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，以设计新的策略来防止损伤和致癌。在这项提案中，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。我们的研究集中在黑素细胞及其细胞表面受体，黑皮质素-1受体（Mc 1 r），它调节黑色素的合成。我们假设，Mc 1 r保护黑素细胞免受紫外线介导的诱变和转化，通过调节黑化和恢复紫外线介导的细胞损伤。Mc 1 r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，而强大的Mc 1 r功能与深色皮肤和抗紫外线相关。我们已经开发了一种新的人类皮肤基因匹配的小鼠模型，以及从这些小鼠中生长原代黑素细胞的方案。这些同基因的黑素细胞跨越真黑素，pheomelanotic，和amelanotic黑色素的组成，将成为一个独特的和有效的工具来描绘的作用，Mc 1 r功能响应UV。使用我们独特的动物模型和其衍生的原代黑素细胞，我们提出以下目的：目的1。表征Mc 1 r介导的核苷酸切除修复（NER）增强的机制;目的2。确定Mc 1 r信号传导是否保护免受紫外线介导的氧化损伤;目的3.确定药理学Mc 1 r拯救保护免受UV损伤的能力。相对于目标1，我们将检查的作用，Mc 1 r的紫外线诱导的光损伤的修复西南和流式细胞仪分析，并确定的影响Mc 1 r的核苷酸切除修复酶的细胞水平的qRT-PCR和Western分析。我们将研究Mc 1 r信号传导和增强修复酶水平之间的分子联系，通过检查NER酶的表达基础上，并在紫外线照射的设置，并通过研究cAMP反应转录因子下游的Mc 1 r信号传导（即Mitf和CREB）的能力，结合并诱导NER酶启动子的转录。在目标2中，我们将研究褐黑素，这是由于低Mc 1 r功能，促进氧化损伤的黑素细胞的氧化负荷的补充措施（DCF介导的荧光流式细胞术，西南印迹，hOGG 1适应彗星试验，TBARS试验，并通过GC/MS直接定量的氧化DNA加合物）。重要的是，我们将直接测试 褐黑素通过基于HPRT的正向诱变方法在原代黑素细胞中作为促癌物质发挥作用。最后，我们将直接测试佛司可林的能力，腺苷酸环化酶的激活剂，绕过缺陷Mc 1 r功能，以提高从紫外线介导的DNA损伤的恢复，并拯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化研究奠定基础，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.