Molecular determinants of retinoid metabolism in embryonic tissues

描述(申请人提供)：维生素A代谢物，全反式维甲酸(ATRA)，在生命中的许多生物过程中发挥重要作用，并在几种治疗环境中显示出良好的结果。然而，在怀孕期间暴露于ATRA水平降低或过高都会导致胚胎发育异常。这个项目的具体目标是确定控制ATRA形成的因素 活着。长期目标是开发基于对疾病组织中ATRA内源性水平和活性的操纵的治疗方法。中心假说是，ATRA通过控制视黄醇的氧化和视黄醛的还原的平衡，通过动态平衡反馈机制来调节自身的合成。目前的研究在很大程度上忽略了执行视黄醛还原的酶在调节ATRA水平中的作用。本申请的总体目标是确定DHRS3的作用和调节， 这里显示的是维生素A新陈代谢的关键酶。这一假设是基于对Dhrs3缺陷小鼠模型的初步研究结果。这些结果表明：1)DHRS3是发育过程中负责还原视黄醛的主要酶；2)DHRS3还原视黄醛对ATRA动态平衡和正常胚胎发育是不可或缺的。对中心假说的验证将在三个具体目标上进行：目的1研究将建立1)DHRS3的酶特性，2)ATRA调节Dhrs3的机制，以及3)Dhrs3在发育过程中表达的时空模式。这些知识对于理解DHRS3的生理作用是必不可少的。目标2中描述的研究将基于DHRS3是胚胎发育过程中负责还原视黄醛的主要酶的工作假设，确定DHRS3在维生素A代谢中的作用。目的1)通过LC-UV和LC-MS/MS分析维甲酸水平，以及2)检测ATRA靶基因在Dhrs3-/-胚胎组织中的表达，以确定Dhrs3-/-胚胎对视黄醇和维生素A原类胡萝卜素代谢的影响。目的3将验证这一假设，即消融Dhrs3导致的发育缺陷是ATRA代谢改变的结果。初步研究表明，Dhrs3/-胚胎在前后构型、心脏和骨骼发育方面存在缺陷，不能存活。目标3将通过表征与ATRA形成改变有关的所观察到的发育缺陷的途径来实现。因此，我们将研究饮食、遗传背景和维生素A代谢抑制对Dhrs3消融相关发育缺陷表现的影响。这些研究旨在进一步建立在体内调节全反式维甲酸形成的过程，并为开发预防和治疗与全反式维甲酸信号改变相关的先天性和遗传性疾病的长期目标提供框架。

DESCRIPTION (provided by applicant): The vitamin A metabolite, all-trans-retinoic acid (ATRA), plays essential roles in a multitude of biological processes throughout life and has shown promising results in several therapeutic settings. However, exposure to either reduced or excess levels of ATRA during pregnancy can result in abnormalities in embryonic development. The specific goal of this project is to determine the factors that control the formation of ATRA in vivo. The long-term goal is to develop therapies based on the manipulation of the endogenous levels and activity of ATRA in diseased tissues. The central hypothesis is that ATRA regulates its own synthesis via a homeostatic feedback mechanism by controlling the balance of the oxidation of retinol versus the reduction of retinaldehyde. Current studies have largely ignored the role of enzymes that carry out the reduction of retinaldehyde in the regulation of ATRA levels. The overall objective of this application is to determine the role and regulation of DHRS3, shown here to be a pivotal enzyme in vitamin A metabolism. This hypothesis is based on preliminary results derived from the characterization of a Dhrs3-deficient mouse model. These results indicate that 1) DHRS3 is the predominant enzyme responsible for the reduction of retinaldehyde during development and 2) the reduction of retinaldehyde by DHRS3 is indispensable for ATRA homeostasis and normal embryonic development. Testing of the central hypothesis will be pursued in three specific aims: Aim 1 studies will establish 1) the enzymatic properties of DHRS3, 2) the mechanism of regulation of Dhrs3 by ATRA, and 3) the spatial- temporal pattern of the expression of Dhrs3 during development. This knowledge is essential for understanding the physiological role of DHRS3. Studies described in Aim 2 will determine the role of DHRS3 in vitamin A metabolism based on the working hypothesis that DHRS3 is the main enzyme responsible for reducing retinaldehyde during embryogenesis. Aim 2 will be pursued by establishing the effect of Dhrs3-ablation on the metabolism of retinol and provitamin A carotenoids by 1) LC-UV and LC-MS/MS analysis of the levels of retinoids, and 2) assaying the expression of ATRA-target genes in the tissues of Dhrs3-/- embryos. Aim 3 will test the hypothesis that the developmental defects caused by ablation of Dhrs3 are a result of altered ATRA metabolism. Preliminary studies indicate that Dhrs3-/- embryos have defects in anterior-posterior patterning, cardiac and skeletal development, and are non-viable. Aim 3 will be pursued by characterizing the pathways responsible for the observed developmental defects in relation to altered ATRA formation. Therefore, the effect of diet, genetic background and inhibition of vitamin A metabolism on the manifestation of developmental defects associated with Dhrs3-ablation will be examined. These studies aim to further establish the processes responsible for the regulation of ATRA formation in vivo and contribute to a framework for the long-term goal of developing approaches to prevent and treat congenital and inherited disorders associated with altered ATRA-signaling.