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First published online August 17, 2006
Stem Cells Vol. 24 No. 12 December 2006, pp. 2637 -2648
doi:10.1634/stemcells.2005-0588; www.StemCells.com
© 2006 AlphaMed Press

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EMBRYONIC STEM CELLS: CHARACTERIZATION SERIES

ATP Stimulates Mouse Embryonic Stem Cell Proliferation via Protein Kinase C, Phosphatidylinositol 3-Kinase/Akt, and Mitogen-Activated Protein Kinase Signaling Pathways

Jung Sun Heo, Ho Jae Han

Department of Veterinary Physiology, Biotherapy Human Resources Center, College of Veterinary Medicine, Chonnam National University, Gwangju, Korea

Key Words. ATP • Mitogen-activated protein kinases • Phosphatidylinositol 3-kinase/Akt • Protein kinase C • Embryonic stem cells

Correspondence: Ho Jae Han, D.V.M., Ph.D., Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea. Telephone: 82-62-530-2831; Fax: 82-62-530-2809; e-mail: hjhan{at}chonnam.ac.kr

Received November 24, 2006; accepted for publication August 3, 2006.
First published online in STEM CELLS EXPRESS   August 17, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
This study investigated the effect of ATP and its related signal cascades on the proliferation of mouse ESCs. ATP increased the level of [3H]thymidine/5-bromo-2'-deoxyuridine incorporation and the number of cells in both a time- and dose-dependent manner. AMP-CPP (a P2X1 and P2X3 agonist), ATP-{gamma}S (a P2Y agonist), and 2-methylthio-ATP (a P2X and P2Y agonist) stimulated [3H]thymidine incorporation. P2 purinoceptor antagonists (suramin, reactive blue 2) inhibited the ATP-induced increase in [3H]thymidine incorporation. Reverse transcription-polymerase chain reaction analysis revealed P2X3, P2X4, P2Y1, and P2Y2 expression in mouse ESCs. Adenylate cyclase inhibitor (SQ 22536), phospholipase C inhibitors (neomycin or U 73122), and protein kinase C (PKC) inhibitors (bisindolylmaleimide I or staurosporine) inhibited the ATP-induced increase in [3H]thymidine incorporation. ATP increased the level of intracellular cAMP and inositol phosphates. ATP translocated PKC {alpha}, {delta}, and {zeta} from the cytosol to the membrane compartment. ATP and its agonists increased [Ca2+]i. In addition, the ATP-induced increase in [3H]thymidine incorporation was completely inhibited by a combination of EGTA (extracellular Ca2+ chelator) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (intracellular Ca2+ chelator). ATP phosphorylated Akt and p44/42 mitogen-activated protein kinases (MAPKs) in a time-dependent manner, and either suramin or reactive blue 2 (RB2) blocked the ATP-induced phosphorylation of Akt. Suramin, RB2, the phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin), or the Akt inhibitor inhibited the phosphorylation of p44/42 MAPKs. The ATP-induced increase in [3H]thymidine incorporation was inhibited by wortmannin, the Akt inhibitor, and the MAPK kinase inhibitor (PD 98059). Suramin, RB2, PD 98059, and wortmannin blocked the ATP-induced increase in the cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 levels. In conclusion, ATP stimulates mouse ESC proliferation through PKC, PI3K/Akt, and MAPKs via the P2 purinoceptors.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
ATP is not only a neurotransmitter but also a potent signaling molecule that plays important biological roles in different cell types [1]. Extracellular ATP is an important signaling molecule in many embryonic cell types that increases the intracellular Ca2+ concentration, which can regulate cell proliferation, migration, and differentiation [2, 3]. Therefore, extracellular ATP has the potential to regulate many important processes in embryonic development. Hence, ATP might play an important autocrine/paracrine role in the proliferation of mouse ESCs, which were established as a permanent line of undifferentiated pluripotent cells from early mouse embryos. However, the potential role of purinergic signaling on the proliferation of mouse ESCs is unknown. These effects are mediated by nucleotide receptors known as P2 plasma membrane receptors (P2Rs), which are grouped into two main subfamilies (P2YRs and P2XRs) according to their molecular structure [4, 5]. P2YRs are seven transmembrane-spanning and G-protein-coupled receptors including eight subtypes. Their activation triggers the generation of inositol 1,4,5-trisphosphate, Ca2+ mobilization from the intracellular stores, and in some subtypes, adenylate cyclase stimulation [6, 7]. P2XRs are ligand-gated plasma membrane channels numbering up to seven and are activated directly by extracellular ATP in the absence of additional second messengers besides the monovalent and divalent ion flux [8, 9]. It was reported that the purinergic ATP receptors are expressed in the early stages of embryonic development, which indicates that these receptors play a role in embryogenesis [10, 11]. Although these findings strongly suggest a role of ATP in embryonic development, there are few reports on the function of ATP in the proliferation of mouse embryonic stem cells. Therefore, the precise functions of this receptor in these tissues remain to be determined.

ESCs have the ability to differentiate into all three germ layers and have an unlimited growth potential under certain conditions [12]. The ESCs were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with the leukemia inhibitory factor to maintain their undifferentiated state as well as to support the derivation and expansion of the ESCs [13, 14]. These cells closely resemble their in vivo counterparts and provide a stable in vitro model of embryonic growth and development. In addition, they provide a tool whereby specific signaling systems can be investigated [15, 16]. Therefore, this study examined the effect of ATP on mouse ESC proliferation and its related signaling pathways.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Materials
Mouse ESCs were obtained from the American Type Culture Collection (Manassas, VA, http://www.atcc.org) (ES-E14TG2a). Fetal bovine serum was purchased from BioWhittaker (Walkersville, MD, http://www.cambrex.com). The ATP, AMP-CPP, ATP-{gamma}S, 2-methylthio-ATP (2-MesATP), suramin, reactive blue 2 (RB2), PD 98059, SB 203580, wortmannin, fluorescence isothiocyanate (FITC)-conjugated goat anti-mouse IgM, FITC-conjugated goat anti-rabbit IgM, and ß-actin were obtained from Sigma-Aldrich (St. Louis, http://www.sigmaaldrich.com). [3H]Thymidine and [3H]inositol phosphates were purchased from PerkinElmer (Boston, http://www.perkinelmer.com). Fluo-3/AM was obtained from Molecular Probes Inc. (Eugene, OR, http://probes.invitrogen.com). Anti-Oct4, stage-specific embryonic antigen (SSEA) 1, protein kinase C (PKC) {alpha}, PKC {delta}, PKC {zeta}, cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK4 were acquired from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, http://www.scbt.com). Phospho-p44/42, p44/42, phospho-p38, p38 mitogen-activated protein kinase (MAPK), and phospho-Akt antibody were purchased from New England Biolabs (Hertfordshire, U.K., http://www.neb.com). Goat anti-rabbit IgG was supplied by Jackson Immunoresearch Laboratories (West Grove, PA, http://www.jacksonimmuno.com). Liquiscint was obtained from National Diagnostics (Parsippany, NY, http://www.nationaldiagnostics.com). All other reagents were of the highest purity commercially available.

ESC Culture
The mouse ESCs were cultured in DMEM (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 3.7 g/l sodium bicarbonate, 1% penicillin and streptomycin, 1.7 mM L-glutamine, 0.1 mM ß-mercaptoethanol, 5 ng/ml mouse leukemia inhibitory factor, and 15% fetal bovine serum without a feeder layer for 5 days. The cells were passaged with 0.05% trypsin/EDTA onto gelatinized 12-well plates or into a 60-mm culture dish without a feeder layer and were maintained at 37°C in an air atmosphere containing 5% CO2. After 2–3 days, the cells were washed twice with phosphate-buffered saline (PBS) and maintained in serum-free DMEM including all the supplements. After a 24-hour incubation period, the cells were washed twice with PBS, and incubated with fresh serum-free DMEM including all the supplements and designated agents for the indicated period prior to the experiments.

Alkaline Phosphatase Staining
Approximately 70% confluent mouse ESCs were washed twice with PBS and fixed with 4% formaldehyde (in PBS) for approximately 15 minutes at room temperature. The cells were washed with PBS and incubated with an alkaline phosphatase substrate solution (200 µg/ml naphthol AS-MX phosphate, 2% N,N-dimethylformamide, 0.1 M Tris [pH 8.2], and 1 mg/ml Fast Red TR salt [4-chloro-2-methylbenzenediazonium salt; zinc chloride]) for 10 minutes at room temperature. After being washed with PBS, the cells were photographed.

[3H]Thymidine Incorporation
The [3H]thymidine incorporation experiments were carried out as described by Brett et al. [17]. Zhang et al. [18] reported that most ESCs could be arrest in the G0/G1 phase using a serum deprivation culture. Furthermore, the synchronized ESCs could successfully reenter a normal cell cycle after the serum was resupplied. In this study, the cells were cultured in one well until they reached 50% confluence, washed twice with PBS, and maintained in serum-free DMEM including all supplements. After a 24-hour incubation, the cells were washed twice with PBS and incubated with fresh serum-free DMEM including all the supplements and indicated agents. After the indicated incubation period, 1 µCi of methyl-[3H]thymidine (specific activity: 74 GBq/mmol, 2.0 Ci/mmol; Amersham Biosciences, Buckinghamshire, U.K., http://www.amersham.com) was added to the cultures. Incubation with [3H]thymidine continued for 1 hour at 37°C. The cells were then washed twice with PBS, fixed in 10% trichloroacetic acid (TCA) at 23°C for 15 minutes, and then washed twice with 5% TCA. The acid-insoluble material was dissolved in 2 N NaOH for 12 hours at 23°C. Aliquots were removed to determine radioactivity using a liquid scintillation counter (LS 6500; Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com). The control levels of [3H]thymidine incorporation were determined under the conditions in which the cells were cultured in serum-free DMEM without purinergic agonists. The values are expressed as counts per minute (cpm).

To determine the number of cells, the cells were washed twice with PBS and trypsinized from the culture dishes. The cell suspension was mixed with a 0.4% (wt/vol) trypan blue solution, and the number of live cells was determined using a hemocytometer. Cells failing to exclude the dye were considered nonviable.

5-Bromo-2'-Deoxyuridine Incorporation
The level of 5-bromo-2'-deoxyuridine (BrdU) (a thymidine analogue) incorporation was measured to determine the level of DNA synthesis. The ESCs were serum-starved for 24 hours prior to ATP stimulation. The ESCs were then treated with ATP for 8 hours. Fifteen µM BrdU was then added, and incubation was continued for an additional 1 hour. After several washes with PBS, the cells were fixed with methanol (10% [vol/vol] for 10 minutes at 4°C), followed by incubation in 1 N HCl for 30 minutes at room temperature. The cells were then washed and incubated with 0.1 M sodium tetraborate for 15 minutes. Alexa Fluor 488-conjugated mouse anti-BrdU monoclonal antibody (mAb) (diluted 1:200; Molecular Probes) in 2% bovine serum albumin (BSA)-PBS was incubated overnight at 4°C. After being washed in PBS, coverslips were mounted onto glass slides with a DAKO Fluorescent (DAKO, Glostrup, Denmark, http://www.dako.com) mounting medium using gelvatol and examined under an optical microscope (Fluoview 300; Olympus, Tokyo, http://www.olympus-global.com). The mean ± SE number of BrdU-positive cells per field of vision was determined. At least 10 fields of vision per coverslip were counted.

For the double-labeling experiments, the cells were fixed in acid alcohol and processed for Oct4 staining, followed by BrdU staining. The fixed cells were incubated with rabbit anti-Oct4 antibody (1:100; Santa Cruz Biotechnology) for 1 hour at room temperature and Alexa Fluor 555 anti-rabbit IgG (1:100; Molecular Probes) for 1 hour at room temperature. This was followed by incubation in 1 N HCl, neutralization with 0.1 M sodium tetraborate, and incubation with Alexa Fluor 488-conjugated mouse anti-BrdU mAb for 1 hour at room temperature. After washing with PBS, the BrdU/Oct4-stained cells were examined under confocal microscopy (Fluoview 300; Olympus).

Fluorescence-Activated Cell Sorter Analysis
Cells were incubated with ATP (10–4 M) for 24 hours, and then cells were dissociated in trypsin/EDTA, pelleted by centrifugation, and resuspended at approximately 106 cells per milliliter in PBS containing 0.1% BSA. When required (for Oct4 staining), cells were fixed in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. The cells were labeled with the rabbit anti-Oct4 (1:50) or the mouse anti-SSEA 1 antibody (1:50; Santa Cruz Biotechnology) and then incubated with FITC-conjugated secondary antibodies (1:50). The cells were washed and resuspended in PBS and then read by flow cytometry (Beckman Coulter). Samples were analyzed using CXP software (Beckman Coulter).

Measurement of [Ca2+]i
The changes in [Ca2+]i were monitored using Fluo-3/AM dissolved in dimethyl sulfoxide. The mouse ESCs in 35-mm culture dishes were rinsed twice with Bath solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, 5.5 mM HEPES, pH 7.4), incubated in Bath solution containing 3 µM Fluo-3/AM with 5% CO2, 95% O2 at 37°C for 40 minutes, rinsed twice with the Bath solution, mounted on a perfusion chamber, and scanned every second using confocal microscopy (x400) (Fluoview 300; Olympus). Fluorescence was excited at 488 nm, and the emitted light was read at 515 nm. All analyses of [Ca2+]i were processed at a single-cell level and are expressed as the relative fluorescence intensity.

Inositol Phosphate Formation Assay
The assay was performed using a modification of the method reported by Berridge et al. [19]. The cells were labeled with myo-[3H]inositol (2.5 µCi/ml, 2 ml final) for 24 hours and treated with 10 mM LiCl, which was delivered over a 15-minute period. The cells were then treated with the appropriate agent. The medium was removed, the cells were scraped off the dish in 1.2 ml of H2O, and extracted with 1.8 ml of chloroform/methanol (1:2, vol/vol), and the upper phase was applied to a Bio-Rad AG 1-X8 column (Bio-Rad, Hercules, CA, http://www.bio-rad.com). After washing with 5 mM inositol and H2O, the fraction containing the [3H]inositol phosphates (IP1, IP2, and IP3) was eluted with 1 M ammonium formate and 0.1 N formic acid.

RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
The total RNA was extracted from the mouse ESCs using STAT-60, which is a monophasic solution of phenol and guanidine isothiocyanate purchased from Tel-Test, Inc. (Friendwood, TX, http://www.bioresearchonline.com). Reverse transcription was carried out using 3 µl of RNA using a reverse transcription system kit (AccuPower RT PreMix, Bioneer, Daejeon, Korea, http://www.bioneer.com) with the oligo(dT)18 primers. Five µl of the RT products was then amplified using a polymerase chain reaction (PCR) kit (AccuPower PCR PreMix) under the following conditions: denaturation at 94°C for 5 minutes and 30 cycles at 94°C for 45 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by 5 minutes of extension at 72°C. The primers used were 5'-CGTGAGACTTTGCAGCC-TGA-3' (sense), 5'-GGCGATGTAAGTGATCTGCTG-3' (antisense) for Oct4 (519 base pair [bp]); 5'-TCTTACATCGCGCTCATCAC-3' (sense), 5'-TCTTGACGAAGCAGTCGTTG-3' (antisense) for FOXD3 (171 bp); 5'-GTGGAAACTTTTGTCCGAGAC-3' (sense), 5'-TGGAGTGGGAGGAAGAGGTAAC-3' (antisense) for SOX2 (550 bp); 5'-TGGGTGGGTGTTTGTCTATG-3' (sense), 5'-TGAAGTTGAAGCCTGGAGAC-3' (antisense) for P2X1R (739 bp); 5'-TCCATCATCACCAAAGTCAA-3' (sense), 5'-TTGGGGTAGTGGATGCTGTT-3' (antisense) for P2X2R (392 bp); 5'-GCTTCGGACGCTATGCCAACAA-3' (sense), 5'-AACCACGTCCCCTACCCTCAAGAT-3' (antisense) for P2X3R (470 bp); 5'-TCGGCTCCTCGGACACCCACAG-3' (sense), 5'-CCTAGGAGCGCCAAGCCAGAGC-3' (antisense) for P2X4R (559 bp); 5'-ACGTCAGATGAGTACCTGCG-3' (sense), 5'-CCCTGTCGTTGAAATCACAC-3' (antisense) for P2Y1R (289 bp); and 5'-CTGGTCCGCTTTGCCCGAGATG-3' (sense), 5'-TATCCTGAGTCCCTGCCAAATGAGA-3' (antisense) for P2Y2R (311 bp). PCR for ß-actin was also carried out as a control for the quantity of RNA.

Real-Time Reverse Transcription-Polymerase Chain Reaction
Mouse ESCs were treated with 10–4 M ATP for 24 hours prior to total RNA extraction. The real-time quantification of RNA targets was performed in the Rotor-Gene 2000 real-time thermal cycling system (Corbett Research, New South Wales, Australia, http://www.corbettlifescience.com) using the QuantiTect SYBR Green reverse transcription-polymerase chain reaction (RT-PCR) kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). The primers were 5'-CGTGAGACTTTGCAGCCTGA-3' (sense), 5'-GGCGATGTAAGTGATCTGCTG-3' (antisense) for Oct4. The reaction mixture (20 µl) contained 200 ng of total RNA, 0.5 µM each primer, appropriate amounts of enzymes, and fluorescent dyes as recommended by the supplier. The Rotor-Gene 2000 cycler was programmed as follows: 30 minutes at 50°C for reverse transcription; 15 minutes at 95°C for DNA polymerase activation; 15 seconds at 95°C for denaturing; 45 cycles of 15 seconds at 94°C, 30 seconds at 55°C, and 30 seconds 72°C. The data collection was carried out during the extension step (30 seconds at 72°C). The PCR was followed by a melting cure analysis to verify specificity and identity of the RT-PCR products, which can distinguish the specific PCR products from the nonspecific PCR product resulting from primer-dimer formation. The temperature of PCR products was elevated from 65°C to 99°C at a rate of 1°C per 5 seconds, and the resulting data were analyzed by using the software provided by the manufacturer.

cAMP Assay
The mouse ESCs were preincubated with 100 µM 3-isobutyl-1-methylxanthine for 30 minutes at 37°C to inhibit cAMP degradation and incubated with 10–4 M ATP for 8 hours at 37°C in a humidified 5% CO2, 95% air environment. The samples were homogenized in DMEM containing 4 mM EDTA to inhibit cAMP phosphodiesterase activity using a Polytron PT 1200 (Brinkmann Instruments, Westbury, NY, http://www.brinkmann.com), followed by 5 minutes of incubation at 100°C. After centrifugation at 900g for 5 minutes, the supernatants were transferred into new tubes and stored at 4°C. The intracellular cAMP levels in the samples were examined using a [3H]cAMP assay system kit. The values are expressed as picomoles of cAMP per milligram of protein.

Preparation of Cytosolic and Total Membrane Fractions
The cytosolic and total membrane fractions were prepared using a slight modification of the method reported by Mackman et al. [20]. The medium was removed and replaced with serum-free DMEM including all the supplements contained in the LIF for 12 hours prior to the experiments. After removing the medium, the cells were washed twice with ice-cold PBS, scraped, harvested by microcentrifugation, and resuspended in buffer A (137 mM NaCl, 8.1 mM Na2HPO4, 2.7 mM KCl, 1.5 mM KH2PO4, 2.5 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.5 mM sodium orthovanadate, pH 7.5). The resuspended cells were then mechanically lysed on ice by trituration with a 21.1-gauge needle. The cell lysates were initially centrifuged at 1,000g for 10 minutes at 4°C. The supernatants were collected and centrifuged at 100,000g for 1 hour at 4°C to prepare the cytosolic and total particulate fractions. The supernatant (cytosolic fraction) was then precipitated with 5 volumes of acetone, incubated on ice for 5 minutes, and centrifuged at 20,000g for 20 minutes at 4°C. The resulting pellet was resuspended in buffer A containing 1% (vol/vol) Triton X-100. The particulate fractions containing the membrane fraction were washed twice and resuspended in buffer A containing 1% (vol/vol) Triton X-100. The protein in each fraction was quantified using the Bradford procedure [21].

Western Blot Analysis
The cells were harvested, washed twice with PBS, and lysed with a buffer (20 mM Tris [pH 7.5], 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 mg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM sodium orthovanadate) for 30 minutes on ice. The lysates were then cleared by centrifugation (10 minutes at 15,000 rpm, 4°C), and the protein concentration was determined using the Bradford method [21]. Equal amounts of protein (20 µg) were resolved by electrophoresis by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The blots on the membrane were washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk for 1 hour, and incubated with the appropriate primary antibody at the dilutions recommended by the supplier. The membrane was then washed, and the primary antibodies were detected with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase. The bands were then visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Statistical Analysis
The results are expressed as a mean ± SE. All the experiments were analyzed by analysis of variance, and some experiments were examined by a comparison of the treatment means with the control using the Bonferroni-Dunn test. A p value of <.05 was considered significant.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Effect of ATP on Cell Proliferation
The undifferentiated state of the mouse ESCs used in this experiment was confirmed by examining the expression of the undifferentiated stem cells markers, including the Oct4, FOXD3, and SOX2 expression levels and alkaline phosphatase activity. The mouse ESCs in both the presence and the absence of ATP expressed Oct4, FOXD3, and SOX2 mRNA (Fig. 1A). We used real-time RT-PCR to analyze Oct4 gene expression. As shown in Figure 1B, no significant difference in the gene expression levels of Oct4 was identified in cells in both the presence and the absence of ATP. Moreover, cells in the presence of ATP expressed an Oct4 protein level equivalently to that in the control (Fig. 1C) and maintained the alkaline phosphatase enzyme activity (Fig. 1D). Flow cytometry analysis also showed that cells in the presence of ATP expressed 92% Oct4-positive (control, 90%) and 88% SSEA1-positive (control, 90%), respectively (Fig. 1E). Therefore, the results demonstrate that mouse ESCs maintained an undifferentiated state under the experimental conditions used in this study.


Figure 1
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  		Figure 1. Effect of ATP on the characterization of mouse ESCs. (A): Oct4 (519 bp), FOXD 3 (171 bp), SOX 2 (550 bp), and ß-actin (350 bp) mRNA expression levels in the presence or absence of ATP. (B): Real-time reverse transcription-polymerase chain reaction analysis of Oct4 in the presence or absence of ATP. (C): Oct4 and ß-actin protein expression levels in the presence or absence of ATP. The bands represent 50–60 kDa of Oct4 and 41 kDa of ß-actin. (D): The alkaline phosphatase enzyme activity was measured cells in the presence or absence of ATP (10–4 M), as described in Materials and Methods. (E): Flow cytometry analysis to monitor the percentage of Oct4 and SSEA 1 positive in cells in the presence or absence of ATP. The left panel shows Oct4 staining, and the right panel shows SSEA 1 staining. Abbreviations: FITC, fluorescein isothiocyanate; SSEA, stage-specific embryonic antigen.

 
The effect of ATP on cell proliferation was examined by treating mouse ESCs with ATP (10–4 M) for various time periods (0–12 hours) or doses (0–5 x 10–4 M) for 8 hours. As shown in Figure 2A, the maximum increase in the level of [3H]thymidine incorporation was observed after an 8-hour incubation with ATP (10–4 M) (74% increase compared with the control; p < .05). ATP, at ≥10–6 M, also increased the level of [3H]thymidine incorporation in a dose-dependent manner over an 8-hour incubation period (Fig. 2B). In addition, the number of BrdU-labeled cells was also increased in response to ATP, which is consistent with the observed increase in the level of [3H]thymidine incorporation (Fig. 2C). To validate these results, double labeling for Oct4 and BrdU expression was performed to determine whether ATP exerts its growth-promoting effect on undifferentiated ESCs. In these experiments, the ESC population contained more than 90% of undifferentiated (Oct4-positive) cells, which was identified by flow cytometry analysis. The observed effects reflect the role of ATP in the undifferentiated ESCs, not in the spontaneously differentiated progeny (Fig. 2D). Finally, a significant increase in the number of cells was observed after incubation with ATP (10–6 to 5 x 10–4 M) for 24 hours (Fig. 2E). To determine whether the P2 purinoceptors mediated the effect of ATP on cell proliferation, the mouse ESCs were treated with the selective agonists or antagonists. As shown in Figure 3A, AMP-CPP (P2X1 and P2X 3 receptor agonist), ATP-{gamma}S (P2Y receptor agonist), and 2-MesATP (P2X and P2Y receptor agonist; 10–4 M) stimulated [3H]thymidine incorporation, which suggests that both P2X and P2Y receptors can mediate ATP-induced cell proliferation. Suramin (a nonselective P2X and P2Y receptor antagonist; 10–5 M) and reactive blue 2 (P2Y receptor antagonist; 10–6 M) inhibited the ATP-induced increase in [3H]thymidine incorporation (Fig. 3B). In addition, the mouse ESCs expressed the P2X3, P2X4, P2Y1, and P2Y2 receptors but not P2X1 or P2X2 (Fig. 3C).


Figure 2
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  		Figure 2. Effects of ATP on [3H]thymidine and BrdU incorporation. (A): Mouse ESCs were incubated in the presence of ATP (10–4 M) for various times (0–12 hours) under serum-free conditions and subsequently pulsed with 1 µCi of [3H]thymidine for 1 hour prior to counting. (B): Mouse ESCs were incubated with various ATP concentrations (0–5 x 10–4 M) for 8 hours and pulsed with 1 µCi of [3H]thymidine for 1 hour. (C): BrdU-positive cells in response to different ATP concentrations (0–5 x 10–4 M) for 24 hours. The mean ± SE number of BrdU-positive cells per field of vision was determined. At least 10 fields of vision per coverslip were counted. (D): Mouse ESCs were incubated with ATP (10–4 M) for 24 hour and double-labeled with Oct4 and BrdU antibody. Scale bars = 20 µm. (E): Mouse ESCs were treated with ATP (0–5 x 10–4 M) for 24 hours, and the number of cells was counted using a hemocytometer. The values represent mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus control. Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; hr, hour.

 


Figure 3
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  		Figure 3. Effect of ATP agonists and P2 purinoceptor antagonists on [3H]thymidine incorporation and mRNA expression of the P2 purinoceptors. (A): Mouse ESCs were treated with ATP, AMP-CPP, ATP-{gamma}S, and 2-MesATP (10–4 M) for 8 hours and then pulsed with 1 µCi of [3H]thymidine for 1 hour. (B): Mouse ESCs were pretreated with either suramin (10–5 M) or RB2 (10–6 M) for 30 minutes prior to the ATP treatment for 8 hours, and then pulsed with 1 µCi of [3H]thymidine for 1 hour. The values represent mean ± SE of five independent experiments with triplicate dishes. *, p < .05 versus control; #, p < .05 versus ATP alone. (C): The total RNA from mouse ESCs was reverse transcribed, and the P2X and P2Y receptor cDNA was amplified by polymerase chain reaction as described in Materials and Methods. The example shown is a representative of three experiments. Abbreviations: 2-MesATP, 2-methylthio-ATP; RB2, reactive blue 2.

 
Involvement of cAMP/Phospholipase C/PKC in ATP-Induced Cell Proliferation
The effect of SQ 22536 (adenylate cyclase inhibitor; 10–6 M), neomycin or U 73122 (phospholipase C [PLC] inhibitors; 10–6 M), and bisindolylmaleimide I or staurosporine (PKC inhibitors; 10–7 M) on ATP-induced cell proliferation was examined to determine the pathway involved in ATP-induced cell proliferation. All these inhibitors completely blocked the ATP-induced increase in [3H]thymidine incorporation (Fig. 4A). Moreover, ATP increased the intracellular cAMP content (15 ± 5 [control] vs. 25 ± 4 [ATP] pmol of cAMP per mg of protein) (Fig. 4B). An increase in IP3 formation was observed in a time-dependent manner, with the maximum effect being observed at 60 seconds (98 ± 8% compared with the control) (Fig. 4C). Subsequently, ATP induced the translocation of PKC {alpha}, {delta}, and {zeta} from the cytosol to the membrane compartment (Fig. 4D).


Figure 4
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  		Figure 4. Effect of AMP and phospholipase C/PKC inhibitors on ATP-induced [3H]thymidine incorporation. (A): Mouse ESCs were pretreated with SQ 22536 (10–6 M), neomycin (10–4 M), or U 73122 (10–6 M) and with staurosporine or bisindolylmaeimide I (10–6 M) for 30 minutes prior to the ATP (10–4 M) treatment for 8 hours and then pulsed with 1 µCi of [3H]thymidine for 1 hour. (B): Mouse ESCs were treated with ATP for 24 hours before the cAMP assay. (C): The formation of inositol phosphates was measured after the ATP treatment for various times (0–120 seconds). The values represent mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus control; #, p < .05 versus ATP alone. (D): PKC {alpha}, {delta}, and {zeta} isoforms present in either the cytosolic or membrane compartments were detected by Western blotting as described in Materials and Methods. The bands represent 80–90 kDa for PKC {alpha}, {delta}, and {zeta} and 41 kDa for ß-actin. Each example shown is a representative of three experiments. Abbreviations: IPs, inositol phosphates; PKC, protein kinase C; sec, seconds.

 
Involvement of [Ca2+]i in ATP-Induced Cell Proliferation
The intracellular Ca2+ mobility in response to ATP and its agonists was examined to determine whether ESC proliferation involves the ATP-induced increase in [Ca2+]i. As shown in Figure 5A, ATP induced an increase in [Ca2+]i. In addition, [Ca2+]i was increased by AMP-CPP, ATP-{gamma}S, and 2-MesATP. The mechanisms for the ATP-induced increase in [Ca2+]i were determined. BAPTA-AM (an intracellular calcium chelator; 10–5 M) or EGTA (an extracellular calcium chelator; 4 x 10–3 M) partially blocked the ATP-induced increase in [Ca2+]i (Fig. 5B). A pretreatment with EGTA alone (35% decrease; p < .05) or EGTA plus BAPTA-AM (43% decrease; p < .01) inhibited the ATP-induced increase in [3H]thymidine incorporation but BAPTA-AM had no effect (Fig. 5C).


Figure 5
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  		Figure 5. Effect of ATP and ATP agonists on [Ca2+]i. (A): Mouse ESCs were loaded with 2 µM fluo-3-AM in serum-free medium for 40 minutes and treated with ATP (10–4 M). (B): Mouse ESCs were pretreated with either EGTA (4 x 10–3 M) or BAPTA-AM (10–5 M) for 30 minutes, and the ATP-induced Ca2+ influx was then measured. (C): Mouse ESCs were pretreated with EGTA (4 x 10–3 M), BAPTA-AM (10–5 M), or a combined treatment of EGTA and BAPTA-AM for 30 minutes prior to the ATP treatment for 8 hours and pulsed with 1 µCi of [3H]thymidine for 1 hour. Changes in [Ca2+]i were monitored using confocal microscopy and are expressed as the relative fluorescence intensity. Each example shown is a representative of five experiments. The values represent mean ± SE of three independent experiments with triplicate dishes. *, p < .05 versus control; #, p < .05; {diamondsuit}, p < .01 versus ATP alone. Abbreviations: 2-MesATP, 2-methylthio-ATP; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM; RFI, relative fluorescence intensity; sec, seconds.

 
Involvement of PI3K/MAPKs in ATP-Induced Cell Proliferation
ATP induced the phosphorylation of Akt in a time-dependent manner, which was inhibited by either suramin or RB2 (Fig. 6A, 6B). The ATP-induced phosphorylation of p44/42 MAPKs was also observed, which first appeared 10 minutes after stimulation with ATP (Fig. 6C). Suramin, RB2, wortmannin, or the Akt inhibitor inhibited the ATP-induced phosphorylation of p44/42 MAPKs (Fig. 6D, 6E). In an attempt to determine whether the ATP-induced increase in [3H]thymidine incorporation is involved in the phosphatidylinositol 3-kinase (PI3K) or MAPKs pathways, the cells were pretreated with wortmannin (PI3K inhibitor; 10–6 M), the Akt inhibitor (10–5 M), or PD 98059 (MEK inhibitor; 10–5 M) prior to ATP (10–4 M) treatment. These inhibitors blocked the ATP-induced increase in [3H]thymidine incorporation (Fig. 6F). The alteration of the cell cycle regulators in response to ATP was examined. ATP increased the levels of the cyclin D1, cyclin E, CDK2, and CDK4 proteins in a time-dependent manner (Fig. 7A). However, suramin, RB2, wortmannin, or PD 98059 inhibited the ATP-induced increase in these protein levels (Fig. 7B, 7C).


Figure 6
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  		Figure 6. Effect of ATP on the phosphorylation of Akt and mitogen-activated protein kinases (MAPKs). Mouse ESCs were treated with ATP (10–4 M) for various times (0–90 minutes), and the phosphorylation of Akt (A) and p44/42 MAPKs (C) was detected. Mouse ESCs were pretreated with either suramin (10–5 M) or RB2 (10–6 M) for 30 minutes prior to the ATP treatment, and the phosphorylation of Akt (B) and p44/42 MAPKs (D) was detected. Mouse ESCs were pretreated with either wortmannin (10–6 M) or Akt inhibitor (10–5 M) for 30 minutes prior to the ATP treatment, and the phosphorylation of p44/42 MAPKs was then detected (E). Each example shown is a representative of three experiments. The lower panels (bars) denote the mean ± SE of three experiments for each condition determined from densitometry relative to the total Akt or total p44/42 MAPKs, respectively. (F): Mouse ESCs were pretreated with wortmannin (10–6 M), Akt inhibitor (10–5 M), or PD 98059 (10–6 M) for 30 minutes prior to the ATP treatment for 8 hours and then pulsed with 1 µCi of [3H]thymidine for 1 hour. The values represent mean ± SE of four independent experiments with triplicate dishes. *, p < .05 versus control; #, p < .05 versus ATP alone. Abbreviations: min, minute; RB2, reactive blue 2.

 


Figure 7
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  		Figure 7. Effect of ATP on cell cycle regulators. Mouse ESCs were treated with ATP (10–4 M) for various times (0–9 hours) (A) or pretreated with suramin (10–5 M), RB2 (10–6 M), wortmannin (10–6 M), or PD 98059 (10–6 M) for 30 minutes prior to incubating the cells with ATP (10–4 M) for 3 hours (B, C). The total lysates were then subjected to SDS-polyacrylamide gel electrophoresis and blotted with the cyclin D1, cyclin E, CDK2, or CDK4 antibody. Each example shown is a representative of four experiments. The lower panels (bars) denote the mean ± SE of four experiments for each condition determined from densitometry relative to ß-actin. *, p < .05 versus control; #, p < .05 versus ATP alone. Abbreviations: CDK, cyclin-dependent kinase; hr, hours; RB2, reactive blue 2.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
Although many studies have examined the functional role of P2 receptors on extracellular ATP using various cell types, there are no previous pharmacological or expression reports of the P2 receptors on mouse ESCs. Mouse ESCs have unusual proliferative properties [22]. Their derivation does not rely on any immortalizing agent, they do not undergo senescence, and they can proliferate with no apparent limit. These cells can also multiply in the absence of serum and are not subject to contact inhibition or anchorage dependence. In previous reports, various concentrations of ATP were used in various cell types and under a wide variety of experimental conditions. The intracellular ATP concentrations were in the millimolar range (1–10 mM). However, the extracellular ATP concentrations rely on a balance between its release and degradation, and estimates range from nanomolar to micromolar concentrations, with the maximum extracellular ATP concentration being measured at 10 µM [23]. Moreover, the EC50 values for ATP range from 0.7 to 15 µM for the P2X1–6 receptors but the P2X7 receptor requires 300 µM to millimolar ATP to elicit the signal transduction pathways [24]. In addition, many studies have reported the application of 10–5-10–4 M ATP [25, 26]. In this study, ATP at ≥10–6 M significantly increased the level of [3H]thymidine incorporation in a dose-dependent manner over an 8-hour incubation period. The differences in the effectiveness of the various ATP concentrations can be attributed to the unknown ATP quality, the difference in the cell types, marker indices, or the experimental conditions (such as in vitro vs. in vivo, serum vs. serum-free media).

Among the P2X receptors examined, the P2X3 receptor had the highest expression level followed by P2X4. This suggests the potential role of these receptors in the regulation of mouse ESCs. Recently, the expression of the P2Y1 receptor mRNA and protein was demonstrated during chick embryonic development [11, 27]. Meyer et al. [11] reported strong expression of P2Y1 receptor mRNA in undifferentiated limb mesenchyme cells. However, the expression was lost when the cells differentiated. A previous study identified all the P2XR subtypes but identified only P2Y1 and P2Y2 among the P2YRs subtypes in human hematopoietic stem cells [28]. This demonstrates that P2Y1 and P2Y2 are expressed in mouse ESCs where they have the potential to regulate cell proliferation. Furthermore, P2X and P2Y agonists, AMP-CPP, and ATP-{gamma}S or 2-MesATP induced mouse ESC proliferation. On the other hand, it was reported that various cellular responses are regulated by the degradation products of ATP, including AMP [29]. However, AMP is not associated with the P2R-induced biological effects of the extracellular nucleotides.

Although there is evidence of a link between purinergic receptor activation and the PKC pathway, there is no report of a link between the activation of these pathways by the stimulation of the purinergic receptors and the proliferation of mouse ESCs. PKC is involved in the transducing signals from the purinergic receptors in RBA-2 cells [30]. In U138-MG cells, the inhibition of the Ca2+-dependent and -independent PKCs with GF 109203X caused significant inhibition of the ATP-induced increase in [3H]thymidine incorporation, as well as in the number of cells [31]. These results also suggest that PKC is an important mediator of the mitogenic stimuli triggered by ATP in mouse ESCs. In chick embryonic retinal cells, ATP increased the level of DNA synthesis by mediating the P2Y1 receptor linked to PLC/ PKC activation [32]. In addition, calcium influx via the P2 purinoceptor was also correlated with the level of cell proliferation of an embryonic chick retina [33]. The increase in [Ca2+]i is an important intracellular signal in various cell types. It was reported that [Ca2+]i in mouse ESCs is increased by the store-operated Ca2+ channels as well as by the inositol 1,4,5-triphosphate-induced release from the endoplasmic reticulum [34]. ATP and its analogues have been demonstrated to induce the accumulation of inositol phosphate and increase in [Ca2+]i in several cell types [35], which is consistent with the finding obtained in this study. The ATP-induced Ca2+ response is due to Ca2+ entry through the plasma membrane [36], and the ATP-induced increase in intracellular Ca2+ occurs via both the ATP-gated nonspecific cation channels (P2X ion channels) and the P2Y receptors [26]. These results suggest that the increase in [Ca2+]i stimulates the mitogenic activity, which is supported by the observation that the removal of Ca2+ by BAPTA-AM plus EGTA decreased the level of [3H]thymidine incorporation in C6 glioma cells [25].

This study also showed that the P2X and P2Y receptors are linked to the activation of the PI3K/Akt pathway in mouse ESCs. The PI3K pathway is important for the proliferation, survival, and maintenance of pluripotency in ESCs [37]. Therefore, this study examined the effects of extracellular ATP on PI3K/Akt phosphorylation, which is the downstream pathway of PI3K, as well as the correlation between this effect and the proliferation of mouse ESCs. In some cells, PKCs occur upstream of Akt and either activate or inactivate Akt depending on the cell type and the PKC isoform involved [38, 39]. Moreover, Ca2+ influx is also involved in the P2X7 receptor-mediated phosphorylation of Akt in astrocytes [40]. It was previously reported that Ca2+ influx activates the Ca2+-dependent tyrosine kinases, and the subsequent Ras activation could lead to Akt phosphorylation [41]. On the other hand, it was reported that Ca2+ could also trigger Akt phosphorylation directly through Ca2+/calmodulin-dependent protein kinase [42]. Akt is recognized as a downstream PI3K target, which plays a key role in a variety of biological effects, cell survival, and the cell cycle [37]. In addition to its well-known role in various cell survival and metabolic responses, Akt was reported to play an important role in regulating the cell cycle [43, 44]. However, the contribution of Akt to cell proliferation was suggested to be cell type-specific and stimulus-dependent.

Many studies using somatic cells have demonstrated that PI3K and p44/42 MAPKs are essential for mediating the mitogen-induced growth responses. Moreover, p44/42 MAPKs are involved in the signaling cascades that regulate several major cellular functions, including cell proliferation and differentiation. The p44/42 MAPKs lie downstream of PI3K in many cell systems and mitogenic signaling pathways [45–48]. Previous studies have demonstrated that the ATP-induced phosphorylation of extracellular signal-related kinase (ERK)1/2 in human monocytic cells [49] and HeLa cells [50] is mediated by PI3K linked to the P2Y2 receptor. In other cell systems, ERK1/2 and PI3K act independently but in parallel to promote cell growth [51, 52]. The latter situation appears to be the case for ATP-stimulated proliferation of adventitial fibroblasts. In ESCs, p44/42 MAPKs signaling inhibits self-renewal and promotes cell differentiation [22, 53]. However, in contrast to previous reports, it was found that ATP-induced signaling of p44/42 MAPKs contributes to ESC proliferation without differentiation. Similarly, basic fibroblast growth factor contributes to the maintenance of human ESCs, at least in part, through the MEK1/ERK pathway [54]. The ERK1/2 signaling pathway was reported to play an important role in the mitogenic/survival effect on central nervous system stem cells by PDGF-AA, but not in the initial steps of neuronal differentiation [55]. Moreover, MEKK1 plays a key role in self-renewal and STAT3 activation in Bcr-Abl-transformed ESCs [56]. Although it is unclear how p44/42 MAPKs contributes to the proliferation of mouse ESCs, these results demonstrate that p44/42 MAPKs signaling is essential for regulating the process of self renewal and propagation in mouse ESCs under the conditions examined in this study.

There is evidence showing that extracellular ATP enhances the expression of the cell cycle regulator proteins [1, 57]. In various cell types, the cyclin D-associated CDK activities are essential for the G1 to S phase transition [58]. In ESCs, cyclin D-CDK4/6 complexes are inactive and cyclin A/E-CDK2 complexes are constitutively active throughout the cell cycle [59]. However, the recent reports have demonstrated that the cyclin D-associated CDK activities are regulated by mitogenic signaling and are involved in the G1 to S phase transition in ESCs [16, 60, 61]. Cyclin D/CDK4 has a noncatalytic function, which sequestrates CDK inhibitors (CKIs), including p27Kip1 and p21Cip1, which are complexed to cyclin D-dependent kinases [62]. Once the Cip/Kip sequestration downregulates the CKI levels, cyclin E/CDK2 can facilitate its activation by phosphorylation of p27Kip1 to induce its degradation [63, 64]; that is, the CDK2 activity requires inactivation of the Cip/Kip proteins and is therefore dependent on prior activation of the cyclin D/CDK4 pathway. Once CDK2 becomes active, it reinforces CDK4 to complete Rb phosphorylation and also triggers the degradation of p27Kip1. This changes the program to reduce the dependence of the cell on mitogens for completion of the cell cycle and, in this sense, results in an irreversible commitment of cells to enter S phase. This reestablishes the requirement for cyclin D and reinstitutes a period of mitogen dependence in the ensuing G1 phase of the next cell cycle [58]. Thus, the functional significance of cyclin D/CDK4 complexes in ESCs might be identified to sequester p27Kip1 and prevent this inhibitor acting on cyclin E/CDK2 [65]. Therefore, cyclin D-dependent kinases may play a role in controlling the cell cycle of ESCs. The regulation of cyclin D1 expression is mediated by the Ras/ERK signaling pathways [66]. Raf/MEK/ERK and PI3K/Akt signaling pathways can act in synergy to promote the G1-S phase cell cycle progression in both normal and cancer cells [67]. The promoter for cyclin D1 contains an AP-1 site, and the ectopic expression of either c-fos or c-jun induces cyclin D1 mRNA expression [1]. In ESCs, the PI3-K-dependent signaling pathways also regulate cyclin D1 expression [60]. In this study, the control of the cell cycle regulatory proteins (cyclin D1, cyclin E, CDK2, and CDK4) were dependent on the PI3K and p44/42 MAPK pathways. This suggests that extracellular ATP alone is sufficient to induce cell cycle progression beyond the G1 phase of the cell cycle. This also suggests that once the ATP receptors are activated, PKC transmits signals to the nucleus through one or more of the MAPK cascades, which may include Raf-1, MEK, and ERK, and activate transcription factors such as myc, max, fos, and jun. Therefore, these results show that extracellular ATP plays an important physiological role during mammalian embryo development by stimulating the proliferation of mouse ESCs and might be a novel and powerful tool for modulating the mouse ESCs functions. In conclusion, P2X and P2Y purinergic receptors can mediate the proliferation of mouse ESCs through cellular pathways that are dependent on PKC, PI3K/Akt, and p44/42 MAPKs.


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
References
 
This research was supported by Grant SC 2210 from the Stem Cell Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology. We acknowledge a graduate fellowship provided by the Ministry of Education and Human Resources Development through the Brain Korea 21 project, Republic of Korea.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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