Regulation of the Tryptophan Hydroxylase-2 Promoter by Estrogen

描述（由申请人提供）：卵巢激素，如雌激素，被认为在调节情感障碍中起重要作用。这可能是通过调节中脑中缝背核（DRN）发出的5-羟色胺（5-HT）系统而发生的。为了支持这一假设，我们发现，慢性雌二醇（E2）治疗卵巢切除大鼠增加色氨酸羟化酶-2（TPH 2）的表达，5-HT生物合成的限速酶，选择性地在尾部DRN，这种增加是关键的抗焦虑作用的E2。E2诱导的TpH 2 mRNA的增加是通过雌激素受体β（ER 2）介导的，DRN中局部ER 2的激活足以使TpH 2 mRNA增加，并使E2具有抗抑郁作用。由于在尾侧DRN的E2能神经元上发现了ER 2，这些研究为E2直接作用于5-HT神经元以通过ER 2增加TpH 2 mRNA的假设提供了证据。然而，E2对TpH 2影响的确切机制尚未阐明。 因此，拟议的研究将调查总体假设，即雌激素直接通过ER 2增加TpH 2 mRNA，并解决雌激素通过与ER 2及其共调节蛋白相互作用激活TpH 2启动子转录区域的可能性。我们将测试这一想法，首先检查雌激素与ER 2的相互作用的TpH启动子荧光素酶构建体使用报告基因在体外的功能影响。其次，我们将通过使用TpH 2启动子的渐进5'缺失，随后通过潜在调控元件的定点诱变来鉴定雌激素和ER 2诱导的TpH 2启动子活化的调控区。最后，我们将通过染色质免疫沉淀分析、三重标记免疫组织化学和反义寡核苷酸敲除来探讨ER 2介导的TpH 2激活的一系列可能的共调节因子和机制。

DESCRIPTION (provided by applicant): Ovarian Hormones, such as estrogen, are thought to play a significant role in regulation of affective disorders. This may occur through modulation of serotonin (5-HT) system emanating from the midbrain dorsal raphe nucleus (DRN). In support of this hypothesis, we have found that chronic estradiol (E2) treatment in ovariectomized rats increases the expression of tryptophan hydroxylase-2 (TpH2), the rate-limiting enzyme for 5-HT biosynthesis, selectively in the caudal DRN and this increase is critical for the anxiolytic effects of E2. E2- induced increases of TpH2 mRNA are mediated via estrogen receptor beta (ER2), and activation of local ER2 in the DRN is sufficient for this increase in TpH2 mRNA and for antidepressant effects of E2. As ER2 is found on serotonergic neurons in the caudal DRN, these studies lend credence to the hypothesis that E2 acts directly on the 5-HT neurons to increase TpH2 mRNA via ER2. However, exact mechanisms underlying this E2 effect on TpH2 are yet to be elucidated. Therefore, the proposed study will investigate the overarching hypothesis that that estrogen increases TpH2 mRNA directly via ER2, and address the possibility that estrogen activates the transcriptional region of the TpH2 promoter via interaction with ER2 and its co-regulator proteins. We will test this idea by first examining the functional effects of estrogen interaction with ER2 on the TpH promoter-luciferase construct using reporter gene assay in vitro. Second, we will identify the regulatory regions of the estrogen and ER2 induced activation of the TpH2 promoter by using progressive 5' deletions of the TpH2 promoter, followed by site directed mutagenesis of potential regulatory elements. Finally, we will explore the array of possible co- regulatory factors and mechanisms involved in ER2 mediated activation of TpH2 by using chromatin immunoprecipitation assay, triple labeled immunohistochemistry and antisense oligonucleotide knockdown.