Genetic and Biochemical Dissection of Prolactin Actions

描述（由申请人提供）：使用包括基因破坏小鼠、表达谱和体外和体内药理学在内的方法的组合，我们最近做出了令人惊讶的发现，即色氨酸羟化酶（TPH）基因是乳腺功能的重要调节因子。TPH催化L-色氨酸转化为5-羟色氨酸，5-羟色氨酸是5-羟色胺（5-HT）合成的限速底物。我们表明TPH是由乳汁停滞引起的，而5-HT参与了抑制哺乳的自分泌/旁分泌反馈循环。这些发现表明，生物源性单胺（特别是5-HT）是重要的乳腺源性信号分子。 我们计划的中心目标是探索5-HT在乳腺内作用的机制的关键细节（目标1-3）。一个辅助目标（目标4）是确定5-HT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，我们既不知道乳腺生理学和发育的哪些具体方面受到5-HT的影响，也不知道乳腺中5-HT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雌激素能药物来研究乳腺基因调控，增殖和细胞凋亡的器官型培养和原代培养的解离细胞。 由于药物对乳腺以外的组织（特别是大脑）具有混淆作用，因此进行乳腺TPH功能体内研究的方法非常有限。为了解决这些问题，我们需要遗传模型来进行生理和发育研究，但其他实验室还没有建立有用的模型，如TPH基因敲除。因此，我们建议，使小鼠中的TPH基因被破坏的乳腺，和转基因小鼠表达的人血清素转运蛋白（hSERT）的乳腺。这些模型将使我们能够检查乳腺表达的TPH在自分泌/旁分泌5-HT暴露中断的动物中的发育和生理学中的作用。

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.