Gasdermin A3Gene Affecting The Expression Of Wnt5a, E-cadherin And NF-κB In The Mouse Skin Keratinocytes

Background, Gsdma3gene is a member of Gsdm gene family, which belongs to theGsdma gene sub-family, located at the mouse chromosome11, closely linked to the retinoicacid receptor α and keratin1genes. Six mutants of Gsdma3can be derived from theGsdma3gene hotspots, which could result in irregular hair follicle cycling, skin hyperplasiaand skin inflammation. On the other hand, canonical Wnt／β-catenin pathway play thecritical role in mastering the proliferation and differentiation of mouse skin keratinocyte.Since our previous investigation indicated that Gsdma3might influence the canonical Wnt／β-catenin pathway, following objectives have been established:Objective,1)To ascertain the effect of Gsdma3on the canonical Wnt／β-cateninpathway inhibitor Wnt5a in the mouse skin keratinocyte.2) To ascertain the effect of Gsdma3on the canonical Wnt／β-catenin pathwayinhibitor E-cadherin in the mouse skin keratinocyte.3) To ascertain the effect of Gsdma3on the NF-κB in the mouse skin keratinocyte.NF-κB is a co-activator of β-catenin in the hair follicle morphogenesisMethods,1) Plasmid transfection, tissue slide, IHC, RT-PCR, WB were exploited tovalidate whether the associations can be established between Gsdma3andWnt5a/E-cadherin/NF-κB in vivo.2) Cell culture, plasmid transfection, ICC, RT-PCR, WBwere exploited to validate whether the associations can be established between Gsdma3andWnt5a/E-cadherin/NF-κB in vitro.Results,1) In vivo, IHC assays showed that, in the skins of Gsdma3mutant mice,stronger positive signals of Wnt5a were detected. Gene rescue assays represented thatweaker positive signals of Wnt5a were detected in the Gsdma3overexpression skins ofmutant mice, as well as the synchronized hair follicle cycling mice did. In vitro, Wnt5aexpression was down-regulated when Gsdma3gene was overexpressed in the skinkeratinocytes. 2) In vivo, IHC, RT-PCR, WB assays revealed that, E-cadherin expression in theGsdma3mutant mice was significantly increased. Gsdma3gene rescue assays demonstratedthat E-cadherin expression was inhibited in the skin of Gsdma3mutant mice when Gsdma3gene was up-regulated, as well as the synchronized hair follicle cycling mice did. In vitro,ICC, RT-PCR, WB assays showed a negative correlation between the expression of Gsdma3and E-cadherin.3) In vivo, IHC, RT-PCR, WB assays revealed that, NF-κB expression in the Gsdma3mutant mice was significantly decreased. Gsdma3gene rescue assays demonstrated thatNF-κB expression was activated in the skin of Gsdma3mutant mice when Gsdma3genewas up-regulated. In vitro, ICC, RT-PCR, WB assays showed a positive correlation betweenthe expression of Gsdma3and E-cadherin.Conclusion，1) Gsdma3can inhibit the expression of Wnt5a in the mouse skinkeratinocyte.2) Gsdma3can inhibit the expression of Wnt5a in the mouse skin keratinocyte.3) Gsdma3can activate the expression of NF-κB in the mouse skin keratinocyte.