The Role Of Wnt5a In Regulating Hair Follicle Differentiation

Hair follicle-related diseases including polytrichosis,alopecia and pseudofolliculitis barbae have been increasingly emerged as the pace of people’s life is speeding up and their living stress is also getting higher.This largely influences one’s daily life and even mental health.So,investigation on hair follicle development,growth and cycling is significant.Hair follicle is one of the most important appendages of the skin.According to the different morphological changes of hair follicles,during hair cycling,hair follicles can be divided into three stages:Anagen,Catagen and Telogen.Hair follicle is a mini-organ that consists of complex but well-organized structures,which are differentiated from hair follicle progenitor or stem cells.Hair follicle consists of two main components including epithelia and mesenchyme.The epithelia are composed of inner root sheath,outer root sheath,hair shaft,hair matrix at the hair bulb and hair follicle stem cells which locate at the bulge region.The mesenchyme is composed of dermal sheath and dermal papilla(DP),a group of mesenchymal cell condensate engulfed by the epithelial matrix cells.DP plays a crucial role in hair follicle development,growth and regeneration.Known studies show that there are many similarities between hair follicle development and regeneration.Thus,investigating embryonic hair follicle development is useful for treating hair follicle-related diseases,such as alopecia.However,during hair cycling,the mechanism by which DP regulates epithelial-mesenchymal interactions remains unknown.The Wnt family as the secreted glycoproteins family includes 19 Wnt members.These 19 Wnt ligands have been discovered in human and mouse tissues.Wnt signaling emerges as one of the earliest and the most-important pathways in development of many tissues.These Wnt ligands are classified into mediate canonical Wnt signaling pathway or non-canonical Wnt signaling pathway,both of which exert extensive physiological roles in governing cellular behaviors through an autocrine or paracrine mechanism during hair follicle development.As a representative of the non-canonical Wnt signaling pathway,Wnt5a plays a key role in regulating cell behaviors in many tissues,such as cell proliferation,differentiation,migration,and polarity.Previous studies showed that,during hair follicle development,Wnt5a is first expressed in the mesenchymal cells,then in dermal condensation and dermal papilla.During anagen phase,Wnt5a is expressed in the inner root sheath and peak at mid-anagen.These studies indicate that Wnt5a widely functions in various cell types and at different time points during hair follicle development.In addition,the dermal papilla plays an important role in stimulating activation of hair follicle stem cells,which generate the transient amplifying cells that differentiate into the mature epithelial components of the hair follicle.These indicate that Wnt5a may regulate hair follicle development through dermal papilla and epithelia interaction.Moreover,our group has investigated a new gene GasderminA3(Gsdma3),which influences hair follicle differentiation through regulating hair cuticle formation.We observed an apparent co-localization of Wnt5a and Gsdma3 in the epithelia but not in the mesenchyme of the hair follicle.It is known that epithelial-mesenchymal interaction is required for hair follicle development and regeneration.Thus,it is tempting to speculate that there might be a regulation mechanism between Wnt5a and Gsdma3 in controlling hair follicle differentiation.To understand the mechanism by which Wnt5a regulates cell differentiation,in the present study,we first observed the expression of Wnt5a in the embryonic developing hair follicles.Second,we established the mouse immortalized DP cell line.By applying adenovirus-mediated overexpression of Wnt5a to the isolated dermal papilla cells,we evaluated the downstream targets of Wnt5a that mediate the non-canonical Wnt signaling in the DP cells and its related molecular mechanism.Last,we compared the expression of Wnt5a and Gsdma3 in the hair follicle at postnatal stages.Because of the similar expression pattern of Wnt5a and Gsdma3 in the hair follicle,we further evaluated Wnt5a expression in wild type C57BL/6J mice and Gsdma3 mutation mice in the hair follicle of dorsal skin in mice by immunohistochemistry and RT-PCR.The main results are as follows:(1)The expression of Wnt5a was detected during embryonic hair development by immunofluorescence,RT-PCR,Western Blot and whole-mount in situ hybridization.The results show that Wnt5a is expressed in the mesenchymal components,particularly in the dermal condensation(DC)of the developing hair follicle.The expression of Wnt5a is consistently increasing from embryonic day 14.5(E14.5)to E19.5.(2)We then set up a stable dermal papilla culture system,to establish an immortalized dermal papilla cell line.We overexpressed Wnt5a by applying Wnt5a-expressing adenovirus(AdWnt5a)into the DP cells passaged for 1 time,to test the expression of genes involved in Wnt signaling pathway.Real-time PCR results show that the mRNA expression of Wnt pathway receptors including Fzds 1,2,3,4,6,7,8,9,and 10,Lrps 5 and 6 are not significantly changed in the AdWnt5a-treated group,compared to the AdGFP-treated group 24h and 48h after culture.This indicates that Wnt5a cannot increase the expression of frizzled receptors in cultured DP cells.Intriguingly,RT-PCR and its analysis show that the expression of key genes in canonical Wnt signaling pathway such asβ-catenin and Lef1 is significantly decreased in DP cells after AdWnt5a treatment,when compared to the control group.Overexpression of Wnt5a significantly decreased P53 expression but didn’t influence P21 expression.In addition,overexpression of Wnt5a results in significantly decreased expression of cell cycle genes such as CyclinB1 and CDK1 expression in DP cells,when compared to the AdGFP control group.These data suggest that the non-canonical Wnt5a may antagonize the canonical Wnt signaling pathway to regulate cell cycle-related genes,which regulate the dermal papilla cell behavior that then functions on controlling hair follicle differentiation through epithelial-mesenchymal interation.(3)We harvested skin from WT mice and Gsdma3-mutant mice at the full anagen phase at P8.RT-PCR and its analysis show that the mRNA expression of Wnt5a is significantly decreased in the Gsdma3-mutant mice when compared to the WT mice.Immunohistochemistry staining shows that Wnt5a is decreased in its expression to the IRS,ORS and hair matrix regions of the hair follicle in Gsdma3-mutant mice,compared to that of the WT mice.To examine if Gsdma3 regulates Wnt5a in epithelial cells,we constructed Gsdma3 overexpression plasmids and transfected them into the cultured JB6 cells.Green fluorescence shows successful transfection of Gsdma3-expressing plasmid(Gsdma3~+)and the control plasmid(Gsdma3~-)into JB6 cells 24h after culture.At this time point,RT-PCR and its analysis show that Wnt5a expression is significantly increased in Gsdma3-overexpressed JB6 cells,when compared to the control group.(4)Taking together,our findings demonstrate that Gsdma3 promotes hair follicle differentiation by positively regulating Wnt5a expression in the epithelia of the hair follicle.Epithelia-derived Wnt5a further influences DP cells which govern the adjacent epithelial transient amplifying cells to differentiate into the hair shaft and IRS.How the molecular change of DP cells influences DP-epithelial interaction remains further investigation.Our study provides new clues not only for the relationship between canonical and non-canonical Wnt signaling pathways in regulating hair follicle growth,but also enhances the mechanism of epithelial-mesenchymal interactions in governing hair follicle differentiation.