LHPP-Mediated Histidine Dephosphorylation Suppresses the Self-Renewal of Mouse Embryonic Stem Cells

Abstract

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 Oct4 and Lefty1, 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 G0/G1 phase arrest in mESCs, suggesting LHPP was implicated in cell proliferation and cell cycle. Conversely, silencing of Lhpp 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-catenin and the cell cycle-related genes CDK4 and CyclinD1, while it up-regulated the cell cycle suppressor genes P21 and P27. 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.

LHPP suppresses gastric cancer progression via the PI3K/AKT/mTOR signaling pathway

“…Thus, we decided to detect histidine phosphorylation levels following LHPP expression alteration in GC cells. Consistent with previous reports 17 , fewer 1-pHis proteins were detected compared with 3-pHis proteins. However, our data indicated that the enzymatic active site of LHPP is neither the cysteine residue at position 226 nor at position 53 in GC.…”

“…Moreover, neither the an-oncogenic effect of LHPP nor the PI3K/AKT/mTOR pathway was not been disrupted with LHPP site mutation (Figure 5C-G). Inconsistent with previous articles [2,17], our data revealed that the enzymatic active site of LHPP may not be present in the cysteine residues at positions 53 and 226.…”

“…Until recently, technical difficulties were circumvented by the development of 1-pHis or 3-pHis monoclonal antibodies 30 . Moreover, some researchers indicated that dysregulated histidine phosphorylation is associated with tumorigenesis and self-renewal ability of embryonic stem cells 6 , 17 . Thus, we decided to detect histidine phosphorylation levels following LHPP expression alteration in GC cells.…”

Does the LHPP gene share a common biological function in pancancer progression?

“…Mechanistically, LHPP suppresses cancer cell proliferation, metastasis, and energy metabolism and promote cell apoptosis and autophagy. Interestingly, Xia et al illustrated [ 31 ] that LHPP -mediated histidine dephosphorylation repressed self-renewal of mouse embryonic stem cells by mediating the mRNA or protein expression of β-catenin, CDK4 and CyclinD1. Therefore, we chose to explore LHPP functions systematically, in addition to other molecular features and genetic alterations.…”

“…Mechanistically, as a kind of histidine phosphatase with homodimerization and diphosphatase activity, the LHPP protein is mainly correlated with substance metabolism, which has been proved in colitis, hepatocellular carcinoma [ 5 ] and embryonic stem cells [ 31 ]. Additionally, the dephosphorylation function of LHPP protein has been also widely studied in multiple cancers.…”