Expression Profile And Function Of Frizzled4in Hair Follicle Keratinocytes

Hair follicle is an important skin appendages with a complex structure,includingepithelial part and dermal part. The epithelial part contains hair matrix, outer root sheathand inner root sheath. The dermal part contains dermal papilla and connective tissue sheath.Hair follicle contains over twenty cell types, such as keratinocyte, dermal papilla cell,melanocyte and Langerhans cell. The development, growth and periodic variation of hairfollicle is achieved by the interaction of epithelium and dermis. Hair follicles keep ongrowth and self-renewal by periodic hair cycle that consists of three phases: anagen,catagen and telogen. The hair cycle is strictly regulated by various growth factors,hormones, signal molecules and signaling pathways, such as Wnt, Bmp, Shh and Notch, ofwhich Wnt pathway is one of the most important pathways.The Wnt protein binds to theFrizzled receptor to active the downstream signaling and target gen. Localization of Wntreceptor in hair follicles reflects the Wnt signal that acts on it, thus Frizzled receptors alsoplay an important role in the cyclical variation in the process. The Frizzled family containsat least10members, but the research on them is not clear. There is research about Frizzled1、2、3、5、6、7、10in anagen but it does not further study the changes in the hair folliclecycle，and studies have shown that Frizzled4was expressed on melanocyte stem cells, butwhether Frizzled4erxpresss in the keratinocytes of hair follicle or not has not been reportedObjectives:In order to study the expression and roles of Frizzled4in keratinocytes during hairfollicle cycle.Methods:Firstly, the expression profile of Frizzled4was observed during the postnatal hair cycle(P1d, p3d, p8d, p16d, p21) by using the Immunofluorescence, Q-PCR and Western blot, inorder to provide morphological evidence for revealing the relation between Frizzled4and hair follicle keratinocytes. Secondly, JB6cl30-7b cells were used as the experimental objectin order to study the function in vitro.The expression of Frizzled4protein in JB6cl30-7bcells was detected by immunofluorscece technique and the epithelial cell proliferation wasintervened by anti-Frizzled4antibody with MTT and BrdU labeling method. Finally,Plucking model of mice was established to induce hair follicle regeneration,immunofluorescence was used to test expression of Frizzled4in the regeneration of hairfollicle cycle (after plucked P5d, P10d, P20d and P25d).Results:1. Expression Profile of Frizzled4during Hair Follicle Cycle1). Localization of Frizzled4protein was detected by immunofluorescence test and theresult showed that the expression of Frizzled4protein was in hair follicle keratinocytes andchanged with hair cycle. The expression of Frizzled4protein was detected mainly in Bulge,out root sheath (ORS), lateral hair bulb, inner rootsheath (IRS) and precortex hair follicle.As hair follicles grew, the expression level gradually increased. At catagen, expression ofFrizzled4was decreased significantly and mainly located in Bulge while little in out rootsheath (ORS) and inner root sheath (IRS).At telogen, expression of Frizzled4was in lowestlevel and restricted in Bulge.2). Protein of Frizzled4was detected in hair follicle by Western blot. The resultsshowed that Frizzled4protein at P1d mouse skin was obvious, on the third day theexpression of Frizzled4protein was significantly increased, compared with P1d theexpression of Frizzled4protein in P8d continued to rise. At catagen, Frizzled4proteinsignificantly decreased. At telogen, the expression of Frizzled4protein was weak,significantly lower than that of P1d, P3d, P8d, P16d.3). Q-PCR and semiquantitative RT-PCR were showed the same expression tendencyof Frizzled4mRNA and protein, the most in anagen, less in catagen, lowest in telogen.2. Effects of Frizzled4on the proliferation of keratinocytes in hair follicle at thecellar level1). Expression of Frizzled4protein was detected by immunofluorescence and resultshowed that the Frizzled4were expressed on the membrane of JB6cl30-7b cell.2).5ug/mL,10ug/mL and20ug/mL anti-Frizzled4antibody were added in culture ofJB6cl30-7b with the same concentration of IgG as the experimental group, the absorbance value with MTT was detected. MTT results showed that: compared with the control group,the absorbance of5ug/mLgroup was not significantly change, while the10ug/mL groupsignificantly decreased, and the20ug/mL group decrease to a minimum. The resultsshowed that the anti-Frizzled4antibody made proliferation of JB6cl30-7b decreased.Withthe increase of concentration, the inhibition of proliferation was more obvious.3). The effect of anti-Frizzled4antibody on the proliferation of JB6cl30-7b was alsoevaluated by BrdU.Results showed anti-Frizzled4antibody also lowered the JB6cl30-7bcells proliferation.3. The expression of Frizzled4in regenerated hair follicles induced by externalfactorsThe expression of Frizzled4protein in hair follicle regeneration induced by externalfactors was consistent with hair follicle growth and also consistent with the expression ofnatural cycle.Conclusions:This study is the first to examine the expression of Frizzled4protein in thekeratinocytes during murine hair follicle cycle. We explored its role in murine hair folliclekeratinocytes in vitro and in vivo, which paves the way to continue in-depth study todetermine combination of Frizzled4and Wnt protein and its downstream signalingpathways, also providing a theoretical basis for the control of hair growth and hairregeneration.