Genetic and Biochemical Dissection of Prolactin Actions

描述(申请人提供)：利用多种方法，包括基因干扰小鼠、表达谱和体外和体内药理学，我们最近取得了令人惊讶的发现，色氨酸羟基酶(TPH)基因是乳腺功能的重要调节因子。茶多酚催化L-色氨酸转化为5-羟色氨酸，5-羟色氨酸是合成5-羟色胺的限速底物。我们发现TPH是由乳汁淤积引起的，5-羟色胺参与了抑制哺乳的自分泌/旁分泌反馈环路。这些发现表明，生物来源的单胺类(特别是5-羟色胺)是重要的乳腺信号分子。 我们计划的中心目标是探索5-羟色胺在乳腺内作用机制的关键细节(目标1-3)。辅助目标(目标4)是确定5-羟色胺是否从乳腺输出到母体循环和/或乳汁中。 我们现在面临的情况是，我们既不知道5-羟色胺影响乳腺生理和发育的哪些特定方面，也不知道乳腺中5-羟色胺调节功能的药理学特征(受体类型)。为了填补这些知识空白，我们将利用丰富的可用的5-羟色胺能药物来检测器官型培养和分离细胞的原代培养中的乳腺基因调控、增殖和凋亡。 对乳腺TPH功能进行体内研究的手段非常有限，因为药物对乳腺以外的组织(尤其是乳腺)具有混杂作用。大脑)。为了绕过这些问题，我们需要进行生理和发育研究的遗传模型，但其他实验室还没有制作出有用的模型，如TPH基因敲除。因此，我们建议建立在乳腺中TPH基因被破坏的小鼠，以及在乳腺中表达人5-羟色胺转运体(HSERT)的转基因小鼠。这些模型将使我们能够检验乳腺表达的TPH在自分泌/旁分泌5-羟色胺暴露中断的动物的发育和生理中的作用。

DESCRIPTION (provided by applicant): Using a combination of approaches, including gene-disrupted mice, expression profiling, and pharmacology in vitro and in vivo, we recently made the surprising discovery that the tryptophan hydroxylase (TPH) gene is an important regulator of mammary gland function. TPH catalyzes the conversion of L-tryptophan to 5-hydroxytryptophan, which is the rate-limiting substrate for serotonin (5-HT) synthesis. We showed that TPH is induced by milk stasis, and that 5-HT participates in an autocrine/paracrine feedback loop that inhibits lactation. These discoveries imply that biogenic monoamines (esp. 5-HT) are important mammary-derived signaling molecules. The central objective of our plan is to explore critical details of the mechanisms by which 5-HT acts within the mammary gland (aims 1-3). A subsidiary objective (aim 4) is to determine whether 5-HT is exported from the mammary gland into the maternal circulation and/or milk. The situation we now confront is that we know neither what specific aspects of mammary physiology and development are influenced by 5-HT, nor do we know the pharmacological profile (receptor types) for the 5-HT regulated functions in the mammary glands. To fill these knowledge gaps we will make use of the wealth of available serotonergic agents to examine mammary gene regulation, proliferation, and apoptosis in organotypic cultures and primary cultures of dissociated cells. The means to do in vivo studies of mammary TPH functions are very limited because of the confounding effects that pharmacological agents have on tissues other than the mammary gland (esp. the brain). To circumvent these problems we need genetic models in which to do the physiological and developmental studies, but useful models, such as TPH gene knockouts, have not been made by other labs. Consequently, we propose to make mice in which the TPH gene is disrupted in the mammary glands, and transgenic mice expressing the human serotonin transporter (hSERT) in the mammary glands. These models will allow us to examine the role of mammary-expressed TPH in development and physiology in animals that have interruptions in their autocrine/paracrine 5-HT exposure.