A2Lox-Cre mouse embryonic stem cells (mESCs) which is kindly donated by Kyba et al. (2002; Iacovino et al., 2014) were seeded onto an irradiated MEFs at the density of 5 × 10⁵ cells. R1 mESCs were obtained from the American Type Culture Collection (ATCC). A2Lox-Cre mESCs and R1 mESCs were cultured in DMEM (Gibco, Grand Island, NY, United States) containing 15% FBS (Hyclone, Utah, United States), 0.1 mM non-essential amino acids (Gibco, Grand Island, NY, United States), 1 mM sodium pyruvate (Gibco, Grand Island, NY, United States), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, Saint Louis, MO, United States), 100 U/ml penicillin (Gibco, Grand Island, NY, United States), 100 μg/ml streptomycin (Gibco, Grand Island, NY, United States) and 1000 U/ml leukemia inhibitory factor (LIF) (ESGRO, Millipore, Chemicon, United States). The cells were incubated at 37°C (5% CO₂) in a humidified incubator, and the culture medium was changed every 24 h.

In this study, the construction of a Doxycycline (DOX)-induced human LHPP-overexpressing mouse ESC line was performed as described previously (Zhang et al., 2016). Briefly, human Lhpp (hLhpp) was sequenced and cloned into the p2Lox-Rbm24-Flag-IRES-GFP plasmid to replace Rbm24, which resulted in the p2Lox-hLHPP-IRES-GFP recombinant plasmid. To induce the expression of Cre recombinant enzyme prior to electroporation, the cells were cultured in mouse ESC medium containing 1 μg/ml DOX. The next day, the cells were adjusted to a concentration of 1 × 10⁶ cells per sample and resuspended in 90 μl Opti-MEM (Gibco, Grand Island, NY, United States). The Opti-MEM was equilibrated at room temperature before use. 10 μl p2Lox-hLHPP-IRES-GFP plasmid (10 μg) was used for each sample, which was transferred into a sterile cuvette and electroporated using the NEPA21 Super Electroporator (NEPA GENE, Japan). The procedure was conducted using the following parameters: 130 V, 5 ms; three pulses. Afterward, the cuvettes were immediately placed on ice for 5 min. The mixture was then transferred to a plate containing MEFs. Two days after inoculation, the cells were screened with 300 μg/ml of G418 for a further 7 days, and single clones were selected for amplification and identification.

Short hairpin RNA (shRNA) targeting Lhpp was used to construct LHPP-silenced ESC line. Briefly, sh1 Lhpp sequence (5′-TGGGAAAAGGACGCTATTACAAG-3′); sh2 Lhpp sequence (5′-GCTCAGAATTTGATCAGAT-3′); sh3 Lhpp sequence (5′-GGGAAAAGGACGCTATTACAAGG-3′) was cloned into lentivirus plasmid (plvx), and then transfected into A2Lox-Cre mESCs, followed by incubation with 10 μg/mL puromycin for 3 days. Finally, shLHPP-A2Lox mESCs was validated by qPCR and western blotting. Besides, R1 mESCs infected the above virus were subjected to the same treatment to obtain LHPP-silenced R1 mESCs.

To construct R1 mESCs overexpressing human LHPP, the CDS region of human Lhpp was cloned into the polyclonal site of the plvcs plasmid as described above and transfected into 293T cells together with a lentiviral packaging plasmid to produce lentiviral particles. Afterward, R1 mESCs were infected using these lentiviruses. Forty-eight hours later, the cells were selected with 10 μg/mL of puromycin for 3 days to generate stable cell lines. The lentivirus vector carrying a non-targeting shRNA sequence was used as a control.

To construct the plasmid for transient overexpression of Lhpp, the CDS region of Lhpp was cloned into the pXJ-40 plasmid with the following primers. Lhpp upstream primer (XhoI restriction enzyme site): 5′-CCGCTCGAGATGGCACCGTGGGGCAAG-3′; Lhpp downstream primer (Pst1 restriction enzyme site): 5′-GCTGCAGCTTGTCGGCGTGCTGCAGC-3′. Subsequently, to construct plasmid contained a point mutation in the Lhpp sequence, the mutant Lhpp (C53S) and Lhpp (C226S) were generated using forward primer and reverse primer carrying the mutation, respectively. The primers were as follows: hLhpp (c.158G > C) forward: TGAAGGTGAGGTTCTcCACCAACGAG, hLhpp (c.158G > C) reverse: gAGAACCTCACCTTCAGCCGGGAAC; hLhpp (c.677G > C) forward: GCGGTGCCCAGCGGTcTGGAAT GAGA, hLhpp (c.677G > C) reverse: gACCGCTGGGCACCG CCGACGTCGCC. The products were digested with DMT enzyme for 1 h at 37°C, and then transformed into Escherichia coli for amplification. Mutagenesis was detected by Sanger sequencing. After successful construction of the plasmids, the plasmid vectors were transfected into 293T cells. Forty-eight hours later, western blotting assay was carried out to detect the intracellular pHis content as described below.

mESCs were digested with trypsin and resuspended in mESC differentiation medium containing the following: DMEM supplemented with 10% FBS, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM β-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin and 50 μg/ml ascorbic acid (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The cell suspension was seeded into gelatin-coated (0.1%) culture dishes at the density of 5 × 10⁶ cells and left to rest for 20 min to remove the irradiated MEFs; the non-adherent cells were then collected and adjusted to 40 cells/μl in mESC differentiation medium. In order to prepare suspended droplets, the cells were transferred into a 50 ml sterile container, and 25 μl homogenous cell suspension was dropped onto the lid of a bacterial culture dish (150 mm) using a multi-channel pipette. A2Lox-Cre mESCs were then incubated at 37°C (5% CO₂) to differentiate into embryonic bodies (EBs) via the hanging drop method (Han et al., 2013); each drop contained an average of 1,000 aggregated cells. After 4 days, the EBs were harvested from the droplets and seeded into a gelatin-coated petri dish (0.1%) for further differentiation.

Total RNA was extracted from the cells or tissues using TRIzol^® reagent (Life Technologies, CA, United States) according to the manufacturer’s instructions. The purity and concentration of the RNA were determined using an ultraviolet spectrophotometer, and an A260/A280 ratio in the range of 1.8–2.0 was considered to indicate acceptable purity. 1 μg total RNA was reverse transcribed into cDNA at 37°C for 15 min, 85°C for 5 s, and then stored at 4°C for 30 min. The cDNA was subsequently used as the template for qPCR, which was conducted using the ABI7500 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Inc.) with GAPDH as the internal reference. The thermocycling conditions were as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s; annealing at 60°C for 15 s; all for 40 cycles. Each sample was run in triplicate and relative expression of the target genes was calculated using the 2^–ΔΔCt method (Livak and Schmittgen, 2001). The sequences of all primers are represented in Table 1.

Standard western blot assay: Briefly, the total protein was extracted from cells using RIPA buffer (Solarbio, Beijing, China) on ice, and protein concentration was determined via the BCA method. Following denaturation, the proteins (30 μg of protein loaded per lane) were separated by 10% SDS-PAGE, and then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat dried milk for 2 h, washed 3 times with TBST, and incubated overnight at 4°C with the following primary antibodies: anti-β-actin (dilution, 1:4000; Catalog No. BM0627; Boster Biological Technology), anti-LHPP (dilution, 1:1000; Catalog No. sc-376648, Santa Cruz Biotechnology, Inc.), anti-β-catenin (6B3) Rabbit Monoclonal Antibody (dilution, 1:1000; #9582, Cell Signaling Technology), anti-CDK4 Mouse Monoclonal Antibody (dilution, 1:1000; Catalog No. 66950-1-Ig; Eenchmark Proteintech Group, Wuhan, China), Anti-p21 antibody (dilution, 1:1000; Catalog No. ab188224; Abcam), Anti-p27 Antibody (dilution, 1:1000; Catalog No. ab92741; Abcam), anti-CyclinD1 Mouse Monoclonal Antibody (dilution, 1:1000; Catalog No. 60186-1-Ig; Eenchmark Proteintech, Wuhan, China). The membranes were washed with TBST (5 min × 10 min) and subsequently incubated with Goat Anti-Mouse IgG (H + L) horseradish peroxidase-conjugated Secondary Antibody (dilutions 1:5000; Catalog No. BM3895; Boster Biological Technology) or Goat Anti-Rabbit IgG (H + L) horseradish peroxidase-conjugated Secondary Antibody (dilutions 1:5000; Catalog No. BA1054; Boster Biological Technology) for 3 h at room temperature. Following further washing with TBST (5 min × 10 min), the bands were visualized using an enhanced chemiluminescence detection kit (Advansta, Menlo Park, CA, United States). Quantity One Version 4.6.7 (Bio-Rad Laboratories, Inc., Hercules, CA, United States) was used to quantify the gray values of bands.

Detection of histidine phosphorylation: All procedures were conducted at 4°C, and all reagents were pre-cooled before use. The same number of cells was directly lysed in 2× pHis buffer (250 mM Tris-HCl at pH 8.8, 0.02% bromophenol blue, 50% glycerol, 50 mM EDTA, 500 mM DTT, and 10% SDS) on ice, followed by ultrasonication (3 times for 15 s each). The sediment was removed by centrifugation (10,000 × g for 15 min, 4°C). The supernatant was immediately separated at 4°C by modified SDS-PAGE (using a modified stacking gel at pH 7.2, and 10% separation gel at pH 8.8), and then transferred onto PVDF membranes. The membranes were blocked in 2.5% BSA buffer (pH 8.8) (Solarbio, Beijing, China) and then incubated overnight at 4°C with the following primary antibodies: anti-N3-Phosphohistidine (3-pHis) Monoclonal Antibody (Catalog No. MABS1352; Merk KGaA, Germany) and anti-N1-Phosphohistidine (1-pHis) Monoclonal Antibody (Catalog No. MABS1330; Merk KGaA, Germany), diluted in BSA buffer (0.1% Tween-20) to 0.5 μg/ml. The membranes were washed three times (at 4°C for 10 min) with TBST (pH8.8) and incubated with an anti-rabbit HRP-conjugated secondary antibody (dilutions 1:5000) at 4°C. As the control, cell lysates were heated at 100°C for 20 min in loading buffer (pH 8.8) to remove phosphohistidine. Lanes loaded with the heated lysates indicated either heat-resistant phosphohistidine or non-specific signals. Absolute gray value of intracellular phosphohistidine = gray value of samples - gray value of the control sample. Relative gray value = absolute gray value/gray value of reference gene.

Firstly, sterile slides were placed into each well of a 6-well plate. Cells in the logarithmic growth phase were digested with 0.25% trypsin, and then seeded into the plates at a density of 1 × 10⁵ cells per well. The following day, the slides were washed 3 times with PBS, and then fixed with 4% paraformaldehyde for 30 min at room temperature prior to additional washing as aforementioned. The slides were then treated with 0.25% Triton for 10 min, and washed again prior to blocking with 5% BSA solution for 1 h. Following overnight incubation with the primary antibodies: anti-Oct3/4 (C-10) Mouse monoclonal Antibody (dilution, 1:300; Catalog No. sc-5279; Santa Cruz Biotechnology, Inc.) and anti-Lefty (F-11) Mouse monoclonal Antibody (dilution, 1:300; Catalog No. sc-166708; Santa Cruz Biotechnology, Inc.) at 4°C, the slides were washed again with PBS (3 times), and incubated with Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (dilution, 1:300; Catalog No. A-21147; Life Technology) and Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (dilution, 1:300; Catalog No. A-21428; Life Technology) at room temperature for 3 h. The slides were then washed with PBS 3 more times, counterstained with DAPI solution (dilution, 1:10,000; Catalog No. C0060; Solarbio, Beijing, China) for 5 min and washed with PBS a further 3 times. Finally, the slides were sealed with 50% glycerol and observed under a fluorescence microscope (Olympus Corporation, Tokyo, Japan). Magnification, 100×.

mESCs were collected by centrifugation at 200 × g for 3 min at room temperature, and fixed with 70% pre-cooled ethanol at 4°C overnight. The following day, the cells were centrifuged at 700 × g for 3 min at room temperature, washed with pre-cooled PBS and resuspended (also in pre-cooled PBS) to a final concentration of 1.0 × 10⁶ cells/ml. RNase A (10 μg/ml) was added to 100 μl cell suspension and the samples were harvested by centrifugation (as aforementioned). The cells were washed with pre-cooled PBS, resuspended and filtered using a 75 μm nylon mesh; Cell cycle analysis was detected using a flow cytometer (Guava EasyCyte 8, Merk), and the data were analyzed using FlowJo 7.6 Software (FlowJo LLC, Ashland, OR, United States).

mESCs in the logarithmic growth phase were digested with trypsin, resuspended in culture medium, and then seeded into 96-well plates at a density of 1 × 10⁴ cells per well. 10% of Cell Counting Kit-8 (CCK-8) reagent (Cat. C0038, Beyotime Biotechnology, China) was added at 12, 24, 36, 48, 60, and 72 h time points, followed by incubation at 37°C for 2 h. Absorbance values were detected with a microplate reader (Bio-Rad) at 450 nm.

Stem cell properties were validated by AP staining. The cells treated with RA were used as a positive control, while untreated cells (receiving neither RA nor DOX) were used as a negative control. Briefly, mESCs were fixed with 4% formaldehyde for 2 min at room temperature. The cells were subsequently stained using the Alkaline Phosphatase Kit (SiDanSai Biotechnology, Shanghai, China) according to the manufacturer’s instructions. After washing 3 times with PBS, the cells were observed under an optical microscope (Leica DM750). Magnification, 100×.

mRSCs were inoculated into 6-well plates at a density of 500 cells per well and incubated for 10 consecutive days. Culture medium was changed every 2 days. Clones were observed, and the culture was terminated when the clones grew to the naked eye. After fixation with 75% ethanol for 10 min at room temperature, the cells were stained with 0.1% crystal violet solution for 15 min. Images were photographed under a microscope (Leica DM750).

For BrdU staining, when mESCs grown to 60% confluence, Brdu solution (10 μg/ml, Sigma) was added into culture medium. After 6 h of incubation, the cells were collected and fixed with 75% ethanol for 30 min at room temperature. Next, the cells were treated with 1 nM HCl for 30 min. After discarding the HCl, the cells were rinsed with 10 μM boric acid, treated with 0.1% Triton for 30 min, blocked with 5% BSA for 1 h, incubated with anti-BrdU primary antibody (ab6326, 1:200 dilution, Abcam) diluted with 5% BSA for 12 h at 4°C, and then labeled with corresponding fluorophore-conjugated secondary antibody (#4416, 1:400 dilution, Cell Signaling Technology) for 2 h at room temperature. Flow cytometric data were analyzed using FlowJo 7.6 Software (FlowJo LLC, Ashland, OR, United States).

C57BL/6 mice (n = 10), aged 6–8 weeks and weighing 18.5 ± 1.9 g each, were purchased from the animal center of Xiamen University. The mice were euthanized by cervical dislocation. Adult mouse tissues, including muscle tissue, brain tissue, kidney, gut, spleen, bladder, lung, and liver were carefully excised and kept in liquid nitrogen immediately. All animal experiments were conducted with the approval of the animal ethics and use committee (IACUC) of Xiamen university (Xiamen, Fujian, China; Approval ID: scxk2013-0006).

SPSS 21.0 (IBM Corporation, Armonk, NY, United States) and GraphPad Prism 6.0 (GraphPad Software, Inc., La Jolla, CA, United States) were used for data analysis and graphical representation, respectively. The difference between two groups was determined using the Student’s t-test, and comparisons among multiple groups were measured using one-way ANOVA with Dunnett’s test. Data were expressed as mean ± Standard Deviation (SD). Each experiment was independently repeated three times, and P < 0.05 was considered as statistically significant.

To understand the role of LHPP in mouse ESCs differentiation, differentiation of A2Lox-Cre mESCs to cardiomyocytes was induced via embryoid bodies (EBs). We observed a weakened self-renewal ability and loss of stemness in mESCs after RA treatment for 72 h, in which Lhpp expression was significant upregulated (Figures 1A–C). Besides, the expression of Lhpp was increased during myocardial differentiation of mESCs at transcriptional and translational levels (Figures 1D–F). Furthermore, data indicated that Lhpp was more highly expressed in adult mouse tissues than in mESCs (Figure 1G).

To explore the role of LHPP in mESCs, DOX-induced hLHPP-overexpressing mESC line was constructed via homologous recombination (Figure 2A). Following DOX treatment, immunofluorescence, qPCR, and western blotting revealed that the successful construction of human Lhpp (hLhpp)-overexpressing mESC line, in which the expression of DOX-induced hLhpp was up-regulated, compared with that in the untreated cells at different time points (Figures 2B,C); meanwhile, flow cytometry analysis suggested that DOX induction influenced the fluorescence intensity of GFP in a time dependent manner (Figure 2D). To determine the biological function of endogenous LHPP in mESCs, we constructed three shRNAs targeting mouse Lhpp and the silencing efficiency of these 3 shRNAs in A2Lox-Cre ESCs cells was verified. sh2 RNA with the highest silencing efficiency was selected for subsequent experiments (Figure 2E). These results confirmed the successful construction of DOX-induced Lhpp-overexpressing mESCs and Lhpp knockdown mESCs.

To unveil the mechanism by which LHPP regulates the biological behavior of mESCs, the level of intracellular histidine phosphorylation was measured using modified SDS-PAGE and immunofluorescence assay. As illustrated in Figures 3A,B, the levels of 1-pHis and 3-pHis were found to be reduced in mESCs overexpressing LHPP, compared with those not induced with DOX. Protein samples that were pretreated at 100°C for 20 min exhibited non-specific bands, which may have been due to the low specificity of the antibody, or an excessive incubation time. For loading control, Ponceau’s staining was conducted (Figures 3C,D). Besides, similar results of 1-pHis and 3-pHis levels were obtained using immunofluorescence (Figures 3E,F).

To further understand the role of LHPP in mESC self-renewal, stem cell proprieties were validated by AP staining. Seventy-two hours after DOX induction (Figure 4A) or RA treatment (Figure 4B), the cell colonies were decentralized with irregular edges, and AP staining was reduced compared with the control (Figures 4C,D). Colony formation experiments further confirmed that the clonogenic capacity of both DOX-induced A2Lox-Cre mESCs and R1 mESCs overexpressing LHPP by lentiviral infection was significantly reduced (Figures 4E,F). Immunofluorescence, qPCR and western blotting analysis revealed that the expression levels of stem cell marker genes Oct4 and Lefty1 were significantly down-regulated (Figures 4G–I and Supplementary Figures 1A–C, 2A) in mESCs with Lhpp overexpression compared with the control cells, where there was no significant change in endogenous mouse Lhpp expression following the exogenous overexpression of human Lhpp. Meanwhile, the mRNA levels of T-Bra, Mesp1, Nestin, SOX1, SOX17, Afp were significantly elevated in LHPP-overexpressed mESCs (Supplementary Figure 1D). Conversely, silencing of Lhpp in A2Lox-Cre mESCs and R1 mESCs enhanced the clonogenic ability of mESCs (Figures 4J,K). Besides, Lhpp knockdown upregulated Lefty1 expression at the mRNA and protein levels, but reduced the mRNA levels of T-Bra, Mesp1, Nestin, SOX1, SOX17, Afp in mESCs as compared to the control group (Figures 4L–N and Supplementary Figures 1E,F, 2B). No significant difference in Oct4 expression was observed in DOX-induced LHPP overexpressing mESCs, while Lhpp knockdown by shRNA upregulated Oct 4 mRNA expression in R1 mESCs compared to the control. Overall, these findings suggested that LHPP plays role in differentiation of mESCs.

Although previous literature has speculated that the specific enzymatic active site of LHPP is the cysteine residues at position 53 and 226 (Yokoi et al., 2003), so far, no study has confirmed this. In present study, we constructed two mutant plasmids of Lhpp as described in the methods, resulting in mutant proteins LHPP (C53S) and LHPP (C226S) (Supplementary Figures 3A,B). After overexpression of these two mutants in human 293T cells, respectively, we detected that the LHPP (C53S) mutant was able to significantly reduce intracellular histidine phosphorylation, but the LHPP (C226S) mutant did not produce this effect (Figures 5A–D).

To determine the effects of overexpression of these two catalytic mutants induced by DOX on the self-renewal of mESCs, CCK-8 assay, qPCR analysis for pluripotency and differentiation markers and colony formation assay was performed in A2Lox-Cre mESCs. We found that overexpression of LHPP (C53S) mutant significantly attenuated cell proliferative ability, while there was no significant difference between LHPP (C226S) mutant-overexpressed mESCs and the control group (Figure 5E). Besides, colony formation assay confirmed that overexpression of LHPP (C53S) mutant resulted in significantly reduced colony formation capability in mESCs; however, mutating LHPP (C226S) had no significant effect (Figures 5F,G). Meanwhile, qPCR analysis indicated that both mutants were successfully overexpressed in the mESCs. Similar to the aforementioned results, LHPP (C53S) mutant markedly decreased the mRNA expression of pluripotency gene Lefty1, but elevated the expression of differentiation-associated genes T-box transcription factor Brachyury (T-Bra), Mesp1, Nestin, SOX1, SOX17, and Afp (Figure 5H). In contrast, although LHPP (C226S) mutant was also significantly upregulated in mESCs, it did not produce similar results. Cumulatively, these data suggest that the enzymatic active site of LHPP is the cysteine residue at position 226, which is essential for maintaining its function to regulate mESCs self-renewal.

To investigate how LHPP exerts its biological effects, flow cytometry was performed to detect changes of cell-cycle progression. It was revealed that when cultured with medium containing LIF, the proliferative capacity of DOX-induced LHPP-overexpressing mESCs was dramatically attenuated (Figures 6A,C), and that the proliferation of mESCs slowed down, compared with cells of the control group (Figures 6D,E), which was consistent with what was observed in RA treated (RA+) group (Figures 6F,G). Conversely, proliferation of Lhpp knockdown mESCs was significantly enhanced compared with that in the control cells (Figure 6B). Moreover, compared with wild-type cells, Lhpp knockdown mESCs cultured in LIF medium showed significant changes in cell cycle distribution, with an increase in the proportion of G₂/M cells, but a decrease in the proportion of G₀/G₁ cells (Figures 6H,I). In addition, DOX-induced overexpression of LHPP exhibits a lower proportion of pHH3-positive cells than the control cells. These results were consistent with the results from RA-induced ESCs differentiation. Conversely, Lhpp knockdown caused an increased proportion of pHH3-positive cells (Figure 6J). Further, Brdu staining showed that overexpression of LHPP in A2Lox-Cre mESCs and R1 mESCs significantly reduced the percentage of Brdu-positive cells, whereas silencing of Lhpp in both cells yielded opposing effects (Figures 6K–N).

We further repeated the above experiments with LIF-free culture medium. Data indicated that the proliferation and cell cycle of mESCs were affected after culturing with LIF-free medium for 3 days, in comparison to cells treated with medium containing LIF; besides, when the cells were cultured in LIF-free medium, the inhibitory effect of DOX-induced LHPP-overexpression on the cell cycle was enhanced, resulting in a slower proliferation of mESCs (Supplementary Figures 4A,C,D). Nevertheless, the proliferation of Lhpp knockdown mESCs cultured in LIF-free medium were promoted (Supplementary Figure 4B) and cell cycle was changed significantly as in LIF medium (Supplementary Figures 4E,F). Collectively, our findings suggest that LHPP suppresses the proliferation of mESCs and induces cell cycle arrest, and that these effects are enhanced in the absence of LIF treatment.

Mechanistically, we detected the expression of Wnt signaling pathway-related molecules and cell cycle-related genes. Data showed that the mRNA (Supplementary Figure 5A) and protein (Figure 7) expression levels of β-catenin, and the cell cycle-promoting genes CDK4 and CyclinD1 were significantly attenuated, whilst those of the cell cycle-inhibitory genes P21 and P27 were markedly increased in DOX-induced LHPP-overexpressing mESCs, in comparison to the control cells (when both were cultivated with medium containing LIF). By contrast, Lhpp knockdown produced the opposite results (Supplementary Figure 5B).