Abstract：
BACKGROUND: Keratinocytes are recipients of melanosomes. Although the chemical basis of melanogenesis is well documented, the molecular mechanism of melanosome transfer must be elucidated. TRPA1 is a member of the transient receptor potential A subfamily. Previous studies have shown that inhibition of TRPA1 activity reduces melanin synthesis in human epidermal melanocytes; however, the mechanism remains unknown.
OBJECTIVE: This study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes.
METHODS: The correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human epidermal keratinocytes and keratinocyte cell lines HaCaT were established using adenovirus-expressing shRNAs against TRPA1. The effects of TRPA1 on keratinocytes and HaCaT cells were determined using cell-based analyses, including light stimulation, calcium imaging, melanin phagocytosis, immunoblotting, and co-immunoprecipitation assays. The degree of epidermal pigmentation was determined in a guinea pig model.
RESULTS: TRPA1 mediated the phagocytic activity of keratinocytes. TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. Mechanistically, TRPA1 was required for PAR-2-induced melanosome phagocytosis in keratinocytes. Furthermore, TRPA1 activation indirectly stabilized microtubules by promoting the competitive binding of CYLD and acetylated α-tubulin. In addition, bortezomib (PS-341), a proteasome inhibitor, increased TRPA1 and CYLD expression and promoted phagocytic activity both in vitro and in vivo.
CONCLUSIONS: Our findings firstly suggest that TRPA1 promotes melanosome transport in keratinocytes and reveal that TRPA1 is a regulator of PAR-2 activation and microtubule stability via the PAR-2/CYLD axis.
OBJECTIVE: This study aimed to investigate the roles and mechanism(s) of action of TRPA1 in keratinocytes.
METHODS: The correlation between TRPA1 expression levels and the ability of keratinocytes to phagocytize melanosomes was examined using melanin silver staining. TRPA1 depleted human epidermal keratinocytes and keratinocyte cell lines HaCaT were established using adenovirus-expressing shRNAs against TRPA1. The effects of TRPA1 on keratinocytes and HaCaT cells were determined using cell-based analyses, including light stimulation, calcium imaging, melanin phagocytosis, immunoblotting, and co-immunoprecipitation assays. The degree of epidermal pigmentation was determined in a guinea pig model.
RESULTS: TRPA1 mediated the phagocytic activity of keratinocytes. TRPA1 knockdown markedly suppressed melanosome transport to keratinocytes. Mechanistically, TRPA1 was required for PAR-2-induced melanosome phagocytosis in keratinocytes. Furthermore, TRPA1 activation indirectly stabilized microtubules by promoting the competitive binding of CYLD and acetylated α-tubulin. In addition, bortezomib (PS-341), a proteasome inhibitor, increased TRPA1 and CYLD expression and promoted phagocytic activity both in vitro and in vivo.
CONCLUSIONS: Our findings firstly suggest that TRPA1 promotes melanosome transport in keratinocytes and reveal that TRPA1 is a regulator of PAR-2 activation and microtubule stability via the PAR-2/CYLD axis.
摘要：
背景：角质形成细胞是黑色素体的受体。虽然黑素生成的化学基础是有据可查的，必须阐明黑素体转移的分子机制。TRPA1是瞬时受体电位A亚家族的成员。先前的研究表明，抑制TRPA1活性会降低人表皮黑素细胞中黑色素的合成；然而，机制仍然未知。
目的：本研究旨在探讨TRPA1在角质形成细胞中的作用和作用机制。
方法：使用黑色素银染色检查TRPA1表达水平与角质形成细胞吞噬黑色素体能力之间的相关性。使用表达针对TRPA1的腺病毒的shRNA建立了TRPA1耗尽的人表皮角质形成细胞和角质形成细胞系HaCaT。使用基于细胞的分析确定TRPA1对角质形成细胞和HaCaT细胞的影响，包括光刺激，钙成像,黑色素吞噬，免疫印迹,和免疫共沉淀试验。在豚鼠模型中确定表皮色素沉着的程度。
结果：TRPA1介导角质形成细胞的吞噬活性。TRPA1敲低显著抑制黑素体向角质形成细胞的转运。机械上,TRPA1是PAR-2诱导的角质形成细胞中黑体吞噬作用所必需的。此外，TRPA1激活通过促进CYLD和乙酰化α-微管蛋白的竞争性结合来间接稳定微管。此外,硼替佐米（PS-341），蛋白酶体抑制剂,增加TRPA1和CYLD的表达，并在体外和体内促进吞噬活性。
结论：我们的发现首先表明TRPA1促进角质形成细胞中的黑素体运输，并揭示TRPA1是通过PAR-2/CYLD轴调节PAR-2活化和微管稳定性的调节剂。
目的：本研究旨在探讨TRPA1在角质形成细胞中的作用和作用机制。
方法：使用黑色素银染色检查TRPA1表达水平与角质形成细胞吞噬黑色素体能力之间的相关性。使用表达针对TRPA1的腺病毒的shRNA建立了TRPA1耗尽的人表皮角质形成细胞和角质形成细胞系HaCaT。使用基于细胞的分析确定TRPA1对角质形成细胞和HaCaT细胞的影响，包括光刺激，钙成像,黑色素吞噬，免疫印迹,和免疫共沉淀试验。在豚鼠模型中确定表皮色素沉着的程度。
结果：TRPA1介导角质形成细胞的吞噬活性。TRPA1敲低显著抑制黑素体向角质形成细胞的转运。机械上,TRPA1是PAR-2诱导的角质形成细胞中黑体吞噬作用所必需的。此外，TRPA1激活通过促进CYLD和乙酰化α-微管蛋白的竞争性结合来间接稳定微管。此外,硼替佐米（PS-341），蛋白酶体抑制剂,增加TRPA1和CYLD的表达，并在体外和体内促进吞噬活性。
结论：我们的发现首先表明TRPA1促进角质形成细胞中的黑素体运输，并揭示TRPA1是通过PAR-2/CYLD轴调节PAR-2活化和微管稳定性的调节剂。
