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 G_0/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.

Keywords: LHPP; embryonic stem cell; histidine dephosphorylation; post-translational modification; self-renewal.

FIGURE 1

Up-regulation of Lhpp expression during mES cell differentiation. (A–C) Up-regulated expression of mLhpp in mESCs treated with 10 mM of RA for 3 days. (A) Expression of mLhpp mRNA; (B) representative blots of mLHPP expression; (C) statistical analysis of the protein expression of mLHPP. (D–F) Increased expression of mLhpp in mESCs during myocardial differentiation, as validated by qPCR and western blotting analysis. (D) Statistical results of qPCR; (E) representative blots; (F) quantification of protein expression. (G) Up-regulated transcription of mLhpp in adult mouse tissues, compared with mESCs. GAPDH was used as the internal control. All experiments were independently repeated at least three times, and the data are presented as the mean ± SD. *P < 0.05. LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase; mLhpp, mouse Lhpp; mESCs, mouse embryonic stem cells; RA, retinoic acid.

FIGURE 2

Construction and identification of DOX-induced hLhpp-overexpressing and -silenced mESCs. (A) Schematic diagram depicting the production of DOX-induced hLHPP-overexpressing mESCs. (B) Fluorescence detection of the mESCs following DOX treatment (1 μg/ml) for 48 h. Scale bar = 100 μm. (C) The mRNA and protein expression levels of Lhpp following DOX treatment (1 μg/ml) at different time points. (D) GFP expression in mESCs following DOX treatment (1 μg/ml) for 24 or 48 h was detected by flow cytometry. (E) The mRNA and protein expression levels of Lhpp were determined by qPCR and western blotting analysis in Lhpp-silenced mESCs. All experiments were independently repeated at least three times, and the data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. DOX, doxycycline; LHPP, human phospholysine phosphohistidine inorganic pyrophosphate phosphatase; hLhpp, human Lhpp; mESCs, mouse embryonic stem cells; RA, retinoic acid.

FIGURE 3

Effect of human LHPP-overexpression on histidine phosphorylation in mESCs. (A) Intracellular 1-pHis levels were detected by modified western blotting in mESCs treated with 1 μg/ml DOX for 72 h. (B) Intracellular 3-pHis levels in mESCs were detected by modified western blotting following treatment with 1 μg/ml DOX for 72 h. (C,D) Ponceau’s staining was used to validate the amount of protein loaded per well. (E,F) Immunofluorescence was carried out to observe the (E) 1-pHis and (F) 3-pHis levels in mESCs treated with 1 μg/ml DOX for 72 h. Red, 1-pHis or 3-pHis; green, GFP; blue, DAPI. Scale bar = 100 μm. All experiments were independently repeated at least three times, and the data are presented as the mean ± SD. *P < 0.05. LHPP, human phospholysine phosphohistidine inorganic pyrophosphate phosphatase; mESCs, mouse embryonic stem cells; DOX, doxycycline.

FIGURE 4

Effects of LHPP on the expression of cellular pluripotency and differentiation-related genes in mESCs. (A) mES cell colonies were observed with or without DOX treatment (1 μg/ml) for 72 h. (B) mES cell colonies after incubation with or without 10 mM RA for 72 h. (C,D) AP staining was performed in mESCs with or without (C) DOX (1 μg/ml) or (D) 10 mM RA treatment for 72 h. (E,F) Colony-formation assay was performed to detected the effects of Lhpp overexpression on (E) A2Lox-Cre mESCs and (F) R1 mESCs. (G,H) Immunofluorescent labeling of (G) Oct4 and (H) Lefty1 in DOX-induced hLhpp-overexpressing mESCs. Red, Oct4 or Lefty1; green, GFP; blue, DAPI. Scale bar = 100 μm. (I) qPCR quantification of the mRNA levels of human Lhpp, mouse Lhpp, Oct4 and Lefty1 in mESCs treated with or without DOX (1 μg/ml). (J,K) Colony-formation assay was performed to evaluate the effects of Lhpp knockdown on (J) A2Lox-Cre mESCs and (K) R1 mESCs. (L) qPCR detection of mLhpp expression in mESCs. (M,N) The mRNA and protein levels of (L) Oct4 and (M) Lefty1 in Lhpp-silenced mESCs. All experiments were independently repeated at least three times, and the data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. DOX, doxycycline; LHPP, human phospholysine phosphohistidine inorganic pyrophosphate phosphatase; hLhpp, human Lhpp; mESCs, mouse embryonic stem cells; RA, retinoic acid; AP, alkaline phosphatase; sh, short hairpin RNA.

FIGURE 5

The cysteine at position 226 of LHPP is essential for its histidine phosphatase activity. (A,B) Western blotting analysis for intracellular (A) 1-pHis and (B) 3-pHis levels after overexpression of LHPP (C53S) in 293T cells. (C,D) Western blotting analysis for intracellular (C) 1-pHis and (D) 3-pHis levels after overexpression of LHPP (C226S) in 293T cells. (E) Cell proliferative ability was detected by CCK-8 assay after overexpression of LHPP (C53S) mutant or LHPP (C226S) mutant in A2Lox-Cre mESCs. (F,G) Cell colony formation capability was assayed by colony formation assay after overexpression of (F) LHPP (C53S) mutant or (G) LHPP (C226S) mutant in A2Lox-Cre mESCs. (H) After overexpression of LHPP (C53S) mutant or LHPP (C226S) mutant in A2Lox-Cre mESCs, the expression levels of pluripotency and differentiation-associated genes were evaluated by qPCR analysis. All experiments were independently repeated at least three times, and the data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant difference. LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase.

FIGURE 6

Effect of LHPP on mESC proliferation and cell cycle. (A,B) The Cell Counting Kit-8 assay was used to detect the proliferation of (A) mESCs following treatment with DOX (1 μg/ml) and (B) shRNA targeting Lhpp. (C) mES cell colonies following DOX treatment (1 μg/ml) for 72 h. Scale bar = 100 μm. (D,E) Flow cytometry was conducted to detect changes to the cell cycle in mESCs treated with DOX (1 μg/ml) for 3 days. (E) Quantification. (F,G) RA-treated (10 mM) cells were used as the positive control. Cells without DOX treatment (1 μg/ml) were used as the negative control group. (G) Quantification. (H,I) Flow cytometry was used to determine the changes in the cell cycle in Lhpp knockdown mESCs cultured for 3 days, respectively. (I) Quantification. (J) The percentage of pHH3-positive cells in mESCs. (K,L) The percentage of Brdu-positive cells in (K) DOX-induced (1 μg/ml) LHPP overexpressing mESCs and (L) R1 mESCs with LHPP overexpression. (M,N) The percentage of Brdu-positive cells in Lhpp knockdown (M) A2Lox-Cre mESCs and (N) R1 mESCs. All experiments were independently repeated at least three times, and the data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase; mESCs, mouse embryonic stem cells; DOX, doxycycline; RA, retinoic acid; shRNA, short hairpin RNA.

FIGURE 7

Effect of LHPP-overexpression on the expression levels of Wnt signaling pathway-related molecules and cell cycle-related genes. (A–F) The protein expression levels of (A) hLHPP, (B) β-catenin, (C) CDK4, (D) CyclinD1, (E) P21, and (F) P27 in A2Lox-Cre mESCs treated with DOX (1 μg/ml) for 72 h were detected by western blotting. (G–L) The protein expression levels of (G) mLHPP, (H) β-catenin, (I) CDK4, (J) CyclinD1, (K) P21, and (L) P27 in Lhpp-silenced A2Lox-Cre mESCs cultured for 72 h. All experiments were independently repeated at least three times, and the data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. LHPP, phospholysine phosphohistidine inorganic pyrophosphate phosphatase; hLHPP, human LHPP; mLHPP, mouse LHPP; mESCs, mouse embryonic stem cells; DOX, doxycycline; shRNA, short hairpin RNA.