The Key Laboratory of Pathobiology, Ministry of Education, Department of Pathology, College of Basic Medical Sciences, Jilin University, 126 Xinmin Avenue, Changchun, 130021, China.
Department of Ophthalmology, The Second Hospital of Jilin University, Changchun, 130021, China.
Stem Cell Res Ther. 2019 Aug 23;10(1):268. doi: 10.1186/s13287-019-1382-y.
PBX 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.
A lentiviral vector designed to carry the PBX1 sequence or PBX1 short 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 the NANOG promoter. 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.
Overexpression 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 G/G to 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 upregulated Nanog transcription by activating the promoter and promoted the expression of endogenous SOX2 and OCT4. 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.
PBX1 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 the NANOG promoter, upregulated NANOG, and inhibited mitochondrion-mediated apoptosis via the AKT/GSK3β pathway during the early stages of reprogramming.
PBX 同源盒 1（PBX1）参与维持人类胚胎和造血干细胞的多能性；然而，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。AKT/糖原合成酶激酶（GSK）3β通路调节细胞代谢、增殖、凋亡和重编程，该通路下游的 p16 和 p21 调节重编程诱导的细胞增殖、细胞周期和细胞凋亡。在这里，我们旨在阐明 PBX1 在调节 HF-MSCs 增殖和重编程中的作用。
设计携带 PBX1 序列或 PBX1 短发夹 RNA 序列的慢病毒载体，过表达或敲低 PBX1。通过流式细胞术研究 PBX1 对增殖和凋亡的作用。实时聚合酶链反应评估多能基因表达。双荧光素酶报告基因检测评估 NANOG 启动子的转录活性。Western blot 鉴定参与增殖和重编程的 PBX1 下游分子。检测 caspase3 活性评估 HF-MSC 重编程。使用磷脂酰肌醇 3-激酶/AKT 抑制剂 LY294002 抑制 AKT 的磷酸化和活性。
HF-MSCs 中 PBX1 的过表达增加了 AKT 的磷酸化和 β-catenin 的核转位，导致细胞周期从 G1/G0 期进入 S 期。此外，与转染四个转录因子（SOMK）的 HF-MSCs 相比，转染五个转录因子（SOMKP）的 HF-MSCs 中碱性磷酸酶染色集落的形成增强。PBX1 通过激活启动子上调 Nanog 转录，并促进内源性 SOX2 和 OCT4 的表达。此外，PBX1 表达通过激活 AKT/GSK3β 通路并减少重编程早期的细胞凋亡来促进细胞增殖。磷酸化 AKT 的抑制或 PBX1 的敲低促进了线粒体介导的细胞凋亡并降低了重编程效率。
PBX1 通过激活 AKT/GSK3β 信号通路增强 HF-MSC 增殖，并促进 HF-MSCs 生成诱导多能干细胞（iPSC）。在 HF-MSCs 重编程为 HF-iPSC 的过程中，PBX1 通过 AKT/GSK3β 通路在重编程早期激活 NANOG 启动子，上调 NANOG，并抑制线粒体介导的细胞凋亡。
