Study Of β-catenin On Proliferation And Differentiation Of Hair Follicle Stem Cells

Hair follicle is one of the important appendages in skin with a complex structure, including outer root sheath (ORS),inner root sheath (IRS)and hair shaft. Recently people confirmed that a repository of progenitor cell was exist in the bulge region, specialized portion in the outer root sheath of upper hair follicle, which is located below the opening of the sebaceous duct and serves as the insertion site of arrector pili muscle. A great deal of investigation revealed that the stem cells resided in the bulge area (hair follicle stem cells, HFSCs) had a proven role in hair follicle cycling and organ formation. Moreover, the bulge, with pluripotent differentiation potential, is able to not only migrate downwards to become the matrix keratinocytes of bulb, but also migrate upwards to maintain the epidermis and involve in the formation of hair follicles and sebaceous gland.In previous studies of both mouse and human skin, the HFSCs have been observed with superior clonogenicity and proliferative capacity on stimulation. So HFSCs were though as the greatest proliferative potential stem cells in skin and acted as an essential cell resource of epidermis, hair follicle and sebaceous gland. But up to now, the exact characteristic of cells from bulge region has not been examined in detail. In this report, we described a modified method to harvest intact hair follicles. The bulge, microdissected on the basis of morphologic feature, were cultured and passaged successfully, which promoted the further study on the biological characterization of hair follicle stem cells.β-catenin is a cytosolic protein with dual functions, belonging to the armadillo family proteins. One is as a structural component of adherens junction, which forms the cadherin-catenin complex to mediate intercellular adhesion. The other is as a downstream effector of Wnt signaling pathway, which controls essential steps in many aspects of embryogenesis and tumorigenesis. Binding of wnt proteins to their receptors will activates the signaling pathway and causes stabilization of cytoplamicβ-catenin. Accumulatedβ-catenin may translocate into the nucleus and co-activated with Lef1, where it serves as a transcriptional factor to activate various target genes. Recently, investigator found that β-catenin was required for the initiation of hair follicle development. Whenβ-catenin is mutated during embryogenesis, formation of the hair follicles is blocked. Meanwhile, dermal papilla (DP) cells have the inductive activity to promote differentiation of HFSCs. Andβ-catenin, as regulative factor, is also essential for fate decisions of skin stem cells. In order to make it clear about the roles ofβ-catenin on the proliferation and differentiation of hair follicle stem cells, this research was designed from follow aspects: The expression ofβ-catenin and its related protein during hair cycle; HFSCs were cultured in vitro and the biological feature was observed; The translocation expression ofβ-catenin in HFSCs in vitro and the relationship between Lef1 and nuclear translocation; The effect ofβ-catenin on cell proliferation of HFSCs, involving cyclin D1 and COX2 expression; The role ofβ-catenin on the differentiation of HFSCs induced by DP cells; Also, the mixtures of HFSCs and DP cells were transplanted into nude mice, the related factor were detected in the hair follicle-like structures.The main results are listed following:1. After the hair follicle cycles of wistar rats were definited, IHC was used to detectβ-catenin and its related protein expression in different phages of hair follicle cycles. The results suggested that: (1) The hair follicle cycle of wistar rats were last 28d, composed of anagen (1-18d),catagen (19-22d) and telogen (23-28d).β-catenin was strongly expressed in anagen, while weakly in catagen and telogen,displaying an great correlation between expression ofβ-catenin and the cycle maintance. (2) The different expression ofβ-catenin in the development of hair follicle indicated thatβ-catenin might play a possible role in the formation and development of hair follicles, meanwhile the negative expression of DKK4 implied that Wnt signaling pathway was activited. (3) E-cadherin andα-catenin displayed a cyclic expression in hair cycle, coincidence with that ofβ-catenin. (4) Lef1 promoted hair growth, functioning as transcription factor ofβ-catenin.2. After a modified method to harvest HFSCs was establish successfully,we detect the translocation expression ofβ-catenin in HFSCs in culture, and discuss the relationship between Lef1 andβ-catenin in the translocation mechanism. The results suggested that: (1) The HFSCs were obtained from primary culture of bulge region. (2) A translocation expression ofβ-catenin was detected in the cells, companied with E-cadherin translocated into nuclear, but no obviously change inα-catenin expression. (3) Lef1 can promote β-catenin immigration into nuclear. (4) Translocation ofβ-catenin maybe related to the cell proliferation of HFSCs.3. The effect ofβ-catenin on cell proliferation and the related mechanism were discussed by methods of transfection, MTT assay, RT-PCR and Western blot. The results suggested that: (1) LiCl promoted cell proliferation and decreased the adhersion of HFSCs by enhancingβ-catenin level. (2) Compared with control, the ability of proliferation increased after the cells transfected withβ-catenin. (3) RNAi inhibits proliferative ability of HFSCs by decreasing expression ofβ-catenin. (4) Up-regulatedβ-catenin promoted the expression of Lef1, cyclin D1 and COX2, while down-regulatedβ-catenin was not. The interaction betweenβ-catenin and Lef1 were detected by luciferase report assay, indicating thatβ-catenin play role on cell proliferation by binding with Lef1 and related to cyclinD1 and COX2 activity.4. The differentiate potency of HFSCs induced by DP cells were observed in vivo and in vitro, and the role ofβ-catenin and its target gene on the process of orient differentiation were discussed. The results suggested that: (1) DP cell have the ability to induce HFSCs differentiation in vitro, and promote hair regeneration after co-transplanting DP cells and HFSCs in nude mice. (2)β-catenin is an important regulated factor to impact on cell differentiation; RNAi inhibits the hair follicle regeneration. (3) DP cells could stimulate the expression of c-myc andβ-catenin, that meantβ-catenin regulate cell differentiation byβ-catenin/Lef1/c-myc signaling pathway.In conclusion, the research first studied the expression rules ofβ-catenin and its related protein in hair follicle. Then, on the base of HFSCs obtained successfully in vitro, we focused on the effect ofβ-catenin on cell proliferation and orient differentiation induced by DP cells, including its related signaling molecules. This research intends to provide a theoretical ground in implications for hair growth, tissue engineering and cell therapy.