Hu Qing-Mei, Yi Wen-Juan, Su Meng-Yun, Jiang Shan, Xu Shi-Zheng, Lei Tie-Chi
Department of Dermatology, Renmin Hospital of Wuhan University, Wuhan, China.
Cell Prolif. 2017 Dec;50(6). doi: 10.1111/cpr.12372. Epub 2017 Aug 23.
The transfer of melanosomes from melanocytes to neighbouring keratinocytes is critical to protect the skin from the deleterious effects of ultraviolet A (UVA) and ultraviolet B (UVB) irradiation; however, the initial factor(s) that stimulates melanosome transfer remains unclear. In this study, we investigated the induction of retinal-dependent calcium (Ca ) influx in melanocytes (MCs) by UVA or UVB irradiation and the effect of transient receptor potential cation channel subfamily M member 1 (TRPM1) (melastatin1)-related Ca influx on melanosome transfer.
Primary human epidermal MCs were exposed to physiological doses of UVB or UVA light and loaded with a calcium indicator Fluo-4 dye. The change of intracellular calcium of MCs was monitored using a two-photon confocal fluorescence microscopy. MCs were co-cultured with human epidermal keratinocytes (KCs) in the absence or presence of voriconazole (a TRPM1 blocker) or calcium chelators. MCs were also transfected with TRPM1 siRNA for silencing the expression of TRPM1 gene. The melanosome transfer in the co-cultured cells was quantitatively analysed using flow cytometry and was further confirmed by immunofluorescent double-staining. The protein levels and distributions of TRPM1, OPN3 and OPN5 in MCs were measured by Western blotting or immunofluorescent staining.
The retinal-dependent Ca influx of UVA-exposed melanocytes differed greatly from that of UVB-exposed melanocytes in the timing-phase. The protein expression of TRPM1 in mono- and co-cultured MCs was dose-dependently up-regulated by UVA and UVB. TRPM1 siRNA-mediated knockdown and the blockage of TRPM1 channel using a putative antagonist (voriconazole) significantly inhibited melanosome transfer in co-cultures following UVA or UVB exposure.
The distinct time-phases of Ca influx in MCs induced by UVA or UVB contribute to the consecutive stimulation of melanosome transfer, thereby providing a potent photoprotection against harmful UV radiation.
黑素小体从黑素细胞转移至邻近角质形成细胞对于保护皮肤免受紫外线A（UVA）和紫外线B（UVB）辐射的有害影响至关重要；然而，刺激黑素小体转移的初始因素仍不清楚。在本研究中，我们调查了UVA或UVB照射对黑素细胞（MCs）中视网膜依赖性钙（Ca）内流的诱导作用，以及瞬时受体电位阳离子通道亚家族M成员1（TRPM1）（褪黑素1）相关的Ca内流对黑素小体转移的影响。
将原代人表皮MCs暴露于生理剂量的UVB或UVA光下，并用钙指示剂Fluo-4染料加载。使用双光子共聚焦荧光显微镜监测MCs细胞内钙的变化。在不存在或存在伏立康唑（一种TRPM1阻滞剂）或钙螯合剂的情况下，将MCs与人表皮角质形成细胞（KCs）共培养。MCs也用TRPM1 siRNA转染以沉默TRPM1基因的表达。使用流式细胞术对共培养细胞中的黑素小体转移进行定量分析，并通过免疫荧光双重染色进一步证实。通过蛋白质印迹或免疫荧光染色测量MCs中TRPM1、OPN3和OPN5的蛋白质水平和分布。
UVA照射的黑素细胞中视网膜依赖性Ca内流在时间阶段上与UVB照射的黑素细胞有很大差异。UVA和UVB剂量依赖性地上调了单培养和共培养MCs中TRPM1的蛋白质表达。TRPM1 siRNA介导的敲低以及使用推定拮抗剂（伏立康唑）阻断TRPM1通道显著抑制了UVA或UVB照射后共培养物中的黑素小体转移。
UVA或UVB诱导的MCs中Ca内流的不同时间阶段有助于连续刺激黑素小体转移，从而为有害紫外线辐射提供有效的光保护。
