Role and regulation of cytochrome P450 27C1 in human skin

摘要 调节角质形成细胞的增殖和分化对维持皮肤功能非常重要。维甲酸， 维甲酸的活性形式，通过诱导或抑制数百个 基因。维甲酸活性是通过与维甲酸受体结合而介导的，维甲酸受体结合基因内的区域 推动者。由于其活性，类维甲酸已被用于治疗各种皮肤病。 包括粉刺、牛皮癣、鱼鳞病、角化皮肤病和皮肤癌，并用于治疗以减少 光老化效应。临床处方的维甲酸是用来调节内源性代谢的。 维甲酸或直接激活维甲酸受体。正因为如此，了解这两个活动非常重要 和内源性维甲酸的代谢。目前，这些信息中的大部分都是3，4- 脱氢类维甲酸，约占整个表皮类维A酸池的25%。3，4-脱氢视黄醇是一种 角质形成细胞中产生的全反式视黄醇的代谢物，我们的实验室最近才发现 细胞色素P450(CYP)27c1是负责该反应的去饱和酶。3，4-脱氢视黄醇可以进一步 代谢形成一种活性的3，4-脱氢维甲酸，与维甲酸受体结合，但很少有研究 研究了脱氢维甲酸特异的基因调控。此外，3，4-的浓度增加 皮肤中的脱氢视黄醇与皮肤疾病有关，包括牛皮癣，鳞状细胞癌， 和角化棘皮瘤。总体而言，这项研究的目标是更好地了解监管和 脱氢维甲酸通过表征Cyp27c1的功能。我们的实验室最近确定人类 Cyp27c1优先在皮肤中表达，并催化全反式维甲酸的去饱和形成3，4- 脱氢维甲酸在体外存在线粒体P450氧化还原伙伴。Cyp27c1的许多方面 催化机制已被充分了解，但关于这种酶的生物学功能仍然存在问题。 我推测线粒体Cyp27c1通过以下途径调节角质形成细胞分化和增殖 催化3，4-脱氢维甲酸的形成。生化、分析和分子生物学的结合体 将使用包括原代细胞培养、蛋白质组学、RNA测序和免疫分析在内的工具来解决 我的假设。我提出了三个目标：1)Cyp27c1在皮肤中的定位和绝对定量；2) 研究紫外光和维甲酸对Cyp27c1表达的影响，以及3)生理评价 Cyp27c1和脱氢维甲酸在皮肤中的作用。原代人表皮中Cyp27c1的研究 在这项拟议的工作中，角质形成细胞将增加生理性维甲酸动态平衡和 可能有助于皮肤病新疗法的开发。

ABSTRACT Regulation of keratinocyte proliferation and differentiation is important to maintain skin function. Retinoic acids, the active form of retinoids, play a role in this regulation by inducing or repressing the expression of hundreds of genes. Retinoic acid activity is mediated by binding to retinoic acid receptors that bind to regions within gene promoters. Because of their activity, retinoids have been used in the treatment of a variety of skin disorders including acne, psoriasis, ichthyoses, keratodermas, and skin cancers, and are utilized in treatments to reduce photoaging effects. Clinically prescribed retinoids are developed to modulate the metabolism of endogenous retinoids or to directly activate retinoid receptors. Because of this, it is important to understand both the activity and metabolism of endogenous retinoids. Currently, much of this information is not known for 3,4- dehydroretinoids, which make up approximately 25% of the total epidermal retinoid pool. 3,4-Dehydroretinol is a metabolite of all-trans retinol produced in keratinocytes, and our laboratory has only recently identified cytochrome P450 (CYP) 27C1 as the desaturase responsible for this reaction. 3,4-Dehydroretinol can be further metabolized to form an active 3,4-dehydroretinoic acid that binds to retinoic acid receptors, but few studies have investigated dehydroretinoid-specific gene regulation. Additionally, increased concentrations of 3,4- dehydroretinol in the skin have been associated with skin diseases including psoriasis, squamous cell carcinoma, and keratoacanthoma. Overall, the goal of this study is to provide a better understanding of the regulation and function of dehydroretinoids through characterizing CYP27C1. Our lab recently determined that human CYP27C1 is preferentially expressed in the skin and catalyzes the desaturation of all-trans retinoids to form 3,4- dehydroretinoids in vitro in the presence of mitochondrial P450 redox partners. Many aspects of the CYP27C1 catalytic mechanism are well understood, but questions remain regarding the biological function of this enzyme. I hypothesize that mitochondrial CYP27C1 regulates keratinocyte differentiation and proliferation through catalyzing the formation of 3,4-dehydroretinoids. A combination of biochemical, analytical, and molecular biology tools including primary cell culture, proteomics, RNA-sequencing, and immunoassays will be used to address my hypothesis. I propose three aims: 1) localization and absolute quantification of CYP27C1 within the skin, 2) investigate UV light and retinoids as effectors of CYP27C1 expression, and 3) evaluation of the physiological function of CYP27C1 and dehydroretinoids in the skin. Studying CYP27C1 in primary human epidermal keratinocytes in this proposed work will add to the knowledge-base of physiological retinoid homeostasis and may aid in the development of new therapies for dermatological diseases.