Laboratory of Stem Cell Biology, Department of Biomedical Science, College of Health Science, Korea University, Jeongneung-dong, Sungbuk-gu, Seoul 136-703, Republic of Korea; Department of Health Science, Korea University Graduate School, Jeongneung-dong, Sungbuk-gu, Seoul 136-703, Republic of Korea.
J Dermatol Sci. 2013 Dec;72(3):274-83. doi: 10.1016/j.jdermsci.2013.08.006. Epub 2013 Aug 23.
Melanin for skin pigmentation is synthesized from tyrosine via an enzymatic cascade that is controlled by tyrosinase (TYR), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase/tyrosinase related protein 2 (Dct/TRP2), which are the targets of microphthalmia-associated transcription factor (MITF). MITF is a master regulator of pigmentation and a target of β-catenin in Wnt/β-catenin signaling during melanocyte differentiation. Stem cells have been used in skin pigmentation studies, but the mechanisms were not determined for the conditioned medium (CM)-mediated effects.
In this study, the inhibition and mechanisms of melanin synthesis were elucidated in B16 melanoma cells and UV-B irradiated C57/BL-6 mice that were treated with human neural stem cell-conditioned medium (NSC-CM).
B16-F10 melanoma cells (1.5×10(4)cells/well) and the shaved dorsal skin of mice were pretreated with various amount (5, 10, 20, 50, and 100%) of NSC-CM. Melanin contents and TYR activity were measured by a Spectramax spectrophotometer. The expression of TYR, TRP1, Dct/TRP2, MITF, β-catenin and Wnt inhibitors were evaluated by RT-PCR and western blot. The dorsal skin samples were analyzed by immunofluorescence with various antibodies and compared with that control of tissues.
Marked decreases were evident in melanin content and TYR, TRP1, DCT/TRP2, MITF, and β-catenin expression in B16 cells and C57/BL-6 mice. NSC-CM negatively regulated Wnt/β-catenin signaling by decreasing the expression of β-catenin protein, which resulted from robust expression of Wnt inhibitors Dickkopf-1 (DKK1) and secreted frizzled-related protein 2 (sFRP2).
These results demonstrate that NSC-CM suppresses melanin production in vitro and in vivo, suggesting that factors in NSC-CM may play an important role in deregulation of epidermal melanogenesis.
皮肤色素沉着的黑色素是由酪氨酸通过酶级联反应合成的，该反应受酪氨酸酶（TYR）、酪氨酸酶相关蛋白 1（TRP1）和多巴色素互变异构酶/酪氨酸酶相关蛋白 2（Dct/TRP2）的控制，它们是小眼相关转录因子（MITF）的靶点。MITF 是色素沉着的主要调节因子，也是 Wnt/β-catenin 信号通路中黑色素细胞分化时β-catenin 的靶标。干细胞已被用于皮肤色素沉着研究，但条件培养基（CM）介导的作用的机制尚不清楚。
本研究阐明了人神经干细胞条件培养基（NSC-CM）处理的 B16 黑色素瘤细胞和 UV-B 照射的 C57/BL-6 小鼠中黑色素合成的抑制和机制。
B16-F10 黑色素瘤细胞（1.5×10(4)个细胞/孔）和小鼠剃毛的背部皮肤用不同量（5%、10%、20%、50%和 100%）的 NSC-CM 预处理。通过分光光度计测量黑色素含量和 TYR 活性。通过 RT-PCR 和 Western blot 评估 TYR、TRP1、Dct/TRP2、MITF、β-catenin 和 Wnt 抑制剂的表达。用各种抗体对背部皮肤样本进行免疫荧光分析，并与组织对照进行比较。
B16 细胞和 C57/BL-6 小鼠的黑色素含量和 TYR、TRP1、DCT/TRP2、MITF 和 β-catenin 表达明显下降。NSC-CM 通过降低β-catenin 蛋白的表达来负调控 Wnt/β-catenin 信号通路，这是由于 Wnt 抑制剂 Dickkopf-1（DKK1）和分泌型卷曲相关蛋白 2（sFRP2）的强烈表达所致。
这些结果表明，NSC-CM 可抑制体外和体内黑色素的产生，提示 NSC-CM 中的因子可能在表皮黑色素生成失调中发挥重要作用。
