Yu Nanlan, Hu Tianxing, Yang Haichao, Zhang Lian, Song Qin, Xiang Fei, Yang Xichuan, Li Yuhong
Department of Dermatology, The First Affiliated Hospital of the Army Medical University, Chongqing, China.
Department of Cell Biology, Army Medical University, Chongqing, China.
Front Cell Dev Biol. 2020 Aug 19;8:824. doi: 10.3389/fcell.2020.00824. eCollection 2020.
During hair follicle regeneration, hair follicle stem cells (HFSCs) are regulated by signals from dermal papilla cells (DPCs). Previously we found that Tcf4 could promote the proliferation of DPCs. In this study, we focused on whether and how the biological properties of Tcf4-induced DPCs were regulated by Twist1.
Twist1 was overexpressed or knocked down in DPCs following different adenovirus or lentivirus infection. Phase-contrast microscopy was used to observe the agglutinative growth of DPCs. The CCK-8 assay was used to test the proliferation of DPCs. Western blot and qPCR experiments were used to determine the expression of HGF, IGF-1, VEGF, c-myc, survivin, and CyclinD1 in DPCs. ELISAs were used to test the growth factors secreted by DPCs. Conditional medium culture was used to detect the inductive ability of DPCs. Co-immunoprecipitation and immunofluorescence were used to test the binding of Twist1, Tcf4, and β-catenin in DPCs. Immunofluorescence was also used to test the expression of Twist1, Tcf4, and KRT15 in hair follicles.
Twist1 induced DPC agglutinative growth and proliferation. Twist1 upregulated the expression of downstream target genes downstream of Tcf4, c-myc, survivin, in Tcf4-induced DPCs, as well as the expression and secretion of growth factors HGF, IGF-1, VEGF, which had the ability to induce hair follicle growth. The conditional medium from Twist1-treated DPCs increased the expression of KRT40 and MSX2 in HaCaT cells. Twist1 and Tcf4 co-localized in DPCs both and Anti-Twist1 precipitated Tcf4 and β-catenin.
These results indicate that Tcf4 and Twist1 play a synergistic role in regulating the hair follicle induction ability of DPCs. Twist1 functions by forming a ternary complex with Tcf4 and β-catenin. Thus, we report new data that elucidate whether and how Twist1 regulates some biological properties of DPCs.
在毛囊再生过程中，毛囊干细胞（HFSCs）受来自真皮乳头细胞（DPCs）的信号调控。此前我们发现Tcf4可促进DPCs的增殖。在本研究中，我们聚焦于Twist1是否以及如何调控Tcf4诱导的DPCs的生物学特性。
通过不同的腺病毒或慢病毒感染，在DPCs中过表达或敲低Twist1。利用相差显微镜观察DPCs的凝集生长。采用CCK-8法检测DPCs的增殖。通过蛋白质免疫印迹法和定量聚合酶链反应实验确定DPCs中肝细胞生长因子（HGF）、胰岛素样生长因子-1（IGF-1）、血管内皮生长因子（VEGF）、原癌基因c-myc、生存素和细胞周期蛋白D1（CyclinD1）的表达。采用酶联免疫吸附测定法检测DPCs分泌的生长因子。利用条件培养基培养检测DPCs的诱导能力。采用免疫共沉淀和免疫荧光法检测DPCs中Twist1、Tcf4和β-连环蛋白的结合情况。免疫荧光法还用于检测毛囊中Twist1、Tcf4和角蛋白15（KRT15）的表达。
Twist1诱导DPCs凝集生长和增殖。Twist1上调Tcf4诱导的DPCs中Tcf4下游靶基因c-myc、生存素的表达，以及具有诱导毛囊生长能力的生长因子HGF、IGF-1、VEGF的表达和分泌。来自Twist1处理的DPCs的条件培养基增加了HaCaT细胞中角蛋白40（KRT40）和肌节同源盒基因2（MSX2）的表达。Twist1和Tcf4在DPCs中均共定位，抗Twist1沉淀出Tcf4和β-连环蛋白。
这些结果表明，Tcf4和Twist1在调控DPCs的毛囊诱导能力中发挥协同作用。Twist1通过与Tcf4和β-连环蛋白形成三元复合物发挥作用。因此，我们报告了新的数据，阐明了Twist1是否以及如何调控DPCs的某些生物学特性。
