跨膜蛋白106B通过ERK/CREB信号通路调控黑色素生成

TMEM106B Induces Melanogenesis by Regulating ERK/CREB Signaling Pathway

TMEM106B是一种二型跨膜蛋白质,定位于神经元细胞树突中的核内体和溶酶体中,能够调控神经元细胞树突中溶酶体的逆向转运,对于神经元树突的分支和维持发挥重要作用。哺乳动物的黑色素细胞来源于神经嵴细胞,胞内黑素体来源于早期核内体,但TMEM106B是否在黑色素细胞中发挥功能未有报道。现有研究表明,TFEB与溶酶体的合成和功能有关,在神经元细胞中,TMEM106B表达促进了TFEB的核转位,而在黑色素瘤细胞中,TFEB和MITF存在互相调控的作用。因此,本研究通过TMEM106B在黑色素细胞过表达的方法,研究TMEM106B调控黑色素的生成作用及其可能的作用机制。研究表明,TMEM106B定位于黑色素细胞的细胞质中,提示其可能在黑色素细胞中有重要作用。将构建的TMEM106B表达载体转染到黑色素细胞中,与对照组相比,CREB和MITF的mRNA水平有明显的增加(P<0.001),其中,CREB增加最明显;通过免疫印迹分析,p-ERK蛋白表达水平明显增加(P<0.001),导致黑色素生成相关蛋白MITF、TYR、TYRP1和TYRP2表达上调。黑色素含量分析显示,TMEM106B过表达可使真黑素(P<0.05)和总黑素(P<0.001)含量增加,然而褐黑素减少(P<0.001)。以上结果表明,在黑色素细胞中,TMEM106B通过调控CREB/ERK信号通路调控黑色素生成。

The TMEM106 B protein is a type-II transmembrane protein,which localizes in the endosome and lysosome of dendrites in primary neurons.TMEM106 B is essential for maintaining and branching of dendrites,and thus regulates retrograde lysosomal trafficking of dendrites in primary neurons.Mammalian melanocytes are derived from neural cells,while melanosomes are originated from early endosome.However,the function of TMEM106 B protein in melanocytes and its potential molecular mechanism in melanogenesis still remain unknown.Recently it was reported that transcription factor EB(TFEB)was the regulator of lysosome synthesis and TMEM106 B protein overexpression promoted TFEB translocation into the nucleus.However,MITF(microphthalmia-associated transcription factor)and TFEB regulate each other in melanoma cells in vitro.Here in,plasmid containing gene for TMEM106 B overexpression was transfected into melanocytes to investigate the regulation of TMEM106 B on melanogenesis.The results showed that TMEM106 B protein was localized in the cytoplasm of melanocytes.Compared with the negative control(NC),the mRNA levels of cyclic AMP-responsive element-binding protein(CREB)and MITF,especially CREB,were significantly increased in melanocytes with TMEM106 B overexpression P<0.001).Western blot analysis showed that the expression of phosphorylated MAP kinase(p-ERK)was apparently increased(P<0.001)and resulted in the up-regulation of melanogenic regulatory proteins,including MITF,tyrosinase(TYR),tyrosinase-related protein-1(TYRP1)and 2(TYRP2).MassonFontana method showed that TMEM106 B influenced the production of melanin in melanocytes.The spectrophotometry assay indicated that the amount of total melanin(ASM)(P<0.001)and eumelanin(EM)(P<0.05)were increased in alpaca melanocytes transfected with TMEM106 B,while pheomelanin(PM)(P<0.001)was decreased.These results demonstrated that TMEM106 B played a vital role in melanogenesis in melanocytes by regulating ERK/CREB signaling pathway.