Regulation Of Hair Color By Mammalian Pigment Epithelium-Derived Factors

The synthesis of melanin in animal skin tissue plays a crucial role in the formation of animal hair color.Pigment epithelium-derived factor(PEDF)is widely expressed in mammals and affects the biological activity of melanocytes.Exploring the expression characteristics of PEDF in mammals and exploring the relationship between PEDF and melanin synthesis can provide a theoretical basis for the application of PEDF to regulate animal hair color,and also contribute to the research and development of drugs for the treatment of melanin biosynthesis related diseases.This study first took sheep as an animal model to clarify the expression and distribution of PEDF in mammals,furthermore,the regulatory effect of PEDF on melanin synthesis was explored,to provide a basis for application of PEDF.The main research contents and results are as follows:(1)PEDF m RNA and protein were expressed to varying degrees in all tissues of sheep,and the expression level of PEDF in lung was significantly higher than that in heart,liver,spleen,kidney,brain and gonad tissues(P < 0.001).In the brain tissue,the expression level of PEDF in the cerebrum was significantly higher than that in the cerebellum and hypothalamus(P < 0.001).In gonadal tissues,the expression level of PEDF in caput epididymidis was significantly higher than that in corpora epididymidis,caudal epididymidis and testis(P < 0.001).The results of immunohistochemical localization of PEDF in different tissues of sheep were analyzed by optical density values,which were consistent with the results of m RNA level and protein level tests.(2)The expression level of PEDF in different color skin tissue of sheep have obvious difference,the result showed that the expression level of PEDF in the white sheep skin tissue is significantly higher than in the brown sheep skin tissue and black sheep skin tissue(P < 0.01);the expression level of PEDF in brown sheep skin tissue was significantly higher than that in black sheep skin tissue(P < 0.001).(3)Mice melanocytes were treated with different concentrations(10-7 mol/L,10-6 mol/L and 10-5 mol/L)of α-MSH,The cell viability was significantly higher than that of the control group(P < 0.05),and the cell viability was the highest when the concentration was 10-6 mol/L.When different concentrations of α-MSH(10-9 mol/L,10-8 mol/L and 10-7 mol/L)were used to treat mouse melanocytes,the content of melanin was extremely significantly higher than that of the control group(P < 0.01),and the content of melanin was the highest when the concentration of α-MSH was 10-7 mol/L.The expression level of PEDF in mouse melanocytes treated with different concentrations of α-MSH(10-9 mol/L,10-8 mol/L,10-7 mol/L)was significantly lower than that in the control group(P < 0.001),and with the increase of α-MSH concentration,the expression of PEDF showed a downward trend;When the concentration of α-MSH was 10-8 mol/L,the expression of MC1 R was significantly higher than that of the control group(P < 0.05),the expression of MC1 R was significantly higher than that of the control group when the concentration of α-MSH was 10-7 mol/L(P < 0.001);The relative expression levels of MC1 R protein were significantly higher than those in the control group(P < 0.001),the expression of MC1 R increased with the increase of α-MSH concentration.In conclusion,α-MSH can enhance the activity of melanocytes,promote melanin synthesis and the expression of MC1 R,and inhibit the expression of PEDF.(4)PEDF-si RNA interference melanin cells of mice,compared with control group,the melanin cell vitality significantly enhanced(P < 0.001),the melanin content significantly increased(P < 0.001),the expression level of the downstream genes: Wnt-1,Wnt-3α,MITF and β-catenin were significantly increased(P < 0.01),cell immunofluorescence test results show the same trend.After PEDF was overexpressed,compared with control group,the vitality of melanocytes significantly reduced(P < 0.001),the melanin content was significantly reduced(P < 0.01),the expression of downstream genes: Wnt–1,Wnt-3α,MITF,β-catenin were extremely significant(P < 0.001)reduced in m RNA and protein level,cellular immune fluorescence detection were show the same trend with protein and m RNA level detection.(5)Transcriptome sequencing of melanocytes after silencing and overexpression of PEDF genes showed that PEDF was in the Wnt signaling pathway and had negative regulation effects on downstream genes including Wnt-1,Wnt-3α,MITF,β-catenin and MC1 R mainly through Wnt 3α.PEDF participates in the classic pathway of α-MSH promoting melanin synthesis,and α-MSH plays a negative regulatory role on PEDF.This study clarified that PEDF gene plays an important regulatory role in the synthesis of melanin in animal skin tissues,and PEDF plays a role through the Wnt signaling pathway by down-regulating the expression of genes that promote melanin synthesis represented by Wnt-3α and MC1 R,thereby inhibiting the synthesis of melanin and reducing the activity of melanocytes.The gene is located in the upstream of Wnt-1 and other genes in the Wnt signaling pathway,and interacts with a variety of genes to form a cascade reaction and jointly regulate the synthesis of melanin.This study revealed that PEDF can regulate the formation of melanin in animal skin,and PEDF is expected to be applied to regulate animal hair color and treat melanin-related diseases.