The Experimental Study Of The Molecular Regulation Mechanism Of Wnt10b On The Human Hair Follicle

BackgroundThe structure and cell component of hair follicle is the most complex miniorgan of the skin and one of the defining features of the human body. The most obvious function of hair follicle is to produce the hair shaft, which exerts a wide range of function including physical protection, thermal insulation, camouflage, dispersion of sweat and sebum, sensory and tactile functions, and social interactions. It represents a prototypic neuroectodermal-mesodermal interaction system that is provided by three different stem cell sources:epithelial, neural crest and mesenchymal. Depending on their origin, these stem cells differentiate into distinct epithelial lineages (sebocytes and keratinocytes of the hair matrix, which go on to develop into outer and inner root sheath cells, or into trichocytes), or mesenchymal lineages, such as specialized inductive fibroblasts of the follicular dermal papilla and the hair foliccle connective tissue sheath. Others (neural crest cells), in turn, give rise to melanocytes of the hair follicle pigmentary unit.Increased evidences suggest that the initiation of a new growth phase in the postnatal hair follicles and the hair follicle morphogenesis and development show many similar features. Many growth factors and their receptors that are involved in signaling exchanges between the epithelium and the mesenchyme during hair follicle development also regulate the cyclic activity of postnatal hair follicles. So the molecular principles of hair follicle induction and morphogenesis are also suitable for hair renewal.The formation of hair follicles occurs during embryogenesis and relies on a series of signals that cause fate changes between dermal cells and overlying surface epithelial cells, ultimately resulting in differentiation of the inner root sheath, outer root sheath, hair shaft, and dermal papilla. These consist of secreted molecules and changes in the expression of cognate receptors, topobiological alterations in the expression of key adhesion molecules and integrins and underlie epigenetic controls.The evolutionary ancestry of these signaling partners can be traced back millions of years to molecules like Wnt/wingless and members of the Sonic hedgehog family of secreted glycoproteins, which govern the development of simple organisms such as Drosophila. In addition, many other key regulatory molecules involved in proliferation, morphogenesis and patterning during embryonic development of metazoan organisms are also re-utilized in the control of hair follicle development, such as members of the TGF-β/BMP and FGF families, and even TNF family members-Eda/Edar.Among which the most important signal is Wnt/wingless family. Many researchers have demonstrated by far that the Wnt signaling operates as the key stimulator of the hair follicle morphogenesis and development through multiple knockout and transgenic mouse models.Wnts is alarge family of secreted signaling molecules with homology to the fly Wingless Protein that activate cell-surface receptor-mediated signal transduction pathways to regulate a variety of cellular activities,including cell fate determination, proliferation, migration,polarity, and gene expression. Thes proteins are heavily involved in morphogenesis during embryogenesis, as well as in the regeneration of various adult tissues including skin and hair follicles. The binding of Wnt proteins to their receptors, frizzled (FZ) proteins, and low-density lipoprotein receptor-related protein (LRP) family members, results in activation of a conserved "canonical" signaling pathway. Cytoplasmic β-catenin is known to translocate to the nucleus and forms active transcription complexes with members of the T cell factor/lymphoid enhancer factor(TCF/LEF-1) family.By controlling the complex formation with TCF/LEF-1transcription factors, Wnt proteins play fundamental roles in various cells and tissues.The canonical Wnt/β-catenin signaling pathway provides the critical switch for hair follicle fate, as ectopic epithelial expression of the secreted Wnt inhibitor DKK1or lack of epidermal P-catenin expression results in the absence of hair follicle induction. Conversely, the over expression of the β-catenin stabilized form causes the strongly enhanced placode formation, due to the epidermal keratinocytes globally adopt a hair follicle fate. Wnt signaling also have very important mediative function in the postnatal hair shaft growth. Millar et.al demonstrated that a role for Wnt signaling in hair shaft formation is supported by the observations that Wnt3and DVL2are expressed in hair shaft precursor cells and that ectopic expression of Wnt3in the outer root sheath disrupts hair shaft differentiation. Kishimoto et.al showed that the freshly isolated dermal papilla cells are capable of inducing epithelial cells to form a new hair follicle, but lose this property when maintained in culture. In the presence of cononical Wnt proteins, the inductive abilities of cultured dermal papilla cells are retained, suggesting that, in vivo, Wnt signals from the follicular epithelium act to maintain the function of the dermal papilla.However, up to this date, there are19known family members in humans and mice, and it is still unkown exactly which member of the Wnt family is responsible for the postnatal human hair follicle growth regulation and whether it arises intradermally or intraepidermally. Seshamma Reddy et.al had been carried out a comprehensive survey of all currently identified mouse Wnt genes expression in embryonic and postnatal skin and been idendified that Wnt10b and Wnt5a, whose expression were significantly upregulated in hair follicles at early stage of hair follicle morphogenesis and development and was also specifically expressed in the postnatal hair follicles at the onset of a new cycle of hair growth. Furthermore, other researches showed that Wnt10b promoted the differentiation of the primary skin epithelial cells toward to hair shaft and inner root sheath of the hair follicle cells in vitro, elongation of the hair shaft in hair follicle organic cultures, and induction of hair follicle formation in the cultured embryonic skin tussue, whereas Wnt3a,5a and11did not elicit such actions. Therefore, WntlOb is a strong candidate for the accumulation of β-catenin in the epithelium and mesenchyme cells, and then transfer into the nucleus to initiate the hair follicle morphogenesis and development. Progress in this area of research may ultimately permit the development of novel and effective therapies for the human hair follicle disorders.In conclusion, WntlOb was shown to play various roles in a wide range of biological actions, such as adipogenesis, bone formation, tumor development. In the formation of embryogenesis hair follicle placode stage, WntlOb is one of the earliest and most prominent expression marker, indicated that Wnt10b signal can promote the differentiation of the epithelium cells and the earlier hair follicle development. Meanwhile, WntlOb also can promote the differentiation of the primary skin epithelial cells toward to hair shaft and inner root sheath of the hair follicle cells in vitro. However, there is still not evidence show that whether Wnt10b expresses in the human hair follicle, if it exists, how can WntlOb mediate the human hair follicle? Such as human dermal papilla and the growth of hair shaft, whether WntlOb have positive or negative influences on dermal papilla cells and hair shaft? In this experiment research, we had discussed and studied the mediation molecular mechanism of the Wnt10b on the human hair follicle growth. First of all, we identified the expression of Wnt3a、Wnt5a、WntlOb and the canonical Wnt signal pathway downstream effector proteins β-catenin and LEF-1in the anagen human hair follicle, and then detected the expression of specific marker molecules and cell proliferation ability of long-term cultured human dermal papilla cells in vitro. After that, we studied the effects of Wnt10b on the hair follicle induction and proliferation of long-term cultured human dermal papilla cells (HDPCs) in vitro. At last, we discussed the impact and molecular regulation mechanism of Wnt10b on the human hair follicle shaft growth.Chapter1:The expression of Wnt3a、Wnt5a、Wnt10b、β-catenin and LEF-1in the human hair follicleObjectiveTo identify the expression of Wnt3a、Wnt5a、Wnt10b and the canonical Wnt signal pathway downstream effector proteins β-catenin and LEF-1in the anagen human hair follicle.MethodsThe healthy scalp with intact hair follicles came from the face-lift plastic surgery. After processed the scalp through paraffin embedding and cut into slices, the immunohistochemical staining method was exerted to observed the expression of Wnt3a、Wnt5a、Wnt10b and the canonical Wnt signal pathway downstream effector proteins β-catenin and LEF-1in the anagen human hair follicle.ResultsWnt3a、Wnt5a and Wnt10b were expressed in the human hair follicle at the stage of anagen. The experiment showed that Wnt3a can express in the outer root sheath, inner root sheath, hair matrix and hair shaft precursor cells, and there was weak expression in the hair dermal papilla; but the distribution of Wnt5a was limited than Wnt3a’s, only very weak positive expression can be seen in the hair matrix, hair shaft precursor cells and inner root sheath. The expression location of WntlOb in the anagen human hair follicle was similar to Wnt3a, but is the most prominent. The canonical Wnt signal pathway downstream effector proteins β-catenin and LEF-1can also find express in the anagen human hair follicle. The expression pattern ofα-catenin and LEF-1were almost the same, but in the dermal papilla, the expression of LEF-1was stronger than α-catenin.ConlusionWnt3a、Wnt5a and Wnt10b can be observed to expresse in the human hair follicle at the stage of anagen. The expression of Wnt10b is the most significant, strong level of Wnt10d distributed in the inner root sheath, outer root sheath, hair matrix cells and hair shaft precursor cells of the hair follicle, in dermal papilla also had positive expression. The canonical Wnt signal pathway downstream effector proteins β-catenin and LEF-1can also find express in hair matrix and hair shaft precursor cells of the anagen human hair follicle, indicated that WntlOb may be the most important member of the Wnt family to maintain and mediate the growth of the hair follicle in anagen through the canonical Wnt/β-catenin signal pathway.Chapter2:The analysis of the expression of specific marker molecules and cellproliferation of long-term cultured human dermal papilla cells in vitroObjectiveTo investigate the expression of specific marker molecules and cell proliferation oflong-term cultured human dermal papilla cells in vitro.MethodsAfter dissected and cultured the human hair follicle dermal papilla cells in vitro, the cells of passages1to8were used for experiments. To detect the expression of specific marker molecules of long-term cultured human hair follicle dermal papilla cells, the alkaline phosphatase activity of the HDPCs was examed, and the specific genes alkaline phosphatase and insulin like growth factor-1expression of HDPCs were determined by Real-time quantitative PCR. The proliferation of cultured human dermal papilla cells in different passages were detected by Cell Counting Kit-8method.ResultsAfter long-term cultured in vitro, the ALP and IGF-1expression levels of HDPCs gradually decreased in different passages, as well as the display of the aggregated and cartouche growth and the cell proliferation ability. The ALP and IGF-1expression levels of HDPCs in passage1was the highest, they were almost about6.8-fold and3.5-fold higher than the HDPCs in passage8. The ALP staining of the HDPCs in passage1and passage2were evident, but in passage3～6, the cells’ ALP staining gradually became weaker after the long-term cultured in vitro; the ALP staining of passage7and passage8were far more weaker than the cells in the previous passages.ConclusionAfter long-term cultured in vitro, the specific marker molecules expression of the human hair follicle dermal papilla cells were decreased gradually, and lead to the loss of hair-inducing activity. Moreover, the ability of the human dermal papilla cells’ proliferation also deteriorated as the passages go on during the long-term cultured.Chapter3:The mechanism of Wnt10b to regulate and control the hair-inducingactivity and proliferation of long-term cultured human dermal papilla cellsObjectiveTo investigate the effects of Wnt10b on the hair follicle induction and proliferation oflong-term cultured human dermal papilla cells in vitro.Methods After dissected and cultured the human hair follicle dermal papilla cells (HDPCs) in vitro, the cells of passages8were used for experiments. The recombinant human Wnt10b(1μg/ml) was added to the medium and cultured with HDPCs. To evaluate the effects of WntlOb treatment, β-catenin expression and ALP activity of the HDPCs were examed, the growth character of cells were observed, and the genes expressions of HDPCs markers were determined by Real-time quantitative PCR. The effect of Wnt10b on the proliferation of the long-term cultured human dermal papilla cells was detected through Cell Counting Kit-8method.ResultsWntlOb had a significant effect on the HDPCs through Wnt/β-catenin signaling pathway, it can successfully retrieved the specific expression of HDPCs hair follicle induction marker molecules, such as ALP and insulin like growth factor-1(IGF-1), and recovered the aggregated and cartouche growth appearance in the long-term cultured HDPCs in vitro, also had an evident function to promote the proliferation ability of the HDPCs.ConclusionWnt10b can maintain the hair follicle-inducing ability of the long-term cultured HDPCs through the classic canonical Wnt/β-catenin signaling pathway in vitro, moreover, WntlOb had the ability to promote the proliferation of the long-term cultured dermal papilla cells.Chapter4:The preliminary study of function of Wnt10b on the human hair shaft growthObjectiveTo investigate the effect and the mechanism of WntlOb on the human hair shaft growth.Methods After dissected the single intact human hair follicle, the recombinant human Wnt10b(2μg/ml) was added into the hair follicle organ culture in the experimental group with DMEM medium. After3days changed the medium and readded same dose Wnt10b, keep in culture for5days in all, and then measured the hair shaft length with the Binocular stereoscopic dissecting microscope.ResultsThe human hair follicle cultured in the experimental group had more significantly growth speed, and the length of the hair shaft was longer than the control group.ConclusionWnt10b may induce the hair dermal papilla cells express β-catenin, stimulate the hair matix cells to proliferate and differetiate into inner root sheath and hair shaft, and then promote the growth of hair shaft.ConclusionOur results suggest that WntlOb can significantly express in the anagen human hair follicle, including outer root sheath, inner root sheath, hair matrix, hair shaft precursor cells and dermal papilla. Wnt10b can maintain the War follicle-inducing ability of the long-term cultured HDPCs through the classic canonical Wnt/β-catenin signaling pathway in vitro. Moreover, WntlOb had the ability to promote the proliferation of the long-term cultured dermal papilla cells and the hair shaft growth in the hair follicle organ culture.