| Background:Hair follicle is an important skin accessory organ, composed of types of cells. Murine hair follicle underwent cycling of anagen, catagen and telogen after birth. After growing for a certain time, the murine hair follicle entered catagen phase while hair bulb cells began to apoptosis, forming an epithelial cell strand. During this time, DP was pulled up toward the bulge. When the hair follicle ceased degradating, it entered the telogen. Having rest for a certain time, cells in the second hair germ received growing signals from macro environment and began proliferating, which started the new hair cycle.Abnormals in hair follicle cycling may lead to serious consequences, such as alopecia areata, androgenetic alopecia, alopecia senilis and postpartum alopecia, followed by enormous social pressures. Worse, the treatments today are inefficient and still have a long way to go. So, research on hair follicle cycling may benefits the future clinical studies.A variety of signals take part in regulating the hair follicle cycling, among which Wnt pathway is the most important. 19 Wnts have been identified so far, and have been divided into two pathways—the canonical Wnt pathway(Wnt/β-catenin pathway) and the non canonical Wnt pathway. The Wnt/β-catenin pathway, which has long being interested by researchers, regulates the proliferation and differentiation of hair follicle cells, plays a role in controlling hair follicle morphogenesis and hair cycle. Recent studies focused on the anagen-catagen and telogen-anagen transition, but lack catagen-telogen transition study.Gasdermin A3(Gsdma3) gene belongs to Gasdermin gene super family, specific expressing in murine epidermis and hair follicle. Gsdma3 gene mutant caused abnormal hair cycle as well as high β-catenin expression. Gsdma3 gene located on nucleus and with a Leucine zipper domain which made itself like a transcription factor. So, study about the hair cycle control will provide good experiences for clinical hair loss research.Objective:To examine the hair cycle details of Gsdma3 gene mutant mice and find the signaling pathways behind the abnormal hair cycle. Find the way in which Gsdma3 regulating the hair cycle. Providing experiences for the theoretical and clinical study.Methods:1. Effects of the Gsdma3 gene mutant to the hair cycleBy using H&E staining, observe the hair follicle cycle change. Confirm DP location by immunofluorescence and alkaline phosphatase staining.2. Effects of restore Gsdma3 gene expression on the hair follicle cycle3. Effects of the Gsdma3 gene mutant to the second hair cycleBy observing the color of back skin and H&E staining of Gsdma3 gene mutant mice back skin, found the difference between Gsdma3 gene mutant mice and wild type mice. Using immunofluorescence and TUNEL to study the cell proliferation and apoptosis rate.4. Expression of Wnt/β-catenin signalling pathway members in Gsdma3 gene mutant mice back skin(1)Using PCR array and immunofluorescence staining, study the expression of Wnt signaling members;(2)Using immunofluorescence staining to study the location of Gsdma3 and β-catenin; observe the expression pattern of wnt10 b, β-catenin and lef1;(3)Count the nuclear β-catenin cell number;(4)Restore Gsdma3 gene and mutant form Gsdma3 gene expression; observe Wnt signaling and hair cycle change;(5)Over expressing wnt10 b in catagen wild type mice, observe the hair cycle change.5. Gsdma3 gene regulates hair cycle through Wnt/β-catenin signalling pathway(1)Inducing anagen by depilation-induced HF cycling, then over express Gsdma3 gene, Using H&E and immunofluorescence staining to observe the effects of Gsdma3 gene on endogenic Wnt/β-catenin signalling pathway;(2)Inducing anagen by AdWnt10 b, then over express Gsdma3 gene. Using HE and immunofluorescence staining to observe the effects of Gsdma3 on ectogenic Wnt/β-catenin signalling pathway;(3)Treating JB6 cells with AdGsdma3 and AdWnt10 b, detected the TOP/FOP luciferase activity as well as cell proliferation rate.6. Molecular mechanism of Gsdma3 gene regulating Wnt/β-catenin signalling pathway(1)Using luciferase report gene, analyse the transcription of wnt10b;(2)Using anti-Gsdma3 antibody to do the Ch IP assay, to confirm whether Gsdma3 is a transcription factor and found its target genes;(3)Using RT-PCR to study the expression of retinoic acid receptors. Using AT-RA to study the relationship among Gsdma3 gene, retinoic acid and wnt10 b.Results and discussions:1. Gsdma3 gene mutation caused the deletion of telogen stageGsdma3 gene mutant mice lack of telogen and retained the epithelial cell strand throughout catagen, kept DP away from bulge.2.Restored Gsdma3 gene expression led catagen Gsdma3 gene mutant mice hair follicles enter telogen phase.3. Gsdma3 gene mutation did not affect the second anagen re-entryThe time that Gsdma3 gene mutant mice enter the second anagen was the same with the wild type mice. Cell proliferation rate was high in catagen Gsdma3 gene mutant mice. In late catagen, cells in epithelial cell strand proliferate quickly. No obvious secondary hair follicle germ was detected in Gsdma3 gene mutant mice, but cells in lower part of epithelial cell strand began to proliferate on the second anagen re-entry.4. Wnt molecules expression was upregulated in Gsdma3 gene mutant mice.(1) Expression of canonical Wnt pathway factors were upregulated in Gsdma3 gene mutant mice throughout the hair cycle.(2) Gsdma3 gene and nuclear β-catenin was colocalized in catagen Gsdma3 gene mutant mice hair follicles. Expression of Wnt10 b, β-catenin and lef1 are higher in catagen Gsdma3 gene mutantmice bulb.(3)Number of nuclear β-catenin are higher in Gsdma3 gene mutant mice, prolong of Gsdma3 gene mutant mice catagen were result of Wnt over activating. Under Wnt over activating, the cell proliferation and apoptosis rate changed, ended in a balance in catagen that telogen entry was prevented.(4)Gsdma3 gene but not mutant form Gsdma3 gene downregulated Wnt signal in catagen Gsdma3 gene mutant mice hair follicles which lead hair cycle from catagen to telogen.(5)Raised wnt10 b expression in catagen wild type mice hair follicle caused prolonged catagen.5. Gsdma3 gene regulates hair follicle cycling through Wnt signaling.(1)Gsdma3 gene inhibited expression of endogenic Wnt/β-catenin signal, as well as the raised cell proliferation rate by endogenic Wnt/β-catenin signal activation.(2)Gsdma3 gene depressed expression of ectogenic Wnt/β-catenin signal as well as the cell proliferation.(3)Gsdma3 gene overexpression reduced the TOP/FOP Luciferase activity, and depressed the raised wnt10 b, β-catenin and lef1 expression caused by Wnt10 b.6.Gsdma3 gene depressed Wnt activity through raised the retinoic acid signaling activity.(1)Gsdma3 gene could not downregulated the wnt10 b promoter activity.(2) Ch IP assay ruled out the possibility that Gsdma3 gene might be a transcription factor.(3) Retinoic acid signaling activity was low in Gsdma3 gene mutant mice. Gsdma3 gene depressed wnt10 b expression by upregulated retinoic acid signaling.Conclusion:We demonstrated the first time that Gsdma3 gene mutantation prevent catagen-telogen transformation. The catagen were prolonged but the next anagen were not affected by which the telogen phase was ruled out. Wnt took part in this process. Gsdma3 gene could depress Wnt activity, and thereby affect hair cycling. Our results suggest that Gsdma3 gene depress Wnt though retinoic acid. By upregulated retinoic acid signaling activity, Gsdma3 gene depressed wnt10 b expression. This study improved the hair cycle regulating theory, would provide a useful theory evidence for the further clinical research. |
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