Autophagy in epidermal melanocyte: a protective or a destructive role?

描述(申请人提供)：皮肤中的黑色素保护表皮和底层真皮免受阳光下紫外线辐射的有害影响。因此，黑素细胞的动态平衡对于一个有功能的表皮黑色素单位和健康的皮肤是必不可少的。黑素细胞动态平衡的破坏会导致皮肤疾病。最值得注意的是，由于黑素细胞死亡而导致的黑素细胞丢失会导致退行性白癜风和伴随的皮肤光敏反应，而黑素细胞由于不受控制的增殖而增加，就像皮肤黑色素瘤一样，最终可能导致死亡。在正常和/或恶性黑素细胞死亡后暴露细胞内蛋白导致白癜风和黑色素瘤对几种黑素小体自身抗原的耐受性和免疫反应的破坏。因此，了解调节黑素细胞稳态的机制，特别是黑素细胞死亡，对于制定预防和治疗黑素细胞疾病的策略是重要的。虽然黑素细胞因坏死和凋亡导致的死亡已经得到了很好的研究，但对黑素细胞自噬死亡的机制以及自噬在恶性黑素细胞行为中的作用还不是很清楚。例如，尚不清楚a)皮肤黑色素瘤的易感性是否与黑素细胞致癌转化时激活或抑制自噬的能力有关，b)深色皮肤和浅色皮肤的黑素细胞之间自噬和自噬细胞死亡的激活存在差异，c)自噬激活的差异可以解释黑素细胞对恶性转化的抵抗力。自噬通常是细胞在应激状态下的生存机制，受哺乳动物雷帕霉素靶点(MTOR)信号通路的调控，mTOR信号通路对营养缺乏和其他细胞生存信号做出反应。雷帕霉素抑制mTOR信号转导诱导自噬，这种自噬可以通过形态学(光镜免疫荧光和电子显微镜)和生化方法进行监测。在我们的初步研究中，我们发现欧洲和非洲血统的黑素细胞对mTOR表现出不同的敏感性，癌基因BRAF的过度激活可以诱导恶性黑素细胞的自噬细胞死亡。我们假设自噬在表皮黑色素单位的动态平衡中起作用。在这里，我们建议解决以下问题：自噬细胞死亡是维持表皮黑色素单位的正常动态平衡机制吗？角质形成细胞在黑素细胞对自噬的敏感性中扮演什么角色？ATG5和ATG7在黑素细胞对mTOR抑制的敏感性中起什么作用？这里提出的实验有望产生新的科学知识，可能导致更好地了解黑素细胞疾病的发病机制，并最终可能导致更有效的治疗。

DESCRIPTION (provided by applicant): Melanin in the skin protects the epidermis and the underlying dermis from the deleterious effects of ultraviolet radiation in the sun light. Therefore melanocyte homeostasis is essential for a functional epidermal melanin unit and healthy skin. Disruption of melanocyte homeostasis leads to skin disorders. Most notably, loss due to death of melanocyte causes depigmentary disorder vitiligo and accompanying skin photosensitivity, and gain of melanocytes due to unregulated proliferation as occurs in cutaneous melanoma could eventually lead to death. Exposure of intracellular proteins following death of normal and/or malignant melanocytes leads to break in tolerance and immune response to several melanosomal self antigens in vitiligo and melanoma. Therefore, understanding the mechanisms that regulate melanocyte homeostasis, specifically melanocyte death, is important for developing strategies to prevent and treat melanocytic disorders. While melanocyte death due to necrosis and apoptosis has been well investigated, mechanisms of autophagic death of melanocytes and the role of autophagy in the behavior of malignant melanocytes are not well understood. For example, it is not known whether a) susceptibility to develop cutaneous melanoma is related to ability to activate or inhibit autophagy upon oncogenic transformation of melanocytes, b) differences exist in activation of autophagy and autophagic cell death between melanocytes in darker and lighter skin individuals, and c) differences in activation of autophagy can account for susceptibility of resistance of melanocytes to malignant transformation. Autophagy, which normally serves as cell survival mechanism when cells are under stress, is regulated by mammalian Target of Rapamycin (mTOR) signaling pathway that responds to nutrient deprivation and other cell survival signals. Inhibition of mTOR signaling by rapamycin induces autophagy, which can be monitored by morphological (light microscopic immunofluorescence and electron microscopy) and biochemical methods. In our preliminary studies, we found that melanocytes from European and African ancestry show differential sensitivity to mTOR and autophagic cell death can be induced in malignant melanocytes by hyperactivation of oncogene BRAF. We hypothesized that autophagy plays a role in homeostasis of epidermal melanin unit. Here, we propose to address the following questions: Is autophagic cell death a normal homeostatic mechanism that maintains epidermal melanin unit? What role do keratinocytes play in sensitivity of melanocytes to autophagy? What is the role of Atg5 and Atg7 in sensitivity of melanocytes to mTOR inhibition? Experiments proposed here are expected to generate new scientific knowledge that could lead to better understanding of the pathogenesis of melanocytic disorders and may eventually lead to more effective treatments.