Mechanisms of regulation of retinoic acid homeostasis

描述（申请人提供）：全反式维甲酸（atRA）是膳食维生素A的生物活性代谢产物，对介导肝脏、肾脏、肺和胰腺等多种组织的多种生物功能至关重要。atRA的不同生物学作用是通过atRA的组织浓度梯度来调节的，但是我们对atRA在儿童和成人生活期间如何调节组织特异性信号传导的知识存在相当大的差距。我们提出，atRA浓度梯度产生的调节表达和活性的酶合成atRA（ALDH 1As），代谢atRA（CYP 26）和细胞视黄酸结合蛋白（CRABP）。我们的中心假设是， 这些酶活性或表达改变atRA信号传导和分布，促进特定组织中的疾病发展并导致副作用。为了验证这一假设，我们将首先描述atRA在细胞系统中的代谢，并建立细胞维甲酸代谢的作用。 酸结合蛋白（CRABP）在调节atRA清除、信号传导和分布中的作用。我们将使用基本的生物化学和酶动力学方法和体外细胞实验来建立CYP 26和CRABP之间的直接蛋白质-蛋白质相互作用的作用和动力学。然后，我们将建立组织和细胞类型的ALDH 1A，CYP 26和CRABP酶在维持人体和小鼠的atRA稳态的具体作用。这将使用正常人和小鼠组织、新型高灵敏度质谱法、产生新的CYP 26酶条件性敲除小鼠、检测CYP 26和ALDH 1A抑制剂的药理学作用以及使用基于生理学的药代动力学（PBPK）建模来完成。为了确定这些酶促过程的总体生理重要性，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，以证明改变的CYP 26或ALDH 1A活性损害靶组织中的正常生理atRA信号传导。由于现有的知识，atRA信号在这些组织中起着重要的作用，我们将集中在肝，肾，胰腺和肺中的atRA信号。这些研究将产生一个新的和独特的类维生素A稳态的综合模型，这将是有用的评估和预测外源性物质，新的治疗方法，疾病过程和遗传因素在改变组织类维生素A信号的影响。这将产生重大影响， 改善人类健康，因为它直接应用于改善我们对肝脏和胰腺中脂质和葡萄糖稳态、肾病的发展和治疗以及肺泡健康所涉及的过程的理解。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，并可以扩展到提高我们对atRA在皮肤病（如银屑病和鱼鳞病）和神经退行性疾病（如阿尔茨海默病和痴呆症）中的作用的理解，其中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.