The Molecular Mechanism Of Regulation Of SYT4 On The Melanins Production In Alpaca Melanocytes

The pigmentation of mammals includes melanins production in melanocytes and melanins transferring to the around keratinocytes.Melanins containing eumelanins and phoemelanins are the major determinate for the hair color formation or pigmentation,which is regulated by the molecular net including genes,mi RNAs,and lnc RNAs.In the previous research for the transcriptome profile of melanocytes,it has been found that Synaptotagmin 4(SYT4)was expressed in the melanocytes,which suggested that it might be related to the melanins production.Here,the expression of SYT4 was controlled by over expression or knockdown and the effects of SYT4 on melanins production and the molecular mechanism was investigated by using quantitative real-time PCR assay(q PCR),western blotting,Ca2+ signal detection,and cellular immunochemistry.The results were as follows:1.After SYT4 was overexpressed in alpaca melanocytes,the localization of SYT4 in alpaca melanocytes and its effect on cell morphology were detected by immunocytohemistry.The results showed that SYT4 was mainly expressed in the cytoplasm of alpaca melanocytes and could significantly extend the dendrites of alpaca melanocytes.2.In alpaca melanocytes overexpressed by SYT4,intracellular Ca2+ signaling was detected by Rhod-3.The expression of transient receptor potential cation channel subfamily M member 1(TRPM1)which is the key protein of Ca2+ signaling pathway and the related genes calcium/calmodulin dependent protein kinase IV(CAMK4)and p-ERK were detected by western blotting.The results showed that the overexpression of SYT4 in the alpaca melanocytes significantly increased the intracellular Ca2+ signal intensity,and that SYT4up-regulated the expression levels of CAMK4 and TRPM1,and inhibited the expression of p-ERK(***P<0.001).Therefore,SYT4 regulated intracellular Ca2+ by the TRPM1 signaling pathway,and therefore affected the melanins production.3.After SYT4 was overexpressed and knocked down in alpaca melanocytes,,the m RNA and protein expression levels of the melanin-related genes melanocyte inducing transcription factor(MITF),tyrosinase(TYR),tyrosinase related protein 1(TYRP1),and dopachrome tautomerase(DCT)were detected by q PCR and western blotting.The total melanin,pheomelanin,eumelanin content and tyrosinase activity were detected.After the knockdown of SYT4,the total melanin,pheomelanin,eumelanin,and tyrosinase activity were significantly down-regulated(**P<0.01,***P<0.001).SYT4 overexpression could up-regulate m RNA and protein expression levels of MITF,TYR,TYRP1,and DCT(**P<0.01.***P<0.001).After the knockdown of SYT4,the expression levels of the above genes were decreased(***P<0.001).SYT4 overexpression could significantly increase the total melanin,pheomelanin,eumelanin,and could significantly activate tyrosinase activity(***P<0.001).4.In order to verify the role of SYT4 in the regulation of melanin production,SYT4 was over-expressed and knocked down in mouse melanoma cells.The m RNA and protein expression levels of melanin related genes MITF,TYR,TYRP1,DCT were detected by q PCR and western blotting(**P<0.01,***P<0.001).The results confirmed that SYT4 could still regulate the expression of melanin related genes.Taken together,SYT4 regulated Ca2+ signaling through TRPM1 channel to control the expression of hair color related genes MITF,TYR,TYRP1,DCT and then affected the melanins production.At the same time,SYT4 could extend the dendrites of alpaca melanocytes,which was important for the melanins transfer.