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

描述(申请人提供)：我们寻求了解皮肤暴露在紫外线辐射下时发生的分子反应，以设计新的策略来防止损害和致癌。在这项建议中，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。我们的研究集中在黑素细胞及其细胞表面受体，黑素皮质素-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突变和转化。MC1R功能缺失与白皙皮肤和高黑色素瘤发病率相关，而强健的MC1R功能与深色皮肤和紫外线抵抗力相关。我们开发了一种新的基因匹配的人类皮肤小鼠模型，以及从这些小鼠培养原代黑素细胞的方案。这些等基因黑素细胞包括真黑色素、黑素性黑素和无色素性黑素，将成为一种独特而有效的工具来描述MC1R功能在紫外线反应中的作用。利用我们独特的动物模型及其衍生的原代黑素细胞，我们提出了以下目标：目的1.确定MC1R介导的核苷酸切除修复(NER)增强的机制；目的2.确定MC1R信号是否对紫外线介导的氧化损伤具有保护作用；以及目的3.确定药理学MC1R拯救对紫外线损伤的保护能力。对于目标1，我们将通过西南和流式细胞仪分析来检测MC1R在紫外线诱导的光渗漏修复中的作用，并通过qRT-PCR和Western分析来确定MC1R对细胞核苷酸切除修复酶水平的影响。我们将通过检测NER酶在基础和紫外线照射下的表达，以及MC1R信号下游的cAMP反应转录因子(即MITF和CREB)与NER酶启动子结合和诱导转录的能力，来研究MC1R信号和增强修复酶水平之间的分子联系。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措施(DCF介导的荧光流式细胞术、Southwest blotting、hOGG1适应的彗星试验、TBARS试验和GC/MS直接定量氧化DNA加合物)来促进黑素细胞的氧化损伤。重要的是，我们将直接测试 在原代黑素细胞中，通过基于HPRT的正向诱变方法，褐黑素起到了致癌作用。最后，我们将直接测试腺酰环化酶激活剂Forsklin绕过MC1R功能缺陷的能力，以促进从紫外线介导的DNA损伤中恢复，并挽救黑素细胞和整个皮肤的紫外线保护。总之，这些研究将为未来的转译研究奠定基础，旨在开发新的基于小分子的紫外线和癌症保护方法，以预防许多黑色素瘤病例。

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.