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

描述（由申请人提供）：我们试图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，即调节黑色素合成的黑素皮质素-1受体（Mc1r）。我们假设Mc1r通过调节黑色素化和从紫外线介导的细胞损伤中恢复来保护黑色素细胞免受紫外线介导的突变和转化。Mc1r的功能缺失多态性与白皙皮肤和黑色素瘤的高发病率相关，而Mc1r功能强大与较黑的皮肤和抗紫外线能力相关。我们已经开发了一种新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色素细胞的方案。这些等基因黑色素细胞跨越真黑、异黑和无黑黑色素组成，将成为描述Mc1r功能在紫外线响应中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色素细胞，我们提出以下目标：鉴定mc1r介导的核苷酸切除修复（NER）增强机制；目标2。确定Mc1r信号是否对紫外线介导的氧化损伤有保护作用；和Aim 3。确定药物Mc1r对紫外线损伤的保护能力。关于Aim 1，我们将通过西南和流式细胞分析研究Mc1r在紫外线诱导的光损伤修复中的作用，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。我们将研究Mc1r信号转导和修复酶水平增强之间的分子联系，通过检测基本和紫外线照射下NER酶的表达，以及通过研究Mc1r信号转导下游camp响应转录因子（即Mitf和CREB）结合和诱导NER酶启动子转录的能力。在Aim 2中，我们将通过氧化负荷的补充措施（dcf介导的荧光流式细胞术、西南印迹法、hogg1适应性Comet试验、TBARS试验和GC/MS直接定量氧化DNA加合物），研究由Mc1r功能低下而产生的现象黑色素是否会促进黑素细胞的氧化损伤。重要的是，我们将直接测试是否

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.