Molecular Pharmacology of Estrogen Sulfotransferase

说明(申请人提供)：雌激素磺基转移酶(EST)是一种II相代谢酶，它与类固醇激素雌激素发生磺基偶联并使其失活。虽然对EST及相关类固醇磺基转移酶的生物化学、结构生物学和药物遗传学已有很多研究，但EST及其他类固醇磺基转移酶的分子药理学和体内功能仍不完全清楚。这项提议的目的是验证EST是一种重要的肝外结合酶的假设，它调节组织对雌激素的敏感性，因此在局部雌激素代谢和动态平衡中发挥生理作用。这项研究的长期目标是了解EST活性的变化如何通过遗传(药物遗传学)或环境因素(药物-药物相互作用)在生理或治疗环境下改变组织的雌激素敏感性。在我们的初步研究中，我们已经产生了一只EST基因敲除(KO)小鼠，它将被用来检验我们的假设。在这项提案中，我们将追求以下特定目标：特定目标1：验证子宫和胎盘EST表达防止胎儿雌激素毒性的假设。这一假设得到了我们的初步发现的支持，即雌性EST KO小鼠的生育力降低。在没有EST的情况下，未代谢的雌激素可能会对发育中的胎儿产生毒性或干扰分娩过程。药理学方法，如使用雌激素受体拮抗剂，将被应用于这项研究。具体目的2：验证EST在维持间质细胞胆固醇稳态中起生理作用的假说。我们发现EST-KO小鼠间质细胞异常积聚中性脂类。我们将确定这些细胞中的异常脂质积累是否由清道夫B类I型受体(SRBI)介导，以及它是否依赖于雌激素受体的功能。生物化学、分子生物学和遗传学方法将用来解决这个问题。具体目的3：验证EST保护间质细胞免受雌激素诱导的类固醇合成抑制的假设，以及这种抑制发生在底物动员步骤或P45017a-羟基酶步骤。具体目的4：验证EST以雄激素依赖的方式在男性生殖道的附属结构中表达的假说。我们对EST KO小鼠的初步研究支持了特定目标3和4中的两个假说。这些研究将为理解雌激素磺基转移酶作为典型的II相类固醇磺基结合酶的体内药理和生理学提供分子基础。

DESCRIPTION (provided by applicant): Estrogen sulfotransferase (EST) is a phase II metabolic enzyme that sulfoconjugates and inactivates the steroid hormone estrogen. Although much has been learned on the biochemistry, structural biology and pharmacogenetics of EST and related steroid sulfotransferases, the molecular pharmacology and in vivo function of EST and other steroid sulfotransferases remain incompletely understood. The goal of this proposal is to test the hypothesis that EST is a significant extrahepatic conjugation enzyme that regulates tissue estrogen sensitivity, and as such plays a physiological role in local estrogen metabolism and homeostasis. The long-term objective of this study is to understand how variation in EST activity, through either genetic (pharmacogenetics) or environmental factors (drug-drug interaction), might alter tissue estrogen sensitivity under physiological or therapeutic settings. In our preliminary studies, we have generated an EST knockout (KO) mouse which will be used to test our hypothesis. In this proposal, we will pursue the following specific aims: Specific aim 1: To test the hypothesis that uterine and placental EST expression prevents fetal estrogen toxicity. This hypothesis is supported by our preliminary finding that female EST KO mice have reduced fertility. In the absence of EST, unmetabolized estrogen may become toxic to the developing fetus or disturb the parturition process. Pharmacological approaches such as the use of estrogen receptor antagonists will be applied to this investigation. Specific aim 2: To test the hypothesis that EST plays a physiological role in maintaining cholesterol homeostasis in Leydig cells. We have found that EST KO mouse Leydig cells abnormally accumulate neutral lipids. We will determine if abnormal lipid accumulation in these cells is mediated by the Scavenger Receptor, Class B, Type I (SRBI) and whether it is dependent on estrogen receptor function. Biochemical, molecular biological and genetic approaches will be applied to address this question. Specific aim 3: To test the hypothesis that EST protects Leydig cells from estrogen-induced inhibition of steroidogenesis and that such inhibition occurs either at the substrate mobilization step or at the P450 17a-hydroxylase step. Specific aim 4: To test the hypothesis that EST is expressed in the accessory structures of the male reproductive tract in an androgen-dependent manner. Both hypotheses in Specific aim 3 and 4 are supported by our preliminary study of the EST KO mice. The proposed studies will provide a molecular basis for understanding the in vivo pharmacology and physiology of estrogen sulfotransferase as a prototypical phase II steroid sulfoconjugation enzyme.