Study Of Molecular Mechanisms Regulating The Expression Of Hairless Gene On Hair Growth

Alopecia affects approximately 2%of the general population at some point during their lifetime,as documented by several large epidemiological studies from Europe 12,North America13 and Asia.Although this condition often occurs in healthy people,suggesting that it does not cause harm to a person's physical health,it is true that alopecia influences their life quality due to the anxiety and lack of self-confidence caused,especially in childhood.There are several forms of hereditary human hair loss,such as androgenetic alopecia(AGA),alopecia universalis(AU),and alopecia areata(AA).In these conditions,dysregulation of the cycle of HFs may occur due to the loss of normal epithelial-mesenchymal interactions caused by hereditary disorders.Hair follicles(HFs)-epithelial appendage begins to form during embryogenesis,and undergoes cycles of growth(anagen),apoptosis-mediated regression(catagen),and relative quiescence(telogen).Hair cycling patterns are complex and change over time,and regulated by cell signaling transduction networks.Currently,the major issues are how to obtain extrafollicular factors regulating hair growth.Laboratory mouse has held up as a useful species for mechanistic and preclinical studies.The HR protein which interacts with vitamin D receptor(VDR)and histone deacetylase(HDAC)family members is a nuclear receptor corepressor that functions in hair cycling by regulating gene expression.Hairless(HR)protein is essential for skin homeostasis,since both humans and mice lacking HR activity suffer from congenital hair loss and defects in epidermal proliferation and differentiation.We especially aim to elucidate which signaling molecules regulated by HR are responsible for hair cycle and hair shaft differentiation.In our previous study,YYHL was bred,and Transcription activator-like effector nucleases(TALENs)was used to disrupt the mouse Hr locus,creating heritable mutations that eliminate Hr function to explore effects of Hr on hair development.In the present study,full length cDNAs of hairless gene(Hr)were cloned and a point mutation at 3247(G?A)was introduced to study the function of Hr.The expression plasmid which was constructed by Gateway clone technology was microinjected into the fertilized eggs from C57BL/6 mice.The Hr transgenic mice were generated by microinjection method and the genotype of gene was identified by PCR.The heterozygote mice were gradually eliminated by intercrossing the descendants.Of 26 pups recovered,4(referred to as mice#3,#8,#12,and#32)were found to contain the mutant Hr gene in their chromosomal DNA using PCR analysis(Fig.1B).Seven independent lines were subsequently established by mating with C57BL/6 strains,but only three lines(TG3,TG8,and TG12)were successfully constructed.The other lines could not be established due to death or infertility.The mutant mice grew a first coat of hair and appeared normal until approximately 12 days after birth.Subsequently,hair loss began around the eyelid and progressed caudally,resulting in a completely hairless condition within two weeks with the exception of the vibrissae.Transgenic mice exhibited a pattern of hair loss and regrowth.The striking difference in the expression of Hr in the skin clearly showed that the patterns of hair loss can be deeply affected by its expression level.The mutant Hr expression was significantly increased during hair loss,after which it began to decline when hair regrew.HR protein changes were associated with the alterations at the mRNA levels.It seemed that the mutant Hr gene might have an inhibitory effect on the expression of the endogenous Hr gene.The phenotype of transgenic mice does appear to be dependent on the expression level of Hr.Elevating BMP signaling might have suppressed the canonical Wnt and SHH signalingTo understand which regional differences in signaling mechanisms are key to the uncommon phenotype of model mice,we used microarray analysis to investigate the genes whose expression was specifically affected by the mutation Hr due to the functions of HR as a transcriptional corepressor.We determined the time point at which hair loss first occurred to(P14)in YYHL,KO and TG mice and regrowth(P35)in TG mice to elucidate the events underlying the morphological changes.Using 45,281 mouse probes,we detected differential expression in 2027 and 530 at P14 and P35,respectively in the transgenic skins compared with those of wild-type mice.There were 1839 and 1816 genes expressed differently in YYHL and KO skins versus those of wild-type mice,respectively.The differential expression of BMP2,?-catenin,and Foxel was validated at the transcriptional levels by qRT-PCR assays with mRNAs originally used as templates in the microarray analysis.Pathway analysis showed that elevating BMP signaling might have suppressed the canonical Wnt signaling with a functional change and/or loss of Hr when hair loss began at P14,whereas the results were reversed in transgenic skin at P35 when hair regrew.Immunohistochemical staining showed that the genes expressed differently that were associated with BMP and Wnt signaling pathway were critical to the unusual phenotype.The ultrastructure of the dorsal skin in mutant mouse was also compared with that of wild-type mice.The results showed that in the peripheral part of the cysts,the cytoplasm of the wall epithelial cells that occupied the same position as the initial follicular bulb was filled with lipid droplets when hair loss began.The present results showed that progenitors are unable to adopt the fate of hair keratinocytes and instead,differentiate into epidermis and sebaceous glands in the absence of HR.We show that BMP signaling suppresses WNT signaling,which lead to the disrupted crosstalk of gene network ruling hair growth and cycling with a functional change and/or loss of HR.Conversely,HFs regrowth is induced when epithelial Wnt signaling is activated due to a decline in BMP signaling in the gain of HR function.