Molecular determinants of retinoid metabolism in embryonic tissues

描述（由申请人提供）：维生素A代谢产物全反式维甲酸（ATRA）在整个生命过程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有希望的结果。然而，在怀孕期间暴露于降低或过量的ATRA水平可能导致胚胎发育异常。该项目的具体目标是确定控制ATRA形成的因素， vivo.长期目标是开发基于操纵病变组织中ATRA的内源性水平和活性的疗法。中心假设是ATRA通过控制视黄醇氧化与视黄醇醛还原的平衡，通过稳态反馈机制调节其自身的合成。目前的研究在很大程度上忽略了酶的作用，进行减少的视黄醇在调节ATRA水平。本申请的总体目标是确定DHRS 3的作用和调节， 是维生素A代谢的关键酶。这一假设是基于Dhrs 3缺陷小鼠模型表征的初步结果。这些结果表明：1）DHRS 3是发育过程中负责还原视黄醇的主要酶; 2）DHRS 3还原视黄醇对ATRA稳态和正常胚胎发育是必不可少的。中心假设的检验将在三个具体目标中进行：目标1研究将建立1）DHRS 3的酶性质，2）ATRA调节Dhrs 3的机制，和3）发育期间Dhrs 3表达的时空模式。这些知识对于理解DHRS 3的生理作用至关重要。目标2中描述的研究将基于DHRS 3是负责在胚胎发生期间减少视黄醇的主要酶的工作假设来确定DHRS 3在维生素A代谢中的作用。目的2将通过1）类维生素A水平的LC-UV和LC-MS/MS分析和2）测定Dhrs 3-/-胚胎组织中ATRA靶基因的表达来确定Dhrs 3-消融对视黄醇和维生素A原类胡萝卜素代谢的影响。目的3将检验以下假设：Dhrs 3的消融引起的发育缺陷是ATRA代谢改变的结果。初步研究表明，Dhrs 3-/-胚胎在前后图案、心脏和骨骼发育方面存在缺陷，并且是不可存活的。目标3将通过表征与改变的ATRA形成有关的所观察到的发育缺陷的途径来实现。因此，将检查饮食、遗传背景和维生素A代谢抑制对与Dhrs 3-消融相关的发育缺陷表现的影响。这些研究旨在进一步建立负责体内ATRA形成调控的过程，并为开发预防和治疗与ATRA信号改变相关的先天性和遗传性疾病的方法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.