Expression And Localization Of Dgkζ And PKCβⅡ In Mouse Dorsal Skin With Different Coat Colors

Objective:To explore the expression and location of diacylglycerol kinase ζ(diacylglycerol kinase ζ,DGKζ)and protein kinase C βⅡ(protein kinase CβⅡ,PKCβⅡ)in mouse dorsal skin with different coat colors and their correlation with development of the coat colors.Methods:1.On the NCBI website,to access to human and mouse DGKζ sequence;on a variety of online bioinformatics analysis platform,to predict the expression of human DGKζ in various tissues,its transcription factors,the signal peptide,transmembrane domain and,subcellular localization of its encoding product,and analyze the homology of human DGKζ;to predict transcription factors in mouse DGKζ.2.The dorsal skin of two-month-old mouse with different coat colors,6 for each color,was detected by Real-time PCR,Western blotting and Immunohistochemistry ABC staining.Results:1.The results of bioinformatics analysis showed that: DGKζ was expressed in human skin;among the 22 predicted transcription factors of human DGKζ,ER-alpha,Sp1,p53,c-ets-1 and YYY1 were expressed in melanocytes;the human DGKζ encoding product mainly located in the nucleus and also in the cytoplasm,without signal peptide cleavage site and being a non-secreted protein,no transmembrane domain;DGKζ homology between human and Pan troglodytes,Equus caballus,Felis catus,Cricetulus griseus,Bos tsurus,Cavia porcellus,Sus scrofa,Rattus norvegicus,Mus musculus and Gallus gallus was 99.8%,98.3%,97.7%,96.6%,96.6%,96.4%,96.3%,96.0%,96.0%,94.6%,and 78.6%,respectively;phylogenetic tree analysis found that human had the closest relationship with Pan troglodytes and the farthest relationship with Gallus gallus.ER-alpha,p53 and YYY1 were expressed in the melanocyte among 13 transcription factors predicted to combine to mice DGKζ promoter..2.The results of RT-PCR showed that DGKζ and PKCβⅡ mRNA were both expressed in the dorsal skin of three kinds of coat color mice,with its relative expression amount being: gray group > white group;black group > white group(P<0.05).The results of Western-Blot showed that both DGKζ and PKCβⅡ protein were expressed in the dorsal skin of mice with different hair co lors.The ratio of p-PKCβⅡ to PKCβⅡ showed that the change of PKCβⅡ activity had the same tendency with DGKζ expression: gray group > white group;black group > white group(P <0.05).Immunohistochemistry ABC staining showed that DGKζ and p-PKCβⅡ were positively expressed in germinativum of the epidermis and melanocyte,the outer root sheath of hair follicle,hair matrix cell and melanocyte in the hair matrix in the dorsal skin of gray and black mouse,negative in the epidermal germinativum and hair follicle of white mouse.Conclusion:1.Bioinformatics analysis suggests that DGKζ may be expressed in human melanocyte and highly conservative in mammals.DGKζ may be expressed in melanocyte in mice.2.According to the difference of expression of DGKζ,PKCβⅡ in mRNA and protein in white,grey and black mouse dorsal skin and localization of DGKζ,PKCβⅡ in germinativum of the epidermis and melanocytes in it,hair matrix cells and melanocytes among them in white,grey and black mouse back skin,we suggest that DGKζ and PKCβⅡ may be related to skin pigmentation and the formation of the coat color.