Elucidating melatonin-regulated mechanisms of melanogenesis in human full-thickness skin and human melanocytes exposed to ultraviolet radiation

临床上与皮肤色素沉着相关的疾病包括色素沉着过少或过多，因此非常需要活性和安全的色素调节剂。基于迄今为止所描述的褪黑素的高生物活性，它可能是一种有效控制黑素生成机制的有效物质。褪黑素(N-乙酰-5-甲氧基色胺)是脊椎动物松果体的一种分泌产物，调节多种功能，包括清除自由基、DNA修复或抗细胞凋亡活性。它还与皮肤功能有关，如表皮增殖/分化、头发生长和黑色素瘤/鳞状细胞癌的控制。在这项研究应用中，有人建议评估褪黑素对黑素生成调节机制的潜在影响。到目前为止，申请人在褪黑素领域获得的广泛经验使他能够进行本提案中的所有实验。计划中的研究将在人类体外全层皮肤(第一部分)中进行，第二部分额外使用纯培养的正常人黑素细胞(NHM)，与与反映活体皮肤生理的正常人角质形成细胞(NHEK)共培养进行比较。此外，这种方法可以描述角质形成细胞与黑素细胞的相互作用以及角质形成细胞依赖的黑素生成调节的关键作用。在皮肤方面，褪黑素的作用和作用将从UVR介导的黑素合成方面进行评估，包括褪黑素对酪氨酸酶(TYR)的影响，酪氨酸酶(TYR)是黑素生成的关键酶之一，控制小眼球相关转录因子(MITF)的表达，以及褪黑素对黑色素合成和cAMP-PKA-MITF轴的影响。此外，将对前面提到的细胞系NHM、NHEK进行一组分析，这一部分与第一个细胞株获得的数据一起被认为是关于褪黑素调节黑素生成的分子作用方式的全面知识。在这两个实验模型中，将对黑素生成依赖的基因进行沉默，以研究褪黑素是否会在这些基因发生变化的情况下保持这一过程。其结果无疑将使人们对褪黑素在紫外线照射条件下皮肤黑素生成中的作用有一个全新的认识，并为色素紊乱的治疗提供新的原则。

Clinical disorders related to skin pigmentation include hypo- or hyperpigmentation, therefore active and safe pigment modulatory agents are strongly desired. Based on the so far described high biological activity of melatonin, it can be a potent substance that effectively controls mechanisms of melanogenesis. Melatonin (N-acetyl-5-methoxytryptamine) is a secretory product of the vertebrate pineal gland modulating various functions including scavenging free radicals, DNA repair or anti-apoptotic activity. It is also implicated in skin functions such as epidermal proliferation/differentiation, hair growth and melanoma/squamous cell carcinoma control. In this research application, it is proposed to evaluate the potential impact of melatonin on regulatory mechanisms of melanogenesis. So far wide-range experience gained by the applicant in the field of melatonin allows him to conduct all experiments enclosed in this proposal. The planed investigations will be carried out in human ex vivo full-thickness skin (PART I) with additional PART II using normal human melanocytes (NHM) in pure culture in comparison to co-culture with normal human keratinocytes (NHEK) that will reflect skin physiology in vivo. Additionally, such approach may describe the keratinocyte-melanocyte interaction as well as the key role of keratinocyte-dependent regulation of melanogenesis. In skin, the role and action of melatonin will be evaluated with respect to UVR-mediated melanogenesis including melatonins impact on tyrosinase (TYR), one of the key enzymes in melanogenesis, controlling the expression of microphtalmia-associated transcription factor (MITF) as well as the effect of melatonin on melanin synthesis and cAMP-PKA-MITF axis. Furthermore, a set of analysis will be performed with the earlier mentioned cell lines, i.e. NHM, NHEK, and this part together with data obtained from the first one is supposed to provide a comprehensive knowledge over the molecular way of action of melatonin-regulated melanogenesis. In both, experimental models, silencing of melanogenesis-dependent genes will be performed in order to investigate whether melatonin may maintain this process in case of alterations within these genes. Resulting findings undoubtedly will put in a brand new insight into the role of melatonin in cutaneous melanogenesis under UVR conditions and provide new principles for treatment of pigmentary disorders.