SHORT-CHAIN DEHYDROGENASES IN RETINOL/STEROL METABOLISM

描述（由申请人提供）：维生素A衍生物全反式维甲酸调节530多种不同基因的表达。因此，胚胎发生过程中维甲酸的水平是在空间和时间上精确控制的。然而，这种调控的分子机制尚不完全清楚。该项目的长期目标是确定短链脱氢酶/还原酶（sdr）在健康和疾病中调节视黄酸生物合成中的作用。最近，我们在青蛙中发现了SDR超家族蛋白的一个新成员rd2，它是一种高活性的全反式视黄醇脱氢酶，对非洲爪蟾的胚胎发育至关重要。重要的是，在哺乳动物中似乎存在与青蛙rdh2功能相当的rdh2，它具有全反式视黄醇脱氢酶活性，并在早期胚胎发育期间表达。我们认为这种被命名为RDH-E2S的新酶在哺乳动物胚胎期和成年期的视黄酸生物合成中是必不可少的。为了验证这一假设，我们将表征哺乳动物RDH-E2S的催化特性，并利用转基因小鼠模型确定其对维甲酸生物合成的贡献（Specific Aim 1）。我们的初步研究表明，在人类细胞中，视网膜短链脱氢酶/还原酶1 （retSDR1）基因表达的沉默导致视黄酸和视黄醛水平的显著增加，从而转化为视黄酸应答基因的显著上调。这一发现表明，视黄酸的生物合成速率是由视黄醇脱氢酶和视黄醛还原酶的相对活性决定的，它们共同控制着视黄酸前体视黄醛的水平。为了验证这一假设，我们提出利用转基因小鼠、人类皮肤器官培养和非洲爪蟾早期胚胎发育的体外模型来表征retSDR1的催化特性，并确定其在体内维甲酸水平调控中的作用（Specific Aim 2）。这些研究将通过提供有关类视黄酸调节系统中潜在重要组成部分的新信息，填补我们对维持视黄酸稳态机制的理解空白。这些研究的结果对于理解与类视黄醇稳态破坏相关的疾病的病理生理学非常重要，如胎儿酒精综合征、酒精性肝病、癌变和糖尿病。

DESCRIPTION (provided by applicant): Vitamin A derivative all-trans-retinoic acid regulates the expression of over 530 different genes. Consequently, the levels of retinoic acid during embryogenesis are controlled in a spatially and temporally precise manner. However, the molecular mechanisms underlying this regulation are not yet fully understood. The long-term objective of this project is to determine the role of short-chain dehydrogenases/reductases (SDRs) in the regulation of retinoic acid biosynthesis in health and disease. Recently, we have identified a new member of the SDR superfamily of proteins in frogs, rdhe 2, that is highly active as an all-trans-retinol dehydrogenase and is critical for embryonic development in Xenopus laevis. Importantly, there appears to be a functional equivalent of the frog rdhe2 in mammals, which exhibits an all-trans-retinol dehydrogenase activity and is expressed during early embryonic development. We propose that this novel enzyme, named RDH-E2S, is essential for retinoic acid biosynthesis in mammals during embryogenesis and, possibly, in adulthood. To test this hypothesis, we will characterize the catalytic properties of mammalian RDH-E2S and determine its contribution to retinoic acid biosynthesis in vivo using genetically modified mouse model (Specific Aim 1). Our preliminary studies indicate that silencing of retina short-chain dehydrogenase/reductase 1 (retSDR1) gene expression in human cells results in significant increase in the levels of both retinoic acid and retinaldehyde, which translates into dramatic upregulation of retinoic acid-responsive genes. This finding suggests that the rate of retinoic acid biosynthesis is determined by the relative activities of retinol dehydrogenases and retinaldehyde reductases, which together control the levels of retinoic acid precursor, retinaldehyde. To test this hypothesis, we propose to characterize the catalytic properties of retSDR1 and to determine its role in the regulation of retinoic acid levels in vivo using genetically modified mice, human skin organ culture, and Xenopus laevis in vitro model of early embryonic development (Specific Aim 2). These studies will fill the gaps in our understanding of the mechanisms responsible for the maintenance of retinoic acid homeostasis by providing new information regarding the roles of potentially important components of the retinoid regulatory system. The results of these studies will be important for understanding the pathophysiology of disorders associated with disruptions of retinoid homeostasis, such as fetal alcohol syndrome, alcoholic liver disease, carcinogenesis, and diabetes.