Down-regulation of melanogenesis by phospholipase D2 through ubiquitin proteasome-mediated degradation of tyrosinase
Down-regulation of melanogenesis by phospholipase D2 through ubiquitin proteasome-mediated degradation of tyrosinase
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DOI：
10.1074/jbc.m401786200
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发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Nishigori, C
中科院分区：
文献类型：
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作者：
Kageyama, A;Oka, M;Nishigori, C
研究了磷脂酶D(PLD)对黑素生成的调节作用。用12-O-十四酰佛波醇-13-乙酸酯(TPA)处理B16小鼠黑色素瘤细胞，可激活PLD，降低黑素含量。1-丁醇，而不是2-丁醇，完全阻断了TPA诱导的黑素合成抑制，提示PLD参与了这一事件。逆转录-聚合酶链式反应和免疫印迹分析表明，B16细胞中同时存在PLD同工酶PLD1和PLD2。当通过腺病毒基因转移技术将PLD1或PLD2导入这些细胞时，PLD1和PLD2都被TPA激活。当PLD1和PLD2过表达时，PLD2有效地降低了黑色素含量，而PLD1表达对黑色素含量的影响很小。过表达PLD2本身并不影响蛋白激酶C的活性，通过B16细胞中各亚型的细胞内分布和表达水平来评估。TPA对基础或α-黑素细胞刺激素促进的黑素合成的下调作用几乎完全被表达脂肪酶活性阴性的突变体LN-PLD2所阻断，但不能被LN-PLD1所阻断。此外，PLD2诱导的黑色素含量下降伴随着黑素合成关键酶酪氨酸酶的数量和活性的下降，而PLD2的过度表达对酪氨酸酶的mRNA水平没有影响。此外，蛋白酶体抑制剂的治疗完全阻断了PLD2诱导的黑素合成下调。综上所述，目前的结果表明，TPA诱导的黑素合成下调是由PLD2介导的，而不是由PLD1通过泛素蛋白酶体介导的酪氨酸酶降解而实现的。这提示PLD2可能在体内调节色素沉着中起重要作用。
The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoylphorbol-13- acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decrease in melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or alpha-melanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo.