| Chapter oneA study of Wnt and TGF-? signaling in the hair follicle tissues from patients with androgen alopeciaBackground and Objective:Androgenetic alopecia(AGA),also known as male pattern alopecia and seborrheic alopecia,is the most common type of hair loss in men.This continuous process results in a type of alopecia that follows a definite pattern in those individuals who are genetically predisposed.The prevalence of progressive AGA approaches 50%of Caucasian men beyond the age of 40 years and 71%of all the alopecia.Whereas in other races,the prevalence is lower and balding is less severe.A variety of factors are involved in the pathogenesis of AGA,including age,hormone,signaling molecules,cytokines and neuropeptides.Androgen plays a most important role among all factors.AGA is mediated by androgen which induces shortened hair cycle period due to the advance of the catagen with atrophy follicles and thin hair.Wnt and Transforming growth factor-?(TGF-?)signalings function in numerous developmental processes,and alterations of both signaling pathways are associated with common pathologic conditions,including AGA.The classic Wnt/?-catenin signal which is known as "the first dermal signal"regulates the development and regeneration of the hair follicle.Studies showed that androgen contributes to the pathogenesis of classic AGA through inhibiting Wnt/?-catenin signaling pathway.Dermal papilla cell(DPC)derived from AGA inhibited the keratinocyte growth and the potential transcription mediated by LEF/TCF.Activated Wnt signaling binds Frizzled on the cell membrane and starts a cascade.Activated DSH increases the activation of intracellular ?-catenin.The activated ?-catenin translocates to nuclear and has the mutual effect on N-terminal of LEF/TCF transcription factor and then binds with AR-DHT complexes to block the expression of downstream target genes mediated by ?-catenin.This cascade induces the inhibition of Wnt signaling,prevention of the platecode forming in hair follicles,hair lost after the first catagen and finally the appearance of alopecia.TGF-?signaling pathway is involved in the growth and development of hair follicles and hair cycle.A rescent Study found that follicles stopped differentiation after the first cycle of hair follicle wihtout Smad4.TGF-?2 dominates catagen cascade in AGA,other signaling molecules such as TGF-?1 and TGF-?3 also participates in catagen.Due to the difficulty of the source of donor samples,most of the studies on the Wnt and TGF-? signaling pathways related to the pathogenesis of AGA have been focused on in-vitro experiments.To investigate the expression of Wnt and TGF-?signaling and the possible crosstalk between these two signaling pathways in AGA,we analyzed the expression and activation of downstream molecules of Wnt and TGF-? signalings in both balding and non-balding hair follicles isolated from scalp specimens.It may further reveal the pathogenesis of AGA.Materials and methods:1,Specimen:AGA patients were from departments of dermatology and neurosurgery from September in 2013 to June in 2015.A total of 15 men aged between 22 and 47yr were involved in this study.Scalp specimens of the bald(frontal)area were collected from all 15 AGA patients,and specimens of the non-bald(occipital)area were collected from nine of them.The study was performed after the minimal-risk institutional review board approval.Patients meeting the following criteria were included in the study:1)positive diagnosis as AGA(Hamilton-Norwood classifications III-V anterior),and 2)exclusion of traumatic and inflammatory treatment.2,We evaluated the mRNA level of Wnt10a and LEF1,T?RI and T?RII in the 24 samples of scalp specimen by RT-PCR.3,We detected the protein expression of total TGF-?1 and the active TGF-?1 by ELISA.Results:1,Compared to the non-balding counterparts,the mRNA level of Wnt10a and LEF1 in balding hair follicles was decreased;2,Compared to the non-balding counterparts,the mRNA level of T?R? and T?R?and the protein expression of total TGF-?1 and the active TGF-?1 were elevated in balding hair follicles.Conclusion:The expression of Wnt related signaling molecules decreased in AGA patients,while the expression of TGF-? signaling molecules increased.Our results which showed a reserve expression in these two signaling pathways indicates that there may have an interaction between Wnt and TGF-? signalings.Chapter twoAn investigation of crosstalk between Wnt/?-catenin and transforming growth factor-? signaling in androgenetic alopeciaBackground and Objective:Androgenetic alopecia(AGA)is the most common alopecia.AGA is mediated by androgen which induces shortened hair cycle period due to the premature entry into catagen resulting in atrophy follicles and thin hair and even the complete hair loss.Dermal papilla which is considered as the effect target organ of androgen locates at the bottom of the hair follicles and contains rich androgen receptor(AR)and 5?reductase type ?.DP releases a variety of cytokines to regulate hair growth and hair follicle cycle.A variety of transcription factors and signal transduction pathways are involved in the growth of hair follicles.One or more signal transduction pathways are involved in the incidence of AGA,such as:androgen metabolism pathway,Wnt signaling pathway,TGF-?/Smad pathway,p53 signaling pathway.Wnt signal transduction plays an important role in embryonic development and human disease,and also affects the development of hair follicles,regulates hair growth and hair shaft structure.Wnt/?-catenin signal transduction pathway regulates development and regeneration of hair follicle as "the first dermal signal".Studies showed that androgen could induce the onset of AGA through inhibiting Wnt/?-catenin signaling pathway.Androgen activated the canonical Wnt signal and starts a cascade through blocking the expression of downstream target genes mediated by ?-catenin,prevention of the platecode forming in hair follicles,hair lost after the first catagen and finally the appearance of alopecia.TGF-? signaling pathway is involved in the catagen and rapid fibrosis of hair follicles.In vitro and in vivo TGF-?antagonists can effectively prevent catagen and promote the growth of the volume of hair follicle.Studies found that the presence of a "catagen cascade" in AGA,Including:(1)the conversion of testosterone to DHT by type ? 5-a-reductase;(2)the synthesis of TGF-?2 in dermal papilla cells;and(3)the activationof the intrinsic caspase network.These sequential events contribute to the shortening of the human hair cycle.During the pathogenesis of AGA,DHT stimulates the synthesis of TGF-?2 in dermal papilla cells;TGF-?2 induces epithelial cells to promote up-regulation and activation of caspase-9 and caspase-3 in matrix cells,resulting in the removal of epithelial cells by apoptotic cell death.This series of reactions are named as the"catagen cascade" which is the main path for the onset of AG A.TGF-?2 paly s a central role in this "catagen cascade"and others such as TGF-?1 and TGF-?3 are also involved.In addition,TGF-?1 was able to induce a rapid fibrosis of hair follicules.Studies have shown that the crosstalk between TGF-?/Smad and Wnt/?-catenin signaling pathways have played an important role in the regulation of development,cell differentiation and fibrotic diseases,tumor formation,evolution process and wound healing.It has been confirmed that the crosstalk between androgen/?-catenin and Follistatin(Fst)/TGF-? regulated the differentiation of mesenchymal pluripotent cells.In the pathogenesis of AGA,weather there is a crosstalk between TGF-?/Smad and Wnt/?-catenin signaling pathways is till unknown.The first part of our experiments was conducted to study the expression of Wnt/?-catenin and TGF-? signaling in the balding and non-balding hair follicles in 15 AGA patients.It was found that the expression of some Wnt signaling molecules decreased in balding hair follicles in AGA patients,while some TGF-? signaling molecules increased.Our results showed that these two sigaling pathways were expressed in reverse in AG A.In order to verify whether there is an interaction between these two pathways,we cultured DPCs derived from the balding(balding DPCs)and non-balding(non-balding DPCs)hair follicles from AGA patients and detected the expression of Wnt and TGF-? signaling of both DPCs groups.To investigate the expression of Wnt and TGF-? signaling and the crosstalk between these two signaling pathways in AGA,we evaluated the activation of Wnt and TGF-?signaling after either of them was blocked with the inhibitor in balding and non-balding DPCs.Specimens and methods:1,Scalp specimens were obtained from the same source as described in the first part of our experiments.Balding and non-balding DPCs were separated and amplified from the frontal and occipital scalps from AGA patients respectively in vitro.The DPCs used in this research were in the second and third passage of subculture.2,We detected the expression level of ?-catenin in the cytoplasm and nucleus of the balding and non-balding DPCs in both groups by immunofluorescence assay and western blotting method.3,We detected the phosphorylation levels of Smad2 and Akt by Western blotting method.4,We treated balding DPCs with TGF-? signaling inhibitor SB431542,then detected the phosphorylation of Smad2 and Akt by western blotting,evaluated mRNA expression levels of Wnt10a and LEF1 by RT-PCR,measured ?-catenin expression by immunofluorescence.5,We treated non-balding DPCs with Wnt signaling inhibitor XAV939,then evaluated mRNA expression levels of Wnt10a,LEF1,T?RI and T?RII by RT-PCR,measured ?-catenin expression by immunofluorescence,detected the protein concentration of total TGF-?1 and the active TGF-?1 by ELISA,and the phosphorylation of Smad2 and Akt by western blotting method.Results:1,Compared to the non-balding DPCs,the activity of ?-catenin in balding DPCs was lower not only in the cytoplasm but also in the nuclear and the phosphorylation of Smad2 and Akt in the balding DPCs was up-regulated.2,After the DPCs were treated with SB431542,the phosphorylation level of Smad2 and Akt was significantly down-regulated,while mRNA expression level of Wnt10a and LEF1 increased significantly.The total ?-catenin expression was not significantly altered but ?-catenin nuclear accumulation was remarkably increased.3,The expression of ?-catenin and LEF1 was inhibited in non-balding DPCs which were treated with XAV939,while the mRNA expression of Wnt10a did not change significantly.XAV939 significantly increased the concentration of active TGF-?1 but without affecting the total TGF-?land the expression of T?RI and T?RII,meanwhile the phosphorylation of Smad2 and Akt was up-regulated.Conclusions:Our results showed that compared to the non-balding follicles and DPCs in AGA,Wnt signaling was down-regulated and TGF-? signaling was up-regulated in both balding follicles and DPCs in AGA.Blocking either of these two singalings resulted in the up-regulation of the other signaling.The data suggests that crosstalk between Wnt/?-catenin and TGF-? signaling pathways may exist as one of the important mechanisms contributing to AGA. |
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