The role of Mc1r in melanocytic UV-induced DNA damage and repair responses

Mc1r 在黑素细胞紫外线诱导的 DNA 损伤和修复反应中的作用

• 批准号: 8079709
• 负责人: John A D'Orazio
• 金额: $ 29.62万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079709
• 关键词: Address; Adenylate Cyclase; Animal Model; Automobile Driving; Binding; Biological Assay; Bypass; CREB1 gene; Carcinogens; Cell Line; Cell Surface Receptors; Cells; Comet Assay; Cyclic AMP; DNA; DNA Adducts; DNA Damage; DNA Repair; Defect; Exposure to; Flow Cytometry; Fluorescence; Forskolin; Foundations; Future; Genes; Genetic Polymorphism; Genetic Transcription; Goals; Human; In Vitro; Incidence; Individual; Injury; Link; Malignant - descriptor; Malignant Neoplasms; Measures; Mediating; Melanins; Melanocortin 1 Receptor; Melanocyte stimulating hormone; Methods; Modeling; Molecular; Monophenol Monooxygenase; Mus; Mutagenesis; Nucleotide Excision Repair; Phenotype; Pigmentation physiologic function; Pigments; Primary Cell Cultures; Protocols documentation; Receptor Signaling; Recovery; Research Design; Resistance; Risk Factors; Role; Skin; Skin Cancer; Skin Pigmentation; Southwestern Blotting; Sun Exposure; Surface; System; Testing; The Sun; UV induced; UV induced DNA damage; UV protection; UV response; Ultraviolet B Radiation; Ultraviolet Rays; Variant; alpha-Melanocyte stimulating hormone; base; carcinogenesis; cell injury; congenic; defined contribution; eumelanin; high risk; loss of function; melanocyte; melanoma; novel; novel strategies; oxidative damage; pheomelanin; photolesion; prevent; promoter; public health relevance; receptor; receptor binding; receptor function; repair enzyme; repaired; research study; response; small molecule; synthetic enzyme; tool; transcription factor; translational study; ultraviolet damage; ultraviolet irradiation

DESCRIPTION (provided by applicant): We seek to understand the molecular responses that occur when skin is exposed to UV radiation in order to devise novel strategies to protect against damage and carcinogenesis. In this proposal we will examine two major risk factors for skin cancer: UV radiation and pigment (melanin) phenotype. Our studies focus on the melanocyte, and its cell surface receptor, the melanocortin-1 receptor (Mc1r), which regulates melanin synthesis. We hypothesize that Mc1r protects melanocytes against UV-mediated mutagenesis and transformation by modulating melanization and recovery from UV-mediated cellular injury. Loss-of-function polymorphisms in Mc1r correlate with fair skin and a high incidence of melanoma, while robust Mc1r function correlates with darker skin and UV resistance. We have developed a novel genetically-matched murine model of human skin as well as a protocol for growing primary melanocytes from these mice. These isogenic melanocytes span eumelanotic, pheomelanotic, and amelanotic melanin composition, will serve a unique and potent tool to delineate the role of Mc1r function in response to UV. Using our unique animal model and primary melanocytes derived thereof, we propose the following aims: Aim 1. Characterize the mechanism of Mc1r-mediated enhancement of nucleotide excision repair (NER); Aim 2. Determine whether Mc1r signaling protects against UV-mediated oxidative damage; and Aim 3. Determine the ability of pharmacologic Mc1r rescue to protect against UV damage. With respect to Aim 1, we will examine the role of Mc1r in the repair of UV-induced photolesions by southwestern and flow cytometric analysis and determine the influence of Mc1r on cellular levels of nucleotide excision repair enzymes by qRT-PCR and Western analysis. We will investigate a molecular link between Mc1r signaling and enhanced repair enzyme levels by examining expression of the NER enzymes basally and in the setting of UV irradiation and by investigating the ability of cAMP-responsive transcription factors downstream of Mc1r signaling (namely Mitf and CREB) to bind to and induce transcription of NER enzyme promoters. In Aim 2 we will study whether pheomelanin, which is produced as a result of low Mc1r function, promotes oxidative damage in melanocytes by complementary measures of oxidative load (DCF-mediated fluorescence flow cytometry, Southwestern blotting, hOGG1-adapted Comet assay, TBARS assay, and direct quantification of oxidative DNA adducts by GC/MS). Importantly, we will directly test whether pheomelanin functions as a pro-carcinogen by an HPRT-based forward mutagenesis approach in primary melanocytes. Finally, we will directly test the ability of forskolin, an activator of adenylyl cyclase, to bypass defective Mc1r function to enhance recovery from UV-mediated DNA damage and to rescue UV protection in melanocytes and in whole skin. Together, these studies will lay the foundation for future translational studies designed to develop novel small molecule-based approaches for UV and cancer protection to prevent many cases of melanoma. PUBLIC HEALTH RELEVANCE: Though it has long been known that people with functional defects of the melanocortin 1 receptor (Mc1r) protein are at high risk of developing melanoma, the way(s) in which Mc1r protects against melanoma are not understood. Using pigment cells known as melanocytes, we will study how the Mc1r protein influences UV- mediated DNA and cellular damage in melanocytes, and contributes to melanoma resistance. Our goal is to develop the strategy of pharmacologic Mc1r rescue toward translational applicability so that many cases of melanoma can be prevented.

描述（由申请人提供）：我们寻求了解当皮肤暴露于紫外线辐射时发生的分子反应，以便设计新的策略来防止损伤和致癌。在本提案中，我们将研究皮肤癌的两个主要危险因素：紫外线辐射和色素（黑色素）表型。我们的研究重点是黑素细胞及其细胞表面受体黑皮质素-1 受体 (Mc1r)，它调节黑色素的合成。我们假设 Mc1r 通过调节黑化和从紫外线介导的细胞损伤中恢复来保护黑素细胞免受紫外线介导的诱变和转化。 Mc1r 功能缺失的多态性与白皙皮肤和黑色素瘤的高发病率相关，而强健的 Mc1r 功能则与深色皮肤和抗紫外线能力相关。我们开发了一种新型的基因匹配的人类皮肤小鼠模型，以及从这些小鼠中培养原代黑素细胞的方案。这些同基因黑素细胞涵盖真黑素、褐黑素和无黑素黑色素组成，将成为描述 Mc1r 功能对紫外线反应的作用的独特而有效的工具。利用我们独特的动物模型及其衍生的原代黑素细胞，我们提出以下目标： 目标 1. 表征 Mc1r 介导的核苷酸切除修复 (NER) 增强的机制；目标 2. 确定 Mc1r 信号传导是否可以防止紫外线介导的氧化损伤；目标 3. 确定 Mc1r 药物救援防止紫外线损伤的能力。关于目标 1，我们将通过西南和流式细胞术分析检查 Mc1r 在修复紫外线诱导的光损伤中的作用，并通过 qRT-PCR 和 Western 分析确定 Mc1r 对核苷酸切除修复酶细胞水平的影响。我们将通过在紫外线照射下检查 NER 酶的基础表达以及研究 Mc1r 信号下游 cAMP 响应转录因子（即 Mitf 和 CREB）的能力，研究 Mc1r 信号传导与增强的修复酶水平之间的分子联系。结合并诱导 NER 酶启动子的转录。在目标 2 中，我们将研究由于 Mc1r 功能低下而产生的褐黑素是否通过氧化负荷的补充测量（DCF 介导的荧光流式细胞术、Southwestern 印迹、hOGG1 适应彗星测定、TBARS 测定）促进黑素细胞的氧化损伤，以及通过 GC/MS 直接定量氧化 DNA 加合物）。重要的是，我们将直接测试是否 通过基于 HPRT 的正向诱变方法，褐黑素在原代黑素细胞中发挥前致癌物的作用。最后，我们将直接测试毛喉素（一种腺苷酸环化酶激活剂）绕过有缺陷的 Mc1r 功能的能力，以增强紫外线介导的 DNA 损伤的恢复，并挽救黑素细胞和整个皮肤的紫外线防护。这些研究将为未来的转化研究奠定基础，这些研究旨在开发基于小分子的新型紫外线和癌症防护方法，以预防许多黑色素瘤病例。 公众健康相关性：尽管人们早已知道黑皮质素 1 受体 (Mc1r) 蛋白功能缺陷的人患黑色素瘤的风险很高，但 Mc1r 预防黑色素瘤的方式尚不清楚。使用称为黑素细胞的色素细胞，我们将研究 Mc1r 蛋白如何影响黑素细胞中紫外线介导的 DNA 和细胞损伤，并有助于抵抗黑素瘤。我们的目标是开发 Mc1r 药物拯救策略以实现转化应用，从而预防许多黑色素瘤病例。