PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis
PBX homeobox 1 enhances hair follicle mesenchymal stem cell proliferation and reprogramming through activation of the AKT/glycogen synthase kinase signaling pathway and suppression of apoptosis
复制标题
PBX 同源盒 1 通过激活 AKT/糖原合酶激酶信号通路和抑制细胞凋亡来增强毛囊间充质干细胞增殖和重编程
DOI：
10.1186/s13287-019-1382-y
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发表时间：
2019-08-23
影响因子：
7.5
通讯作者：
Liu, Jinyu
中科院分区：
文献类型：
--
作者：
Jiang, Yixu;Liu, Feilin;Liu, Jinyu
PBX Homeobox 1(PBX Homeobox 1，PBX Homeobox 1)参与维持人胚胎干细胞和造血干细胞的多能性，但Pbx1在毛囊间充质干细胞(HF-MSCs)自我更新和重编程中的作用尚不清楚。β途径调节细胞的代谢、增殖、凋亡和重编程，p16和p21作用于该通路的下游，调节细胞的增殖、细胞周期和重编程诱导的细胞凋亡。在此，我们旨在阐明Pbx1在调控HF-MSCs增殖和重编程中的作用。方法设计携带PBX1序列或PBX1短发夹状RNA序列的慢病毒载体，过表达或敲除Pbx1。用流式细胞仪检测Pbx1在细胞增殖和凋亡中的作用。实时定量聚合酶链式反应检测多潜能基因的表达。用双荧光素酶报告实验检测NANOG启动子的转录活性。Western blotting鉴定Pbx1下游与增殖和重编程有关的分子。检测Caspase3活性以评估HF-MSC重编程。结果在HF-MSCs中过表达Pbx1可增加AKT的磷酸化，增加β-连环蛋白的核转位，使细胞周期由G0/G1期进入S期。此外，联合5种转录因子(SOMKP)的HF-MSCs与联合4种转录因子(SOMK)的HF-MSCs相比，可促进碱性磷酸酶染色集落的形成。PBX1通过激活启动子上调纳米转录，并促进内源性SOX2和OCT4的表达。此外，在重编程的早期阶段，PBX1的表达激活了AKT/糖原合成酶激酶3的β通路，减少了细胞的凋亡。结论PBX1促进HF-MSCs增殖，并通过激活Akt/Gsk3β信号通路诱导多能干细胞的产生。在将HF-MSCs重编程为HF-IPSCs的过程中，在重编程的早期阶段，Pbx1激活NANOG启动子，上调NANOG，并通过Akt/Gsk3β途径抑制线粒体介导的细胞凋亡。
BackgroundPBX homeobox 1 (PBX1) is involved in the maintenance of the pluripotency of human embryonic and hematopoietic stem cells; however, the effects of PBX1 in the self-renewal and reprogramming of hair follicle mesenchymal stem cells (HF-MSCs) are unclear. The AKT/glycogen synthase kinase (GSK) 3β pathway regulates cell metabolism, proliferation, apoptosis, and reprogramming, and p16 and p21, which act downstream of this pathway, regulate cell proliferation, cell cycle, and apoptosis induced by reprogramming. Here, we aimed to elucidate the roles of PBX1 in regulating the proliferation and reprogramming of HF-MSCs.MethodsA lentiviral vector designed to carry thePBX1sequence orPBX1short hairpin RNA sequence was used to overexpress or knock down PBX1. The roles of PBX1 in proliferation and apoptosis were investigated by flow cytometry. Real-time polymerase chain reaction was performed to evaluate pluripotent gene expression. Dual-luciferase reporter assays were performed to examine the transcriptional activity of theNANOGpromoter. Western blotting was performed to identify the molecules downstream of PBX1 involved in proliferation and reprogramming. Caspase3 activity was detected to assess HF-MSC reprogramming. The phosphatidylinositol 3-kinase/AKT inhibitor LY294002 was used to inhibit the phosphorylation and activity of AKT.ResultsOverexpression of PBX1 in HF-MSCs increased the phosphorylation of AKT and nuclear translocation of β-catenin, resulting in the progression of the cell cycle from G0/G1to S phase. Moreover, transfection with a combination of five transcription factors (SOMKP) in HF-MSCs enhanced the formation of alkaline phosphatase-stained colonies compared with that in HF-MSCs transfected with a combination of four transcription factors (SOMK). PBX1 upregulatedNanogtranscription by activating the promoter and promoted the expression of endogenousSOX2andOCT4. Furthermore, PBX1 expression activated the AKT/glycogen synthase kinase (GSK) 3β pathway and reduced apoptosis during the early stages of reprogramming. Inhibition of phospho-AKT or knockdown of PBX1 promoted mitochondrion-mediated apoptosis and reduced reprogramming efficiency.ConclusionsPBX1 enhanced HF-MSC proliferation, and HF-MSCs induced pluripotent stem cells (iPSC) generation by activating the AKT/GSK3β signaling pathway. During the reprogramming of HF-MSCs into HF-iPSCs, PBX1 activated theNANOGpromoter, upregulatedNANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3β pathway during the early stages of reprogramming.