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

描述（由申请人提供）：皮肤中的黑色素保护表皮和下层真皮层免受太阳光线中紫外线辐射的有害影响。因此，黑素细胞的稳态对表皮黑色素单位的功能和皮肤的健康至关重要。黑素细胞稳态的破坏导致皮肤疾病。最值得注意的是，由于黑素细胞死亡而导致的损失会导致色素沉着障碍白癜风和伴随的皮肤光敏性，而由于皮肤黑色素瘤中发生的不受调节的增殖而导致的黑素细胞的增加最终可能导致死亡。在白癜风和黑色素瘤中，正常和/或恶性黑色素细胞死亡后暴露于细胞内蛋白导致对几种黑色素体自身抗原的耐受性和免疫反应中断。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非常重要。虽然黑素细胞坏死和凋亡导致的死亡已经得到了很好的研究，但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尚不清楚。例如，尚不清楚a)发生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)肤色较深和较浅的黑素细胞在自噬激活和自噬细胞死亡方面存在差异，以及c)自噬激活的差异可以解释黑素细胞对恶性转化的抗性易感性。自噬是哺乳动物Rapamycin靶蛋白（Target of Rapamycin, mTOR）信号通路的调控机制，它响应营养剥夺等细胞生存信号，通常是细胞在应激状态下的生存机制。雷帕霉素抑制mTOR信号传导可诱导自噬，可通过形态学（光镜免疫荧光和电镜）和生化方法进行监测。在我们的初步研究中，我们发现来自欧洲和非洲血统的黑素细胞对mTOR表现出不同的敏感性，恶性黑素细胞可以通过癌基因BRAF的过度激活诱导自噬细胞死亡。我们假设自噬在表皮黑色素单位的稳态中起作用。在此，我们提出以下问题：细胞自噬死亡是维持表皮黑色素单位的正常稳态机制吗？角化细胞在黑色素细胞自噬的敏感性中起什么作用？Atg5和Atg7在黑色素细胞对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，从而更好地了解黑素细胞疾病的发病机制，并可能最终导致更有效的治疗。

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. PUBLIC HEALTH RELEVANCE: Pigment producing cells in the skin are maintained in equilibrium with respect to the surrounding keratinocytes to which they export melanin pigment that provides protection from damaging ultraviolet radiation in the sun light. However, if melanocytes undergo unscheduled cell death or fail to die when damaged, it can lead to skin diseases such as vitiligo and skin cancer melanoma. The aim of this proposal to investigate melanocyte cell death mechanism that scontribute to melanocytic disorders.