TY - JOUR
AU - Zhang, Yong
AB - Abstract Vitamin C (Vc), also known as ascorbic acid, is involved in many important metabolic and physiological reactions in the body. Here, we report that Vc enhances the expression of Nanog and inhibits retinoic acid-induced differentiation of embryonic stem cells. We investigated Vc regulation of Nanog through Janus kinase/signal transducer and activator of transcription pathway using cell signaling pathway profiling systems, and further confirmed by specific pathway inhibition. Using overexpression and knockdown strategies, we demonstrated that STAT2 is a new positive regulator of Nanog and is activated by phosphorylation following Vc treatment. In addition, site mutation analysis identified that STAT2 physically occupies the Nanog promoter, which was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift assays. Taken together, our data suggest a role for Vc in Nanog regulation networks and reveal a novel role for STAT2 in regulating Nanog expression. Stem Cells 2014;32:166–176 Embryonic stem cells, Pluripotent stem cells, Nanog, Signaling pathway, Transcription factors, Vitamin C Introduction Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of the blastocyst. They are able to differentiate into all derivatives of the three primary germ layers: the ectoderm, endoderm, and mesoderm [1, 2]. ESCs can sustain the undifferentiated state and have the ability to proliferate indefinitely under specific cultural conditions in vitro. Nanog is a 305 amino acids protein with a conserved homeodomain, and is expressed in ESCs [3]. It is thought to be a key factor in maintaining ESC pluripotency. Over the past few years, efforts have been undertaken to examine how the Nanog gene is regulated in ESCs [4-10]. Thus far, the specific pathways responsible for regulating Nanog expression have not been well-defined. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway was first discovered in the study of the interferon signal transduction pathway [11]. JAKs and STATs transduce extracellular signals to the cytoplasm and then to the nucleus. STATs are implicated in programming gene expression in biological events as diverse as embryonic development, programmed cell death, organogenesis, innate immunity, adaptive immunity, and cell proliferation regulation in organisms [12-17]. Vitamin C (Vc) is a natural small molecular compound. It is essential for humans and several other species that lack l-gulonolactone oxidase in its biosynthetic pathway [18]. Recently, it was reported that Vc promotes the generation of mouse- and human-induced pluripotent stem cells (iPSCs) [19]. There was sufficient evidence to show that the improvement of Vc efficiency in reprogramming had nothing to do with its antioxidant ability. This suggested that Vc might have additional functions other than suppressing free radicals. In this article, we demonstrate that Vc enhances Nanog expression via activation of the JAK/STAT signaling pathway. We also describe the mechanism of Vc signaling through the JAK/STAT pathway following stimulation: STAT2 then binds to the Nanog promoter to result in changes in Nanog expression. All these results not only provide theoretical basis for the application of Vc in ESCs and iPSCs research but also make an important contribution to the understanding of Nanog regulation networks. Materials and Methods Reagents Unless otherwise indicated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO, www.sigmaaldrich.com). The rabbit anti-Nanog polyclonal antibody was obtained from Bethyl Laboratories (Montgomery, TX, www.bethyl.com). The rabbit anti-JAK2 and mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, www.scbt.com). The rabbit anti-STAT2, rabbit anti-STAT3, and rabbit anti-STAT5 antibody were purchased from Merck Millipore (Darmstadt, Germany, www.merckmillipore.com). The rabbit anti-phospho-JAK2, rabbit anti-phospho-STAT2, rabbit anti-phospho-STAT3, and rabbit anti-phospho-STAT5 were purchased from Cell Signaling Technology (Beverly, MA, www.cellsignal.com). Cell Culture, Transient Transfection, and Treatment J1 mouse ESCs were purchased from ATCC (Manassas, VA, www.atcc.org) and were grown on 0.2% (wt/vol) gelatin-coated culture plates with ESC medium comprising Knockout Dulbecco's modified Eagle's medium (DMEM), 15% (vol/vol) knockout serum replacement, 1× nonessential amino acids, 100 μM β-mercaptoethanol, 2 mM glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, and 1,000 U/ml leukemia inhibitory factor (LIF) (ESGRO, Millipore). Mouse F9 embryonal carcinoma (EC) cells were grown on 0.2% gelatin-coated culture plates with DMEM (Invitrogen, CA, www.lifetechnologies.com) supplemented with 10% fetal bovine serum (Gibco, Invitrogen). All cells were maintained in a humidified, 37°C incubator with 5% CO2 and 95% air. Transfections were performed with FuGENE HD reagent (Roche Diagnostics, Basel, Switzerland, www.roche.com) following the manufacturer's instructions. Vc, Retinoic acid (RA), and AG490 were purchased from Sigma (St. Louis, MO). The working concentrations were: 50 μg/ml Vc (unless otherwise indicated), 1 μM RA, and 20 μM AG490. Cells were treated with AG490 for 4 hours before transfections. Vc and RA treatments were performed after transfections, as indicated. Construction of Plasmids Nanog promoters were cloned from genomic DNA of mouse F9 EC cells by polymerase chain reaction (PCR). The NP-2.5kb reporter plasmid contained the Nanog promoter region from −2,301 bp to +294 bp (relative to transcription start site), which was cloned into pGL4.10 construct and confirmed by sequencing. NP-2kb, NP-1.5kb, NP-1kb, and NP-0.5kb constructs containing the Nanog promoter regions −1,894 to +294 bp, −1,209 to +294 bp, −770 to +294 bp, and −176 to 294 bp were created by amplification from the NP-2.5kb plasmid, using the common reverse primer and the different forward primers. All these plasmids were confirmed by sequencing. The full-length coding sequences of the STATs family were amplified from F9 cDNA, then these sequences were inserted into pCMV-HA plasmid by standard molecular cloning methods and confirmed by sequencing. All the primers used for plasmids construction are listed in Supporting Information Table S1. Cellular Proliferation Assay Cellular proliferation was assessed using the Cell Counting Kit-8 (CCK-8) (Beyotime, Shanghai, China, www.beyotime.com), according to the manufacturer's instructions. Briefly, cells were seeded (1 × 104 cells per well) in a 96-well plate (Corning Costar, Acton, MA, www.corning.com) and after 6 hours were treated with 50 μg/ml Vc for different time intervals (12–60 hours). CCK-8 working solution (10 μl) was then added to each well and incubated for 4 hours. To eliminate the effect of Vc on the solution of CCK-8, controls were set up in which culture medium or CCK-8 and Vc were added to wells containing no cells. The absorbance was measured with a microplate reader (Epoch, BioTek, Luzern, Switzerland, www.biotek.com) set at 450 nM. All experiments were performed in triplicate. Cell Cycle Analysis Cell cycle was determined by flow cytometry. Cells were harvested, fixed in cold 70% ethanol (1 hour), and incubated (30 minutes, room temperature) with 50 μg/ml propidium iodide and 1 mg/ml RNase. Cells were then examined with the BD LSR II flow cytometry system (BD Biosciences). Luciferase Assays Luciferase measurements were performed with the dual luciferase reporter (DLR) Assay System (Promega, WI, www.promega.com) according to the manufacturer's instructions. F9 cells were transfected with reporter constructs, and a Renilla luciferase plasmid pRL-SV40 was cotransfected as an internal control. Twenty-four hours after transfection or treatment, cells were lysed with 200 μl/well (12-well plate) passive lysis buffer for 15 minutes with shaking. A total of 20 μl of each lysate was transferred to a 96-well plate and assayed by the addition of 100 μl Luciferase Assay Reagent and 100 μl Stop & Glo Reagent. Data were collected with a VICTOR X5 Multilabel Plate Reader (PerkinElmer). The relative activities of the promoters were measured by firefly luciferase luminescence divided by Renilla luciferase luminescence. Reverse Transcription PCR and Quantitative Real-Time PCR Total RNA was isolated from F9 EC cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Purified RNA was reverse-transcribed using a SYBR PrimeScript RT-PCR Kit (TaKaRa, Japan, www.takaraTissue-SpecificStemCellsbio.com). Real-time PCR was performed with an ABI StepOnePlus PCR system (Applied Biosystems, CA) using SYBR Premix ExTaq II (TaKaRa). The comparative Ct method was used to calculate the relative quantity of the target gene mRNA, normalized to GAPDH and relative to the calibrator, and was expressed as the fold change = 2−ΔΔCt [20]. The following conditions were used for quantitative real-time PCR (qPCR) experiments: 30 seconds at 95°C, followed by 40 cycles of 5 seconds at 95°C, and 30 seconds at 60°C. Primer sequences used for qPCR have been described elsewhere [21] and are listed in Supporting Information Table S2. Western Blot Analysis Cell lysates were fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to poly(vinylidene fluoride) membranes (Millipore, MA) for 2.5 hours at 100 V, and the membranes were blocked in 5% non-fat milk powder/Tris-buffered saline containing 0.05% Tween 20 (TBST) for 2 hours. Then the membranes were incubated with the primary antibody at 4°C overnight. After being washed three times with TBST, the membranes were incubated further with secondary antibody for 2 hours at room temperature. After washing three times for 10 minutes each, immunoblots were revealed by autograph using SuperSignal west pico substrate (Thermo Scientific, IL, www.thermoscientific.com). Electrophoretic Mobility Shift Assay and Supershift Assay Electrophoretic mobility shift assays (EMSAs) were performed using a Electrophoretic Mobility Shift Assay Kit (Molecular Probes, Invitrogen) according to the manufacturer's instructions. The complementary oligonucleotides were annealed in annealing buffer (10 mM Tris-Cl, pH 7.5, 1 mM EDTA, 100 mM NaCl) to form double-stranded oligodeoxynucleotide probes. A total of 50 ng probes was incubated with 10 μg cell lysates from F9 cells that had been transfected with pCMV-HA-STAT2 for 30 minutes at room temperature. For supershift experiments, HA-tagged monoclonal antibody (Sigma) was incubated with cell lysates for 30 minutes at room temperature, followed by incubation with probes for an additional 30 minutes. Then, the mixtures were resolved on 6% nondenaturing polyacrylamide gels. Procedures were performed in strict accordance to the manufacturer's recommendations. Primer sequences used for EMSA are listed in Supporting Information Table S3. Chromatin Immunoprecipitation F9 cells were cultured to a density of 1 × 108 cells for each immunoprecipitation. Cells were cross-linked for 10 minutes at room temperature with 1% (wt/vol) formaldehyde and the reaction subsequently quenched with 125 mM glycine. Genomic DNA was isolated and sheared to average lengths of 300–500 bp by ultrasonic. Rabbit anti-STAT2 or rabbit anti-STAT3 antibody (Millipore) was used for immunoprecipitation. Chromatin immunoprecipitation (ChIP) enrichment was performed by qPCR. Fold-enrichment was determined by normalizing threshold cycle values of STAT2 ChIP against IgG ChIP. Primer sequences used for ChIP-qPCR are listed in Supporting Information Table S4. Results Vc Inhibits RA-Induced Differentiation of J1 Mouse ESCs and F9 ECs In order to gain insight into the function of Vc in mouse ESC (mESC) pluripotency, J1 mESCs were induced to differentiate by treatment with 1 μM RA. Ninety-six hours later, J1 cells had differentiated, as characterized by the loss of tight colony morphology and loss of alkaline phosphatase (AP)-positive staining (Fig. 1A-a,b). In remarkable contrast, J1 cells exhibited colony morphology and partial AP activity when treated with 1 μM RA for 48 hours followed by Vc treatment for an additional 48 hours (Fig. 1A-c). Open in new tabDownload slide Vc inhibits RA-induced differentiation of J1 embryonic stem cells (ESCs) and F9 embryonal carcinomas. (A): J1 mESCs were treated with 1 µM RA or 50 µg/ml Vc. (a): control; (b): 1 µM RA for 48 hours followed by phosphate buffered saline for 48 hours; (c): 1 µM RA for 48 hours followed by Vc for 48 hours; (d): Vc for 96 hours. Alkaline phosphatase staining was performed to examine pluripotency. (B): Schematic representation of Nanog promoter reporter constructs. Right-hand side shows the activities of these promoters relative to NP-0.5kb. (C): F9 cells were transfected with Nanog promoter reporter constructs and treated as indicated for 48 hours. Cell lysates were harvested for dual luciferase reporter assays. (D): Relative levels of Nanog mRNA in response to various treatment of RA or Vc as indicated. (E): Relative levels of Nanog protein in response to treatment with RA, Vc, or DHA as indicated. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05. Abbreviations: DHA, dehydroascorbic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot. Open in new tabDownload slide Vc inhibits RA-induced differentiation of J1 embryonic stem cells (ESCs) and F9 embryonal carcinomas. (A): J1 mESCs were treated with 1 µM RA or 50 µg/ml Vc. (a): control; (b): 1 µM RA for 48 hours followed by phosphate buffered saline for 48 hours; (c): 1 µM RA for 48 hours followed by Vc for 48 hours; (d): Vc for 96 hours. Alkaline phosphatase staining was performed to examine pluripotency. (B): Schematic representation of Nanog promoter reporter constructs. Right-hand side shows the activities of these promoters relative to NP-0.5kb. (C): F9 cells were transfected with Nanog promoter reporter constructs and treated as indicated for 48 hours. Cell lysates were harvested for dual luciferase reporter assays. (D): Relative levels of Nanog mRNA in response to various treatment of RA or Vc as indicated. (E): Relative levels of Nanog protein in response to treatment with RA, Vc, or DHA as indicated. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05. Abbreviations: DHA, dehydroascorbic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot. To test whether Vc promoted the proliferation of residual undifferentiated cells, flow cytometry and the CCK-8 assay were used to detect cell cycle distribution and cellular proliferation, respectively. The results showed that Vc did not significantly affect the cellular proliferation or cell cycle distribution (Supporting Information Fig. S1) of mESCs, suggesting other mechanisms underlying Vc-induced pluripotency. To further quantitatively measure the functional role of Vc in mESC renewal and proliferation, we first focused on the question of whether there is a relationship between Vc and the stem cell markers. To this end, −2,301 to +294 bp Nanog promoter region was amplified by PCR from genomic DNA of mouse F9 EC cells and truncated by ≈500 bp each time. The schematic representation is shown in Figure 1D. These fragments were inserted into pGL4.10 for DLR assays. As shown in Figure 1E, the activities of five of the Nanog promoters were upregulated 60%–120% by Vc treatment. Furthermore, Vc blocked the loss of Nanog promoter activity induced by RA. Next, changes in Nanog transcription and translation levels with Vc treatment were detected using quantitative PCR and Western blot analysis, respectively. Nanog mRNA was upregulated by only about 20%–40% with Vc treatment as well as protein levels. But when F9 cells were induced by RA, Vc showed a remarkable effect on the maintenance of Nanog expression (Fig. 1F, 1G). All these data provide additional support to the conclusion that Vc inhibits RA-induced differentiation and maintains pluripotency. We also tested the effect of dehydroascorbic acid (DHA), the oxidized form of Vc. Our data showed that DHA had the same effect as Vc, suggesting that Vc maintenance of pluripotency had nothing to do with its antioxidant ability, as previously reported [19]. Vc Regulates Nanog Expression Through the JAK/STAT Signaling Pathway In order to investigate the specific mechanism of Vc regulation of Nanog expression, the cell signaling pathway profiling systems (Clontech) were used to determine which pathways were involved in the Vc networks. The plasmids used in the test and the pathway they represent are listed in Supporting Information Table S5. Our results showed that the JAK/STAT pathway was affected by RA treatment and Vc inhibited the effect of RA dramatically, implying that Vc might exert its effect through the JAK/STAT signaling pathway (Fig. 2A). Open in new tabDownload slide Vc regulates the expression of Nanog through the Janus kinase/signal transducer and activator of transcription signaling pathway. (A): Dual luciferase reporter (DLR) assay of interferon stimulated response element reporter from cell signaling pathway profiling results. F9 cells were transfected with plasmids indicated in Supporting Information Table S5, followed by treatment with RA, Vc, or DHA as indicated. Cell lysates were harvested for DLR assays. (B): Phosphorylation state of JAK2 in response to RA, RA+Vc, or Vc treatment. Cells were treated as indicated, and 48 hours later cell lysates were harvested for Western blotting. (C): Relative levels of Nanog mRNA in response to various treatment of RA, Vc, or AG490 (a specific JAK inhibitor), as indicated. (D): Relative levels of Nanog protein in response to various treatment of RA, Vc, or AG490 as indicated. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05. Abbreviations: DHA, dehydroascorbic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-JAK2, phos-JAK2; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot. Open in new tabDownload slide Vc regulates the expression of Nanog through the Janus kinase/signal transducer and activator of transcription signaling pathway. (A): Dual luciferase reporter (DLR) assay of interferon stimulated response element reporter from cell signaling pathway profiling results. F9 cells were transfected with plasmids indicated in Supporting Information Table S5, followed by treatment with RA, Vc, or DHA as indicated. Cell lysates were harvested for DLR assays. (B): Phosphorylation state of JAK2 in response to RA, RA+Vc, or Vc treatment. Cells were treated as indicated, and 48 hours later cell lysates were harvested for Western blotting. (C): Relative levels of Nanog mRNA in response to various treatment of RA, Vc, or AG490 (a specific JAK inhibitor), as indicated. (D): Relative levels of Nanog protein in response to various treatment of RA, Vc, or AG490 as indicated. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05. Abbreviations: DHA, dehydroascorbic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-JAK2, phos-JAK2; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot. As JAKs are the key kinases in the JAK/STAT pathway, in order to verify the pathway profiling result, the phosphorylation of the JAKs was measured by Western blot analysis. We found that Vc promoted the phosphorylation of JAK2 and helped to maintain the phosphorylation of JAK2 in F9 cells induced by RA (Fig. 2B). Subsequently, AG490, a specific inhibitor of the JAK/STAT pathway, was used to confirm the foregoing conclusion. As shown in Figure 2C, in the non-AG490-treated group, Vc upregulated Nanog to 130% over the control, and blocked the loss of Nanog induced by RA, which was consistent with the findings detailed in Figure 1D. However, upon treatment with AG490, Vc no longer helped to maintain the expression of Nanog, suggesting that Vc might regulate Nanog expression through the JAK/STAT signaling pathway. Western blotting was also performed to detect Nanog protein in F9 cells that had been treated with AG490. Unsurprisingly, Nanog protein showed exactly the same changes as mRNA (Fig. 2D). All these data revealed that Vc regulated the expression of Nanog through the JAK/STAT signaling pathway. Vc Stimulates the Phosphorylation of STAT2 To further address which trans-acting factor promoted upregulation of Nanog in the Vc-JAK/STAT signaling pathway, we performed overexpression and knockdown of the STATs. The STATs family comprises STAT1, STAT2, STAT3, STAT4, STAT5α, STAT5β, and STAT6. Among the STATs family, STAT4 and STAT6 are known to have relatively simple functions, focusing primarily on innate immunity, and the remaining STATs are involved in proliferation or development [22, 23]. Based on this, expression vectors for STAT1, STAT2, STAT3, STAT5α, and STAT5β were constructed and independently cotransfected with NP-0.5kb into F9 cells, then DLR assays were performed. As shown in Figure 3A, all the STATs enhanced the activity of the Nanog promoter to varying degrees. Loss of selectivity of the Nanog promoter suggests there may be a cofactor that assists correct STAT-specific binding located beyond the proximal promoter. However, we are able to make several conclusions. First, the Nanog promoter showed more sensitivity to STAT2 and STAT5α in the absence of treatment. Second, Nanog promoter activity was upregulated by Vc treatment in each group, but the STAT2, STAT5α, and STAT5β groups demonstrated more significant increased activity. Third, RA and treatment with Vc resulted in regained Nanog promoter activity, and Nanog promoter activity was almost 100% recovered with addition of STAT2 or STAT3 or STAT5α protein. Taken together, these data revealed that STAT2 or STAT5α may be the most likely trans-acting factors that act downstream of the Vc-JAK/STAT signaling pathway. Open in new tabDownload slide Vc stimulates the phosphorylation of STAT2. (A): Preliminary screening of the transcription factor response to Vc signal. Signal transducer and activator of transcriptions (STATs) overexpression constructs were independently cotransfected with NP-0.5kb into F9 cells, followed by treatment with RA or Vc as indicated. Forty-eight hours later, cell lysates were harvested for dual luciferase reporter (DLR) assays. (B): Verification of STAT2 and STAT5α knockdown. F9 cells were transfected with STAT2-siRNA or STAT5-siRNA. Cell lysates were harvested, and quantitative real-time PCR and Western blotting were performed to check the efficiency of STAT2 or STAT5α knockdown. (C): Relative levels of Nanog promoter activity in STAT2- or STAT5-knockdown F9 cells. Cells were transfected with STAT2-siRNA or STAT5-siRNA, and treated with RA or Vc as indicated. Cell lysates were harvested for DLR assays. (D): Relative levels of Nanog promoter activity in response to overexpressed STAT2 and various small molecule compounds. F9 cells were transfected with pCMV-HA-STAT2 and treated with RA, Vc, or AG490 as indicated. Cell lysates were harvested for DLR assays. (E): Relative levels of Nanog mRNA in response to overexpressed STAT2 and various small molecule compounds. F9 cells were transfected with pCMV-HA-STAT2 and treated with RA, Vc, or AG490 as indicated. (F): Relative levels of Nanog protein and phosphorylation state of JAK2 in response to overexpressed STAT2 and various small molecule compounds. F9 cells were transfected with pCMV-HA-STAT2 and treated with RA, Vc, or AG490 as indicated. (G): Phosphorylation state of STAT2 and STAT5 in response to RA, RA+Vc, or Vc treatment. Cells were treated as indicated for 48 hours. Cell lysates were harvested for Western blotting. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05; **, p < .01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control; p-STAT2, phospho-STAT2; p-STAT5, phospho-STAT5; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot. Open in new tabDownload slide Vc stimulates the phosphorylation of STAT2. (A): Preliminary screening of the transcription factor response to Vc signal. Signal transducer and activator of transcriptions (STATs) overexpression constructs were independently cotransfected with NP-0.5kb into F9 cells, followed by treatment with RA or Vc as indicated. Forty-eight hours later, cell lysates were harvested for dual luciferase reporter (DLR) assays. (B): Verification of STAT2 and STAT5α knockdown. F9 cells were transfected with STAT2-siRNA or STAT5-siRNA. Cell lysates were harvested, and quantitative real-time PCR and Western blotting were performed to check the efficiency of STAT2 or STAT5α knockdown. (C): Relative levels of Nanog promoter activity in STAT2- or STAT5-knockdown F9 cells. Cells were transfected with STAT2-siRNA or STAT5-siRNA, and treated with RA or Vc as indicated. Cell lysates were harvested for DLR assays. (D): Relative levels of Nanog promoter activity in response to overexpressed STAT2 and various small molecule compounds. F9 cells were transfected with pCMV-HA-STAT2 and treated with RA, Vc, or AG490 as indicated. Cell lysates were harvested for DLR assays. (E): Relative levels of Nanog mRNA in response to overexpressed STAT2 and various small molecule compounds. F9 cells were transfected with pCMV-HA-STAT2 and treated with RA, Vc, or AG490 as indicated. (F): Relative levels of Nanog protein and phosphorylation state of JAK2 in response to overexpressed STAT2 and various small molecule compounds. F9 cells were transfected with pCMV-HA-STAT2 and treated with RA, Vc, or AG490 as indicated. (G): Phosphorylation state of STAT2 and STAT5 in response to RA, RA+Vc, or Vc treatment. Cells were treated as indicated for 48 hours. Cell lysates were harvested for Western blotting. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05; **, p < .01. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NC, negative control; p-STAT2, phospho-STAT2; p-STAT5, phospho-STAT5; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot. Subsequently, we performed knockdown of STAT2 and STAT5α using short interfering RNA (siRNA) to examine which STAT is involved in regulating Nanog. qPCR and Western blot analysis of F9 cells transfected with STAT2-siRNA or STAT5α-siRNA efficiently knocked down STAT2 or STAT5α expression (Fig. 3B). STAT2 or STAT5α was not affected in cells transfected with negative control si-RNA, as expected. Next, DLR assays were performed in F9 cells cotransfected with STAT2-siRNA or STAT5α-siRNA and NP-0.5kb to F9 cells, and Nanog promoter activity was calculated as previously described. As shown in Figure 3C, Vc lost its ability to regulate Nanog following knockdown of STAT2, in contrast to knockdown of STAT5α. Next, we tested the effect of overexpressed STAT2 on Nanog promoter activity in the presence of Vc, RA, or AG490. As shown in Figure 3D, Vc and STAT2 both upregulated Nanog promoter activity and exhibited synergistic effects. However, following inhibition of the JAK/STAT pathway with AG490, Vc and STAT2 regulation of Nanog were inhibited. qPCR and Western blotting were also performed to validate the mRNA and protein results (Fig. 3E, 3F). As expected, we observed the synergistic effect of Vc and STAT2, and the inhibitory effect of AG490. Besides, the expression of Nanog was in proportion to the phosphorylation of STAT2 (Fig. 3F). It suggested that STAT2 might be connected with the regulation of Nanog. STATs must be phosphorylated to bind to the promoter of target gene. In order to further confirm our conclusions, the phosphorylation state of STAT2 in F9 cells treated with Vc or RA was detected by Western blot analysis. As shown in Figure 3G, Vc had no impact on total STAT2, but STAT2 phosphorylation levels were remarkably improved by Vc treatment in F9 cells. Furthermore, addition of Vc after induction by RA resulted in moderate recovery of STAT2 phosphorylation levels. In contrast, Vc did not affect the expression and phosphorylation of STAT5. Based on these data, we conclude that Vc regulates Nanog expression via stimulation of STAT2 phosphorylation. STAT2 Binds Directly to Nanog Promoter and Regulates the Expression of Nanog An online tool, PROMO v8.3 (http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB = TF_8.3) was used to predict the potential transcription factors that bind to the proximal region of the Nanog promoter [24, 25]. The prediction results show that the +268 bp to +282 bp region is a STAT-intensive area. Previous studies have shown that phosphorylated STAT2 recognizes a conserved palindromic sequence, TTCNn(n = 2–4)GAA, to bind to the promoter of the target gene. Based on these, we found the +272 to +280 bp fragment of Nanog promoter (TTCTGGGAA) was the most probable candidate. Next, the −176 to +280 bp Nanog proximal promoter was cloned into the luciferase reporter vector, pGL4.10 (Np-WT), and the core sequence TTCTGGGAA was mutated to CCATGGAGG (Np-MUT; primer sequences are listed in Supporting Information Table S6). DLR assays were performed to confirm that this mutated residue is responsible for response to Vc signaling. As shown in Figure 4A, the Np-WT group gave good reproducibility following Vc, RA, or AG490 treatment; in contrast, Vc and AG490 had no effect on the Np-MUT group. In addition, overexpressed STAT2 could not stimulate Nanog promoter activity, and knockdown of STAT2 did not decrease Nanog promoter activity. These data revealed that the +272 to +280 bp fragment of the Nanog promoter responds to the Vc-JAK/STAT signaling pathway. Open in new tabDownload slide STAT2 binds directly to the Nanog promoter. (A): Relative levels of WT and MUT Nanog promoter activity in response to treatment with various small molecule compounds. F9 cells were transfected with Np-WT or Np-MUT, and treated with RA, Vc, or AG490 as indicated. Cell lysates were harvested for dual luciferase reporter assays. Values were normalized with normal cultured WT group. (B): In vitro binding of STAT2 and Nanog promoter was examined by electrophoretic mobility shift assay using cell lysates from F9 cells transfected with pCMV-HA-STAT2. (a): A preliminary experiment was performed to test the binding of STAT2 and WT/MUT probe. (b): The shift band that comprised protein and probe was regulated by RA and Vc as expected. (c): Anti-HA antibody was used for supershift. The second shift band specifically confirmed that the protein binding to the probe was STAT2. (C): In vivo binding of STAT2 and Nanog promoter was examined by ChIP assay. ChIP was performed using anti-STAT2 antibody, anti-IgG as control antibody to detect enriched fragments. The upper part shows agarose gel electrophoresis of polymerase chain reaction (PCR) products. The templates for PCR were indicated. Fold-enrichment was determined by normalizing threshold cycle values of STAT2 ChIP against IgG ChIP. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05; **, p < .01. Abbreviations: ChIP, chromatin immunoprecipitation; MUT, mutation; RA, Retinoic acid; Vc, Vitamin c; WT, wild-type; Ab, antibody. Open in new tabDownload slide STAT2 binds directly to the Nanog promoter. (A): Relative levels of WT and MUT Nanog promoter activity in response to treatment with various small molecule compounds. F9 cells were transfected with Np-WT or Np-MUT, and treated with RA, Vc, or AG490 as indicated. Cell lysates were harvested for dual luciferase reporter assays. Values were normalized with normal cultured WT group. (B): In vitro binding of STAT2 and Nanog promoter was examined by electrophoretic mobility shift assay using cell lysates from F9 cells transfected with pCMV-HA-STAT2. (a): A preliminary experiment was performed to test the binding of STAT2 and WT/MUT probe. (b): The shift band that comprised protein and probe was regulated by RA and Vc as expected. (c): Anti-HA antibody was used for supershift. The second shift band specifically confirmed that the protein binding to the probe was STAT2. (C): In vivo binding of STAT2 and Nanog promoter was examined by ChIP assay. ChIP was performed using anti-STAT2 antibody, anti-IgG as control antibody to detect enriched fragments. The upper part shows agarose gel electrophoresis of polymerase chain reaction (PCR) products. The templates for PCR were indicated. Fold-enrichment was determined by normalizing threshold cycle values of STAT2 ChIP against IgG ChIP. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05; **, p < .01. Abbreviations: ChIP, chromatin immunoprecipitation; MUT, mutation; RA, Retinoic acid; Vc, Vitamin c; WT, wild-type; Ab, antibody. EMSA was then performed to test the in vitro binding of STAT2 and the +272 to +280 bp Nanog fragment. The sequence “gctagTTCTGGGAAcgtcat” repeat was used as a WT nucleic acid probe, and the core sequence (TTCTGGGAA) mutated to CCATGGAGG was used as MUT probe. A preliminary experiment was performed to test the binding of probe (Fig. 4B-a). As shown in Figure 4B-b, there was a shift band in the lane loaded with lysates and WT probe compared with lysates alone, and the binding of protein and probe was regulated by RA and Vc, as expected. Moreover, a specific supershift band was detected using anti-HA antibody, indicating that STAT2 was bound to the probe (Fig. 4B-c). To further confirm the EMSA results and to verify that STAT2 physically occupies the Nanog promoter, we performed ChIP assays. As shown in Figure 4C, a 5.8-fold enrichment of STAT2 was observed. These data collectively indicate that STAT2 directly binds to the Nanog promoter and activates the expression of Nanog. Open in new tabDownload slide Vc signaling pathway is an novel pathway that conserved in mouse embryonic stem cells (ESCs). (A): J1 ESCs were transfected with Nanog promoter reporter construct and treated as indicated for 48 hours. Cell lysates were harvested for dual luciferase reporter (DLR) assays. (B): Relative levels of Nanog mRNA in response to various treatment of RA or Vc as indicated. (C): Relative levels of Nanog protein, and phosphorylation state of JAK2 and STAT2 in response to 48 hours treatment of RA, RA+Vc, or Vc in J1 ESCs. (D, E): J1 ESCs were transfected with pGL3p-S2(D) and pGL3p-S3(E) reporter constructs and treated as indicated for 48 hours. Cell lysates were harvested for DLR assays. (F, G): Verification of STAT3 knockdown. J1 cells were transfected with STAT3-siRNA, cell lysates were harvested, and qPCR(F) and Western blotting (G) were performed to check the efficiency of STAT3 knockdown. (H): Relative levels of Nanog promoter activity in STAT2- or STAT3-knockdown J1 cells. Cells were transfected with STAT2-siRNA or STAT3-siRNA, and treated with Vc as indicated. Cell lysates were harvested for DLR assays. (I, J): Phosphorylation state of STAT2 and STAT3 in response to Vc or LIF treatment. F9(I) or J1(J) cells were treated as indicated for 48 hours. Cell lysates were harvested for Western blotting. (K): Chromatin immunoprecipitation was performed using anti-STAT3 antibody, anti-IgG as control antibody to detect enriched fragments. The upper part shows agarose gel electrophoresis of polymerase chain reaction (PCR) products. The templates for PCR were indicated. (L): Schematic representation of this study. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05; **, p < .01. Abbreviations: LIF, leukemia inhibitory factor; MUT, mutation; pGL3p-S2, pGL3-promoter with STAT2 binding site; pGL3p-S3, pGL3-promoter with STAT3 binding site; p-STAT2, phospho-STAT2; p-STAT3, phospho-STAT3; p-JAK2, phospho-JAK2; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot; WT, wild-type. Open in new tabDownload slide Vc signaling pathway is an novel pathway that conserved in mouse embryonic stem cells (ESCs). (A): J1 ESCs were transfected with Nanog promoter reporter construct and treated as indicated for 48 hours. Cell lysates were harvested for dual luciferase reporter (DLR) assays. (B): Relative levels of Nanog mRNA in response to various treatment of RA or Vc as indicated. (C): Relative levels of Nanog protein, and phosphorylation state of JAK2 and STAT2 in response to 48 hours treatment of RA, RA+Vc, or Vc in J1 ESCs. (D, E): J1 ESCs were transfected with pGL3p-S2(D) and pGL3p-S3(E) reporter constructs and treated as indicated for 48 hours. Cell lysates were harvested for DLR assays. (F, G): Verification of STAT3 knockdown. J1 cells were transfected with STAT3-siRNA, cell lysates were harvested, and qPCR(F) and Western blotting (G) were performed to check the efficiency of STAT3 knockdown. (H): Relative levels of Nanog promoter activity in STAT2- or STAT3-knockdown J1 cells. Cells were transfected with STAT2-siRNA or STAT3-siRNA, and treated with Vc as indicated. Cell lysates were harvested for DLR assays. (I, J): Phosphorylation state of STAT2 and STAT3 in response to Vc or LIF treatment. F9(I) or J1(J) cells were treated as indicated for 48 hours. Cell lysates were harvested for Western blotting. (K): Chromatin immunoprecipitation was performed using anti-STAT3 antibody, anti-IgG as control antibody to detect enriched fragments. The upper part shows agarose gel electrophoresis of polymerase chain reaction (PCR) products. The templates for PCR were indicated. (L): Schematic representation of this study. All data are presented as the mean ± SD and are derived from three independent experiments. *, p < .05; **, p < .01. Abbreviations: LIF, leukemia inhibitory factor; MUT, mutation; pGL3p-S2, pGL3-promoter with STAT2 binding site; pGL3p-S3, pGL3-promoter with STAT3 binding site; p-STAT2, phospho-STAT2; p-STAT3, phospho-STAT3; p-JAK2, phospho-JAK2; RA, Retinoic acid; Vc, Vitamin c; WB, Western blot; WT, wild-type. The Vc-JAK/STAT2-Nanog Pathway Is Independent of the LIF-JAK/STAT3 Pathway and Is Conserved in mESCs To confirm whether regulation of the Vc-JAK/STAT2-Nanog signaling pathway was conserved in mouse ESCs, further experiments were repeated in mouse J1 ESCs. As expected, Nanog promoter activity was upregulated (∼61%) (Fig. 5A), and Vc treatment increased (∼26%) the expression of mRNA of Nanog while antagonizing RA-induced differentiation of ESCs (Fig. 5A, 5B). Most importantly, compared with control, Vc treatment activated JAK2 and STAT2 dramatically (Fig. 5C). Furthermore, the addition of Vc after induction by RA resulted in the recovery of JAK2 and STAT2 phosphorylation levels. These data revealed that Vc regulated the expression of Nanog via the JAK/STAT2 pathway in ESCs. It is well known that the LIF-gp130/JAK-STAT3 signaling pathway is a key factor of mouse ESCs pluripotency [26, 27]. To address the relationship between LIF-JAK-STAT3 and Vc-JAK-STAT2, the binding sites of STAT2 and STAT3 [9] were cloned to the pGL3-promoter vector and transfected to J1 cells for DLR assays. As shown in Figure 5D, 5E, Vc treatment increased the activity of promoter with the STAT2 binding site by 2.67-fold. LIF activated STAT3 and upregulated the activity of promoter with the STAT3 binding site by 3.6-fold, without an effect on STAT2. To rule out the possibility of Vc upregulates Nanog expression through STAT3, a knockdown strategy was used to address this issue. qPCR and Western blot analysis of J1 cells transfected with STAT3-siRNA efficiently knocked down STAT3 expression (Fig. 5F, 5G). As shown in Figure 5H, Vc treatment increased the activity of the Nanog proximal promoter by 1.5-fold when STAT3 was knocked down in ESCs, whereas no effect was observed by knockdown of STAT2. Furthermore, the level of p-STAT2 and p-STAT3 was detected in F9 cells and J1 cells treated with Vc or LIF. As shown in Figure 5I, 5J, LIF increased the phosphorylation of STAT3 without an effect on STAT2, whereas Vc increased the phosphorylation of STAT2 in both F9 and J1 cells. Moreover, STAT3 ChIP was performed to show that STAT3 was not able to occupy the potential site (Fig. 5K). These data suggested that Vc-JAK-STAT2 is a novel pathway, independent of that of LIF-JAK-STAT3. Discussion F9 is a clonal line of mouse teratocarcinoma-derived EC cells which can differentiate into a varied spectrum of cell types. The F9 cell line has been widely used as a model for the analysis of the molecular mechanisms of differentiation and proliferation. The reasons for the use of F9 cells as such a model are: (a) their ability for self-renewal, (b) they maintain an undifferentiated state when cultured under feeder-free conditions in vitro, and (c) they share many similar mechanisms of differentiation and gene regulation with mESCs [28-30]. The LIF-gp130/JAK-STAT3 signaling pathway plays an important role in maintaining the pluripotency of mouse ESCs [26, 27]. F9 cells can maintain an undifferentiated state without the stimulation by LIF. In this study, we performed most of the experiments in F9 cells to investigate the Vc-JAK-STAT2 signaling pathway, which was later confirmed in mESCs, to avoid the interference of the LIF-JAK-STAT3 pathway in mESCs. Vc was found to function in the prevention and treatment of colds, and antivirus, and in antiaging products as well as the prevention and auxiliary treatment of cancer [31-36]. With further developments in research, additional functional roles of Vc have been identified. Wei reported that Vc enhances telomerase activity to promote mesenchymal stem cell sheet formation and tissue regeneration [36]; Huang found that Vc treatment increased the blastocyst formation rate of pig somatic cell nuclear transplantation embryos from 11.5% to 36%, while causing increased histone H4K5 acetylation levels and high expression of stem cell markers Oct4, Sox2, and Klf4 [37]. Recently, it was reported that Vc was able to promote the generation of mouse and human iPSCs [19]; one possible mode of action was that Vc cooperates with histone demethylases Jhdm1a/1b to promote the reprogramming of somatic cells [38]. However, the specific mechanism was unclear. Here, we report that Vc is involved in regulation of Nanog expression in mESCs, which provides new insights to uncover the mechanism of Vc promotion of iPSCs generation. In this study, we discovered a novel signaling pathway that connects Vc to the core circuitry of pluripotency in mESCs (Fig. 5G). Vc is capable of antagonizing RA-induced differentiation and maintaining the pluripotency of ESCs. When ESCs were pretreated with RA for 48 hours they began to differentiate irreversibly, and continued despite the withdrawal of RA. Therefore, RA-induced differentiation of ESCs was shown to be powerful and irreversible. However, treatment with Vc for the following 48 hours reduced the differentiation remarkably. If the cells were treated with RA and Vc simultaneously, the effect of Vc could be mostly covered by the over-powering actions of RA-mediated differentiation, thereby weakening the response of Vc. Also, we show that Vc treatment can significantly improve the activity of the Nanog promoter. Interestingly, another stem marker, Oct4, did not show significant change to Vc treatment, in contrast to Nanog (data not shown). In normally proliferating ESCs, Vc improves the activity of the Nanog promoter dramatically (60%–120%), but only about 20%–40% on mRNA level. This may due to the negative feedback regulation effect of Nanog regulatory network. However, when ESCs were induced to differentiate by RA, the addition of Vc resulted in maintenance of Nanog expression and inhibition of differentiation. Thus, Vc may perform a fine-tuning effect in normally proliferating cells. Similar to Vc treatment, overexpression of STAT2 upregulated Nanog by about 1.5-fold, and knockdown of STAT2 dramatically reduced Nanog expression. Moreover, we discovered STAT2 physically occupies the Nanog promoter and helps to stabilize Nanog expression. Mutation of the STAT2 binding site resulted in loss of Nanog promoter activity to basal levels, suggesting that STAT2 is important for initiation of Nanog transcription. Furthermore, we found that STAT2 is regulated by the Vc-JAK/STAT signaling pathway, and Vc treatment increased the phosphorylation of STAT2. STATs were activated by phosphorylation, and then translocated to nucleus to function as transcription regulators; however, we were unable to see clear evidence of STAT2 translocation because it was mostly localized to the nuclei of normally proliferating ESCs (Supporting Information Fig. S1). This infers that STAT2 is maintained in an activated state in ESCs, consistent with our finding that STAT2 is a important factor for Nanog transcription initiation. Here, we have demonstrated that STAT2 is a positive regulator of Nanog, in vitro and in vivo experiments further demonstrated that STAT2 physically occupies the Nanog promoter. It has been reported that JmjC-domain-containing histone demethylase (JHDM) cooperates with Vc to enhance Oct4 reprogramming [38]. In this study, we found that another pluripotent marker, Nanog, was regulated by Vc. The involvement of JHDM with the Vc-Nanog signaling pathway is potentially interesting for further investigation because JHDM may accelerate DNA demethylation at the promoter region of Nanog, thus helping STAT2 binding, but this needs to be explored further experimentally. Moreover, it is not clear whether its regulatory activity is dependent on other transcription factors. It would be interesting to further examine the association of STAT2 with other factors or regulatory units to determine their interaction and potential for coregulation of downstream target genes. Conclusion This study shows for the first time that Vc regulates the expression of Nanog, and identifies STAT2 as a novel regulator of Nanog that is essential for the initiation of Nanog transcription. We also demonstrate that Vc treatment activates the JAK/STAT signaling pathway, and stimulates the phosphorylation of STAT2. Activated STAT2 binds to the proximal region of the Nanog promoter and maintains high expression levels of Nanog in ESCs. The findings presented here contribute to our understanding of the role of Vc in enhancing the generation of iPSCs. In addition, our study identified a novel transcription regulator that is important for ESC pluripotency, and expanded the regulatory network in ESC maintenance. Acknowledgments We thank Dr. Wenzhong Li for insightful discussions and technical support. This work was funded by the National High Technology Research and Development Program of China (863 Program) (No. 2011AA100303) and Grant (No. 31172279) from National Natural Science Foundation of China. We also thank Prof. Cao of the Fourth Military Medical University for the help in flow cytometry of this work. 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Google Scholar Crossref Search ADS PubMed WorldCat Copyright © 2013 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Vitamin C Enhances Nanog Expression Via Activation of the JAK/STAT Signaling Pathway
JO - Stem Cells
DO - 10.1002/stem.1523
DA - 2014-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/vitamin-c-enhances-nanog-expression-via-activation-of-the-jak-stat-5IxmlJA05u
SP - 166
EP - 176
VL - 32
IS - 1
DP - DeepDyve
ER -