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 (ER), and activation of local ER in the DRN is sufficient for this increase in TpH2 mRNA and for antidepressant effects of E2. As ERis 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 ER. 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 ER, and address the possibility that estrogen activates the transcriptional region of the TpH2 promoter via interaction with ERand its co-regulator proteins. We will test this idea by first examining the functional effects of estrogen interaction with ERon the TpH promoter-luciferase construct using reporter gene assay in vitro. Second, we will identify the regulatory regions of the estrogen and ER 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 coregulatory factors and mechanisms involved in ERmediated activation of TpH2 by using chromatin immunoprecipitation assay, triple labeled immunohistochemistry and antisense oligonucleotide knockdown.
|Effective start/end date||7/1/12 → 6/30/14|
- HHS: National Institutes of Health (NIH): $113,404.00