Mechanisms of regulation of retinoic acid homeostasis

描述(申请人提供)：全反式维甲酸(AtRA)是饮食中维生素A的生物活性代谢物，对调节肝、肾、肺和胰腺等多种组织的多种生物学功能是必不可少的。AtRA的不同生物学作用是通过atRA的组织浓度梯度来调节的，但我们对atRA的组织特异性信号在儿童和成人生活中是如何调控的认识还存在相当大的差距。我们认为全反式维甲酸浓度梯度是由合成全反式维甲酸的酶(ALDH1As)、代谢全反式维甲酸的酶(CYP26s)和细胞维甲酸结合蛋白(CRABPs)的调节表达和活性产生的。我们的中心假设是 这些酶的活性或表达改变了RA的信号和分布，促进了特定组织的疾病发展，并导致了不良反应。为了验证这一假说，我们将首先描述全反式维甲酸在细胞系统中的代谢，并确定细胞维甲酸的作用。 酸结合蛋白(CRABPs)在调节atRA清除、信号和分布中的作用。我们将使用基本的生化和酶动力学方法以及体外细胞实验来确定CYP26和CRABPs之间直接的蛋白质-蛋白质相互作用的作用和动力学。然后，我们将确定ALDH1A、CYP26和CRABP酶在维持全反式维甲酸在人类和小鼠体内稳定中的组织和细胞类型特异性作用。这将使用正常的人和鼠组织，新的高灵敏度质谱学方法，产生新的条件敲除的CYP26酶的小鼠，测试CYP26和ALDH1A抑制剂的药理作用，并使用基于生理的药代动力学(PBPK)建模。为了确定这些酶过程的整体生理重要性，我们将使用基因敲除小鼠和我们的体外和体内药理学工具，证明改变的CYP26或ALDH1A活性会损害靶组织中正常的生理性atRA信号。我们将重点关注atRA信号在肝、肾、胰腺和肺中的作用，因为现有的知识表明atRA信号在这些组织中起着基础性的作用。这些研究将产生一个新的和独特的维甲酸动态平衡的综合模型，该模型将有助于评估和预测外源物质、新的治疗方法、疾病过程和遗传因素在改变组织维甲酸信号方面的作用。这将在以下方面产生重大影响 改善人类健康，因为它直接应用于提高我们对肝脏和胰腺中涉及的脂肪和葡萄糖动态平衡的过程的了解，在肾病的发展和治疗以及肺泡健康方面。此外，通过这些研究获得的知识将提高我们对atRA信号在儿童发育和成熟过程中的作用的理解，并可以扩展到更好地理解atRA在皮肤疾病(如牛皮癣和鱼鳞病)以及神经退行性疾病(如阿尔茨海默病和痴呆症)中的作用，在这些疾病中，atRA信号已被证明是改变的。

DESCRIPTION (provided by applicant): All-trans-retinoic acid (atRA), the biologically active metabolite of dietary Vitamin A, is essential for mediating diverse biological functions in multipl tissues such as the liver, kidney, lung and pancreas. The different biological actions of atRA are regulated through tissue concentration gradients of atRA, but there are considerable gaps in our knowledge on how the tissue specific signaling of atRA is regulated during childhood and adult life. We propose that atRA concentration gradients are generated by regulated expression and activity of the enzymes synthesizing atRA (ALDH1As), those that metabolize atRA (CYP26s) and cellular retinoic acid binding proteins (CRABPs). Our central hypothesis is that alterations in the activity or expression of these enzymes change atRA signaling and distribution, contribute to disease development in specific tissues and result in adverse effects. To test this hypothesis we will first characterize atRA metabolism in cell systems and establish the role of cellular retinoic acid binding proteins (CRABPs) in modulating atRA clearance, signaling and distribution. We will use basic biochemical and enzyme kinetic methods and in vitro cell experiments to establish the role and kinetics of direct protein-protein interactions between CYP26s and CRABPs. We will then establish the tissue and cell type specific roles of ALDH1A, CYP26 and CRABP enzymes in maintaining atRA homeostasis in humans and mice. This will be done using normal human and mouse tissues, novel high sensitivity mass spectrometry methods, generating new conditional knock-out mice of CYP26 enzymes, testing pharmacological effects of CYP26 and ALDH1A inhibitors and using physiologically based pharmacokinetic (PBPK) modeling. To determine the overall physiological importance of these enzymatic processes, we will use the knock-out mice and our pharmacological tools in vitro and in vivo, to demonstrate that altered CYP26 or ALDH1A activity impairs normal physiological atRA signaling in target tissues. We will focus on atRA signaling in the liver, kidney, pancreas and lung due to the existing knowledge that atRA signaling plays a fundamental role in these tissues. Together these studies will generate a new and unique integrative model of retinoid homeostasis that will be useful in evaluating and predicting the effects of xenobiotics, new therapeutic approaches, disease processes and genetic factors in altering tissue retinoid signaling. This will have major impact in improving human health as it has direct application in improving our understanding of the processes involved in lipid and glucose homeostasis in the liver and the pancreas, in development and treatment of nephropathies and in lung alveoli health. In addition, the knowledge gained through these studies will improve our understanding of the role of atRA signaling during childhood development and maturation, and can be extended to improve our understanding of the role of atRA in skin diseases such as psoriasis and ichthyosis and in neurodegenerative diseases such as Alzheimer's and dementia in which atRA signaling has been shown to be altered.