REGULATED EXPRESSION OF CATECHOLAMINE BIOSYNTHESIS GENES

本建议的总体重点是描述导致神经内分泌基因酪氨酸羟化酶（TH）和多巴胺-羟化酶的调控表达的分子和遗传事件。TH是多巴胺、去甲肾上腺素和肾上腺素生物合成的起始酶和限速酶，DBH催化多巴胺向去甲肾上腺素的转化。TH和DBH在去甲肾上腺素能细胞和肾上腺素能细胞中共同表达，TH和DBH基因的表达经常受到环境、神经元和激素影响的协调调节。这个正在进行的研究项目已经确定了几个控制TH和DBH基因转录的遗传调控元件，以及与调控元件相互作用以控制转录速率的转录因子。在当前的提案中，我们将利用生物化学，分子生物学和细胞方法：(1)通过分析Arix磷酸化，研究第二信使和蛋白激酶级联如何与组织特异性转录因子Phox2a/Arix相互作用，影响TH和DBH基因的转录；(2)鉴定和表征不合适组织中抑制DBH基因表达的蛋白；(3)评估转录因子Phox2a/Arix、Phox2b/NBPhox、CREB/ATF和AP1与完整细胞内TH和DBH基因的相互作用。这些实验将确定组织特异性转录因子与普遍存在因子相互作用介导TH和DBH基因选择性表达的机制。了解这些基因调控表达的机制与多巴胺能细胞丢失的研究相关，如帕金森病，以及神经递质异常平衡导致疾病的研究，如高血压和双相精神障碍。

The overall focus of this proposal is to delineate the molecular and genetic events which lead to the regulated expression of the neuroendocrine genes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase. TH is the initial and rate limiting enzyme in the biosynthesis of dopamine, norepinephrine and epinephrine, and DBH catalyzes the conversion of dopamine to norepinephrine. TH and DBH are co-expressed in noradrenergic and adrenergic cells, and the expression of the TH and DBH genes are frequently coordinately regulated by environmental, neuronal and hormonal influences. This ongoing research program has identified several of the genetic regulatory elements which control the transcription of the TH and DBH genes, as well as the transcription factors which interact with the regulatory elements to control the rate of transcription. In this current proposal, we will utilize biochemical, molecular biology and cellular approaches to: (l) investigate how second messengers and protein kinase cascades interact with the tissue specific transcription factor Phox2a/Arix to influence transcription of the TH and DBH genes through analysis of Arix phosphorylation, (2) identify and characterize the proteins which repress DBH gene expression in inappropriate tissues, and (3) evaluate the interaction of transcription factors Phox2a/Arix, Phox2b/NBPhox, CREB/ATF and AP1 with the TH and DBH genes within intact cells. These experiments will define the mechanisms by which tissue specific transcription factors interact with ubiquitous factors to mediate selective expression of the TH and DBH genes. Knowledge of the mechanisms underlying the regulated expression of these genes is relevant to studies where dopaminergic cells are lost, such as Parkinson's disease, and where aberrant balance of neurotransmitters lead to disease, such as hypertension and bipolar mental disorders.