紫外线通过TRPA1通路影响表皮黑素小体内钙稳态及转运等生物学功能的机制研究

Ultraviolet (UV) is the main pathogenesis or predisposing factors of pigmented dermatosis. Up to now, there is a little understanding of the upstream mechanisms of UV-induced epidermal biology. Recently, we found that transient receptor potential A1 (TRPA1) pathway , the only known extraocular phototransduction pathway in keratinocytes，and UV involved in the regulation of intracellular calcium homeostasis and endocytosis through the TRPA1 ion channels. In this study, we will analyze the effects of UV on the photocurrent and calcium homeostasis in melanocytes and keratinocytes through TRPA1 ion channels by patch clamp and fluorescence probe, determine the interaction sites of membrane signal PIP2 and TRPA1 by TRPA1 mutants, investigate the effects of TRPA1 pathway on the calcium homeostasis, pH of melanosome and melanin synthesis. We will construct melanosome-localized Ca2+ sensor and pH sensor to detect calcium concentration and pH changes in the melanosome lumen of living cells. Moreover, we will study dynamic mechanism of the dynamic binding of TRPA1 to microtubule-binding protein CYLD in melanosome transport. We will setup conditional TRPA1 knockout mice models to observe the effects of TRPA1 pathway in vivo and screen operative medicine. The expected results of this study will has great significance in understanding photobiological pathogenesis of pigmented dermatosis，and in development of TRPA1-targeted drugs for prevention and treatment by acting on initial step of disease.

紫外线为多种色素障碍性皮肤病的主要病因或诱因。目前关于紫外线诱发表皮生物学变化的上游机制了解甚少。我们前期发现目前唯一已知的眼外光通道蛋白——瞬时受体电位A1（TRPA1) 在角质形成细胞中亦有表达；且紫外线通过该通道参与调节细胞钙稳态及胞吞作用。本研究拟采用膜片钳及荧光探针技术，分析紫外线通过TRPA1通道对黑素细胞及角质形成细胞膜光电流及钙稳态影响；构建TRPA1突变体，确定脂膜信号PIP2与TRPA1作用位点；构建黑素小体钙离子及pH指示剂，了解TRPA1对黑素小体腔内钙稳态、pH及黑素合成影响；明确TRPA1与微管结合蛋白CYLD动态结合在黑素小体转运中动力学机制；建立TRPA1条件性基因敲除鼠等动物模型，明确体内机制、筛选有效药物。本研究预期结果将对深入了解紫外线相关色素障碍性皮肤病的光生物学发病机制、开发以TRPA1通路为靶向的、针对发病始动环节的预防及治疗药物具有重要意义。

目的：探讨瞬时受体电位TRPA1通道调控黑素小体转运、黑素小体腔内钙和pH稳态、黑素生成的作用及其相关机制。.方法：原代培养人表皮角质形成细胞（KC）及黑素细胞（MC），构建带有HA和GFP标签的TRPA1真核载体，用TRPA1-shRNA腺病毒感染，分别上调或下调TRPA1，检测黑素转运及合成相关蛋白的表达。建立MC/KC共培养的体外模型，采用gp100荧光标记MC，构建载体EGFP-CYLD和CYLD-siRNA，经UVR生理剂量照射，利用共聚焦显微镜观察乙酰化的α-tubulin与CYLD共定位的变化以及黑素小体的转运。构建黑素小体Ca2+指示剂和黑素小体pH指示剂，分别用UVR照射有/无预处理TRPA1的MC，采用激光共聚焦显微镜、流式细胞术等方法检测TRPA1对[Ca2+]im和pH的影响。建立紫外线诱导豚鼠色沉模型，观察不同的TRPA1激动剂、拮抗剂以及中药制剂对于色素沉着的疗效及机制。 .结果：1.UVR激活人MC及KC中TRPA1通道，引起细胞钙离子内流。2.UVR通过激活TRPA1促进HaCaT细胞吞噬黑素小体。3.UVR通过激活TRPA1促进B16F10细胞黑素生成。4.UVR激活TRPA1是由于TRPA1与CYLD复合体解离，CYLD 核转位介导的。5.CYLD 在核周抑制HDAC6的去乙酰化酶活性，导致α微管乙酰化增加，AC α-tubulin与CYLD直接作用调控CYLD在细胞内重新定位。6.UVR促进PAR-2与TRPA1相互作用，PAR-2激活受TRPA1表达水平的调控。7.抑制TRPA1可使黑素小体内pH降低。8.TRPA1激动剂JT010可促进紫外线诱导的豚鼠皮肤色沉增加，TRPA1抑制剂HC-030031可降低紫外线诱导的豚鼠皮肤色素沉着。.结论：1.UVR激活KC内的TRPA1通路，后者与下游分子PAR-2直接作用，增强KC对黑素小体的吞噬能力。2.UVR诱导TRPA1与微管结合蛋白CYLD复合体解离，CYLD向核周聚集，稳定微管聚合，从而促进KC吞噬，促进黑素小体转运。3.UVR激活TRPA1促进黑素细胞钙离子内流，使黑素小体内pH增高，黑素合成增加。4.TRPA1外用制剂可调节紫外线诱导的豚鼠皮肤色沉变化，提示其具有色素性疾病靶向治疗药物研发的重要意义。