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，被认为在情感性障碍的调节中起重要作用。这可能通过中脑中缝背核（DRN）发出的血清素（5-HT）系统的调节而发生。为了支持这一假设，我们发现，在去卵巢的大鼠中，慢性雌二醇（E2）治疗可以选择性地增加尾侧DRN中色氨酸羟化酶-2 （TpH2）的表达，TpH2是5-羟色胺生物合成的限速酶，这种增加对E2的抗焦虑作用至关重要。E2诱导的TpH2 mRNA的增加是通过雌激素受体β (ER2)介导的，DRN中局部ER2的激活足以导致TpH2 mRNA的增加和E2的抗抑郁作用。由于ER2在尾侧DRN的5-羟色胺能神经元上被发现，这些研究证实了E2直接作用于5-HT神经元，通过ER2增加TpH2 mRNA的假设。然而，E2对TpH2影响的确切机制尚未阐明。因此，本研究将研究雌激素直接通过ER2增加TpH2 mRNA的总体假设，并探讨雌激素通过与ER2及其协同调节蛋白的相互作用激活TpH2启动子转录区域的可能性。我们将首先通过体外报告基因试验检测雌激素与ER2相互作用对TpH启动子-荧光素酶结构的功能影响，来验证这一观点。其次，我们将确定雌激素和ER2诱导的TpH2启动子激活的调控区域，方法是对TpH2启动子进行渐进的5'缺失，然后对潜在的调控元件进行定点诱变。最后，我们将通过染色质免疫沉淀法、三重标记免疫组织化学和反义寡核苷酸敲除来探索ER2介导的TpH2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.