Study On The Molecular Mechanism Of G Protein-Coupled Receptor 54 (GPR54) Gene Deletion To Promote Hair Growth

With the acceleration of life rhythm and the increasing pressure of work,the incidence of hair loss in the population is getting higher and higher.At present,the drugs used to treat hair loss are limited.The specific reasons are that people do not have a comprehensive understanding of the molecular mechanisms and targets of treating hair loss.It has been reported that G protein-coupled receptors(GPCRs)as popular targets for drugs research are related to many diseases,such as cancer and obesity.However,there are few studies related to hair loss.G protein-coupled receptor 54(GPR54)is belongs to the rhodopsin family and is a receptor for the Gn RH-dependent precocious puberty-related.The ligand of GPR54 gene is KISS-1and both are expressed in multiple tissues.As reported,GPR54 gene is related to many diseases,such as reproduction,tumor,immunity and obesity.Its inhibitor peptide-234 has also been reported to have a certain therapeutic effect on some diseases,such as precocious puberty,uterine fibroids and polycystic ovary syndrome.But the relationships between GPR54 gene,peptide-234 and hair growth have not been reported.In this paper,in the process of studying hair growth of C57BL/6 mice,it was found that the GPR54 gene was closely related to hair growth.Hair growth is cyclical,including three stages: Anagen,Catagen,and Telogen phase.The maintenance of normal hair cycle ensures certain numbers of hair sharfts,but once the balance of hair cycle is broken,hair loss will occur.Dermal papilla cell(DPC)located at the base of hair follicle and hair follicle stem cell(HFSC)located in the Bulge area of hair follicle are two important cell types,and closely related to hair growth.The dermal papilla cell is the signaling center for hair growth and controls the proliferation,migration and differentiation of surrounding epidermal stem cell/progenitor cell for completing hair regeneration.The HFSC is pluripotent and is able to differentiate into other epithelial cell lineages and could maintain circulating hair regeneration through constant hair circulation.Wnt/β-catenin signaling pathway has been shown to be essential in process of hair morphogenesis and circulation,and its activation in DPC promotes the proliferation and differentiation of HFSC,which initiates the prolongation of Anagen.In this paper,we first collected the skin tissues from the back of wild mice in three stages of Anagen,Catagen and Telogen,extracted RNA and made paraffin sections,then used some qPCR and immunofluorescence technologies.It was found that the expression of GPR54 gene and its ligand KISS1 increased gradually with the hair cycle going at the transcription level.What was more,the GPR54 gene was mainly distributed in the dermal papilla DP,hair matrix HM(Hair matrix)or hair germ HG(Hair germ)and Bulge region in different growth stages of both mouse skin hair follicle and normal human scalp hair follicle,but the most in the DP region.Through immunofluorescence co-staining experiment,we found that GPR54 gene co-localized with ALP and CD34 in the DP and Bulge regions of mice hair follicle,respectively.Then,we constructed a mouse model of hair regeneration through natural shaving using GPR54 knockout and wild-type mice and observed the hair growth from the day of birth,the structure of hair follicle at various growth stages.It was found that the Telogen could be shortened and Anagen be extended in GPR54 knockout mice compared with wild mice.Moreover,the result was verified by a mouse model of synchronization-induced hair regeneration.Finally,we constructed a model of hair regeneration after skin transplantation in nude mice,which showed that the capability of hair regeneration could be improved and the length of hair shaft could be increased significantly after the GPR54 gene was knocked out.Then,we isolated and cultured mouse dermal papilla cell and and epidermal cell by flow cytometry or sorting technology and deeply explored the molecular mechanism of GPR54 gene affecting hair growth through Immunofluorescence of paraffin section tissues or cells,Immunohistochemistry,Western blot,qPCR and other technologies.It was found that the deletion of GPR54 gene could not only affect the NFATc3/sFRP1/Wnt/β-catenin signaling pathway in DPC,such as inhibiting the expression,nuclear translocation of NFATc3 and expression of sFRP1 which is a inhibitor of Wnt/β-catenin signaling pathway,thereby activating the Wnt/β-catenin signaling pathway in DPC,increasing the activity of DPC and promoting hair growth.Moreover,it could also directly inhibit the NFATc1/CDK4 signaling pathway in HFSC,such as inhibiting the expression,nuclear shift of NFATc1 and promoting the expression of CDK4,the proliferation and activity of HFSC and the signaling transmission of DPC to HFSC.Moreover,Wnt/β-catenin signaling pathway was activated in HFSC and the abilities of proliferation and differentiation of other hair follicle lineage cells were increased,such as HM/HG cell.But,the ability of differentiation was reduced referred to epithelial-like cell.Thus,the integrity of hair follicle structure was maintained and hair growth was promoted.In conclusion,the results of this paper showed that the activities of dermal papilla cell and hair follicle stem cell were activated through NFATc3/sFRP1/Wnt/β-catenin,NFATc1/CDK4 signaling pathways respectively after GPR54 gene was deleted,and hair growth was also promoted.The hair growth-promoting effect of peptide-234 as GPR54 inhibitor was also studied by culturing mouse tentacle hair follicle,human scalp hair follicle and animal model of synchronization induction and the androgenetic alopecia(AGA)model.The results showed that peptide-234 could not only promote hair growth at the level of cultured organs in vitro,but also promote hair regeneration of mice induced by synchronization,and delay the development of AGA significantly.Therefore,we found that GPR54 gene may be a new target regulating hair cycle and hair regeneration in this paper.The results not only revealed the molecular mechanism regulating hair growth through dermal papilla cell and hair follicle stem cell after GPR54 gene deletion,but also confirmed the effect of GPR54 inhibitor peptide-234 promoting hair growth in vivo and in vitro.Moreover,it was proved that GPR54 gene could be used as a new treatment target for androgenetic alopecia,which provides new idea and theoretical basis for the research and development of drugs treating hair loss in the future.