Xia Rong Mu, Yao Dong Bo, Cai Xue Min, Xu Xiu Qin
Institute of Stem Cell and Regenerative Medicine, School of Medicine, Xiamen University, Xiamen, China.
Front Cell Dev Biol. 2021 Mar 16;9:638815. doi: 10.3389/fcell.2021.638815. eCollection 2021.
Self-renewal of embryonic stem cells (ESCs) is orchestrated by a vast number of genes at the transcriptional and translational levels. However, the molecular mechanisms of post-translational regulatory factors in ESC self-renewal remain unclear. Histidine phosphorylation, also known as hidden phosphorylation, cannot be detected by conventional experimental methods. A recent study defined phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) as a histidine phosphatase, which regulates various biological behaviors in cells via histidine dephosphorylation. In this study, the doxycycline (DOX)-induced hLHPP-overexpressing mouse ESCs and mouse LHPP silenced mESCs were constructed. Quantitative polymerase chain reaction (qPCR), western blotting analysis, immunofluorescence, Flow cytometry, colony formation assays, alkaline phosphatase (AP) and bromodeoxyuridine (Brdu) staining were performed. We found that the histidine phosphorylation level was strikingly reduced following LHPP overexpression. Besides, the expression of and , indispensable genes in the process of ESCs self-renewal, was significantly down-regulated, while markers related to the differentiation were markedly elevated. Moreover, LHPP-mediated histidine dephosphorylation induced GG phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of promoted the self-renewal of mESCs and reversed the RA induced increased expression of genes associated with differentiation. Mechanistically, our findings suggested that the enzymatic active site of LHPP was the cysteine residue at position 226, not 53. LHPP-mediated histidine dephosphorylation lowered the expression levels of β and the cell cycle-related genes and , while it up-regulated the cell cycle suppressor genes and . Taken together, our findings reveal that LHPP-mediated histidine dephosphorylation plays a role in the self-renewal of ESCs. LHPP-mediated histidine dephosphorylation inhibited the self-renewal of ESCs by negatively regulating the Wnt/β-catenin pathway and downstream cell cycle-related genes, providing a new perspective and regulatory target for ESCs self-renewal.
胚胎干细胞（ESC）的自我更新在转录和翻译水平上由大量基因精心调控。然而，翻译后调控因子在ESC自我更新中的分子机制仍不清楚。组氨酸磷酸化，也称为隐蔽磷酸化，无法通过传统实验方法检测到。最近的一项研究将磷赖氨酸磷组氨酸无机焦磷酸磷酸酶（LHPP）定义为一种组氨酸磷酸酶，它通过组氨酸去磷酸化调节细胞中的各种生物学行为。在本研究中，构建了强力霉素（DOX）诱导的hLHPP过表达小鼠ESC和小鼠LHPP沉默的mESC。进行了定量聚合酶链反应（qPCR）、蛋白质免疫印迹分析、免疫荧光、流式细胞术、集落形成试验、碱性磷酸酶（AP）和溴脱氧尿苷（Brdu）染色。我们发现，LHPP过表达后组氨酸磷酸化水平显著降低。此外，ESC自我更新过程中不可或缺的基因 和 的表达显著下调，而与分化相关的标志物则明显升高。此外，LHPP介导的组氨酸去磷酸化诱导mESC进入G2期停滞，表明LHPP与细胞增殖和细胞周期有关。相反， 沉默促进了mESC的自我更新，并逆转了视黄酸诱导的与分化相关基因表达的增加。从机制上讲，我们的研究结果表明，LHPP的酶活性位点是第226位的半胱氨酸残基，而不是第53位。LHPP介导的组氨酸去磷酸化降低了β和细胞周期相关基因 和 的表达水平，同时上调了细胞周期抑制基因 和 。综上所述，我们的研究结果表明，LHPP介导的组氨酸去磷酸化在ESC的自我更新中发挥作用。LHPP介导的组氨酸去磷酸化通过负调控Wnt/β-连环蛋白通路和下游细胞周期相关基因抑制ESC的自我更新，为ESC自我更新提供了新的视角和调控靶点。
