The Molecular Mechanism Of Regulation Of DKK3 On The Melanogenesis In Melanocytes Of Alpaca

Wnt/β-catenin signaling pathway plays an important role in melanogenesis.Dickkopf-3(DKK3),as a critical inhibitor of the Wnt/β-catenin signaling pathway,may be involved in melanogenesis.In the current study,we investigated the effects of DKK3 on melanogenesis in melanocytes of alpaca.The expression of DKK3 was controlled by over expression or silencing.The effect of DKK3 on melanin production and the molecular mechanism was investigated by using quantitative real-time PCR assay(q RT-PCR),Western blotting and cellular immunohistochemistry.The results are as follows:1.Using q RT-PCR to detect the experssion of DKK3 m RNA in skins of alpaca with white and black coat color.The result showed the level of DKK3 m RNA was significantly higher in white skin than in black skin(P<0.001).2.Using q RT-PCR,Western blotting and cellular immunocytochemistry techniques to detect the effect of DKK3 overexpression or silencing in melanocytes on the expression level of DKK3.The results showed the m RNA and protein levels of DKK3 were significantly increased in melanocytes with the overexpression of DKK3;The levels of DKK3 m RNA and proteins were significantly decreased in melanocytes of DKK3 silencing(P<0.001).Immunocytochemical analysis also showed that the DKK3 protein was increased in melanocytes with the overexpression of DKK3 and reduced in DKK3-silenced melanocytes.3.Using q RT-PCR and Western blotting techniques to detect the effect of DKK3 overexpression or silencing in melanocytes on the expression level of key genes of Wnt signaling pathway and melanogenic genes.The results showed that in melanocytes with the overexpression of DKK3,the m RNA and protein levels of Wnt1,Lef1 and Myc were significantly decreased(P<0.05),and the m RNA and protein levels of MITF,TYR,TYRP1 and TYRP2 were significantly decreased(P<0.05);In DKK3-silenced melanocytes,the m RNA and protein levels of Wnt1,Lef1,Myc,MITF,TYR,TYRP1 and TYRP2 were significantly increased(P<0.05).4.To investigate whether DKK3 affects melanin production,total alkali melanin,eumelanin and pheomelanin were measured in melanocytes of alpaca after overexpression and silencing of DKK3.In alpaca melanocytes with the overexpression of DKK3,the production levels of total alkali melanin,eumelanin and pheomelanin were significantly decreased(P<0.05).And in melanocytes of DKK3 silencing,the production levels of total alkali melanin,pheomelanin and eumelanin were significantly increased(P<0.05).Taken together,DKK3 regulates melanogenesis via the Wnt/β-catenin signaling pathway that mediates MITF downregulation,and results in downregulating the production of total alkali melanin,pheomelanin and eumelanin through TYR,TYRP1 and TYRP2 in alpaca melanocytes.