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: 8655736
• 负责人: John A D'Orazio
• 金额: $ 5.23万
• 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/8655736
• 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; 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.

描述（由申请人提供）：我们试图了解皮肤暴露于紫外线辐射时发生的分子反应，以设计新型策略以防止损害和癌变。在此提案中，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。我们的研究集中于黑素细胞及其细胞表面受体，黑色素皮质素1受体（MC1R），该受体调节黑色素合成。我们假设MC1R通过调节紫外线化和从紫外线介导的细胞损伤中恢复紫外线介导的诱变和转化来保护黑素细胞。 MC1R的功能丧失多态性与皮肤白皙和黑色素瘤的高发病率相关，而强大的MC1R功能与较深的皮肤和抗UV耐药性相关。我们已经开发了一种新型的人类皮肤的基因匹配鼠模型，以及一种从这些小鼠生长的原发性黑素细胞的方案。这些异源性黑色素细胞涵盖了Eumelanatotic，Pheomelanotic和Omelanotic黑色素组成，将提供独特而有效的工具，以描绘MC1R功能在响应UV中的作用。使用我们衍生出的独特动物模型和原代黑素细胞，我们提出了以下目的：目标1。表征MC1R介导的核苷酸切除修复（NER）的机理； AIM 2。确定MC1R信号传导是否可以防止UV介导的氧化损伤；和目标3。确定药物MC1R救援防止紫外线损伤的能力。对于目标1，我们将通过西南和流式细胞仪分析来检查MC1R在修复紫外线诱导的光子中的作用，并确定MC1R对通过QRT-PCR和Western Anallyaws核苷酸切除修复酶的细胞水平的影响。我们将通过基本和在紫外线照射的情况下检查NER酶的表达，并研究MC1R信号（即MITF和CREB）与Nererzeme Enerzeme prosercription结合并诱导CAMP响应转录的能力，从而研究MC1R信号传导与增强修复酶水平之间的分子联系。在AIM 2中，我们将研究由于较低的MC1R功能而产生的苯烷蛋白是否通过氧化负荷的互补度量（DCF介导的荧光流式细胞仪，Southwestern Plotting，HogG1适应性尺寸的Comet Comet Assay，Tbars Assay和Directights of MSEDIFEDS）促进黑素细胞的氧化损伤。重要的是，我们将直接测试是否 原素黑素化学方法中基于HPRT的正向诱变方法，苯烷蛋白充当促癌动物。最后，我们将直接测试腺苷酸环化酶激活剂Forskolin的能力，绕过有缺陷的MC1R功能，以增强紫外线介导的DNA损伤的恢复并在黑素细胞和整个皮肤中挽救紫外线保护。这些研究将共同​​为未来的翻译研究奠定基础，旨在开发新型的基于小分子的紫外线和癌症保护方法，以防止许多黑色素瘤病例。