A Study Of Wnt5a On Inhibition Of Growth Of Hair Follicles In Vitro And In Vivo

Hair follicle is one of the important appendages in skin with a complex structure, including epithelial part (matrix and concentrical multiple layers structures) and dermal part (dermal papilla and connective tissue sheath). It contains over twenty cell types, such as keratinocyte, melanocyte, Langerhans cell and dermal papilla cell. All these cells participate together in regulation of development, growth, differentiation and hair cycle through their complex interaction. Hair follicles keep on growth and self-renewal by periodic hair cycle which consists of three phases: growth (anagen), regression (catagen) and rest (telogen). During each anagen phase, follicles produce an entire hair shaft from tip to root; during catagen and telogen, follicles reset and prepare their stem cells so that they can receive the signal to start the next growth phase and make the new hair shaft. The hair cycle is strictly regulated by various growth factors, hormones, signal molecules and signaling pathways, such as Wnt, Bmp, Shh, of which canonical Wnt/β-catenin pathway is one of the most important pathways. Up to now, 19 Wnt members have been identified in mice and humans. They are classified into two groups: canonical Wnts and non-canonical Wnts, which activate different signaling pathways, namely the canonical Wnt/β-catenin signaling pathway and the non-canonical Wnt signaling pathway, respectively. Several Wnts exist simultaneously during development and growth of hair follicle in murine. However, whether the effects of noncanonical and canonical Wnts in these processes are similar or not is still largely unknown.As a typical noncanonical Wnt, Wnt5a(wingless-type MMTV integration site family, member 5A) has an important role in regulation of cell proliferation, differentiation, migration, movement and cell polarity. Wnt5a could activate canonical Wnt/β-catenin pathway and several noncanonical Wnt pathway, including Wnt/Ca2+ pathway, PCP pathway, Wnt/Ror2 pathway, and so on. Recently, it was shown that Wnt5a could inhibit the activation of canonical Wnt/β-catenin pathway through promoting degradation ofβ-catenin. In order to make it clear about the roles of Wnt5a on the regulation of hair cycle, this research was designed from follow aspects: Firstly, the expression profile of Wnt5a was observed during the postnatal hair cycle, using the whole-mount in situ hybridization, immunohistochemistry staining, semiquantitative RT-PCR and Western blot, in order to provide morphological evidence for revealing the relation of Wnt5a and hair cycle. Subsequently, using adenovirus in vibrissae hair follicles culture in vitro, the effect of Wnt5a on the growth of hair follicles was investigated. Lastly, through the overexpression of Wnt5a topically in synchronized hair follicle, the influence of Wnt5a on the growth of hair follicles in vivo was studied, using gene chip assay, quantitative RT-PCR and immunohistochemistry staining. The main results are listed following:1. The expression of Wnt5a in hair cycle in murine1) The expression of Wnt5a mRNA during hair cycle was examined by using whole-mount back skin in situ hybridization. Results show:①In anagen, the positive signal of Wnt5a mRNA was highly detected in hair bulb, including matrix, IRS and DP, especially in matrix cells, displaying a correlation between expression of Wnt5a and the growth of hair follicle.②In catagen, the positive signal reduced in hair bulb which has undergone apoptosis.③In telogen, weak Wnt5a mRNA signal only was detected in DP.2) By immunohistochemistry staining, we observed that:①The expression of Wnt5a protein was detected broadly in anagen hair follicle, including matrix, IRS, DP, precortex cells and ORS. As hair follicles grew, the expression level gradually increased. The distribution of Wnt5a protein was more dispersed than its mRNA, which suggested that Wnt5a may have a role in hair follicle in autocrine and paracrine manner.②In catagen, positive cells in epithelial strand decreased.③In telogen, weak Wnt5a protein was observed in bulge region, secondary hair germ and DP.3) Using semiquantitative RT-PCR and Western blot, we observed the same expression tendency of Wnt5a mRNA and protein.2. The effect of Wnt5a on the growth of hair follicle in vitro1) By immunohistochemistry staining, we examined Wnt5a protein immunolocalization in whisker follicles of three hair cycle stages. Positive cells were detected in multiple layers of whisker follicles in anagen, including matrix, IRS, ORS, precortex cells, DP, with the highest expression levels in anagen, lower in catagen and the lowest in telogen, which was in accorded with the profile of pelage.2) Using whisker follicles in in vitro organ culture, we detected that the elongation length of hair shaft of the cultured hair follicle treated with Wnt5a adenovirus was shortened than control group, which suggested Wnt5a could inhibit the growth of hair follicles in vitro.3. Further identifying the inhibition influence of Wnt5a on the growth of hair follicles in vivo1) The hair plucking model was adopted. After an intradermal inject of adenovirus Wnt5a, there was a depilous region. HE staining displayed the injected region had telogen hair follicle morphology surrounded by anagen hair follicles shape, which revealed Wnt5a repressed the growth of hair follicle induced by plucking.2) Through mouse whole genome chip and quantitative RT-PCR, expression level of several genes changed remarkably compared with control, namely decreasedβ-catenin and increased Rac2, which suggested Wnt5a may activate Rac2 and lead to inhibitβ-catenin.3) Treated with Ad-SimBC group had similar phenotype and HE staining changes with Ad-Wnt5a group, which revealed they may be involved in similar molecular mechanism.4) Using immunohistochemistry staining in Ad-SimBC group and Ad-Wnt5a group, we observed that the expression ofβ-catenin, Lef1, Dvl, Ki67 and AE15 protein dereased.In conclusion, the research first studied the expression rules of Wnt5a in hair cycle. Then, we focused on the regulation effect and mechanism of Wnt5a on the growth of hair follicles in vitro and in vivo, respectively. This research intends to provide a theoretical ground in implications for hair growth, skin tissue engineering and an experiment basement for further investigating the roles of different Wnt signals on the hair cycle.