Molecular determinants of retinoid metabolism in embryonic tissues

摘要 维生素A代谢产物全反式维甲酸(ATRA)在多种生物过程中发挥着重要作用。 他终生接受治疗，并在几种治疗环境中显示出了令人振奋的结果。然而，暴露于 怀孕期间ATRA水平降低或过高都会导致胚胎发育异常- 门槛。该项目的具体目标是确定在体内控制ATRA形成的因素。这个 长期目标是开发基于对内源性AT-2水平和活性的控制的治疗方法。 RA在病变组织中的表达。中心假说是ATRA通过体内平衡调节自身的合成。 控制视黄醇氧化与视黄醇还原平衡的反馈机制 海德。目前的研究在很大程度上忽略了在体内执行视黄醛还原的酶的作用 ATRA水平的调节。本申请的总体目标是确定 DHRS3，这里显示是维生素A代谢的关键酶。这一假设是基于初步的 结果来源于Dhrs3缺陷小鼠模型的特征。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去分化是不可或缺的 发展。对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)DHRS3的酶性质；2)ATRA调节Dhrs3的机制； Dhrs3在发育过程中表达的时间模式。这方面的知识对于理解 DHRS3的生理作用。AIM 2中描述的研究将确定DHRS3在维生素A-Me中的作用。 基于DHRS3是负责减少视黄醛的主要酶的工作假说的合成代谢。 海德在胚胎发育过程中。目标2将通过建立Dhrs3-消融对代谢的影响来实现。 视黄醇和维生素A原类胡萝卜素的LC-UV和LC-MS/MS分析 2)检测Dhrs3-/-胚胎组织中ATRA靶基因的表达。目标3将检验这一假设-- Dhrs3消融导致的发育缺陷是ATRA代谢改变的结果。Pre- 初步研究表明，Dhrs3/-胚胎在前后构型、心脏和骨骼等方面存在缺陷。 塔尔的发展，是不可行的。目标3将通过描述导致 观察到的发育缺陷与ATRA形成的改变有关。因此，饮食的影响，遗传 维生素A代谢抑制对相关发育缺陷表现的背景和抑制 使用Dhrs3-消融将被检查。这些研究的目的是进一步确定负责 在体内调节ATRA的形成，并有助于为发展AP的长期目标提供框架。 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。

SUMMARY The vitamin A metabolite, all-trans-retinoic acid (ATRA), plays essential roles in a multitude of biological pro- cesses 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 develop- ment. 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 AT- RA 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 retinalde- hyde. 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 de- velopment. 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 me- tabolism based on the working hypothesis that DHRS3 is the main enzyme responsible for reducing retinalde- hyde during embryogenesis. Aim 2 will be pursued by establishing the effect of Dhrs3-ablation on the metabo- lism 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 hypoth- esis that the developmental defects caused by ablation of Dhrs3 are a result of altered ATRA metabolism. Pre- liminary studies indicate that Dhrs3-/- embryos have defects in anterior-posterior patterning, cardiac and skele- tal 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 ap- proaches to prevent and treat congenital and inherited disorders associated with altered ATRA-signaling.